WO2003064628A2

WO2003064628A2 - Novel proteins and nucleic acids encoding same

Info

Publication number: WO2003064628A2
Application number: PCT/US2003/003401
Authority: WO
Inventors: John P. Ii Alsobrook; Joel S. Bader; Constance Berghs; Catherine E. Burgess; Stacie J. Casman; Elina Catterton; Amitabha Chaudhuri; Shlomit R. Edinger; Karen Ellerman; Valerie L. Gerlach; Linda Gorman; Xiaojia Guo; John L. Herrmann; Weizhen Ji; Nikolai V. Khramtsov; Li Li; Charles E. Miller; Tatiana Ort; Meera Patturajan; Luca Rastelli
Original assignee: Curagen Corporation
Priority date: 2002-02-01
Filing date: 2003-02-03
Publication date: 2003-08-07
Also published as: WO2003064628A3

Abstract

The present invention provides novel isolated polynucleotides and small molecule target polypeptides encoded by the polynucleotides. Antibodies that immunospecifically bind to a novel small molecule target polypeptide or any derivative, variant, mutant or fragment of that polypeptide, polynucleotide or antibody are disclosed, as are methods in which the small molecule target polypeptide, polynucleotide and antibody are utilized in the detection and treatment of a broad range of pathological states. More specifically, the present invention discloses methods of using recombinantly expressed and/or endogenously expressed proteins in various screening procedures for the purpose of identifying therapeutic antibodies and therapeutic small molecules associated with diseases. 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

NOVEL PROTEINS AND NUCLEIC ACIDS ENCODING SAME

RELATED APPLICATIONS

This application is a CIP of USSN 09/783436, filed 14-Feb-01; USSN 09/981566, filed 16-Oct-01; USSN 10/023681, filed 18-Dec-01; and claims priority to USSN 60/356371, filed 12-Feb-02; USSN 60/366802, filed 22-Mar-02; USSN 60/389910, filed 19-Jun-02; USSN 60/402395, filed 9-Aug-02; USSN 60/359848, filed 27-Feb-02; USSN 60/389531, filed 18-Jun-02; USSN 60/402867, filed 12-Aug-02; USSN 60/353287, filed l-Feb-02; USSN 60/365049, filed 15-Mar-02; USSN 60/405820, filed 23-Aug-02; USSN 60/356531, filed 13-Feb-02; USSN 60/405401, filed 23-Aug-02, USSN 60/359603, filed 26-Feb-02; USSN 60/353301, filed l-Feb-02; USSN 60/381666, filed 17-May-02; USSN 60/393265, filed 2-Jul-02; USSN 60/401825, filed 7-Aug-02; USSN 60/391516, filed 25-Jun-02; USSN 60/356424, filed 12-Feb-02; USSN 60/358239, filed 20-Feb-02; and USSN 60/359860, filed 27-Feb-02, each of which is incoφorated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel polypeptides that are targets of small molecule drugs and that have 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

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

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

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

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

Small molecule targets have been implicated in various disease states or pathologies. These targets may be proteins, and particularly enzymatic proteins, which are acted upon by small molecule drugs for the purpose of altering target function and achieving a desired result. Cellular, animal and clinical studies can be performed to elucidate the genetic contribution to the etiology and pathogenesis of conditions in which small molecule targets are implicated in a variety of physiologic, pharmacologic or native states. These studies utilize the core technologies at CuraGen Corporation to look at differential gene expression, protein-protein interactions, large-scale sequencing of expressed genes and the association of genetic variations such as, but not limited to, single nucleotide polymorphisms (SNPs) or splice variants in and between biological samples from experimental and control groups. The goal of such studies is to identify potential avenues for therapeutic intervention in order to prevent, treat the consequences or cure the conditions.

In order to treat diseases, pathologies and other abnormal states or conditions in which a mammalian organism has been diagnosed as being, or as being at risk for becoming, other than in a normal state or condition, it is important to identify new therapeutic agents. Such a procedure includes at least the steps of identifying a target component within an affected tissue or organ, and identifying a candidate therapeutic agent that modulates the functional attributes ofthe target. The target component may be any biological macromolecule implicated in the disease or pathology. Commonly the target is a polypeptide or protein with specific functional attributes. Other classes of macromolecule may be a nucleic acid, a polysaccharide, a lipid such as a complex lipid or a glycolipid; in addition a target may be a sub-cellular structure or extra-cellular structure that is comprised of more than one of these classes of macromolecule. Once such a target has been identified, it may be employed in a screening assay in order to identify favorable candidate therapeutic agents from among a large population of substances or compounds.

In many cases the objective of such screening assays is to identify small molecule candidates; this is commonly approached by the use of combinatorial methodologies to develop the population of substances to be tested. The implementation of high throughput screening methodologies is advantageous when working with large, combinatorial libraries of compounds.

SUMMARY OF THE INVENTION

The invention includes nucleic acid sequences and the novel polypeptides they encode. The novel nucleic acids and polypeptides are referred to herein as NOVX, or NOV1, NOV2, NOV3, etc., nucleic acids and polypeptides. These nucleic acids and polypeptides, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as "NOVX" nucleic acid, which represents the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 79, or polypeptide sequences, which represents the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 79.

In one aspect, the invention provides an isolated polypeptide comprising a mature form of a NOVX amino acid. One example is a variant of a mature form of a NOVX amino acid sequence, wherein any amino acid in the mature form is changed to a different amino acid, provided that no more than 15% ofthe amino acid residues in the sequence of the mature form are so changed. The amino acid can be, for example, a NOVX amino acid sequence or a variant of a NOVX amino acid sequence, 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 includes fragments of any of these. In another aspect, the invention also includes an isolated nucleic acid that encodes a NOVX polypeptide, or a fragment, homolog, analog or derivative thereof.

Also included in the invention is a NOVX polypeptide that is a naturally occurring allelic variant of a NOVX sequence. In one embodiment, the allelic variant includes an amino acid sequence that is the translation of a nucleic acid sequence differing by a single nucleotide from a NOVX nucleic acid sequence. In another embodiment, the NOVX polypeptide is a variant polypeptide described therein, wherein any amino acid specified in the chosen sequence is changed to provide a conservative substitution. In one embodiment, the invention discloses a method for determining the presence or amount of the NOVX polypeptide in a sample. The method involves the steps of: providing a sample; introducing the sample to an antibody that binds immunospecifically to the polypeptide; and determining the presence or amount of antibody bound to the NOVX polypeptide, thereby determining the presence or amount of the NOVX polypeptide in the sample. In another embodiment, the invention provides a method for determining the presence of or predisposition to a disease associated with altered levels of a NOVX polypeptide in a mammalian subject. This method involves the steps of: measuring the level of expression of the polypeptide in a sample from the first mammalian subject; and comparing the amount of the polypeptide in the sample of the first step to the amount of the polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, the disease, wherein an alteration in the expression level of the polypeptide in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.

In a further embodiment, the invention includes a method of identifying an agent that binds to a NOVX polypeptide. This method involves the steps of: introducing the polypeptide to the agent; and determining whether the agent binds to the polypeptide. In various embodiments, the agent is a cellular receptor or a downstream effector.

In another aspect, the invention provides 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 NOVX polypeptide. The method involves the steps of: providing a cell expressing the NOVX polypeptide 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 ofthe substance, the substance is identified as a potential therapeutic agent. In another aspect, the invention describes a method for screening for a modulator of activity or of latency or predisposition to a pathology associated with the NOVX polypeptide. This method involves the following steps: administering a test compound to a test animal at increased risk for a pathology associated with the NOVX polypeptide, wherein the test animal recombinantly expresses the NOVX polypeptide. This method involves the steps of measuring the activity of the NOVX polypeptide in the test animal after administering the compound of step; and comparing the activity of the protein in the test animal with the activity of the NOVX polypeptide in a control animal not administered the polypeptide, wherein a change in the activity of the NOVX 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 NOVX polypeptide. In one embodiment, the test animal is a recombinant test animal that expresses a test protein transgene or expresses the transgene under the control of a promoter at an increased level relative to a wild-type test animal, and wherein the promoter is not the native gene promoter of the transgene. In another aspect, the invention includes a method for modulating the activity of the NOVX polypeptide, the method comprising introducing a cell sample expressing the NOVX polypeptide with a compound that binds to the polypeptide in an amount sufficient to modulate the activity of the polypeptide.

The invention also includes an isolated nucleic acid that encodes a NOVX polypeptide, or a fragment, homolog, analog or derivative thereof. In a preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence of a naturally occurring allelic nucleic acid variant. In another embodiment, the nucleic acid encodes a variant polypeptide, wherein the variant polypeptide has the polypeptide sequence of a naturally occurring polypeptide variant. In another embodiment, the nucleic acid molecule differs by a single nucleotide from a NOVX nucleic acid sequence. In one embodiment, the NOVX nucleic acid molecule hybridizes under stringent conditions to the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 79, or a complement of the nucleotide sequence. In another aspect, the invention provides a vector or a cell expressing a NOVX nucleotide sequence.

In one embodiment, the invention discloses a method for modulating the activity of a NOVX polypeptide. The method includes the steps of: introducing a cell sample expressing the NOVX polypeptide with a compound that binds to the polypeptide in an amount sufficient to modulate the activity of the polypeptide. In another embodiment, the invention includes an isolated NOVX nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising a NOVX amino acid sequence or a variant of a mature form of the NOVX amino acid sequence, 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. In another embodiment, the invention includes an amino acid sequence that is a variant of the NOVX amino acid sequence, 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.

In one embodiment, the invention discloses a NOVX nucleic acid fragment encoding at least a portion of a NOVX polypeptide 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. In another embodiment, the invention includes the complement of any ofthe NOVX nucleic acid molecules or a naturally occurring allelic nucleic acid variant. In another embodiment, the invention discloses a NOVX nucleic acid molecule that encodes a variant polypeptide, wherein the variant polypeptide has the polypeptide sequence of a naturally occurring polypeptide variant. In another embodiment, the invention discloses a NOVX nucleic acid, wherein the nucleic acid molecule differs by a single nucleotide from a NOVX nucleic acid sequence.

In another aspect, the invention includes a NOVX nucleic acid, wherein one or more nucleotides in the NOVX nucleotide sequence is changed to a different nucleotide provided that no more than 15% of the nucleotides are so changed. In one embodiment, the invention discloses a nucleic acid fragment of the NOVX nucleotide sequence and a nucleic acid fragment wherein one or more nucleotides in the NOVX nucleotide sequence is changed from that selected from the group consisting ofthe chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed. In another embodiment, the invention includes a nucleic acid molecule wherein the nucleic acid molecule hybridizes under stringent conditions to a NOVX nucleotide sequence or a complement of the NOVX nucleotide sequence. In one embodiment, the invention includes a nucleic acid molecule, wherein the sequence is changed such that no more than 15% ofthe nucleotides in the coding sequence differ from the NOVX nucleotide sequence or a fragment thereof.

In a further aspect, the invention includes a method for determining the presence or amount of the NOVX nucleic acid in a sample. The method involves the steps of: providing the sample; introducing the sample to a probe that binds to the nucleic acid molecule; and determining the presence or amount ofthe probe bound to the NOVX nucleic acid molecule, thereby determining the presence or amount of the NOVX nucleic acid molecule in the sample. In one embodiment, the presence or amount ofthe nucleic acid molecule is used as a marker for cell or tissue type.

In another aspect, the invention discloses a method for determining the presence of or predisposition to a disease associated with altered levels of the NOVX nucleic acid molecule of in a first mammalian subject. The method involves the steps of: measuring the amount of NOVX 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 NOVX 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.

In an aspect, the present invention provides a method of identifying a candidate therapeutic agent for treating a disease, pathology, or an abnormal state or condition using a target entity having a specific association with the disease. This method includes: /

(1) identification of a target biopolymer associated with the disease, pathology, or abnormal state or condition;

(2) contacting the biopolymer with at least one chemical compound; and

(3) identifying a compound that binds to the biopolymer as a candidate therapeutic agent.

In some embodiments of this method, the chemical compound is a member of a combinatorial library of compounds; the contacting in step (b) is conducted on one or more replicate samples of the biopolymer; and the replicate sample is contacted with at least one member of the combinatorial library. In additional embodiments of this method, the biopolymer is included within a cell and is functionally expressed therein. In still a further advantageous embodiment, the binding of the compound modulates the function ofthe biopolymer, and it is the modulation that provides the identification that the compound is a potential therapeutic agent. In yet further significant embodiments of this method, the target biopolymer is a polypeptide.

In a second aspect of the invention, a method for identifying a pharmaceutical agent for treating a disease, pathology, or an abnormal state or condition is provided. The second method includes the steps of:

(1) identifying a candidate therapeutic agent for treating said disease, pathology, or abnormal state or condition by the method described in the preceding paragraph;

(2) contacting a biological sample associated with the disease, pathology, or abnormal state or condition with the candidate therapeutic agent;

(3) determining whether the candidate induces an effect on the biological sample associated with a therapeutic response therein; and

(4) identifying a candidate exerting such an effect as a pharmaceutical agent.

In some embodiments of the second method, the biological sample includes a cell, a tissue or organ, or is a nonhuman mammal.

The present invention discloses novel associations of proteins and polypeptides and the nucleic acids that encode them with various diseases or pathologies. The proteins and related proteins that are similar to them, are encoded by a cDNA and/or by genomic DNA. The proteins, polypeptides and their cognate nucleic acids were identified by CuraGeη Corporation in certain cases. The human Sulfonylurea 2A protein encoded by CGI 54077 and any variants, thereof, are suitable as diagnostic markers, targets for an antibody therapeutic and targets for small molecule drugs. As such the current invention embodies the use of recombinantly expressed and/or endogenously expressed protein in various screens to identify such therapeutic antibodies and/or therapeutic small molecules.

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 incoφorated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

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

DETAILED DESCRIPTION OF THE INVENTION

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

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

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

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

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

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

The NOVX nucleic acids and polypeptides are also useful for detecting specific cell types. Details 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 differentia] expression in normal vs. diseased tissues, e.g. detection of a variety of cancers. SNP analysis for each NOVX, if applicable, is presented in Example D. Additional utilities for NOVX nucleic acids and polypeptides according to the invention are disclosed herein.

NOVX clones

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

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

In one specific embodiment, the invention includes an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) a mature form of the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 79; (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 79, 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 79; (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 79 wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed; and (e) a fragment of any of (a) through (d).

In another specific embodiment, the invention includes an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: (a) a mature form of the amino acid sequence given SEQ ID NO: 2n, wherein n is an integer between 1 and 79; (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 79 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% ofthe 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 79; (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 79, in which any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% ofthe amino acid residues in the sequence are so changed; (e) a nucleic acid fragment encoding at least a portion of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 79 or any variant of said polypeptide wherein any amino acid of the chosen sequence is changed to a different amino acid, provided that no more than 10% of the amino acid residues in the sequence are so changed; and (f) the complement of any of said nucleic acid molecules.

In yet another specific embodiment, the invention includes an isolated nucleic acid molecule, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 79; (b) a nucleotide sequence wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 79 is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed; (c) a nucleic acid fragment of the sequence selected from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 79; and (d) a nucleic acid fragment wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 79 is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed.

NOVX Nucleic Acids and Polypeptides

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

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

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

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

A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79, 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-l, wherein n is an integer between 1 and 79, as a hybridization probe, NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et ah, (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2^nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)

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

As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment ofthe invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes. In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO:2n-l, wherein n is an integer between 1 and 79, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of a NOVX polypeptide). A nucleic acid molecule that is complementary to the nucleotide sequence of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79, is one that is sufficiently complementary to the nucleotide sequence of SEQ ID NO:2ra-l, wherein n is an integer between 1 and 79, that it can hydrogen bond with few or no mismatches to the nucleotide sequence shown in SEQ ID NO:2rc-l, wherein n is an integer between 1 and 79, thereby forming a stable duplex.

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

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

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

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

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

A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences include those sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed in different tissues ofthe same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for a NOVX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NO:27z-l, wherein n is an integer between 1 and 79, as well as a polypeptide possessing NOVX biological activity. Various biological activities of the NOVX proteins are described below.

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

The nucleotide sequences determined from the cloning of the human NOVX genes allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX homologues from other vertebrates. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79; or an anti-sense strand nucleotide sequence of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79; or of a naturally occurring mutant of SEQ ID NO:2?ι-l, wherein n is an integer between 1 and 79.

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

"A polypeptide having a biologically-active portion of a NOVX polypeptide" refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically-active portion of NOVX" can be prepared by isolating a portion of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79, that encodes a polypeptide having a NOVX biological activity (the biological activities of the NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of NOVX.

NOVX Nucleic Acid and Polypeptide Variants

The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79, 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:2«-l, wherein n is an integer between 1 and 79. In another embodiment, an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence of SEQ ID NO:2n, wherein n is an integer between 1 and 79.

In addition to the human NOVX nucleotide sequences of SEQ ID NO:2π-l, wherein n is an integer between 1 and 79, it will be appreciated by those skilled in the art that DNA sequence polymoφhisms that lead to changes in the amino acid sequences of the NOVX polypeptides may exist within a population (e.g., the human population). Such genetic polymoφhism in the NOVX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF) encoding a NOVX protein, preferably a vertebrate NOVX protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the NOVX genes. Any and all such nucleotide variations and resulting amino acid polymoφhisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the NOVX polypeptides, are intended to be within the scope ofthe invention.

Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from a human SEQ ID NO:2n-l, wherein n is an integer between 1 and 79, are intended to be within the scope ofthe invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the NOVX cDNAs of the invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 65% homologous to each other typically remain hybridized to each other.

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

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

Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al, (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65 °C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a sequence of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Reinhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55 °C, followed by one or more washes in IX SSC, 0.1% SDS at 37 °C. Other conditions of moderate stringency that may be used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Krieger, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.

In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci USA 78: 6789-6792.

Conservative Mutations

In addition to naturally-occurring allelic variants of NOVX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO:2«-l, wherein n is an integer between 1 and 79, 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:2rc, wherein n is an integer between 1 and 79. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences ofthe NOVX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the NOVX proteins ofthe invention are not particularly amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.

Another aspect of the invention pertains to nucleic acid molecules encoding NOVX proteins that contain changes in amino acid residues that are not essential for activity. Such NOVX proteins differ in amino acid sequence from SEQ ID NO:2/z-l, wherein n is an integer between 1 and 79, 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 79. Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NO:2«, wherein n is an integer between 1 and 79; more preferably at least about 70% homologous to SEQ ID NO:2/z, wherein n is an integer between 1 and 79; still more preferably at least about 80% homologous to SEQ ID NO:2rc, wherein n is an integer between 1 and 79; even more preferably at least about 90% homologous to SEQ ID NO:2zι, wherein n is an integer between 1 and 79; and most preferably at least about 95% homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 79.

An isolated nucleic acid molecule encoding a NOVX protein homologous to the protein of SEQ ID NO:2ra, wherein n is an integer between 1 and 79, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.

Mutations can be introduced any one of SEQ ID NO:2«-l, wherein n is an integer between 1 and 79, by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more 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 non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity. Following mutagenesis of a nucleic acid of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.

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

In one embodiment, a mutant NOVX protein can be assayed for (i) the ability to form protein :protein interactions with other NOVX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant NOVX protein and a NOVX ligand; or (iii) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins).

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

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

According to the methods 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 Shaφ (1999), Genes & Dev. 13: 3191-3197, incoφorated herein by reference in its entirety. When synthesized, a typical 0.2 micromolar-scale RNA synthesis provides about 1 milligram of siRNA, which is sufficient for 1000 transfection experiments using a 24-well tissue culture plate format.

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

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

In a specific embodiment, siRNAs are transcribed intracellularly by cloning the NOVX gene templates into a vector containing, e.g., a RNA pol III transcription unit from the smaller nuclear RNA (snRNA) U6 or the human RNase P RNA HI . One example of a vector system is the GeneSuppressor™ RNA Interference kit (commercially available from Imgenex). The U6 and HI promoters are members ofthe type III class of Pol III promoters. The +1 nucleotide of the U6-like promoters is always guanosine, whereas the +1 for HI promoters is adenosine. The termination signal for these promoters is defined by five consecutive thymidines. The transcript is typically cleaved after the second uridine. Cleavage at this position generates a 3' UU overhang in the expressed siRNA, which is similar to the 3' overhangs of synthetic siRNAs, Any sequence less than 400 nucleotides in length can be transcribed by these promoter, therefore they are ideally suited for the expression of around 21 -nucleotide siRNAs in, e.g., an approximately 50-nucleotide RNA stem-loop transcript.

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

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

A NOVX mRNA region to be targeted by siRNA is generally selected from a desired NOVX sequence beginning 50 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, polymoφhisms, allelic variants or species-specific variations when targeting a desired gene.

In one embodiment, a complete NOVX siRNA experiment includes the proper negative control. A negative control siRNA generally has the same nucleotide composition as the NOVX siRNA but lack significant sequence homology to the genome. Typically, one would scramble the nucleotide sequence of the NOVX siRNA and do a homology search to make sure it lacks homology to any other gene.

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

A targeted NOVX region is typically a sequence of two adenines (AA) and two thymidines (TT) divided by a spacer region of nineteen (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: 66-200, incoφorated by reference herein in its entirely. The modification ofthe overhang ofthe sense sequence of the siRNA duplex is not expected to affect targeted mRNA recognition, as the antisense siRNA strand guides target recognition. Alternatively, if the NOVX target mRNA does not contain a suitable AA(N21) sequence, one may search for the sequence NA(N21). Further, the sequence of the sense strand and antisense strand may still be synthesized as 5' (N19)TT, as it is believed that the sequence ofthe 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, incoφorated by reference in its entirety.

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

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

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

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

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

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

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

Production of RNAs

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

Lysate Preparation

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

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

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

RNA Preparation

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

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

Cell Culture

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

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

Antisense Nucleic Acids

Another aspect ofthe invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2π-l, wherein n is an integer between 1 and 79, 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:2ra, wherein n is an integer between 1 and 79, or antisense nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ID NO:2«-l, wherein n is an integer between 1 and 79, are additionally provided.

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

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

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

The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a NOVX protein to thereby inhibit expression ofthe protein (e.g., by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove 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.

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

Ribozymes and PNA Moieties

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

In one embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave NOVX mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme having specificity for a NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of a NOVX cDNA disclosed herein (i.e., SEQ ID NO:2rc-l, wherein n is an integer between 1 and 79). For example, a derivative of a Tetrahymena L-19 TVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a NOVX-encoding mRNA. See, e.g., U.S. Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al. NOVX mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.

Alternatively, NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe 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. NY. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.

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

PNAs of NOVX can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of NOVX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S\ nucleases (See, Hyrup, et al., 1996.supra); or as probes or primers for DNA sequence and hybridization (See, Hyrup, et al., 1996, supra; Perry-O'Keefe, et ah, 1996. supra). In another embodiment, PNAs of NOVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNA pol merases) 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 ah, 1996. supra. Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, e.g., Petersen, et ah, 1975. Bioorg. Med. Chem. Lett. 5: 1119-11124.

In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et ah, 1989. Proc. Nath Acad. Sci. U.S.A. 86: 6553-6556; Lemairre, et ah, 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 ah, 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988. Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like. NOVX Polypeptides

A polypeptide according to the invention includes a polypeptide including the amino acid sequence of NOVX polypeptides whose sequences are provided in any one of SEQ ID NO:2«, wherein n is an integer between 1 and 79. 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 79, while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.

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

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

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

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

Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences ofthe NOVX proteins (e.g., the amino acid sequence of SEQ ID NO:2π, wherein n is an integer between 1 and 79) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of a NOVX protein. Typically, biologically-active portions comprise a domain or motif with at least one activity ofthe NOVX protein. A biologically-active portion of a NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.

Moreover, other biologically-active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native NOVX protein. In an embodiment, the NOVX protein has an amino acid sequence of SEQ ID NO:2n, wherein n is an integer between 1 and 79. In other embodiments, the NOVX protein is substantially homologous to SEQ ID NO:2n, wherein n is an integer between 1 and 79, and retains the functional activity of the protein of SEQ ID NO:2n, wherein n is an integer between 1 and 79, 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:2π, wherein n is an integer between 1 and 79, and retains the functional activity of the NOVX proteins of SEQ ID NO:2n, wherein n is an integer between 1 and 79.

Determining Homology Between Two or More Sequences

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

The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. 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:27z-l, wherein n is an integer between 1 and 79. The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.

Chimeric and Fusion Proteins

The invention also provides NOVX chimeric or fusion proteins. As used herein, a NOVX "chimeric protein" or "fusion protein" comprises a NOVX polypeptide operatively-linked to a non-NOVX polypeptide. An "NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a NOVX protein of SEQ ID NO:2n, wherein n is an integer between 1 and 79, whereas a "non-NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism. Within a NOVX fusion protein the NOVX polypeptide can correspond to all or a portion of a NOVX protein. In one embodiment, a NOVX fusion protein comprises at least one biologically-active portion of a NOVX protein. In another embodiment, a NOVX fusion protein comprises at least two biologically-active portions of a NOVX protein. In yet another embodiment, a NOVX fusion protein comprises at least three biologically-active portions of a NOVX protein. Within the fusion protein, the term "operatively-linked" is intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame with one another. The non-NOVX polypeptide can be fused to the N-terminus or C-terminus of the NOVX polypeptide.

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

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

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

A NOVX chimeric or fusion protein ofthe invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be W 03

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

NOVX Agonists and Antagonists

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

Variants of the NOVX proteins that function as either NOVX agonists (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 ah, 1984. Annu. Rev. Biochem. 53: 323; Itakura, et ah, 1984. Science 198: 1056; Ike, et ah, 1983. Nucl. Acids Res. 11: 477.

Polypeptide Libraries

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

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

Anti-NOVX Antibodies

Included in the invention are antibodies to NOVX proteins, or fragments of NOVX proteins. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F_ab, F_ab- and F(_aιy)₂ fragments, and an F_ab expression library. In general, antibody molecules obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG_l5 IgG₂, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.

An isolated protein of the invention intended to serve as an antigen, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence of SEQ ID NO:2rc, wherein n is an integer between 1 and 79, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.

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

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

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

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

Polyclonal Antibodies

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

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

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

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

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

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

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

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

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

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

Humanized Antibodies

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

Human Antibodies

Fully human antibodies essentially relate to antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein. Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-13 (1994)); Fishwild et al,( Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)).

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

An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker. A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.

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

F_ab Fragments and Single Chain Antibodies

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

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit. Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

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

According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')₂ bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')₂ fragments. These fragments are reduced in the presence ofthe dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ofthe other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

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

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

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

Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen 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).

Heterocon jugate Antibodies

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

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

Immunoconjugates

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

Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include Bi, I, In, Y, and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science. 238: 1098 (1987). Carbon-14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.

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

Immunoliposomes

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

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

Diagnostic Applications of Antibodies Directed Against the Proteins of the Invention

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

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

An antibody specific for a NOVX protein of the invention (e.g., a monoclonal antibody or a polyclonal antibody) can be used to isolate a NOVX polypeptide by standard techniques, such as immunoaffinity, chromatography or immunoprecipitation. An antibody to a NOVX polypeptide can facilitate the purification of a natural NOVX antigen from cells, or of a recombinantly produced NOVX antigen expressed in host cells. Moreover, such an anti-NOVX antibody can be used to detect the antigenic NOVX protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the antigenic NOVX protein. Antibodies directed against a NOVX protein can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (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 ^,25I, ¹³¹I, ³⁵S or ³H.

Antibody Therapeutics

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

Alternatively, the effect may be one in which the antibody elicits a physiological result by virtue of binding to an effector binding site on the target molecule. In this case the target, a receptor having an endogenous ligand which may be absent or defective in the disease or pathology, binds the antibody as a surrogate effector ligand, initiating a receptor-based signal transduction event by the receptor. A therapeutically effective amount of an antibody of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target, and in other cases, promotes a physiological response. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg kg body weight to about 50 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.

Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington : The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa. : 1995; Drug Absoφtion Enhancement : Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

If the antigenic protein is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain 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 puφose intended.

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

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

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

ELISA Assay

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

NOVX Recombinant Expression Vectors and Host Cells

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

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

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

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

Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three puφoses: (0 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 ofthe recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et ah, (1988) Gene 69:301-315) and pET 1 Id (Studier et ah, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

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

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

Alternatively, NOVX can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et ah, 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39). In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et ah, 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 ah, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et ah, 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et ah, 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 ah, 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 -fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).

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

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

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

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

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

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

Transgenic NOVX Animals

The host cells ofthe 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 ofthe 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 ofthe transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NOVX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. The human NOVX cDNA sequences, i.e., any one of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79, can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue ofthe human NOVX gene, such as a mouse NOVX gene, can be isolated based on hybridization to the human NOVX cDNA (described further supra) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably-linked to the NOVX transgene to direct expression of NOVX protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the NOVX transgene in its genome and/or expression of NOVX mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene-encoding NOVX protein can further be bred to other transgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a NOVX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NOVX gene. The NOVX gene can be a human gene (e.g., the cDNA of any one of SEQ ID NO:2π-l., wherein n is an integer between 1 and 79), but more preferably, is a non-human homologue of a human NOVX gene. For example, a mouse homologue of human NOVX gene of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79, can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NOVX gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).

Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NOVX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression ofthe endogenous NOVX protein). In the homologous recombination vector, the altered portion of the NOVX gene is flanked at its 5'- and 3'-termini by additional nucleic acid 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 ah, 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 ah, 1992. Cell 69: 915.

The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. Curr. Opin. Biotechnoh 2: 823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169. In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI. For a description ofthe cre/loxP recombinase system, See, e.g., Lakso, et ah, 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 ah, 1991. Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et ah, 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.

Pharmaceutical Compositions

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

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

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeabihty 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 absoφtion of the injectable compositions can be brought about by including in the composition an agent which delays absoφtion, for example, aluminum monostearate and gelatin.

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

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

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

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

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Coφoration and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject W

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

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

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

Screening and Detection Methods

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

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

Screening Assays

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

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

A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention. Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et ah, 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb, et ah, 1994. Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al, 1994. J. Med. Chem. 37: 2678; Cho, et ah, 1993. Science 261: 1303; Carrell, et ah, 1994. Angew. Chem. Int. Ed. Engl. 33: 2059; Carell, et ah, 1994. Angew. Chem. Int. Ed. Engh 33: 2061; and Gallop, et ah, 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (e.g., Houghten, 1992. Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et ah, 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 ah, 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).

In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability ofthe test compound to bind to a NOVX protein determined. The cell, for example, can of mammalian origin or a yeast cell. Determining the ability 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 ofthe test compound to the NOVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with ¹²⁵I, ³⁵S, ^UC, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with a NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability ofthe test compound to preferentially bind to NOVX protein or a biologically-active portion thereof as compared to the known compound.

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

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

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

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

In yet another embodiment, the cell-free assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX protein to form an assay mixture, contacting the assay mixture with a test W 03

compound, and determining the ability of the test compound to interact with a NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the NOVX protein to preferentially bind to or modulate the activity of a NOVX target molecule.

The cell-free assays ofthe invention are amenable to use of both the soluble form or the membrane-bound form of NOVX protein. In the case of cell-free assays comprising the membrane-bound form of NOVX protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of NOVX protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton X-100, Triton^® X-l 14, Thesit^®, Isotridecypoly(ethylene glycol ether)_n,

N-dodecyl— N,N-dimethyl-3-ammonio-l -propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-l -propane sulfonate (CHAPSO).

In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either NOVX protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to NOVX protein, or interaction of NOVX protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-NO VX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or NOVX protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra. Alternatively, the complexes can be dissociated from the matrix, and the level of NOVX protein binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the NOVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated NOVX protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX protein or target molecules, but which do not interfere with binding of the NOVX protein to its target molecule, can be derivatized to the wells of the plate, and unbound target or NOVX protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the NOVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NOVX protein or target molecule.

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

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

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for NOVX is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a NOVX-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g.,

that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with NOVX.

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

Detection Assays

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

Chromosome Mapping

Once the sequence (or a portion ofthe sequence) of a gene has been isolated, this sequence can be used to map the location ofthe gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the NOVX sequences of SEQ ID NO:2rc-l, wherein n is an integer between 1 and 79, or fragments or derivatives thereof, can be used to map the location ofthe NOVX genes, respectively, on a chromosome. The mapping ofthe NOVX sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.

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

Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. See, e.g., D'Eustachio, et ah, 1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions. PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the NOVX sequences to design oligonucleotide primers, sub-localization can be achieved with panels of fragments from specific chromosomes.

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

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

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland, et al, 1987. Nature, 325: 783-787. Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NOVX gene, can be determined. If a mutation is observed in some or all ofthe affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent ofthe particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymoφhisms.

Tissue Typing

The NOVX sequences 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 polymoφhisms," described in U.S. Patent No. 5,272,057).

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

Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences ofthe invention can be used to obtain such identification sequences from individuals and from tissue. The NOVX sequences of the invention uniquely represent portions ofthe human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymoφhisms (SNPs), which include restriction fragment length polymoφhisms (RFLPs). Each ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification puφoses. Because greater numbers of polymoφhisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If coding sequences, such as those of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79, are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

Predictive Medicine

The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) puφoses to thereby treat an individual prophylactically. Accordingly, one aspect 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 puφose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity.

Another aspect 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 ofthe invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX in clinical trials.

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

Diagnostic Assays

An exemplary method for detecting the presence or absence of NOVX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is detected in the biological sample. An agent for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length NOVX nucleic acid, such as the nucleic acid of SEQ ID NO:2«-l, wherein n is an integer between 1 and 79, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays ofthe invention are described herein.

An agent for detecting NOVX protein is an antibody capable of binding to NOVX protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')₂) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (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 fluorescentiy-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescentiy-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method 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.

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

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

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

Prognostic Assays

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

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

The methods of the invention can also be used to detect genetic lesions in a NOVX gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding a NOVX-protein, or the misexpression ofthe 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, (z^'v) a chromosomal rearrangement of a NOVX gene; (v) an alteration in the level of a messenger RNA transcript of a NOVX gene, (vϊ) aberrant modification of a NOVX gene, such as of the methylation pattern of the genomic DNA, (vzϊ) the presence of a non-wild-type splicing pattern of a messenger RNA transcript of a NOVX gene, (viii) a non-wild-type level of a NOVX protein, (ix) allelic loss of a NOVX gene, and (x) inappropriate post-translational modification of a NOVX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in a NOVX gene. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.

In certain embodiments, detection ofthe lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et αl, 1988. Science 241: 1077-1080; and Nakazawa, et αl, 1994. Proc. Nαth Acαd. Sci. USA 91: 360-364), the latter of which can be particularly useful for detecting point mutations in the NOVX-gene (see, Abravaya, et αl., 1995. Nucl. Acids Res. 23: 675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to a NOVX gene under conditions such that hybridization and amplification of the NOVX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

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

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

In other embodiments, genetic mutations in NOVX can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays containing hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et ah, 1996. Human Mutation 7: 244-255; Kozal, et ah, 1996. Nat. Med. 2: 753-759. For example, genetic mutations in ΝOVX can be identified in two dimensional arrays containing light-generated DΝA probes as described in Cronin, et^'ah, supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DΝA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the 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 ah, 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 ah, 1993. Appl. Biochem. Biotechnoh 38: 147-159).

Other methods for detecting mutations in the NOVX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA DNA heteroduplexes. See, e.g., Myers, et ah, 1985. Science 230: 1242. In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S\ nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, et ah, 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba, et ah, 1992. Methods Enzymol. 217: 286-295. In an embodiment, the control DNA or RNA can be labeled for detection. In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in NOVX cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches. See, e.g., Hsu, et al, 1994. Carcino genesis 15: 1657-1662. According to an exemplary embodiment, a probe based on a NOVX sequence, e.g., a wild-type NOVX sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in NOVX genes. For example, single strand conformation polymoφhism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids. See, e.g., Orita, et αl, 1989. Proc. Nαtl Acαd. Sci. USA: 86: 2766; Cotton, 1993. Mutαt. Res. 285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-79. Single-stranded DNA fragments of sample and control NOVX nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e.g., Keen, et al., 1991. Trends Genet. 7: 5.

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

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

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

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

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

Pharmacogenomics

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

In conjunction with such treatment, the pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual's response to a foreign compound or 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 ofthe 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 ofthe individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.

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

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

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

Monitoring of Effects During Clinical Trials

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

By way of example, and not of limitation, genes, including NOVX, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates NOVX activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of NOVX and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one ofthe methods as described herein, or by measuring the levels of activity of NOVX or other genes. In this manner, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment ofthe individual with the agent. In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a NOVX protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (zv) detecting the level of expression or activity ofthe NOVX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity 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 (vz) 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, z.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of NOVX to lower levels than detected, i.e., to decrease the effectiveness of the agent.

Methods of Treatment

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

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

Diseases and Disorders

Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (z'.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: (z) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (z'v) 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 ( z.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between an aforementioned peptide and its binding partner.

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

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

Prophylactic Methods

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

Therapeutic Methods

Another aspect ofthe invention pertains to methods of modulating NOVX expression or activity for therapeutic puφoses. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of NOVX protein activity associated with the cell. An agent that modulates NOVX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a NOVX protein, a peptide, a NOVX peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more NOVX protein activity. Examples of such stimulatory agents include active NOVX protein and a nucleic acid molecule encoding NOVX that has been introduced into the cell. In another embodiment, the agent inhibits one or more NOVX protein activity. Examples of such inhibitory agents include antisense NOVX nucleic acid molecules and anti-NOVX antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a NOVX protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up-regulates or down-regulates) NOVX expression or activity. In another embodiment, the method involves administering a NOVX protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NOVX expression or activity. Stimulation of NOVX activity is desirable in situations in which NOVX is abnormally downregulated and/or in which increased NOVX activity is likely to have a beneficial effect. One example of such a situation is where a subject has a disorder characterized by aberrant cell proliferation and/or differentiation (e.g., cancer or immune associated disorders). Another example of such a situation is where the subject has a gestational disease (e.g., preclampsia).

Determination of the Biological Effect of the Therapeutic

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

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

Prophylactic and Therapeutic Uses of the Compositions of the Invention

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

As an example, a cDNA encoding the NOVX protein of the invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the invention will have efficacy for treatment of patients suffering from diseases, disorders, conditions and the like, including but not limited to those listed herein.

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

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

EXAMPLES

Example A: Polynucleotide and Polypeptide Sequences, and Homology Data

Example 1. Macrophage colony stimulating factor receptor.

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

CAACCTGCAGTTTGGTAAGACCCTCGGAGCTGGAGCCTTTGGGAAGGTGGTGGAGGCC ACGGCCTTTGGTCTGGGCAAGGAGGATGCTGTCCTGAAGGTGGCTGTGAAGATGCTGA AGTCCACGGCCCATGCTGATGAGAAGGAGGCCCTCATGTCCGAGCTGAAGATCATGAG CCACCTGGGCCAGCACGAGAACATCGTCAACCTTCTGGGAGCCTGTACCCATGGAGGC CCTGTACTGGTCATCACGGAGTACTGTTGCTATGGCGACCTGCTCAACTTTCTGCGAA GGAAGGCTGAGGCCATGCTGGGACCCAGCCTGAGCCCCGGCCAGGACCCCGAGGGAGG CGTCGACTATAAGAACATCCACCTCGAGAAGAAATATGTCCGCAGGGACAGTGGCTTC TCCAGCCAGGGTGTGGACACCTATGTGGAGATGAGGCCTGTCTCCACTTCTTCAAATG ACTCCTTCTCTGAGCAAGACCTGGACAAGGAGGATGGACGGCCCCTGGAGCTCCGGGA CCTGCTTCACTTCTCCAGCCAAGTAGCCCAGGGCATGGCCTTCCTCGCTTCCAAGAAT TGCATCCACCGGGACGTGGCAGCGCGTAACGTGCTGTTGACCAATGGTCATGTGGCCA AGATTGGGGACTTCGGGCTGGCTAGGGACATCATGAATGACTCCAACTACATTGTCAA GGGCAATGCCCGCCTGCCTGTGAAGTGGATGGCCCCAGAGAGCATCTTTGACTGTGTC TACACGGTTCAGAGCGACGTCTGGTCCTATGGCATCCTCCTCTGGGAGATCTTCTCAC TTGGGCTGAATCCCTACCCTGGCATCCTGGTGAACAGCAAGTTCTATAAACTGGTGAA GGATGGATACCAAATGGCCCAGCCTGCATTTGCCCCAAAGAATATATACAGCATCATG CAGGCCTGCTGGGCCTTGGAGCCCACCCACAGACCCACCTTCCAGCAGATCTGCTCCT TCCTTCAGGAGCAGGCCCAAGAGGACAGGAGAGAGCGGGACTATACCAATCTGCCGAG CAGCAGCAGAAGCGGTGGCAGCGGCAGCAGCAGCAGTGAGCTGGAGGAGGAGAGCTCT AGTGAGCACCTGACCTGCTGCGAGCAAGGGGATATCGCCCAGCCCTTGCTGCAGCCCA ACAACTATCAGTTCTGCTGA

ORF Start: ATG at 2 ORF Stop: TGA at 2918

SEQ ID NO: 2 972 aa MW at l08001.7kD

NOVla, MGPGVL LLVATAWHGQGIPVIEPSVPELWKPGATVTLRCVGNGSVEWDGPPSPH TLYSDGSSSILST ATFQNTGTYRCTEPGDP GGSAAIH Y π PARPW VLAQEVV CG103404-02 VTEDQDALLPC TDPv_,EAGVSLVRVRGRPLMRHT_JYSFSP HGFTIHRAKFIQSQD Protein YQCSALMGGRKVMSISIR KVQKVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVN Sequence FDWLQHSttCTKLAIPQQSDFHlrøRYQK^

MFFRVVESAYLL^SSEQNLIQEVTVGEGLNLK VEAYPGLQGFN TYLGPFSDHQPΞ PKLANATT DTYRRTFT SLPR KPSEAGRYSF AR PGGWRALTFELTLRYPPEVSV IW FINGSGTLLCAASGYPQP1W LQCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFH KV VQS VETLEH QTYECRAHNSVGSGSWAFIPISAGAHTHPPDEF FTPVVVAC MSIMA LLLL L LYKYKQKPKYQVR KIIESYEGNSYTFIDPTQ PYNEKWEFPR LQFGKT GAGAFGK ATEATAFG GKEDAV KVAVKMLKSTAHADΞKEA MSELKI S HLGQHENIV LGACTHGGPV VITEYCCYGD FLRRKAEAMLGPSLSPGQDPEGG VΌYKJSΓIH EKKYVRRDSGFSSQGVΌTYVE RPVSTSSNDSFSEQD DKEDGRP ELRD LHFSSQVAQGMAFLASKNCIHRDVAARN Π^ TNGHVAKIGDFGLARDIL__S YIVK GNARLPV LFFL_-PESIFDCVY VQSDV SYGILL EIFSLGIINPYPGILV SKFYKL^'VK DGYQMAQPAFAPK IYSIMQACWA EPTHRPTFQQICSFLQEQAQEDRRERDYTN PS SSRSGGSGSSSSELEEESSSEHLTCCEQGDIAQPLLQPN YQFC

SEQ ID NO: 3 3992 bp

NOVlb, GGCTTCAGGAAGGGCAGACAGAGTGTCCAAAAGCGTGAGAGCACGAAGTGAGGAGAAG GTGGAGAAGAGAGAAGAGGAAGAGGAAGAGGAAGAGAGGAAGCGGAGGGAACTGCGGC CG103404-01 CAGGCTAAAAGGGGAAGAAGAGGATCAGCCCAAGGAGGAGGAAGAGGAAAACAAGACA DNA Sequence AACAGCCAGTGCAGAGGAGAGGAACGTGTGTCCAGTGTCCCGATCCCTGCGGAGCTAG TAGCTGAGAGCTCTGTGCCCTGGGCACCTTGCAGCCCTGCACCTGCCTGCCACTTCCC CACCGAGGCCATGGGCCCAGGAGTTCTGCTGCTCCTGCTGGTGGCCACAGCTTGGCAT GGTCAGGGAATCCCAGTGATAGAGCCCAGTGTCCCCGAGCTGGTCGTGAAGCCAGGAG CAACGGTGACCTTGCGATGTGTGGGCAATGGCAGCGTGGAATGGGATGGCCCCGCATC ACCTCACTGGACCCTGTACTCTGATGGCTCCAGCAGCATCCTCAGCACCAACAACGCT ACCTTCCAAAACACGGGGACCTATCGCTGCACTGAGCCTGGAGACCCCCTGGGAGGCA GCGCCGCCATCCACCTCTATGTCAAAGACCCTGCCCGGCCCTGGAACGTGCTAGCACA GGAGGTGGTCGTGTTCGAGGACCAGGACGCACTACTGCCCTGTCTGCTCACAGACCCG GTGCTGGAAGCAGGCGTCTCGCTGGTGCGTGTGCGTGGCCGGCCCCTCATGCGCCACA CCAACTACTCCTTCTCGCCCTGGCATGGCTTCACCATCCACAGGGCCAAGTTCATTCA GAGCCAGGACTATCAATGCAGTGCCCTGATGGGTGGCAGGAAGGTGATGTCCATCAGC ATCCGGCTGAAAGTGCAGAAAGTCATCCCAGGGCCCCCAGCCTTGACACTGGTGCCTG CAGAGCTGGTGCGGATTCGAGGGGAGGCTGCCCAGATCGTGTGCTCAGCCAGCAGCGT TGATGTTAACTTTGATGTCTTCCTCCAACACAACAACACTAAGCTCGCAATCCCTCAA CAATCTGACTTTCATAATAACCGTTACCAAAAAGTCCTGACCCTCAACCTCGATCAAG TAGATTTCCAACATGCCGGCAACTACTCCTGCGTGGCCAGCAACGTGCAGGGCAAGCA CTCCACCTCCATGTTCTTCCGGGTGGTAGAGAGTGCCTACTTGAACTTGAGCTCTGAG CAGAACCTCATCCAGGAGGTGACCGTGGGGGAGGGGCTCAACCTCAAAGTCATGGTGG AGGCCTACCCAGGCCTGCAAGGTTTTAACTGGACCTACCTGGGACCCTTTTCTGACCA CCAGCCTGAGCCCAAGCTTGCTAATGCTACCACCAAGGACACATACAGGCACACCTTC ACCCTCTCTCTGCCCCGCCTGAAGCCCTCTGAGGCTGGCCGCTACTCCTTCCTGGCCA GAAACCCAGGAGGCTGGAGAGCTCTGACGTTTGAGCTCACCCTTCGATACCCCCCAGA GGTAAGCGTCATATGGACATTCATCAACGGCTCTGGCACCCTTTTGTGTGCTGCCTCT GGGTACCCCCAGCCCAACGTGACATGGCTGCAGTGCAGTGGCCACACTGATAGGTGTG ATGAGGCCCAAGTGCTGCAGGTCTGGGATGACCCATACCCTGAGGTCCTGAGCCAGGA GCCCTTCCACAAGGTGACGGTGCAGAGCCTGCTGACTGTTGAGACCTTAGAGCACAAC CAAACCTACGAGTGCAGGGCCCACAACAGCGTGGGGAGTGGCTCCTGGGCCTTCATAC CCATCTCTGCAGGAGCCCACACGCATCCCCCGGATGAGTTCCTCTTCACACCAGTGGT GGTCGCCTGCATGTCCATCATGGCCTTGCTGCTGCTGCTGCTCCTGCTGCTATTGTAC AAGTATAAGCAGAAGCCCAAGTACCAGGTCCGCTGGAAGATCATCGAGAGCTATGAGG GCAACAGTTATACTTTCATCGACCCCACGCAGCTGCCTTACAACGAGAAGTGGGAGTT CCCCCGGAACAACCTGCAGTTTGGTAAGACCCTCGGAGCTGGAGCCTTTGGGAAGGTG GTGGAGGCCACGGCCTTTGGTCTGGGCAAGGAGGATGCTGTCCTGAAGGTGGCTGTGA AGATGCTGAAGTCCACGGCCCATGCTGATGAGAAGGAGGCCCTCATGTCCGAGCTGAA GATCATGAGCCACCTGGGCCAGCACGAGAACATCGTCAACCTTCTGGGAGCCTGTACC CATGGAGGCCCTGTACTGGTCATCACGGAGTACTGTTGCTATGGCGACCTGCTCAACT TTCTGCGAAGGAAGGCTGAGGCCATGCTGGGACCCAGCCTGAGCCCCGGCCAGGACCC CGAGGGAGGCGTCGACTATAAGAACATCCACCTCGAGAAGAAATATGTCCGCAGGGAC AGTGGCTTCTCCAGCCAGGGTGTGGACACCTATGTGGAGATGAGGCCTGTCTCCACTT CTTCAAATGACTCCTTCTCTGAGCAAGACCTGGACAAGGAGGATGGACGGCCCCTGGA GCTCCGGGACCTGCTTCACTTCTCCAGCCAAGTAGCCCAGGGCATGGCCTTCCTCGCT TCCAAGAATTGCATCCACCGGGACGTGGCAGCGCGTAACGTGCTGTTGACCAATGGTC ATGTGGCCAAGATTGGGGACTTCGGGCTGGCTAGGGACATCATGAATGACTCCAACTA CATTGTCAAGGGCAATGCCCGCCTGCCTGTGAAGTGGATGGCCCCAGAGAGCATCTTT GACTGTGTCTACACGGTTCAGAGCGACGTCTGGTCCTATGGCATCCTCCTCTGGGAGA TCTTCTCACTTGGGCTGAATCCCTACCCTGGCATCCTGGTGAACAGCAAGTTCTATAA ACTGGTGAAGGATGGATACCAAATGGCCCAGCCTGCATTTGCCCCAAAGAATATATAC AGCATCATGCAGGCCTGCTGGGCCTTGGAGCCCACCCACAGACCCACCTTCCAGCAGA TCTGCTCCTTCCTTCAGGAGCAGGCCCAAGAGGACAGGAGAGAGCGGGACTATACCAA TCTGCCGAGCAGCAGCAGAAGCGGTGGCAGCGGCAGCAGCAGCAGTGAGCTGGAGGAG GAGAGCTCTAGTGAGCACCTGACCTGCTGCGAGCAAGGGGATATCGCCCAGCCCTTGC TGCAGCCCAACAACTATCAGTTCTGCTGAGGAGTTGACGACAGGGAGTACCACTCTCC

CCTCCTCCAAACTTCAACTCCTCCATGGATGGGGCGACACGGGGAGAACATACAAACT

CTGCCTTCGGTCATTTCACTCAACAGCTCGGCCCAGCTCTGAAACTTGGGAAGGTGAG

GGATTCAGGGGAGGTCAGAGGATCCCACTTCCTGAGCATGGGCCATCACTGCCAGTCA

GGGGCTGGGGGCTGAGCCCTCACCCCCCGCCTCCCCTACTGTTCTCATGGTGTTGGCC

TCGTGTTTGCTATGCCAACTAGTAGAACCTTCTTTCCTAATCCCCTTATCTTCATGGA

AATGGACTGACTTTATGCCTATGAAGTCCCCAGGAGCTACACTGATACTGAGAAAACC

AGGCTCTTTGGGGCTAGACAGACTGGCAGAGAGTGAGATCTCCCTCTCTGAGAGGAGC AGCAGATGCTCACAGACCACACTCAGCTCAGGCCCCTTGGAGCAGGATGGCTCCTCTA AGAATCTCACAGGACCTCTTAGTCTCTGCCCTATACGCCGCCTTCACTCCACAGCCTC

ACCCCTCCCACCCCCATACTGGTACTGCTGTAATGAGCCAAGTGGCAGCTAAAAGTTG

GGGGTGTTCTGCCCAGTCCCGTCATTCTGGGCTAGAAGGCAGGGGACCTTGGCATTGG

CTGGCCACACCAAGCAGGAAGCACAAACTCCCCCAAGCTGACTCATCCTAACTAACAG

ITCACGCCGTGGGATGTCTCTGTCCACATTAAACTAACAGCATTAATGC

ORF Start: ATG at 301 ORF Stop: TGA at 3217

SEQ ID NO: 4 972 aa MW at 107956.6kD

NOVlb, MGPGVLLLL VATA HGQGIPVIEPSVPELVVKPGATV LRCVGNGSVEWDGPASPH CG103404-01 TLYSDGS SS I STNNATFQNTGTYRCTEPGDPLGGS AAIHLYVKDPARP NVLAQEW

VFEDQDAL PCLLTDPVLEAGVSLVRVRGRP MRHT YSFSP HGFTIHRAKFIQSQD Protein YQCSALMGGRKVMSISIRLKVQKVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVN Sequence FDWLQHNOTKLAIPQQSDFHΪ_RYQ

_?FRVVESAYLl^SSEQl_IQEV VGEGLNLKVMvΕAYPGLQGFNWTYLGPFSDHQPE

PKLANATTKDTYRHTFTLSLPR KPSEAGRYSFLAR PGGWRALTFE TIiRYPPEVSV

IWTFINGSGT LCAASGYPQPNVT QCSGHTDRCDΞAQV QVWDDPYPEVLSQEPFH

K TVQSLLTVET EH QTYECRAHNSVGSGS AFIPISAGAHTHPPDEF FTPVVVAC

MSI1_ALL LLLLLLLYKYKQKPKYQVRV^IIESYEGNSYTFIDPTQLPYNEK EFPRN

NLOFGKT GAGAFGKvΛtEATAFGLGKEDAVLKVAV M KSTAHADEKEALMSELKIMS HLGQHENIVTS_LGACTHGGPVLVITEYCCYGD 1^ RRKAEAMLGPSLSPGQDPEGG

VDYKNIHLEKKY /RRDSGFSSQGvOTYVEMRPVSTSSNDSFSEQDLDKEDGRPLELRD

LLHFSSQVAQGI_?_ LASK-SrCIHRDVAARNvX TNGHVA IGDFGLARblMSro

GNARLPVKWMAPESIFDCVY VQSDV SYGI LWEIFSLG NPYPGILVNS FYK VK

DGYQMAQPAFAPKNIYSIMQAC ALEPTHRPTFQQICSFLQEQAQEDRRERDYTN PS

SSRSGGSGSSSSELEEESSSEHLTCCEQGDIAQPLLQPNNYQFC

SEQ ED NO: 5 2920 bp

NOVlc, CA GGGCCCAGGAGTTCTGCTGCTCCTGCTGGTGGCCACAGCTTGGCATGGTCAGGGA ATCCCAGTGATAGAGCCCAGTGTCCCTGAGCTGGTCGTGAAGCCAGGAGCAACGGTGA 246863892 CCTTGCGATGTGTGGGCAATGGCAGCGTGGAATGGGATGGCCCCCCATCACCTCACTG DNA Sequence GACCCTGTACTCTGATGGCTCCAGCAGCATCCTCAGCACCAACAACGCTACCTTCCAA AACACGGGGACCTATCGCTGCACTGAGCCTGGAGACCCCCTGGGAGGCAGCGCCGCCA TCCACCTCTATGTCAAAGACCCTGCCCGGCCCTGGAACGTGCTAGCACAGGAGGTGGT CGTGTTCGAGGACCAGGACGCACTACTGCCCTGTCTGCTCACAGACCCGGTGCTGGAA GCAGGCGTCTCGCTGGTGCGTGTGCGTGGCCGGCCCCTCATGCGCCACACCAACTACT CCTTCTCGCCCTGGCATGGCTTCACCATCCACAGGGCCAAGTTCATTCAGAGCCAGGA CTATCAATGCAGTGCCCTGATGGGTGGCAGGAAGGTGATGTCCATCAGCATCCGGCTG AAAGTGCAGAAAGTCATCCCAGGGCCCCCAGCCTTGACACTGGTGCCTGCAGAGCTGG TGCGGATTCGAGGGGAGGCTGCCCAGATCGTGTGCTCAGCCAGCAGCGTTGATGTTAA CTTTGATGTCTTCCTCCAACACAACAACACCAAGCTCGCAATCCCTCAACAATCTGAC TTTCATAATAACCGTTACCAAAAAGTCCTGACCCTCAACCTCGATCAAGTAGATTTCC AACAGCCGGCAACTACTCCTGCGTGGCCAGCAACGTGCAGGGCAAGCACTCCACCTC CATGTTCTTCCGGGTGGTAGAGAGTGCCTACTTGAACTTGAGCTCTGAGCAGAACCTC ATCCAGGAGGTGACCGTGGGGGAGGGGCTCAACCTCAAAGTCATGGTGGAGGCCTACC CAGGCCTGCAAGGTTTTAACTGGACCTACCTGGGACCCTTTTCTGACCACCAGCCTGA GCCCAAGCTTGCTAATGCTACCACCAAGGACACATACAGGCGCACCTTCACCCTCTCT CTGCCCCGCCTGAAGCCCTCTGAGGCTGGCCGCTACTCCTTCCTGGCCAGAAACCCAG GAGGCTGGAGAGCTCTGACGTTTGAGCTCACCCTTCGATACCCCCCAGAGGTAAGCGT CATATGGACATTCATCAACGGCTCTGGCACCCTTTTGTGTGCTGCCTCTGGGTACCCC CAGCCCAACGTGACATGGCTGCAGTGCAGTGGCCACACTGATAGGTGTGATGAGGCCC AAGTGCTGCAGGTCTGGGATGACCCATACCCTGAGGTCCTGAGCCAGGAGCCCTTCCA CAAGGTGACGGTGCAGAGCCTGCTGACTGTTGAGACCTTAGAGCACAACCAAACCTAC GAGTGCAGGGCCCACAACAGCGTGGGGAGTGGCTCCTGGGCCTTCATACCCATCTCTG CAGGAGCCCACACGCATCCCCCGGATGAGTTCCTCTTCACACCAGTGGTGGTCGCCTG CATGTCCATCATGGCCTTGCTGCTGCTGCTGCTCCTGCTGCTATTGTACAAGTATAAG CAGAAGCCCAAGTACCAGGTCCGCTGGAAGATCATCGAGAGCTATGAGGGCAACAGTT ATACTTTCATCGACCCCACGCAGCTGCCTTACAACGAGAAGTGGGAGTTCCCCCGGAA CAACCTGCAGTTTGGTAAGACCCTCGGAGCTGGAGCCTTTGGGAAGGTGGTGGAGGCC ACGGCCTTTGGTCTGGGCAAGGAGGATGCTGTCCTGAAGGTGGCTGTGAAGATGCTGA AGTCCACGGCCCATGCTGATGAGAAGGAGGCCCTCATGTCCGAGCTGAAGATCATGAG CCACCTGGGCCAGCACGAGAACATCGTCAACCTTCTGGGAGCCTGTACCCATGGAGGC CCTGTACTGGTCATCACGGAGTACTGTTGCTATGGCGACCTGCTCAACTTTCTGCGAA GGAAGGCTGAGGCCATGCTGGGACCCAGCCTGAGCCCCGGCCAGGACCCCGAGGGAGG CGTCGACTATAAGAACATCCACCTCGAGAAGAAATATGTCCGCAGGGACAGTGGCTTC TCCAGCCAGGGTGTGGACACCTATGTGGAGATGAGGCCTGTCTCCACTTCTTCAAATG ACTCCTTCTCTGAGCAAGACCTGGACAAGGAGGATGGACGGCCCCTGGAGCTCCGGGA CCTGCTTCACTTCTCCAGCCAAGTAGCCCAGGGCATGGCCTTCCTCGCTTCCAAGAAT TGCATCCACCGGGACGTGGCAGCGCGTAACGTGCTGTTGACCAATGGTCATGTGGCCA AGATTGGGGACTTCGGGCTGGCTAGGGACATCATGAATGACTCCAACTACATTGTCAA GGGCAATGCCCGCCTGCCTGTGAAGTGGATGGCCCCAGAGAGCATCTTTGACTGTGTC TACACGGTTCAGAGCGACGTCTGGTCCTATGGCATCCTCCTCTGGGAGATCTTCTCAC TTGGGCTGAATCCCTACCCTGGCATCCTGGTGAACAGCAAGTTCTATAAACTGGTGAA GGATGGATACCAAATGGCCCAGCCTGCATTTGCCCCAAAGAATATATACAGCATCATG CAGGCCTGCTGGGCCTTGGAGCCCACCCACAGACCCACCTTCCAGCAGATCTGCTCCT TCCTTCAGGAGCAGGCCCAAGAGGACAGGAGAGAGCGGGACTATACCAATCTGCCGAG CAGCAGCAGAAGCGGTGGCAGCGGCAGCAGCAGCAGTGAGCTGGAGGAGGAGAGCTCT AGTGAGCACCTGACCTGCTGCGAGCAAGGGGATATCGCCCAGCCCTTGCTGCAGCCCA ACiVACTATCAGTTCTGCTGA

ORF Start: ATG at 2 ORF Stop: TGA at 2918

SEQ ID NO: 6 972 aa MW at lO8001.7kD

NOVlc, MGPGVLLL VATAWHGQGI PVIEPSVPEL KPGATVTLRC VGNGSVEWDGPPS PH 246863892 TLYSDGSSSI STI ATFQIWGTYRCTEPGDPLGGSAAIHLYVT-PARPWNVLAQEVV

VTEDQDA LPCLLTDPVLEAGVS VRVRGRP MRHTNYSFSP HGFTIHRAKFIQSQD

Protein YQCSA MGGRKVMSISIR KVQKVIPGPPALTLVPAE VRIRGEAAQIVCSASSVDVN

Sequence FDVFLQH Sl KLAIPQQSDFHlSπ YQKλt TLN DQVDFQHAGNYSCVASNVQ

MFFRWESAYI_>_SSEQ]^_QEvWGEGI__KvT^

PKLANATTKDTYRRTFTLSLPPΛKPSEAGRYSFLARNPGGWRALTFELTLRYPPEVSV IWTFINGSGTLLCAASGYPQP VT LQCSGHTDRCDEAQVLQV DDPYPEV SQEPFH KVΓVQSL _VETLEHNQTYECP__ SVGSGSWAFIPISAGAHTHPPDEFLFTPVVVAC MSIMA LLL LLYKYKQKPKYQVR KIIESYEGNSYTFIDPTQLPYEKWEFPR NLQFGKTLGAGAFGKVVEATAFGLGKEDAV KΛTAVKMLKSTAHADEKEA MSELKIMS HLGQHENIVNLLGACTHGGPVLVITEYCCYGDL NFLRRKAEAMLGPSLSPGQDPEGG VΌYK IH EKKYVRRDSGFSSQGVDTYVEMRPVSTSSNDSFSEQDLDKEDGRP ELRD LHFSSQVAQG_^ ASK_ICIHRDVAAR1WLLTNGHVAKIGDFGLARDI_TOSNYIVK GNARLPVK MAPESIFDCVYTVQSDV SYGIL EIFS GLNPYPGILVWSKFYKLV DGYQMAQPAFAPKNIYSIMQACWALEPTHRPTFQQICSFLQEQAQEDRRERDYTNLPS SSRSGGSGSSSSE EEESSSEHLTCCEQGDIAQPLLQPNNYQFC

SEQ ID NO: 7 1290bp

NOVld, TACCAGGTCCGCTGGAAGATCATCGAGAGCTATGAGGGCAACAGTTATACTTTCATCG ACCCCACGCAGCTGCCTTACAACGAGAAGTGGGAGTTCCCCCGGAACAACCTGCAGTT 259359394 TGGTAAGACCCTCGGAGCTGGAGCCTTTGGGAAGGTGGTGGAGGCCACGGCCTTTGGT DNA Sequence CTGGGCAAGGAGGATGCTGTCCTGAAGGTGGCTGTGAAGATGCTGAAGTCCACGGCCC ATGCTGATGAGAAGGAGGCCCTCATGTCCGAGCTGAAGATCATGAGCCACCTGGGCCA GCACGAGAACATCGTCAACCTTCTGGGAGCCTGTACCCATGGAGGCCCTGTACTGGTC ATCACGGAGTACTGTTGCTATGGCGACCTGCTCAACTTTCTGCGAAGGAAGGCTGAGG CCATGCTGGGACCCAGCCTGAGCCCCGGCCAGGACCCCGAGGGAGGCGTCGACTATAA GAACATCCACCTCGAGAAGAAATATGTCCGCAGGGACAGTGGCTTCTCCAGCCAGGGT GTGGACACCTATGTGGAGATGAGGCCTGTCTCCACTTCTTCAAATGACTCCTTCTCTG AGCAAGACCTGGACAAGGAGGATGGACGGCCCCTGGAGCTCCGGGACCTGCTTCACTT CTCCAGCCAAGTAGCCCAGGGCATGGCCTTCCTCGCTTCCAAGAATTGCATCCACCGG GACGTGGCAGCGCGTAACGTGCTGTTGACCAATGGTCATGTGGCCAAGATTGGGGACT TCGGGCTGGCTAGGGACATCATGAATGACTCCAACTACATTGTCAAGGGCAATGCCCG CCTGCCTGTGAAGTGGATGGCCCCAGAGAGCATCTTTGACTGTGTCTACACGGTTCAG AGCGACGTCTGGTCCTATGGCATCCTCCTCTGGGAGATCTTCTCACTTGGGCTGAATC CCTACCCTGGCATCCTGGTGAACAGCAAGTTCTATAAACTGGTGAAGGATGGATACCA AATGGCCCAGCCTGCATTTGCCCCAAAGAATATATACAGCATCATGCAGGCCTGCTGG GCCTTGGAGCCCACCCACAGACCCACCTTCCAGCAGATCTGCTCCTTCCTTCAGGAGC AGGCCCAAGAGGACAGGAGAGAGCGGGACTATACCAATCTGCCGAGCAGCAGCAGAAG CGGTGGCAGCGGCAGCAGCAGCAGTGAGCTGGAGGAGGAGAGCTCTAGTGAGCACCTG ACCTGCTGCGAGCAAGGGGATATCGCCCAGCCCTTGCTGCAGCCCAACAACTATCAGT TCTGCTGAGCAGGT

ORF Start: at 1 ORF Stop: TGA at 1282

SEQ ID NO: 8 427 aa MW at 47782.4kD

NOVld, YQVRWKIIESYEGNSYTFIDPTQLPY EKMEFPR-SttlLQFGKTLGAGAFG VVEATAFG

LGKEDAVLKVAVKMKSTAHADEKEALMSE KIMSHLGQHENIV LLGACTHGGPVLV 259359394 ITEYCCYGDLL1^LRRK__-AMLGPSLSPGQDPEGGVX>YKNIH EKKYVRRDSGFSSQG Protein VDTY^l^PVSTSSKroSFSEQDLDKEDGRPLELRDLLHFSSQVAQGI_AF ASKNCIHR Sequence DVAARNVL_TNGHVAKIGDFGLARDI1_^

SDV SYGILLWΞIFSLG NPYPGI VNSKFYK VKDGYQMAQPAFAPKNIYSIMQAC

ALEPTHRPTFQQICSFLQEQAQEDRRERDYT LPSSSRSGGSGSSSSELEEESSSEH

TCCEQGDIAQP LQPNNYQFC

SEQ ID NO: 9 1290 bp

NOVle, TACCAGGTCCGCTGGAAGATCATCGAGAGCTATGAGGGCAACAGTTATACTTTCATCG ACCCCACGCAGCTGCCTTACAACGAGAAGTGGGAGTTCCCCCGGAACAACCTGCAGTT 259359398 TGGTAAGACCCTCGGAGCTGGAGCCTTTGGGAAGGTGGTGGAGGCCACGGCCTTTGGT DNA CTGGGCAAGGAGGATGCTGTCCTGAAGGTGGCTGTGAAGATGCTGAAGTCCACGGCCC Sequence ATGCTGATGAGAAGGAGGCCCTCATGTCCGAGCTGAAGATCATGAGCCACCTGGGCCA GCACGAGAACATCGTCAACCTTCTGGGAGCCTGTACCCATGGAGGCCCTGTACTGGTC ATCACGGAGTACTGTTGCTATGGCGACCTGCTCAACTTTCTGCGAAGGAAGGCTGAGG CCATGCTGGGACCCAGCCTGAGCCCCGGCCAGGACCCCGAGGGAGGCGTCGACTATAA GAACATCCACCTCGAGAAGAAATATGTCCGCAGGGACAGTGGCTTCTCCAGCCAGGGT GTGGACACCTATGTGGAGATGAGGCCTGTCTCCACTTCTTCAAATGACTCCTTCTCTG AGCAAGACCTGGACAAGGAGGATGGACGGCCCCTGGAGCTCCGGGACCTGCTTCACTT CTCCAGCCAAGTAGCCCAGGGCATGGCCTTCCTCGCTTCCAAGAATTGCATCCACCGG GACGTGGCAGCGCGTAACGTGCTGTTGACCAATGGTCATGTGGCCAAGATTGGGGACT TCGGGCTGGCTAGGGACATCATGAATGACTCCAACTACATTGTCAAGGGCAATGCCCG CCTGCCTGTGAAGTGGATGGCCCCAGAGAGCATCTTTGACTGTGTCTACACGGTTCAG AGCGACGTCTGGTCCTATGGCATCCTCCTCTGGGAGATCTTCTCACTTGGGCTGAATC CCTACCCTGGCATCCTGGTGAACAGCAAGTTCTATAAACTGGTGAAGGATGGATACCA AATGGCCCAGCCTGCATTTGCCCCAAAGAATATATACAGCATCATGCAGGCCTGCTGG GCCTTGGAGCCCACCCACAGACCCACCTTCCAGCAGATCTGCTCCTTCCTTCAGGAGC AGGCCCAAGAGGACAGGAGAGAGCGGGACTATACCAATCTGCCGAGCAGCAGCAGAAG CGGTGGCAGCGGCAGCAGCAGCAGTGAGCTGGAGGAGGAGAGCTCTAGTGAGCACCTG ACCTGCTGCGAGCAAGGGGATATCGCCCAGCCCTTGCTGCAGCCCAACAACTTTCAGT TCTGCTGAGCAGGT

ORF Start: at 1 ORF Stop: TGA at 1282

SEQ ID NO: 10 427 aa MW at 47766.4kD

NOVle, YQVRVKIIESYEGNSYTFIDPTQLPYlrEKWEFPRM^QFGKTLGAGAFGKVVEATAFG GKEDAVLKVAVKML STAHADEKEA MSE KI SHLGQHENIVNLLGACTHGGPVLV 259359398 ITEYCCYGDL NF RRKJ—AMLGPSLSPGQDPEGGVDYIO riHLEiα YVRRDSGFSSQG Protein VDTYVEi PVSTSSlrøSFSEQDLDKEDGRPLELRDLLHFSSQVAQGMAFLASKNCIHR Sequence DVAARWL TNGHVAKIGDFG ARDIMrø^

SDV SYGIL WΞIFSLGLNPYPGILVNSKFYKLVKDGYQMAQPAFAPKNIYSIMQAC

ALEPTHRPTFQQICSFLQEQAQEDRRERDYTNLPSSSRSGGSGSSSSELEEESSSEHL

NOVlf, TACCAGGTCCGCTGGAAGATCATCGAGAGCTATGAGGGCAACAGTTATACTTTCATCG ACCCCACGCAGCTGCCTTACAACGAGAAGTGGGAGTTCCCCCGGAACAACCTGCAGTT CG103404-03 TGGTAAGACCCTCGGAGCTGGAGCCTTTGGGAAGGTGGTGGAGGCCACGGCCTTTGGT DNA Sequence CTGGGCAAGGAGGATGCTGTCCTGAAGGTGGCTGTGAAGATGCTGAAGTCCACGGCCC ATGCTGATGAGAAGGAGGCCCTCATGTCCGAGCTGAAGATCATGAGCCACCTGGGCCA GCACGAGAACATCGTCAACCTTCTGGGAGCCTGTACCCATGGAGGCCCTGTACTGGTC ATCACGGAGTACTGTTGCTATGGCGACCTGCTCAACTTTCTGCGAAGGAAGGCTGAGG CCATGCTGGGACCCAGCCTGAGCCCCGGCCAGGACCCCGAGGGAGGCGTCGACTATAA GAACATCCACCTCGAGAAGAAATATGTCCGCAGGGACAGTGGCTTCTCCAGCCAGGGT GTGGACACCTATGTGGAGATGAGGCCTGTCTCCACTTCTTCAAATGACTCCTTCTCTG AGCAAGACCTGGACAAGGAGGATGGACGGCCCCTGGAGCTCCGGGACCTGCTTCACTT CTCCAGCCAAGTAGCCCAGGGCATGGCCTTCCTCGCTTCCAAGAATTGCATCCACCGG GACGTGGCAGCGCGTAACGTGCTGTTGACCAATGGTCATGTGGCCAAGATTGGGGACT TCGGGCTGGCTAGGGACATCATGAATGACTCCAACTACATTGTCAAGGGCAATGCCCG CCTGCCTGTGAAGTGGATGGCCCCAGAGAGCATCTTTGACTGTGTCTACACGGT CAG AGCGACGTCTGGTCCTATGGCATCCTCCTCTGGGAGATCTTCTCACTTGGGCTGAATC CCTACCCTGGCATCCTGGTGAACAGCAAGTTCTATAAACTGGTGAAGGATGGATACCA AATGGCCCAGCCTGCATTTGCCCCAAAGAATATATACAGCATCATGCAGGCCTGCTGG GCCTTGGAGCCCACCCACAGACCCACCTTCCAGCAGATCTGCTCCTTCCTTCAGGAGC AGGCCCAAGAGGACAGGAGAGAGCGGGACTATACCAATCTGCCGAGCAGCAGCAGAAG CGGTGGCAGCGGCAGCAGCAGCAGTGAGCTGGAGGAGGAGAGCTCTAGTGAGCACCTG ACCTGCTGCGAGCAAGGGGATATCGCCCAGCCCTTGCTGCAGCCCAACAACTATCAGT TCTGCTGAGCAGGT ■ _________

ORF Start: at 1

SEQ ID NO: 12 427 aa MW at 47782.4kD

NOVlf, YQVRWKIIESYEGNSYTFIDPTQ PΗSfEKWEFPR N QFGKTLGAGAFGKVVEATAFG GKEDAVLKVAVKMLKSTAHADEKEALMSELKIMSH GQHENIVWLLGACTHGGPV V CG103404-03 ITEYCCYGD NFLPJIKAEAMLGPS SPGQDPEGGVDYJCNIHLE KYVRRDSGFSSQG Protein VDTYVErø^PVSTSSKTOSFSEQD DKEDGRPLELRDLLHFSSQVAQGMAFLASKNCIHR Sequence DVAARNV TNGHVAKIGDFGLARDIMrøS^^

SDV SYGI WEIFSLGLNPYPGILV SKFYKLVKDGYQMAQPAFAPKNIYSIMQAC

ALEPTHRPTFQQICSFLQEQAQEDRRERDYTNLPSSSRSGGSGSSSSELEEESSSEHL

TCCEQGDIAQPLLQPN YQFC

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

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

Table 1C. Protein Sequence Properties NOVla

SignalP analysis: Cleavage site between residues 20 and 21

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

N-region: length 0; pos.chg 0; neg.chg 0 H-region: length 23; peak value 10.88 PSG score: 6.47

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 0.57 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 =-13.75 Transmembrane 518 - 534 PERIPHERAL Likelihood = 1.59 (at 404) ALOM score: -13.75 (number of TMSs: 1)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 9 Charge difference: -2.5 C(-1.5) - N( 1.0) N >= C: N-terminal side will be inside

>» membrane topology: type la (cytoplasmic tail 535 to 972)

MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75) : 2.57 Hyd Moment (95) : 2.20 G content: 4 D/E content: 1 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 : none pat7 : none bipartite: none content of basic residues : 8.4% 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: found LL at 535 LL at 536 LL at 649 LL at 671 LL at 755 LL at 785 LL at 844 checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none

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

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

Final Results (k = 9/23):

44.4 %: endoplasmic reticulum

22.2 %: Golgi

22.2 %: extracellular, including cell wall

11.1 %: plasma membrane

» prediction for CG103404-02 is end (k=9)

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

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

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

Example 2. Cation-transporting ATPase 1

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: 15 3840 bp

NOV2a, GCGCCGGGGCCGGCGATGAGCGCGAGGAGCCGGCATGAGCGCAGACAGCAGCCCTCTC CG108945-01 GTGGGCAGCACGCCCACCGGTTATGGGACCCTGACGATAGGGACATCAATAGATCCCC TCAGCTCCTCAGTTTCATCCGTGAGGCTCAGCGGCTACTGTGGCAGTCCATGGAGGGT DNA Sequence CATCGGCTATCACGTCGTGGTCTGGATGATGGCTGGGATCCCTTTGCTGCTCTTCCGT TGGAAGCCCCTGTGGGGGGTGCGGCTGCGGCTCCGGCCCTGCAACCTGGCCCACGCCG AAACACTCGTTATCGAAATAAGAGACAAAGAGGATAGTTCCTGGCAGCTCTTCACTGT CCAGGTGCAGACTGAGGCCATCGGCGAGGGCAGCCTGGAGCCGTCCCCACAGTCCCAG GCAGAGGATGGCCGGAGCCAGGCGGCAGTTGGGGCGGTACCAGAGGGTGCCTGGAAGG ATACGGCCCAGCTCCACAAGAGCGAGGAGGCGGTGAGTGTCGGACAGAAGCGGGTGCT GCGGTATTACCTCTTCCAGGGCCAGCGCTATATCTGGATCGAGACCCAGCAAGCCTTC TACCAGGTCAGCCTCCTGGACCATGGCCGCTCTTGTGACGACGTCCACCGCTCCCGCC ATGGCCTCAGCCTCCAGGACCAAATGGTGAGGAAGGCCATTTACGGCCCCAACGTGAT CAGCATACCGGTCAAGTCCTACCCCCAGCTGCTGGTGGACGAGGCACTGAACCCCTAC TATGGGTTCCAGGCCTTCAGCATCGCGCTGTGGCTGGCTGACCACTACTACTGGTACG CCCTGTGCATCTTCCTCATTTCCTCCATCTCCATCTGCCTGTCGCTGTACAAGACCAG AAAGCAAAGCCAGACTCTAAGGGACATGGTCAAGTTGTCCATGCGGGTGTGCGTGTGC CGGCCAGGGGGAGAGGAAGAGTGGGTGGACTCCAGTGAGCTAGTGCCCGGAGACTGCC TGGTGCTGCCCCAGGAGGGTGGGCTGATGCCCTGTGATGCCGCCCTGGTGGCCGGCGA GTGCATGGTGAATGAGAGCTCTCTGACAGGAGAGAGCATTCCAGTGCTGAAGACGGCA CTGCCGGAGGGGCTGGGGCCCTACTGTGCAGAGACACACCGGCGGCACACACTCTTCT GCGGGACCCTCATCTTGCAGGCCCGGGCCTATGTGGGACCGCACGTCCTGGCAGTGGT GACCCGCACAGGGTTCTGCACGGCAAAAGGGGGCCTGGTGAGCTCCATCTTGCACCCC CGGCCCATCAACTTCAAGTTCTATAAACACAGCATGAAGTTTGTGGCTGCCCTCTCTG TCCTGGCTCTCCTCGGCACCATCTACAGCATCTTCATCCTCTACCGAAACCGGGTGCC TCTGAATGAGATTGTAATCCGGGCTCTCGACCTGGTGACCGTGGTGGTGCCACCTGCC CTGCCTGCTGCCATGACTGTGTGCACGCTCTACGCCCAGAGCCGACTGCGGAGACAGG GCATTTTCTGCATCCACCCACTGCGCATCAACCTGGGGGGCAAGCTGCAGCTGGTGTG TTTCGACAAGACGGGCACCCTCACTGAGGACGGCTTAGACGTGATGGGGGTGGTGCCC CTGAAGGGGCAGGCATTCCTGCCCCTGGTCCCAGAGCCTCGCCGCCTGCCTGTGGGGC CCCTGCTCCGAGCACTGGCCACCTGCCATGCCCTCAGCCGGCTCCAGGACACCCCCGT GGGCGACCCCATGGACTTGAAGATGGTGGAGTCTACTGGCTGGGTCCTGGAGGAAGAG CCGGCTGCAGACTCAGCATTTGGGACCCAGGTCTTGGCAGTGATGAGACCCCCACTTT GGGAGCCCCAGCTGCAGGCAATGGAGGAGCCCCCGGTGCCAGTCAGCGTCCTCCACCG CTTCCCCTTCTCTTCGGCTCTGCAGCGCATGAGTGTGGTGGTGGCGTGGCCAGGGGCC ACTCAGCCCGAGGCCTACGTCAAAGGCTCCCCGGAGCTGGTGGCAGGGCTCTGCAACC CCGAGACAGTGCCCACCGACTTCGCCCAGATGCTGCAGAGCTATACAGCTGCTGGCTA CCGTGTCGTGGCCCTGGCCAGCAAGCCACTGCCCACTGTGCCCAGCCTGGAGGCAGCC CAGCAACTGACGAGGGACACTGTGGAAGGAGACCTGAGCCTCCTGGGGCTGCTGGTCA TGAGGAACCTACTGAAGCCGCAGACAACGCCAGTTATCCAGGCTCTGCGAAGGACCCG CATCCGCGCCGTCATGGTGACAGGGGACAACCTGCAGACAGCGGTGACTGTGGCCCGG GGCTGTGGCATGGTGGCCCCCCAGGAGCATCTGATCATCGTCCACGCCACCCACCCTG AGCGGGGTCAGCCTGCCTCTCTCGAGTTCCTGCCGATGGAGTCCCCCACAGCCGTGAA TGGCGTTAAGGATCCTGACCAGGCTGCAAGCTACACCGTGGAGCCAGACCCCCGATCC AGGCACCTGGCCCTCAGCGGGCCCACCTTTGGTATCATTGTGAAGCACTTCCCCAAGC TGCTGCCCAAGGTCCTGGTCCAGGGCACTGTCTTTGCCCGCATGGCCCCTGAGCAGAA GACAGAGCTGGTGTGCGAGCTACAGAAGCTTCAGTACTGCGTGGGCATGTGCGGAGAC GGCGCCAATGACTGTGGGGCCCTGAAGGCGGCTGATGTCGGCATCTCGCTGTCCCAGG CAGAAGCCTCAGTGGTCTCACCCTTCACCTCGAGCATGGCCAGTATTGAGTGCGTGCC CATGGTCATCAGGGAGGGGCGCTGTTCCCTTGACACTTCGTTCAGCGTCTTCAAGTAC ATGGCTCTGTACAGCCTGACCCAGTTCATCTCCGTCCTGATCCTCTACACGATCAACA CCAACCTGGGTGACCTGCAGTTCCTGGCCATCGACCTGGTCATCACCACCACAGTGGC AGTGCTCATGAGCCGCACGGGGCCAGCGCTGGTCCTGGGACGGGTGCGGCCACCGGGG GCGCTGCTCAGCGTGCCCGTGCTCAGCAGCCTGCTGCTGCAGATGGTCCTGGTGACCG GCGTGCAGCTAGGGGGCTACTTCCTGACCCTGGCCCAGCCATGGTTCGTGCCTCTGAA CAGGACAGTGGCCGCACCAGACAACCTGCCCAACTACGAGAACACCGTGGTCTTCTCT CTGTCCAGCTTCCAGTACCTCATCCTGGCTGCAGCCGTGTCCAAGGGGGCGCCCTTCC GCCGGCCGCTCTACACCAATGTGCCCTTCCTGGTGGCCCTGGCGCTCCTGAGCTCCGT CCTGGTGGGCCTTGTCCTGGTCCCCGGCCTCCTGCAGGGGCCGCTGGCGCTGAGGAAC ATCACTGACACCGGCTTCAAGCTGCTGCTGCTGGGTCTGGTCACCCTCAACTTCGTGG GGGCCTTCATGCTGGAGAGCGTGCTAGACCAGTGCCTCCCCGCCTGCCTGCGCCGCCT CCGGCCCAAGCGGGCCTCCAAGAAGCGCTTCAAGCAGCTGGAACGAGAGCTGGCCGAG CAGCCCTGGCCGCCGCTGCCCGCCGGCCCCCTGAGGTAGTGCAGGCCCACGGGCACCC

CAGACACTGGAACTCCCTGCCTCTGAGCCACCAACTGGACCCCTCTCCAGCAACACCA

CCGCCACCACCTCCCACATCCCTGAGGTTGGCGACTGTCTACACTCCTCCCCCGAGAC

CACCCCCACCCTGGGGAAGCGTTGACTACTGTCCCCTACCTTGGACCATCCCGCGTAGl

GGGTGGCAGCCCCCAGCTCCCCTCAGTGCTGCTGTCAGTGTAGCAAATAAAGTCATGAl

TATTTTCCTGGC

ORF Start: ATG at 35 ORF Stop: TAG at 3575

SEQ ID NO: 16 1180 aa MW at 128792.0kD

NOV2a, MSADSSP VGSTPTGYGT TIGTSIDPLSSSVSSVR SGYCGSPWRVIGYHVVVW MA GIPL LPR KP WGVR RLRPCNLAHAET VIEIRDKEDSSWQ FTVQVQTEAIGEGS CG108945-01 LEPSPQSQAEDGRSQAAVGAVPEGAW DTAQLHKSEEAVSVGQKRV RYYLFQGQRYI Protein WIETQQAF YQVSL DHGRSCDDVHRSRHGLSLQDQMVRKAIYGP VI S I PVKS YPQL Sequence VΌEALNPYΎGFQAFSIAL ADHYYWYALCIF ISSISICLSLYKTRKQSQTLRDMVK SMRVCVCRPGGEEEWVDSSELVPGDCLVLPQEGGLMPCDAA VAGECMV-JESSLTGE SIPVLKTA PEGLGPYCAETHRRHTLFCGT ILQARAYVGPHV AVVTRTGFCTAKGG LVSSILHPRPINF FYKHSL- FVAALSVLA LGTIYSIFILYR RVPL EIVIRALDL VTV ATPPALPAAMTVCTLYAQSRR.RRQGIFCIHPLRINLGGK QLVCFDKTGT TEDG DVMGVVPLKGQAFLP VPΞPRR PVGP LRALATCHALSRLQDTPVGDPMDLK VES TGWV EEEPAADSAFGTQVLAVMRPP WEPQLQA EEPPVPVSVLHRFPFSSALQRMS VWA PGATQPEAYVKGSPELVAGLCNPETVPTDFAQMLQSYTAAGYRWA ASKPLP TVPSLEAAQQLTRDTVEGDLSLLGL VMRØLLKPQTTPVIQALRRTRIRAVMVTGDNL QTAV1VARGCGMVAPQEHLIIVHATHPERGQPASLEFLPMESPTAVNGVKDPDQAASY TVEPDPRSRHLA SGPTFGIIVKHFPK LPKVLVQGTVFARMAPΞQKTELVCE QKLQ YC GMCGDGAISROCGALKAADVGISLSQAEASVVSPFTSSMASIECVPMV^'IREGRCSLD TSFSVFKY __ YSLTOFISV ILYTIN NLGD OF AIDLVITTTVAV MSRTGPA V GRVRPPGALLSVPVLSS L QMVX.VTGVQLGGYFLTIAQP FVPLNRTVAAPD3VT PN YE1< I Λ7FSLSSFQ I A AVSKG PFRRPLY NVPFLVALA LSSVLVG PG QGP A RNITD GFK LLG VTL FVGAFMLESV DQC PACLRRLRPKRASKKRFK QLERELAEQPWPP PAGPLR

SEQ ID NO: 17 3540 bp

NOV2b, GCGCCGGGGCCGGCGATGAGCGCGAGGAGCCGGCATGAGCGCAGACAGCAGCCCTCTC

GTGGGCAGCACGCCCACCGGTTATGGGACCCTGACGATAGGGACATCAATAGATCCCC CG108945-02 TCAGCTCCTCAGTTTCATCCGTGAGGCTCAGCGGCTACTGTGGCAGTCCATGGAGGGT DNA Sequence CATCGGCTATCACGTCGTGGTCTGGATGATGGCTGGGATCCCTTTGCTGCTCTTCCGT TGGAAGCCCCTGTGGGGGGTGCGGCTGCGGCTCCGGCCCTGCAACCTGGCCCACGCCG AAACACTCGTTATCGAAATAAGAGACAAAGAGGATAGTTCCTGGCAGCTCTTCACTGT CCAGGTGCAGACTGAGGCCATCGGCGAGGGCAGCCTGGAGCCGTCCCCACAGTCCCAG GCAGAGGATGGCCGGAGCCAGGCGGCAGTTGGGGCGGTACCAGAGGGTGCCTGGAAGG ATACGGCCCAGCTCCACAAGAGCGAGGAGGCGGTGAGTGTCGGACAGAAGCGGGTGCT GCGGTATTACCTCTTCCAGGGCCAGCGCTATATCTGGATCGAGACCCAGCAAGCCTTC TACCAGGTCAGCCTCCTGGACCATGGCCGCTCTTGTGACGACGTCCACCGCTCCCGCC ATGGCCTCAGCCTCCAGGACCAAATGGTGAGGAAGGCCATTTACGGCCCCAACGTGAT CAGCATACCGGTCAAGTCCTACCCCCAGCTGCTGGTGGACGAGGCACTGAACCCCTAC TATGGGTTCCAGGCCTTCAGCATCGCGCTGTGGCTGGCTGACCACTACTACTGGTACG CCCTGTGCATCTTCCTCATTTCCTCCATCTCCATCTGCCTGTCGCTGTACAAGACCAG AAAGCAAAGCCAGACTCTAAGGGACATGGTCAAGTTGTCCATGCGGGTGTGCGTGTGC CGGCCAGGGGGAGAGGAAGAGTGGGTGGACTCCAGTGAGCTAGTGCCCGGAGACTGCC TGGTGCTGCCCCAGGAGGGTGGGCTGATGCCCTGTGATGCCGCCCTGGTGGCCGGCGA GTGCATGGTGAATGAGAGCTCTCTGACAGGAGAGAGCATTCCAGTGCTGAAGACGGCA CTGCCGGAGGGGCTGGGGCCCTACTGTGCAGAGACACACCGGCGGCACACACTCTTCT GCGGGACCCTCATCTTGCAGGCCCGGGCCTATGTGGGACCGCACGTCCTGGCAGTGGT GACCCGCACAGGGTTCTGCACGGCAAAAGGGGGCCTGGTGAGCTCCATCTTGCACCCC CGGCCCATCAACTTCAAGTTCTATAAACACAGCATGAAGTTTGTGGCTGCCCTCTCTG TCCTGGCTCTCCTCGGCACCATCTACAGCATCTTCATCCTCTACCGAAACCGGGTGCC TCTGAATGAGATTGTAATCCGGGCTCTCGACCTGGTGACCGTGGTGGTGCCACCTGCC CTGCCTGCTGCCATGACTGTGTGCACGCTCTACGCCCAGAGCCGACTGCGGAGACAGG GCATTTTCTGCATCCACCCACTGCGCATCAACCTGGGGGGCAAGCTGCAGCTGGTGTG TTTCGACAAGACGGGCACCCTCACTGAGGACGGCTAGACGTGATGGGGGTGGTGCCC CTGAAGGGGCAGGCATTCCTGCCCCTGGTCCCAGAGCCTCGCCGCCTGCCTGTGGGGC CCCTGCTCCGAGCACTGGCCACCTGCCATGCCCTCAGCCGGCTCCAGGACACCCCCGT GGGCGACCCCATGGACTTGAAGATGGTGGAGTCTACTGGCTGGGTCCTGGAGGAAGAG CCGGCTGCAGACTCAGCATTTGGGACCCAGGTCTTGGCAGTGATGAGACCCCCACTTT GGGAGCCCCAGCTGCAGGCAATGGAGGAGCCCCCGGTGCCAGTCAGCGTCCTCCACCG CTTCCCCTTCTCTTCGGCTCTGCAGCGCATGAGTGTGGTGGTGGCGTGGCCAGGGGCC ACTCAGCCCGAGGCCTACGTCAAAGGCTCCCCGGAGCTGGTGGCAGGGCTCTGCAACC CCGAGACAGTGCCCACCGACTTCGCCCAGATGCTGCAGAGCTATACAGCTGCTGGCTA CCGTGTCGTGGCCCTGGCCAGCAAGCCACTGCCCACTGTGCCCAGCCTGGAGGCAGCC CAGCAACTGACGAGGGACACTGTGGAAGGAGACCTGAGCCTCCTGGGGCTGCTGGTCA TGAGGAACCTACTGAAGCCGCAGACAACGCCAGTTATCCAGGCTCTGCGAAGGACCCG CATCCGCGCCGTCATGGTGACAGGGGACAACCTGCAGACAGCGGTGACTGTGGCCCGG GGCTGTGGCATGGTGGCCCCCCAGGAGCATCTGATCATCGTCCACGCCACCCACCCTG AGCGGGGTCAGCCTGCCTCTCTCGAGTTCCTGCCGATGGAGTCCCCCACAGCCGTGAA TGGCGTTAAGGTCCTGGTCCAGGGCACTGTCTTTGCCCGCATGGCCCCTGAGCAGAAG ACAGAGCTGGTGTGCGAGCTACAGAAGCTTCAGTACTGCGTGGGCATGTGCGGAGACG GTGCCAATGACTGTGGGGCCCTGAAGGCGGCTGATGTCGGCATCTCGCTGTCCCAGGC AGAAGCCTCAGTGGTCTCACCCTTCACCTCGAGCATGGCCAGTATTGAGTGCGTGCCC ATGGTCATCAGGGAGGGGCGCTGTTCCCTTGACACTTCGTTCAGCGTCTTCAAGTACA TGGCTCTGTACAGCCTGACCCAGTTCATCTCCGTCCTGATCCTCTACACGATCAACAC CAACCTGGGTGACCTGCAGTTCCTGGCCATCGACCTGGTCATCACCACCACAGTGGCA GTGCTCATGAGCCGCACGGGGCCAGCGCTGGTCCTGGGACGGGTGCGGCCACCGGGGG CGCTGCTCAGCGTGCCCGTGCTCAGCAGCCTGCTGCTGCAGATGGTCCTGGTGACCGG CGTGCAGCTAGGGGGCTACTTCCTGACCCTGGCCCAGCCATGGTTCGTGCCTCTGAAC AGGACAGTGGCCGCACCAGACAACCTGCCCAACTACGAGAACACCGTGGTCTTCTCTC TGTCCAGCTTCCAGTACCTCATCCTGGCTGCAGCCGTGTCCAAGGGGGCGCCCTTCCG CCGGCCGCTCTACACCAATGAGCGTGCTAGACCAGTGCCTCCCCGCCTGCCTGCGCCG CCTCCGGCCCAAGCGGGCCTCCAAGAAGCGCTTCAAGCAGCTGGAACGAGAGCTGGCC GAGCAGCCCTGGCCGCCGCTGCCCGCCGGCCCCCTGAGGTAGTGCAGGCCCACGGGCA CCCCAGACACTGGAACTCCCTGCCTCTGAGCCACCAACTGGACCCCTCTCCAGCAACA

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

Table 2B. Comparison of NOV2a against NOV2b.

NOV2a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV2b 1..1078 1039/1078 (96%) 1..1039 1039/1078 (96%)

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

Table 2C. Protein Sequence Properties NO 2a

SignalP analysis: Cleavage site between residues 12 and 13

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

N-region: length 4; pos.chg 0; neg.chg 1 H-region: length 21; peak value 0.00 PSG score: -4.40

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -₇.09 possible cleavage site: between 31 and 32

>» 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 C 1.5: 10

INTEGRAL Likelihood = -2.28 Transmembrane 47 - 63

INTEGRAL Likelihood = -6.05 Transmembrane 259 - 275

INTEGRAL Likelihood = -6.48 Transmembrane 431 - 447

INTEGRAL Likelihood = -3.29 Transmembrane 457 - 473

INTEGRAL Likelihood = -0.80 Transmembrane 938 - 954

INTEGRAL Likelihood = -4.25 Transmembrane 963 - 979

INTEGRAL Likelihood = -5.79 Transmembrane 996 -1012

INTEGRAL Likelihood = -2.87 Transmembrane 1049 -1065

INTEGRAL Likelihood =- -11.83 Transmembrane 1082 -1098

INTEGRAL Likelihood = -6.58 Transmembrane 1118 -1134

PERIPHERAL Likelihood = 1.75 (at 379)

ALOM score: -11.83 (number of TMSs: 10)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 54 Charge difference: 3.5 C( 5.0) - N( 1.5) C > N: C-terminal side will be inside

>» membrane topology: type 3b

MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75) : 5.53 Hyd Moment (95) : 4.13 G content: 4 D/E content: 2 S/T content: 10 Score: -6.25

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 88 LRP|CN

NUCDISC: discrimination of nuclear localization signals pat4: RPKR (4) at 1150 pa 7 : none bipartite: none content of basic residues: 8.6%

NLS Score: -0.22

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

ER Membrane Retention Signals : none

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

PTS2: 2nd peroxisomal targeting signal: none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

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

NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: cytoplasmic Reliability: 94.1

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

Final Results (k = 9/23) :

66.7 %: endoplasmic reticulum

11.1 %: mitochondrial

11.1 %: vesicles of secretory system

11.1 %: vacuolar

» prediction for CG108945-01 is end (k=9) 03401

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

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

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

Example 3. Myotonin-protein kinase

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: 19 3407 bp

NOV3a, CAGGGAGGGCTTGGCTCCACCACTTTCCTCCCCCAGCCTTTGGGCAGCAGGTCACCCC

TGTTCAGGCTCTGAGGGTGCCCCCTCCTGGTCCTGTCCTCACCACCCCTTCCCCACCT CGI 19035-01 CCTGGGAAAAAAAAAAAAAAAAAAΆAAAAAGCTGGTTTAΆGCAGAGAGCCTGAGGGC: DNA Sequence TAAATTTAACTGTCCGAGTCGGAATCCATCTCTGAGTCACCCAAGAAGCTGCCCTGGC

CTCCCGTCCCCTTCCCAGGCCTCAACCCCTTTCTCCCACCCAGCCCCAACCCCCAGCC

CTCACCCCCTAGCCCCCAGTTCTGGAGCTTGTCGGGAGCAAGGGGGTGGTTGCTACTGI

GGTCACTCAGCCTCAATTGGCCCTGTTCAGCAATGGGCAGGTTCTTCTTGAAATTCATl

CACACCTGTGGCTTCCTCTGTGCTCTACCTTTTTATTGGGGTGACAGTGTGACAGCTGI

AGATTCTCCATGCATTCCCCCTACTCTAGCACTGAAGGGTTCTGAAGGGCCCTGGAAGl

GAGGGAGCTTGGGGGGCTGGCTTGTGAGGGGTTAAGGCTGGGAGGCGGGAGGGGGGCTl

GGACCAAGGGGTGGGGAGAAGGGGAGGAGGCCTCGGCCGGCCGCAGAGAGAAGTGGCC

AGAGAGGCCCAGGGGACAGCCAGGGACAGGCAGACATGCAGCCAGGGCTCCAGGGCCT

GGACAGGGGCTGCCAGGCCCTGTGACAGGAGGACCCCGAGCCCCCGGCCCGGGGAGGG

GCCATGGTGCTGCCTGTCCAACATGTCAGCCGAGGTGCGGCTGAGGCGGCTCCAGCAG

CTGGTGTTGGACCCGGGCTTCCTGGGGCTGGAGCCCCTGCTCGACCTTCTCCTGGGCG TCCACCAGGAGCTGGGCGCCTCCGAACTGGCCCAGGACAAGTACGTGGCCGACTTCTT GCAGTGGGCGGAGCCCATCGTGGTGAGGCTTAAGGAGGTCCGACTGCAGAGGGACGAC TTCGAGATTCTGAAGGTGATCGGACGCGGGGCGTTCAGCGAGGTAGCGGTAGTGAAGA TGAAGCAGACGGGCCAGGTGTATGCCATGAAGATCATGAACAAGTGGGACATGCTGAA GAGGGGCGAGGTGTCGTGCTTCCGTGAGGAGAGGGACGTGTTGGTGAATGGGGACCGG CGGTGGATCACGCAGCTGCACTTCGCCTTCCAGGATGAGAACTACCTGTACCTGGTCA TGGAGTATTACGTGGGCGGGGACCTGCTGACACTGCTGAGCAAGTTTGGGGAGCGGAT TCCGGCCGAGATGGCGCGCTTCTACCTGGCGGAGATTGTCATGGCCATAGACTCGGTG CACCGGCTTGGCTACGTGCACAGGGACATCAAACCCGACAACATCCTGCTGGACCGCT GTGGCCACATCCGCCTGGCCGACTTCGGCTCTTGCCTCAAGCTGCGGGCAGATGGAAC GGTGCGGTCGCTGGTGGCTGTGGGCACCCCAGACTACCTGTCCCCCGAGATCCTGCAG GCTGTGGGCGGTGGGCCTGGGACAGGCAGCTACGGGCCCGAGTGTGACTGGTGGGCGC TGGGTGTATTCGCCTATGAAATGTTCTATGGGCAGACGCCCTTCTACGCGGATTCCAC GGCGGAGACCTATGGCAAGATCGTCCACTACAAGGAGCACCTCTCTCTGCCGCTGGTG GACGAAGGGGTCCCTGAGGAGGCTCGAGACTTCATTCAGCGGTTGCTGTGTCCCCCGG AGACACGGCTGGGCCGGGGTGGAGCAGGCGACTTCCGGACACATCCCTTCTTCTTTGG CCTCGACTGGGATGGTCTCCGGGACAGCGTGCCCCCCTTTACACCGGATTTCGAAGGT GCCACCGACACATGCAACTTCGACTTGGTGGAGGACGGGCTCACTGCCATGGTGAGCG GGGGCGGGGAGACACTGTCGGACATTCGGGAAGGTGCGCCGCTAGGGGTCCACCTGCC TTTTGTGGGCTACTCCTACTCCTGCATGGCCCTCAGGGACAGTGAGGTCCCAGGCCCC ACACCCATGGAAGTGGAGGCCGAGCAGCTGCTTGAGCCACACGTGCAAGCGCCCAGCC TGGAGCCCTCGGTGTCCCCACAGGATGAAACAGCTGAAGTGGCAGTTCCAGCGGCTGT CCCTGCGGCAGAGGCTGAGGCCGAGGTGACGCTGCGGGAGCTCCAGGAAGCCCTGGAG GAGGAGGTGCTCACCCGGCAGAGCCTGAGCCGGGAGATGGAGGCCATCCGCACGGACA ACCAGAACTTCGCCAGTCAACTACGCGAGGCAGAGGCTCGGAACCGGGACCTAGAGGC ACACGTCCGGCAGTTGCAGGAGCGGATGGAGTTGCTGCAGGCAGAGGGAGCCACAGCT GTCACGGGGGTCCCCAGTCCCCGGGCCACGGATCCACCTTCCCATCTAGATGGCCCCC CGGCCGTGGCTGTGGGCCAGTGCCCGCTGGTGGGGCCAGGCCCCATGCACCGCCGCCA CCTGCTGCTCCCTGCCAGGGTCCCTAGGCCTGGCCTATCGGAGGCGCTTTCCCTGCTC CTGTTCGCCGTTGTTCTGTCTCGTGCCGCCGCCCTGGGCTGCATTGGGTTGGTGGCCC ACGCCGGCCAACTCACCGCAGTCTGGCGCCGCCCAGGAGCCGCCCGCGCTCCCTGAAC CCTAGAACTGTCTTCGACTCCGGGGCCCCGTTGGAAGACTGAGTGCCCGGGGCACGGC

ACAGAAGCCGCGCCCACCGCCTGCCAGTTCACAACCGCTCCGAGCGTGGGTCTCCGCC

CAGCTCCAGTCCTGTGTACCGGGCCCGCCCCCTAGCGGCCGGGGAGGGAGGGGCCGGG TCCGCGGCCGGCGAACGGGGCTCGAAGGGTCCTTGTAGCCGGGAATGCTGCTGCTGCT GCTGCTGCTGCTGCTGCTGCTGGGGGGATCACAGACCATTTCTTTCTTTCGGCCAGGC

TGAGGCCCTGACGTGGATGGGCAAACTGCAGGCCTGGGAAGGCAGCAAGCCGGGCCGT CCGTGTTCCATCCTCCACGCACCCCCACCTATCGTTGGTTCGCAAAGTGCAAAGCTTT CTTGTGCATGACGCCCTGCTCTGGGGAGCGTCTGGCGCGATCTCTGCCTGCTTACTCG

GGAAATTTGCTTTTGCCAAACCCGCTTTTTCGGGGATCCCGCGCCCCCCTCCTCACTT GCGCTGCTCTCGGAGCCCCAGCCGGCTCCGCCCGCTTCGGCGGTTTGGATATTTATTG ACCTCGTCCTCCGACTCGCTGACAGGCTACAGGACCCCCAACAACCCCAATCCACGTT

TTGGATGCACTGAGACCCCGACATTCCTCGGTATTTATTGTCTGTCCCCACCTAGGAC CCCCACCCCCGACCCTCGCGAATAAAAGGCCCTCCATCTGCCC

ORF Start: ATG at 777 ORF Stop: TGA at 2664

SEQ ID NO: 20 629 aa MW at 69370.3kD NOV3a, MSAEVRLRRLQQLVLDPGFLG EPL DLLLGVHQE GASE AQDKYVADFLQWAEPIV

VRLKEVRLQRDDFEILKVIGRGAFSEVAWK QTGQW^ CGI 19035-01 REERDVLVWGDRR ITQLHFAFQDENY YLVMEYYΛtGGDLLT LSKFGERIPAEMARF Protein YLAEIVMAIDSVHR GYVHRDI PDNI LDRCGHIR ADFGSCLKLRADGTVRS VAV Sequence GTPDY SPEILQAVGGGPGTGSYGPECDWWALGVFAYEMFYGQTPFYADSTAETYGKI

VHYKEHLS PLVDEGVPΞEARDFIQR LCPPETR GRGGAGDFRTHPFFFGLD DG R

DSVPPFTPDFEGATDTCNFD VEDG TAMVSGGGET SDIREGAPLGVHLPFVGYSYS

CMALRDSEVPGPTPMEVEAEQ LEPHVQAPSLEPSVSPQDETAEVAVPAAVPAAEAEA

EVT RELQEALEΞEVLTRQSLSREMEAIRTDNQNFASQLREAEARNRDLEAHVRQ QE

R]_3LLQAEGATAVTGVPSPRATDPPSH DGPPAVAVGQCP VGPGPMHRRH PARV

PRPGLSEALSLL FAVVLSRAAALGCIG VAHAGQLTAV RRPGAARAP

SEQ ID NO: 21 3114 bp

NOV3b, GCCACAAGCCTCCACCCCAGCTGGTCCCCCACCCAGGCTGCCCAGTTTAACATTCCTAl CGI 19035-03 GTCATAGGACCTTGACTTCTGAGAGGCCTGATTGTCATCTGTAAATAAGGGGTAGGAC

TAAAGCACTCCTCCTGGAGGACTGAGAGATGGGCTGGACCGGAGCACTTGAGTCTGGG DNA Sequence JATATGTGACCATGCTACCTTTGTCTCCCTGTCCTGTTCCTTCCCCCAGCCCCAAATCC

AGGGTTTTCCAAAGTGTGGTTCAAGAACCACCTGCATCTGAATCTAGAGGTACTGGATl

JACAACCCCACGTCTGGGCCGTTACCCAGGACATTCTACATGAGAACGTGGGGGTGGGG

CCCTGGCTGCACCTGAACTGTCACCTGGAGTCAGGGTGGAAGGTGGAAGAACTGGGTC iTTATTTCCTTCTCCCCTTGTTCTTTAGGGTCTGTCCTTCTGCAGACTCCGTTACCCCA

CCCTAACCATCCTGCACACCCTTGGAGCCCTCTGGGCCAATGCCCTGTCCCGCAAAGG

GCTTCTCAGGCATCTCACCTCTA GGGAGGGCATTTTTGGCCCCCAGAACCTTACACG GTGTTTATGTGGGGAAGCCCCTGGGAAGCAGACAGTCCTAGGGTGAAGCTGAGAGGCA GAGAGAAGGGGAGACAGACAGAGGGTGGGGCTTTCCCCCTTGTCTCCAGTGCCCTTTC TGGTGACCCTCGGTTCTTTTCCCCCACCACCCCCCCAGCGGAGCCCATCGTGGTGAGG CTTAAGGAGGTCCGACTGCAGAGGGACGACTTCGAGATTCTGAAGGTGATCGGACGCG GGGCGTTCAGCGAGGTAGCGGTAGTGAAGATGAAGCAGACGGGCCAGGTGTATGCCAT GAAGATCATGAACAAGTGGGACATGCTGAAGAGGGGCGAGGTGTCGTGCTTCCGTGAG GAGAGGGACGTGTTGGTGAATGGGGACCGGCGGTGGATCACGCAGCTGCACTTCGCCT TCCAGGATGAGAACTACCTGTACCTGGTCATGGAGTATTACGTGGGCGGGGACCTGCT GACACTGCTGAGCAAGTTTGGGGAGCGGATTCCGGCCGAGATGGCGCGCTTCTACCTG GCGGAGATTGTCATGGCCATAGACTCGGTGCACCGGCTTGGCTACGTGCACAGGGACA TCAAACCCGACAACATCCTGCTGGACCGCTGTGGCCACATCCGCCTGGCCGACTTCGG CTCTTGCCTCAAGCTGCGGGCAGATGGAACGGTGCGGTCGCTGGTGGCTGTGGGCACC CCAGACTACCTGTCCCCCGAGATCCTGCAGGCTGTGGGCGGTGGGCCTGGGACAGGCA GCTACGGGCCCGAGTGTGACTGGTGGGCGCTGGGTGTATTCGCCTATGAAATGTTCTA TGGGCAGACGCCCTTCTACGCGGATTCCACGGCGGAGACCTATGGCAAGATCGTCCAC TACAAGGAGCACCTCTCTCTGCCGCTGGTGGACGAAGGGGTCCCTGAGGAGGCTCGAG ACTTCATTCAGCGGTTGCTGTGTCCCCCGGAGACACGGCTGGGCCGGGGTGGAGCAGG CGACTTCCGGACACATCCCTTCTTCTTTGGCCTCGACTGGGATGGTCTCCGGGACAGC GTGCCCCCCTTTACACCGGATTTCGAAGGTGCCACCGACACATGCAACTTCGACTTGG TGGAGGACGGGCTCACTGCCATGGTGAGCGGGGGCGGGGAGACACTGTCGGACATTCG GGAAGGTGCGCCGCTAGGGGTCCACCTGCCTTTTGTGGGCTACTCCTACTCCTGCATG GCCCTCAGGGACAGTGAGGTCCCAGGCCCCACACCCATGGAAGTGGAGGCCGAGCAGC TGCTTGAGCCACACGTGCAAGCGCCCAGCCTGGAGCCCTCGGTGTCCCCACAGGATGA AACAGCTGAAGTGGCAGTTCCAGCGGCTGTCCCTGCGGCAGAGGCTGAGGCCGAGGTG ACGCTGCGGGAGCTCCAGGAAGCCCTGGAGGAGGAGGTGCTCACCCGGCAGAGCCTGA GCCGGGAGATGGAGGCCATCCGCACGGACAACCAGAACTTCGCCAGTCAACTACGCGA GGCAGAGGCTCGGAACCGGGACCTAGAGGCACACGTCCGGCAGTTGCAGGAGCGGATG GAGTTGCTGCAGGCAGAGGGAGCCACAGCTGTCACGGGGGTCCCCAGTCCCCGGGCCA CGGATCCACCTTCCCATGTCCCTAGGCCTGGCCTATCGGAGGCGCTTTCCCTGCTCCT GTTCGCCGTTGTTCTGTCTCGTGCCGCCGCCCTGGGCTGCATTGGGTTGGTGGCCCAC GCCGGCCAACTCACCGCAGTCTGGCGCCGCCCAGGAGCCGCCCGCGCTCCCTGAACCC TAGAACTGTCTTCGACTCCGGGGCCCCGTTGGAAGACTGAGTGCCCGGGGCACGGCAC

AGAAGCCGCGCCCACCGCCTGCCAGTTCACAACCGCTCCGAGCGTGGGTCTCCGCCCA

GCTCCAGTCCTGTGATCCGGGCCCGCCCCCTAGCGGCCGGGGAGGGAGGGGCCGGGTC CGCGGCCGGCGAACGGGGCTCGAAGGGTCCTTGTAGCCGGGAATGCTGCTGCTGCTGC TGCTGCTGCTGCTGCTGCTGGGGGGATCACAGACCATTTCTTTCTTTCGGCCAGGCTG

AGGCCCTGACGTGGATGGGCAAACTGCAGGCCTGGGAAGGCAGCAAGCCGGGCCGTCC GTGTTCCATCCTCCACGCACCCCCACCTATCGTTGGTTCGCAAAGTGCAAAGCTTTCT TGTGCATGACGCCCTGCTCTGGGGAGCGTCTGGCGCGATCTCTGCCTGCTTACTCGGG AAATTTGCTTTTGCCAAACCCGCTTTTTCGGGGATCCCGCGCCCCCCTCCTCACTTGC GCTGCTCTCGGAGCCCCAGCCGGCTCCGCCCGCTTCGGCGGTTTGGATATTTATTGAC CTCGTCCTCCGACTCGCTGACAGGCTACAGGACCCCCAACAACCCCAATCCACGTTTTj

GGATGCACTGAGACCCCGACATTCCTCGGTATTTATTGTCTGTCCCCACCTAGGACCC CCACCCCCGACCCTCGCGAATAAAAGGCCCTCCATCTGCC

ORF Start: ATG at 545 ORF Stop: TGA at 2372

SEQ ID NO: 22 609 aa MW at 67248.5kD

NOV3b, MGGHFWPPEPYTVFMWGSPWEADSPRVKLRGREKGRQTEGGAFPLVSSALSGDPRFFS PTTPPAEPIVVRLKEVRLQRDDFEILKVIGRGAFSEVAVVKJ^QTGQVYAMKI lvπIrø CGI 19035-03 __KRGΞVSCFREERDVLVNGDRRWITQ HFAFQDENYLYLVMEYYVGGDLLT LSKFG Protein ERIPAE_ FYLAEIVTVlAIDSVHRLGYVHRDIKPDNILLDRCGHIRLADFGSCLKLRA Sequence DGTVRS VAVGTPDY SPEILQAVGGGPGTGSYGPECD WALGVFAYEMFYGQTPFYA DSTAETYGKIVHY EHLSLPLVDEGVPEEARDFIQRLLCPPETR GRGGAGDFRTHPF FFGLD DG RDSVPPFTPDFEGATDTCNFD VEDGLTAMVSGGGET SDIREGAPLGV H PFVGYSYSCMA RDSEVPGPTPMEVEAEQLLEPHVQAPSLEPSVSPQDETAEVAVP AAVPAAEAEAEVT RELQEA EEEVLTRQS SRE_miRTDNQNFASQ REAEARNRD EAHVRQLQERME LQAEGATAVTGVPSPRATDPPSHVPRPG SEA SLLLFAWLSR AAALGCIG VAHAGQLTAVWRRPGAARAP

SEQ ID NO: 23 2503 bp

NOV3c, CGGCCCGGGGAGGGGCCATGGTGCTGCCTGTCCAACATCTCAGCCGAGGTGCGGCTGA CGI 19035-02 GGCGGCTCCAGCAGCTGGTGTTGGACCCGGGCTTCCTGGGGCTGGAGCCCCTGCTCGA CCTTCTCCTGGGCGTCCACCAGGAGCTGGGCGCCTCCGAACTGGCCCAGGACAAGTAC DNA Sequence GTGGCCGACTTCTTGCAGTGGGCGGAGCCCATCGTGGTGAGGCTTAAGGAGGTCCGAC TGCAGAGGGACGACTTCGAGATTCTGAAGGTGATCGGACGCGGGGCGTTCAGCGAGGT AGCGGTAGTGAAGATGAAGCAGACGGGCCAGGTGTATGCCATGAAGATCATGAACAAG TGGGACATGCTGAAGAGGGGCGAGGTGTCGTGCTTCCGTGAGGAGAGGGACGTGTTGG TGAATGGGGACCGGCGGTGGATCACGCAGCTGCACTTCGCCTTCCAGGATGAGAACTA CCTGTACCTGGTCATGGAGTATTACGTGGGCGGGGACCTGCTGACACTGCTGAGCAAG TTTGGGGAGCGGATTCCGGCCGAGATGGCGCGCTTCTACCTGGCGGAGATTGTCATGG CCATAGACTCGGTGCACCGGCTTGGCTACGTGCACAGGGACATCAAACCCGACAACAT CCTGCTGGACCGCTGTGGCCACATCCGCCTGGCCGACTTCGGCTCTTGCCTCAAGCTG CGGGCAGATGGAACGGTGCGGTCGCTGGTGGCTGTGGGCACCCCAGACTACCTGTCCC CCGAGATCCTGCAGGCTGTGGGCGGTGGGCCTGGGACAGGCAGCTACGGGCCCGAGTG TGACTGGTGGGCGCTGGGTGTATTCGCCTATGAAATGTTCTATGGGCAGACGCCCTTC TACGCGGATTCCACGGCGGAGACCTATGGCAAGATCGTCCACTACAAGGAGCACCTCT CTCTGCCGCTGGTGGACGAAGGGGTCCCTGAGGAGGCTCGAGACTTCATTCAGCGGTT GCTGTGTCCCCCGGAGACACGGCTGGGCCGGGGTGGAGCAGGCGACTTCCGGACACAT CCCTTCTTCTTTGGCCTCGACTGGGATGGTCTCCGGGACAGCGTGCCCCCCTTTACAC CGGATTTCGAAGGTGCCACCGACACATGCAACTTCGACTTGGTGGAGGACGGGCTCAC TGCCATGGAGACACTGTCGGACATTCGGGAAGGTGCGCCGCTAGGGGTCCACCTGCCT TTTGTGGGCTACTCCTACTCCTGCATGGCCCTCAGGGACAGTGAGGTCCCAGGCCCCA CACCCATGGAACTGGAGGCCGAGCAGCTGCTTGAGCCACACGTGCAAGCGCCCAGCCT GGAGCCCTCGGTGTCCCCACAGGATGAAACAGCTGAAGTGGCAGTTCCAGCGGCTGTC CCTGCGGCAGAGGCTGAGGCCGAGGTGACGCTGCGGGAGCTCCAGGAACCCCTGGAGG AGGAGGTGCTCACCCGGCAGAGCCTGAGCCGGGAGATGGAGGCCATCCGCACGGACAA CCAGAACTTCGCCAGTCAACTACGCGAGGCAGAGGCTCGGAACCGGGACCTAGAGGCA CACGTCCGGCAGTTGCAGGAGCGGATGGAGTTGCTGCAGGCAGAGGGAGCCACAGCTG TCACGGGGGTCCCCAGTCCCCGGGCCACGGATCCACCTTCCCATATGGCCCCCCGGCC GTGGCTGTGGGCCAGTGCCCGCTGGTGGGGCCAGGCCCCATGCACCGCCGCCACCTGC TGCTCCCTGCCAGGGTCCCTAGGCCTGGCCTATCGGAGGCGCTTTCCCTGCTCCTGTT CGCCGTTGTTCTGTCTCGTGCCGCCGCCCTGGGCTGCATTGGGTTGGTGGCCCACGCC GGCCAACTCACCGCAGTCTGGCGCCGCCCAGGAGCCGCCCGCGCTCCCTGAACCCTAG AACTGTCTTCGACTCCGGGGCCCCGTTGGAAGACTGAGTGCCCGGGGCACGGCACAGA

AGCCGCGCCCACCGCCTGCCAGTTCACAACCGCTCCGAGCGTGGGTCTCCGCCCAGCT

CCAGTCCTGTGATCCGGGCCCGCCCCCTAGCGGCCGGGGAGGGAGGGGCCGGGTCCGC GGCCGGCGAACGGGGCTCGAAGGGTCCTTGTAGCCGGGAATGCTGCTGCTGCTGCTGG GGGGATCACAGACCATTTCTTTCTTTCGGCCAGGCTGAGGCCCTGACGTGGATGGGCA

AACTGCAGGCCTGGGAAGGCAGCAAGCCGGGCCGTCCGTGTTCCATCCTCCACGCACC CCCACCTATCGTTGGTTCGCAAAGTGCAAAGCTTTCTTGTGCATGACGCCCTGCTCTG GGGAGCGTCTGGCGCGATCTCTGCCTGCTTACTCGGGAAATTTGCTTTTGCCAAACCC

GCTTTTTCGGGGATCCCGCGCCCCCCTCCTCACTTGCGCTGCTCTCGGAGCCCCAGCC GGCTCCGCCGCCTTCGGCGGTTTGGATATTTATTGACCTCGTCCTCCGACTCGCTGAC AGGCTACAG ORF Start: ATG at 37 |ORF Stop: TAG at 1912

SEQ ID NO: 24 625 aa ^"JMW at 696-73- ^{"" *"}""

NOV3c, MSAΞVRLRRLQQLVLDPGF GLEPL D LLGVHQELGASE AQDKYVADFLQ AΞPIV vTlLKEVR QRDDFΞI E tlGRGAFSEVAVvTα^QTC^ CGI 19035-02 REERDVL\ GDRR ITQLHFAFQDEIr_LYVMEYYVGGD TLLSKFGERIPAEMARF Protein YIAEIVMAIDSVHRLGYVHRDIKPDNILLDRCGHIRLADFGSCLK RADGTVRS VAV Sequence GTPDYLSPEILQAVGGGPGTGSYGPECD WALGVFAYE FYGQTPFYADSTAETYGKI

VHYKEHLSLPLVDEGVPEEARDFIQR CPPETRLGRGGAGDFRTHPFFFGLDWDGLR

DSVPPFTPDFEGATDTCNFDLVEDGLTAMETLSDIREGAPLGVHLPFVGYSYSCMALR

DSEVPGPTPMELEAEQ LEPHVQAPSLEPSVSPQDETAEVAVPAAVPAAEAEAEV R

ELQEPLEEΞVLTRQSLSREMEAIRTDNQNFASQ REAEARRD EAHVRQ QΞRMEL

QAEGATAVTGVPSPRATDPPSHMAPRP LWASAR WGQAPCTAATCCSLPGSLGLAYR

RRFPCSCSPLFCLVPPPWAALGWWPTPANSPQSGAAQEPPALPEP

SEQ ID NO: 25 2503 bp

NOV3d, CGGCCCGGGGAGGGGCCATGGTGCTGCCTGTCCAACATGTCAGCCGAGGTGCGGCTGA CGI 19035-04 GGCGGCTCCAGCAGCTGGTGTTGGACCCGGGCTTCCTGGGGCTGGAGCCCCTGCTCGA CCTTCTCCTGGGCGTCCACCAGGAGCTGGGCGCCTCCGAACTGGCCCAGGACAAGTAC DNA Sequence GTGGCCGACTTCTTGCAGTGGGCGGAGCCCATCGTGGTGAGGCTTAAGGAGGTCCGAC TGCAGAGGGACGACTTCGAGATTCTGAAGGTGATCGGACGCGGGGCGTTCAGCGAGGT AGCGGTAGTGAAGATGAAGCAGACGGGCCAGGTGTATGCCATGAAGATCATGAACAAG TGGGACATGCTGAAGAGGGGCGAGGTGTCGTGCTTCCGTGAGGAGAGGGACGTGTTGG TGAATGGGGACCGGCGGTGGATCACGCAGCTGCACTTCGCCTTCCAGGATGAGAACTA CCTGTACCTGGTCATGGAGTATTACGTGGGCGGGGACCTGCTGACACTGCTGAGCAAG TTTGGGGAGCGGATTCCGGCCGAGATGGCGCGCTTCTACCTGGCGGAGATTGTCATGG CCATAGACTCGGTGCACCGGCTTGGCTACGTGCACAGGGACATCAAACCCGACAACAT CCTGCTGGACCGCTGTGGCCACATCCGCCTGGCCGACTTCGGCTCTTGCCTCAAGCTG CGGGCAGATGGAACGGTGCGGTCGCTGGTGGCTGTGGGCACCCCAGACTACCTGTCCC CCGAGATCCTGCAGGCTGTGGGCGGTGGGCCTGGGACAGGCAGCTACGGGCCCGAGTG TGACTGGTGGGCGCTGGGTGTATTCGCCTATGAAATGTTCTATGGGCAGACGCCCTTC TACGCGGATTCCACGGCGGAGACCTATGGCAAGATCGTCCACTACAAGGAGCACCTCT CTCTGCCGCTGGTGGACGAAGGGGTCCCTGAGGAGGCTCGAGACTTCATTCAGCGGTT GCTGTGTCCCCCGGAGACACGGCTGGGCCGGGGTGGAGCAGGCGACTTCCGGACACAT CCCTTCTTCTTTGGCCTCGACTGGGATGGTCTCCGGGACAGCGTGCCCCCCTTTACAC CGGATTTCGAAGGTGCCACCGACACATGCAACTTCGACTTGGTGGAGGACGGGCTCAC TGCCATGGAGACACTGTCGGACATTCGGGAAGGTGCGCCGCTAGGGGTCCACCTGCCT TTTGTGGGCTACTCCTACTCCTGCATGGCCCTCAGGGACAGTGAGGTCCCAGGCCCCA CACCCATGGAACTGGAGGCCGAGCAGCTGCTTGAGCCACACGTGCAAGCGCCCAGCCT GGAGCCCTCGGTGTCCCCACAGGATGAAACAGCTGAAGTGGCAGTTCCAGCGGCTGTC CCTGCGGCAGAGGCTGAGGCCGAGGTGACGCTGCGGGAGCTCCAGGAACCCCTGGAGG AGGAGGTGCTCACCCGGCAGAGCCTGAGCCGGGAGATGGAGGCCATCCGCACGGACAA CCAGAACTTCGCCAGTCAACTACGCGAGGCAGAGGCTCGGAACCGGGACCTAGAGGCA CACGTCCGGCAGTTGCAGGAGCGGATGGAGTTGCTGCAGGCAGAGGGAGCCACAGCTG TCACGGGGGTCCCCAGTCCCCGGGCCACGGATCCACCTTCCCATATGGCCCCCCGGCC GTGGCTGTGGGCCAGTGCCCGCTGGTGGGGCCAGGCCCCATGCACCGCCGCCACCTGC TGCTCCCTGCCAGGGTCCCTAGGCCTGGCCTATCGGAGGCGCTTTCCCTGCTCCTGTT CGCCGTTGTTCTGTCTCGTGCCGCCGCCCTGGGCTGCATTGGGTTGGTGGCCCACGCC GGCCAACTCACCGCAGTCTGGCGCCGCCCAGGAGCCGCCCGCGCTCCCTGAACCCTAG AACTGTCTTCGACTCCGGGGCCCCGTTGGAAGACTGAGTGCCCGGGGCACGGCACAGA

AGCCGCGCCCACCGCCTGCCAGTTCACAACCGCTCCGAGCGTGGGTCTCCGCCCAGCT

CCAGTCCTGTGATCCGGGCCCGCCCCCTAGCGGCCGGGGAGGGAGGGGCCGGGTCCGC

GGCCGGCGAACGGGGCTCGAAGGGTCCTTGTAGCCGGGAATGCTGCTGCTGCTGCTGG

GGGGATCACAGACCATTTCTTTCTTTCGGCCAGGCTGAGGCCCTGACGTGGATGGGCA

GCTTTTTCGGGGATCCCGCGCCCCCCTCCTCACTTGCGCTGCTCTCGGAGCCCCAGCC GGCTCCGCCGCCTTCGGCGGTTTGGATATTTATTGACCTCGTCCTCCGACTCGCTGAC AGGCTACAG

ORF Start: ATG at 37 ORF Stop: TAG at 1912 SEQ ID NO: 26 625 aa MW at 69617.3kD NOV3d, MSAEVRLRR QQLVLDPGF GLΞPL DLL GVHQΞLGASELAQDKYVADFLQ AΞPIV VR KEVRLQRDDFEILK^IGRGAFSEVAVv^MKQTGQVYAMKIMNKOTDMLKRGEVSCF CGI 19035-04 REERDVLV GDRR ITQLHFAFQDE YLYV EYYVGGD LTLLSKFGERIPAEMARF Protein YliAEIVMAIDSVHRLGYVHRDI PDNIL DRCGHIR ADFGSCLKLRADGTVRSLVAV Sequence GTPDYLSPEILQAVGGGPGTGSYGPECDW ALGVFAYEMFYGQTPFYADSTAETYGKI VHYKEHLSLPLVDEGVPEEARDFIQRLLCPPETRLGRGGAGDFRTHPFFFGLD DG R DSVPPFTPDFEGATDTCNFDLVEDGLTAMETLSDIREGAP GVH PFVGYSYSC ALR DSEVPGPTPMELEAEQ LEPHVQAPSLEPSVSPQDETAEVAVPAAVPAAEAEAEV R ELQEPLEEEV TRQSLSREMEAIRTDNQNFASQLREAEARNRDLEAHVRQ QERMEL QAEGATAVTGVPSPRATDPPSHMAPRP LWASAR GQAPCTAATCCS PGSLGLAYR RRFPCSCSPLFCLVPPPWAALGWWPTPANSPQSGAAQEPPALPEP

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

Table 3B. Comparison of NOV3a against NOV3b through NOV3d.

NOV3a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV3b 54..629 546/576 (94%) 64..609 546/576 (94%)

NOV3c 1..554 543/554 (98%) 1..549 545/554 (98%)

NOV3d 1..554 543/554 (98%) 1..549 545/554 (98%)

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

Table 3C. Protein Sequence Properties NO 3a

SignalP analysis: No Known Signal Sequence Predicted

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

N-regio : length 9 pos .chg ₃ ; neg.chg 1 H-region: length 6; peak value -₃.46 PSG score: -7.86

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -₂.10 possible cleavage site: between ₃8 and 3.9

>» Seems to have no N-terminal signal peptide

Tentative number of TMS(s) for the threshold 0.5: Number of TMS(s) for threshold 0.5: 1 INTEGRAL Likelihood = -6.26 Transmembrane 591 607 PERIPHERAL Likelihood = 2.12 (at 13| ALOM score: -6.26 (number of TMSs: 1)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 598 Charge difference: 0.0 C( 1.5) - N( 1.5) N >= C: N-terminal side will be inside »> Single TMS is located near the C-terminus

>» membrane topology: type Nt (cytoplasmic tail 1 to 590)

MITDISC: discrimination of mitochondrial targeting seq R content: 3 Hyd Momen (75) : 4.89 Hyd Moment (95) : 3.66 G content: 0 D/E content: 2 S/T content: 1 Score: -4.46

NUCDISC: discrimination of nuclear localization signals pat4: none pat7 : none bipartite: none content of basic residues : 9.4% 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 KIVHYKEHL at 289

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: too long tail

Dileucine motif in the tail: found LL at 25 LL at 28 LL at 29 LL at 156 LL at 159 LL at 202 LL at 317 LL at 428 LL at 526 checking 63 PROSITE DNA binding motifs:

Leucine zipper pattern (PS00029) : *** found *** LDPGFLGLEPLLDLLLGVHQEL at 15 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: cyoplasmic Reliability: 94.1

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

458 A 0.62

459 A 0.72

460 E 0.90

461 A 0.99

462 E 1.00

463 A 1.00

464 E 1.00

465 V 1.00

466 T 1.00

467 L 1.00

468 R 1.00

469 E 1.00

470 L 1.00

471 Q 1.00

472 E 1.00

473 A 1.00

474 L 1.00

475 E 1.00

476 E 1.00

477 E 1.00

478 V 1.00

479 L 1.00

480 T 1.00

481 R 1.00

482 Q 1.00

483 S 1.00

484 L 1.00

485 S 1.00

486 R 1.00

487 E 1.00

488 M 1.00

489 E 1.00

490 A 1.00

491 I 1.00

492 R 1.00

493 T 1.00

494 D 1.00

495 N 1.00

496 Q 1.00

497 N 1.00

498 F 1.00

499 A 1.00

500 S 1.00

501 Q 1.00

502 L 1.00

503 R 1.00

504 E 1.00

505 A 1.00

506 E 1.00

507 A 1.00

508 R 1.00

509 N 1.00

510 R 1.00

511 D 1.00

512 L 1.00

513 E 1.00

514 A 1.00

515 H 1.00

516 V 1.00

517 R 1.00

518 Q 1.00

519 L 1.00

520 Q 1.00

521 E 1.00 522 R 1.00

523 M 1.00

524 E 1.00

525 L 1.00

526 L 1.00

527 Q 1.00

528 A 1.00

529 E 1.00

530 G 1.00

531 A 1.00

532 T 0.99

533 A 0.99

534 V 0.80

535 T 0.73 total: 78 residues

Final Results (k = 9/23):

34.8 % : nuclear

21.7 %: cytoplasmic

13.0 %: Golgi

13.0 %: endoplasmic reticulum

8.7 %: mitochondrial

4.3 %: vesicles of secretory system

4.3 %: peroxisomal

» prediction for CG119035-01 is nuc (k=23)

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

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

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

Example 4. Voltage-dependent L-type calcium channel alpha-lS subunit. The NOV4 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 4A.

Table 4A. NO 4 Sequence Analysis

SEQ ID NO: 27 6160 bp

NOV4a, JTCAGGCCGGCAGCGGGGAGCCGAGTGGAGGCTAATTTTACTTGCTGGGAGCGAGGAGA

IGTAATCCTCCTGCCCCCCACTCCTGCCCCGCCCCCTGGCTGGCTCAGCAGGGCAGCTC CG124873-01 AGCCGACAGCCTCAGCCAGCCTAGTCCCCAAGGCGGGGGCATTGGGGACACAGGGAAG DNA Sequence GGAAAGCACTGGGGTGGGGGAGCAGGAGAAAGCCAGATTCCCAGGGAAGCCATGGAGC

CATCCTCACCCCAGGATGAAGGCCTGAGGAAGAAACAGCCCAAGAAGCCAGTTCCTGA GATTCTGCCAAGGCCACCCCGGGCTTTGTTCTGCCTGACCCTGGAGAACCCCCTGAGG AAGGCCTGCATCAGCATTGTAGAATGGAAGCCCTTCGAGACGATCATCTTGCTCACCA TCTTTGCCAATTGTGTGGCCCTGGCCGTGTACCTGCCCATGCCGGAAGATGACAACAA CTCTCTGAACCTCGGCCTGGAGAAGCTGGAGTATTTCTTCCTCATTGTCTTCTCGATT GAAGCCGCCATGAAGATCATTGCCTACGGCTTCTTATTCCACCAGGACGCTTACCTGC GCAGTGGCTGGAATGTGCTGGACTTCACCATTGTCTTCCTGGGGGTCTTCACCGTGAT TCTGGAACAGGTTAACGTCATCCAAAGCCACACAGCCCCAATGAGCAGCAAAGGAGCC GGCTTGGATGTCAAGGCCCTCAGAGCCTTCCGAGTGCTCAGACCCCTCCGGCTGGTGT CGGGGGTGCCTAGCCTGCAGGTGGTCCTGAACTCCATCTTCAAGGCCATGCTCCCCCT CTTTCACATCGCCCTGCTGGTCCTCTTTATGGTCATCATCTATGCCATCATCGGGCTG GAGCTCTTCAAGGGCAAGATGCACAAGACCTGCTACTTCATTGGTACAGATATCGTGG CCACGGTGGAGAATGAAGAGCCATCGCCCTGCGCCAGGACGGGCTCAGGGCGCCGGTG CACCATCAATGGCAGTGAGTGCCGGGGCGGCTGCCCAGGGCCCAACCATGGCATCACC CACTTCGACAACTTCGGCTTCTCCATGCTCACCGTGTACCAGTGCATTACCATGGAGG GATGGACTGACGTCCTTTACTGGGTCAATGATGCCATCGGGAATGAGTGGCCCTGGAT CTATTTTGTCACCCTCATTTTGCTGGGATCCTTCTTCATCCTCAACCTGGTGCTGGGT GTCCTGAGTGGGGAATTCACCAAGGAGCGGGAGAAGGCCAAGTCCAGGGGAACCTTCC AGAAGCTCCGGGAGAAGCAGCAACTAGATGAGGACCTTCGGGGCTACATGAGCTGGAT CACGCAGGGCGAGGTCATGGATGTTGAGGACTTCAGAGAAGGAAAACTGTCTTTGGAT GAAGGTGGCTCTGACACAGAGAGCCTGTATGAAATTGCAGGCTTGAACAAAATCATCC AGTTCATCCGACATTGGAGGCAGTGGAACCGCATCTTTCGCTGGAAGTGCCATGACAT CGTGAAGTCCAAGGTCTTCTATTGGCTGGTGATTCTCATCGTTGCCCTCAACACCCTG TCTATCGCCTCAGAGCACCACAACCAGCCGCACTGGCTGACCCGTTTGCAAGACATTG CCAACCGGGTGCTGCTGTCCCTCTTCACCACTGAGATGCTGATGAAGATGTACGGGCT GGGCCTGCGCCAGTACTTCATGTCTATCTTCAACCGCTTCGACTGCTTCGTGGTGTGC AGCGGTATCCTGGAGATCCTGCTGGTGGAGTCGGGCGCCATGACACCCCTGGGCATCT CCGTGCTCCGCTGCATCCGCCTCCTGAGGATCTTCAAGATCACCAAATATTGGACGTC GCTGAGCAACCTGGTGGCATCCCTGCTCAACTCCATCCGCTCCATCGCCTCCCTGCTG CTGCTGCTCTTCCTCTTCATCGTCATCTTCCGCCTCCTGGGCATGCAGCTCTTTGGGG GGAGGTATGACTTTGAAGACACAGAAGTACGGCGCAGCAACTTTGACAACTTTCCCCA AGCCCTCATCAGCGTCTTCCAGGTACTGACAGGGGAAGACTGGACCTCAATGATGTAC AATGGGATCATGGCCTCGAGCGGGCCGTCCTACCCTGGCATGCTTGTGTGCATTTACT TCATCATCCTTTTCGTCTGTGGCAACTACATCCTGCTCAATGTCTTCCTGGCCATTGC CGTGGACAACCTGGCCGAGGCGGAGAGCCTGACTTCTGCCCAGAAGGCCAAGGCTGAG GAGAAAAAACGCAGGAAGATGTCCAAGGGTCTCCCAGACAAGTCAGAAGAGGAGAAGT CAACGATGGCCAAGAAGCTGGAGCAGAAACCCAAGGGTGAGGGCATCCCCACCACTGC CAAGCTGAAAATCGATGAGTTTGAATCTAATGTCAATGAGGTGAAGGATCCCTACCCC TCAGCCGACTTCCCAGGGGATGACGAGGAAGATGAGCCTGAGATCCCGCTGAGCCCCC GACCACGTCCCCTGGCTGAGCTGCAGCTGAAAGAGAAGGCCGTGCCCATTCCAGAAGC CAGCTCCTTCTTCATCTTCAGCCCCACCAATAAGATCCGTGTCCTGTGTCACCGCATC GTCAATGCCACCTGGTTCACCAACTTCATCCTGCTCTTCATCCTGCTCAGCAGCGCTG CACTGGCTGCGGAAGACCCCATCCGGGCTGATTCCATGAGAAATCAGATCCTTAAACA CTTTGACATCGGGTTCACCTCTGTCT CACTGTGGAGATTGTCCTCAAGATGACGACC TACGGAGCCTTCCTGCACAAGGGTTCCTTCTGCCGCAATTACTTCAACATGCTGGACC TGCTGGTGGTGGCCGTGTCCCTCATCTCCATGGGACTTGAGTCCAGTGCCATCTCCGT GGTGAAGATCCTGAGGGTGCTGAGGGTGCTCCGACCACTCAGAGCCATCAACAGAGCC AAGGGGTTGAAGCACGTGGCTAGGTGCATGTTCGTGGCCATCAGCACCATCGGGAACA TCGTGCTGGTCACTACCCTCCTACAGTTCATGTTTGCCTGCATCGGCGTCCAGCTCTT CAAGGGGAAGTTCT CAGGTGCACCGACTTGTCCAAGATGACAGAGGAGGAGTGCAGG GGCTACTACTACGTGTACAAGGACGGGGACCCCATGCAGATAGAGCTGCGTCACCGCG AGTGGGTACACAGCGACTTCCACTTCGACAATGTGCTCTCAGCCATGATGTCCCTCTT CACGGTCTCCACCTTCGAGGGATGGCCTCAGCTGCTGTACAAGGCCATAGACTCCAAT GCGGAGGACGTGGGTCCCATCTACAACAACCGTGTGGAGATGGCCATCTTCTTCATCA TCTACATCATCCTCATTGCCTTCTTCATGATGAACATCTTTGTGGGCTTCGTCATTGT CACCTTCCAGGAGCAGGGAGAGACTGAGTACAAGAACTGTGAGCTGGACAAGAACCAG CGCCAATGTGTACAGTATGCCCTGAAGGCCCGCCCACTGAGGTGCTACATTCCCAAAA ACCCATACCAGTACCAGGTGTGGTACATTGTCACCTCCTCCTACTTTGAATACCTGAT GTTTGCCCTCATCATGCTCAACACCATCTGCCTCGGCATGCAGCACTACAA.CCAGTCG GAGCAGATGAACCACATCTCAGACATCCTCAATGTGGCCTTCACTATCATCTTCACCC TGGAGATGATCCTCAAGCTCATGGCCTTCAAGGCCAGGGGCTACTTTGGAAACCCCTG GAATGTGTTTGACTTCCTGATTGTCATTGGCAGCATCATTGATGTCATCCTCAGTGAG ATCGACACTTTCCTGGCCTCCAGCGGGGGACTGTATTGCCTGGGTGGAGGCTGCGGGA ACGTTGACCCAGATGAGAGTGCCCGCATCTCCAGCGCCTTCTTCCGCCTGTTCCGTGT CATGAGGCTGATCAAGCTGCTGAGCCGGGCAGAAGGAGTGCGAACCCTCCTGTGGACG TTCATCAAGTCCTTCCAGGCCCTACCCTACGTGGCTCTGCTCATCGTCATGCTCTTCT TCATCTACGCTGTCATCGGCATGCAGATGTTTGGGAAGATCGCCTTGGTGGATGGGAC CCAAATAAA.CCGGAACAACAACTTCCAGACCTTCCCACAAGCTGTGCTACTGCTCTTC AGGTGTGCAACAGGTGAGGCCTGGCAGGAGATCCTACTGGCCTGCAGCTATGGGAAGC TGTGTGACCCAGAGTCGGACTATGCCCCAGGGGAGGAGTACACATGTGGCACCAACTT TGCATACTACTACTTCATCAGCTTCTACATGCTCTGTGCCTTCCTGGTCATCAACCTC TTTGTGGCTGTCATCATGGACAATTTTGACTACCTCACCCGGGACTGGTCCATCCTGG GCCCTCATCACCTGGATGAGTTCAAGGCCATCTGGGCAGAGTATGACCCAGAGGCTAA GGGGAGGATCAAACACCTGGACGTGGTGACCCTGCTGAGAAGGATTCAGCCCCCTCTG GGCTTTGGGAAGTTCTGCCCACATCGGGTAGCTTGTAAGCGGCTGGTGGGCATGAACA TGCCCCTGAACAGCGACGGCACAGTCACCTTCAATGCCACACTCTTTGCCCTGGTCCG CACGGCACTCAAGATCAAGACGGAAGGTAACTTTGAGCAGGCCAACGAGGAGCTGAGG GCCATCATCAAGAAGATCTGGAAGAGAACCAGCATGAAGCTCTTGGACCAGGTCATCC CTCCAATAGGAGATGATGAGGTGACAGTGGGGAAGTTCTACGCCACATTCCTCATCCA GGAGCACTTCCGGAAGTTCATGAAACGCCAAGAGGAGTATTATGGCTATCGGCCCAAG AAGGACATTGTACAGATCCAGGCAGGGCTGCGGACCATTGAGGAAGAGGCAGCCCCCG AGATCTGTCGCACGGTCTCAGGAGACCTGGCTGCTGAGGAGGAGCTGGAGAGAGCCAT GGTGGAGGCTGCGATGGAGGAGGGGATATTCCGGAGGACTGGAGGCCTGTTTGGCCAG GTGGACAACTTCCTGGAAAGGACCAACTCCCTGCCCCCTGTCATGGCCAATCAGAGAC CCCTCCAGTTTGCTGAGATAGAGATGGAAGAGATGGAGTCACCTGTCT CTTGGAGGA CTTCCCACAAGATCCACGCACCAACCCCCTGGCTCGTGCCAATACCAACAATGCCAAC GCCAATGTCGCCTATGCGAACAGCAACCATAGCAACAGCCATGTGTTTTCCAGTGTCC ACTATGAAAGGGAGTTCCCAGAAGAGACAGAGACGCCTGCTACCAGAGGACGAGCCCT TGGCCAACCCTGCAGGTCCCTGGGACCCCACAGCAAACCCTGTGTGGAGATGCTGAAG GGACTGCTGACCCAGAGGGCAATGCCCAGAGGCCAGGCACCTCCTGCCCCCTGCCAGT GCCCCAGGGTGGAGTCCTCCATGCCTGAGGACAGAAAGAGCTCCACACCAGGGTCTCT TCATGAGGAGACACCCCACAGCAGGAGCACCAGGGAGAATACTTCCAGGTGCTCAGCA CCAGCTACAGCCCTGCTGATCCAAAAGGCTCTGGTTCGAGGGGGCCTGGGCACCTTGG CAGCTGATGCAAACTTCATCATGGCAACAGGCCAGGCCCTCGGAGATGCCTGCCAAAT GGAACCAGAGGAAGTGGAGATCATGGCAACAGAGCTACTGAAAGGACGAGAGGCCCCA GACGGCATGGCCAGCTCCCTGGGATGCCTGAACCTCGGGTCCTCCCTGGGCAGCCTCG ACCAACACCAGGGCTCCCAGGAGACCCTTATTCCTCCAAGGCTGTGATGCCCACACAG

CATCAGCATGGGCTTAGAGCTGGCATGACCAATGGGGGTGGGGAAGTTGCTGGGGTGG

AGAAGGGCTAGCCCACCGCAGCAGCCTCCCTCCCTCTCAGCAGCTAGATGCATGCCTG

AGGCAGGGTGGTCAGGAACCACCTCAAAAAGTGCGGAGGAAGTAGCTGGACAGGCCCT GCCCCTCACCAGCAAGAGGCATGATTGGATGGAGCTTCTAATGTCATTCAAAAΑGGCC TGGTCAGTGCCTGTCCCTAGGGCCACTCCCACCTGCAGGACATTAAAATCTCCAGGCC TGTGACACTGGC

ORF Start: ATG at 226 ORF Stop: TGA at 5845

SEQ ID NO: 28 1873 aa MW at 212300.4kD

NOV4a, MEPSSPQDEGLRKKQPKKPVPEI PRPPRALFCLTLENPLRKACISIVE KPFETIIL

LTIFANCVA AVYLPMPEDDI S GLEK EYFF IVFSIEAAMKIIAYGF FHQDA CG124873-01 YLRSGWNVLDFTIVTLGVFTVILEQV1WIQSHTAPMSSKGAGLDVKALRAFRVLRPLR Protein LVSGVPSLQWLNSIFKA PLFHIALLVLFMVIIYAIIGLELFKGKMHKTCYFIGTD Sequence IVATVE EΞPSPCARTGSGRRCTINGSECRGGCPGPITOGITHFDNFGFSM TVYQCIT

MEGWTDVLYWWTOAIGNEWP IYFvTLILLGSFFI l_VLGVLSGEFTKEREKAKSRG

TFQKLREKQQLDEDLRGY S ITQGΞVMDVEDFREGK SLDEGGSDTESLYEIAGL K

IIQFIRH RQlrøRIFRWKCHDIVKSKVFYWLVI IVAI_W SIASEHHNQPH ^

DIAITOV LS FTTE_^MK1_^*GLG R0YFMSIFNRFDCFVVCSGILEI VESGAMTP GISV RCIRLLRIFKITKY TS SNLVASLLNSIRSIASL LLLFLFIVIFRLLGMQL FGGRYDFEDTEVRΛSl^DNFPQA ISVFQVLTGED TSMMYNGIMASSGPSYPGMLVC IYFIILFVCGlSπfl LlvTvTLAIAVO IJ.EAESLTSAQKAKAEEKXRRKMSKGLPDKSEE

EKSTL_^KIIEQKPKGEGIPTTAK KIDEFESNV]VΠ-VKDPYPSADFPGDDEEDEPEIPL SPRPRP AE Q KEKAVPIPEASSFFIFSPTNKIRVLCHRIVNATWFTNFIL FILLS SAA AAEDPIRADSMRNQILKHFDIGFTSVFTVEIVLKMTTYGAFLHKGSFCRNYFI M LDL VVAVSLISMGLESSAISVV^ILRVLRVLRP RAINRAKGLKHVARCMFVAISTI GNIV VTT QFMFACIGVQ FKGKFFRCTDLSKMΓEEECRGYYYVYKDGDPMQIELR

HRE VΗSDFHFD]Srv_SAMMSLFTVSTFEGWPQLLYKAIDSNAEDVGPIYtrøRVEl<aiF FIIYIILIAFFMMNIFVGFVIVTFQEQGETEYK CELDKNQRQCVQYALKARP RCYI PKNPYQYQV YIVTSSYFEYLMFA IM NTICLGMQHYWQSΞQ]_raiSDILlWAFTII FTLEMI KLMAFKARGYFGNPWNVFDF IVIGSIIDVI SEIDTFLASSGGLYCLGGG CG VDPDESARISSAFFRLFRVMR IKLLSRAEGVRTLL TFIKSFQA PYVA IV LFFIYAVIGMQMFGKIALVDGTQINRrøπSlF'QTFPQAVL LFRCATGEA QEILLACSY GKLCDPESDYAPGEEYTCGTWFAYYYFISFYMLCAF VINLFVAVIMDKTFDYLTRDWS I GPHHLDEFKAI AEYDPEAKGRIKH DWTL RRIQPPLGFGKFCPHRVACKRLVG M^PLNSDGTV FNATLFALVRTALKI TEGIWEQANEELRAIIKKIWKRTSMKL DQ VIPPIGDDEVTVGKFYATFLIQEHFR FMKRQEEYYGYRPKKDIVQIQAG RTIEEEA APEICRTVSGDLAAEEE ERAMVEAAMEEGIFRRTGGLFGQx^IWLERTNS PPVMAN QRP QFAEIEMEEMESPVFLEDFPQDPRTNPLARA1W1 ANA1 AYA SNHSNSHVFS SVHYEREFPEΞTETPATRGRA GQPCRSLGPHS PCVEM KGL TQRA PRGQAPPAP CQCPRVΞSSMPEDRKSSTPGS HEETPHSRSTRENTSRCSAPATALLIQKALVRGG G TLAADANFIMATGQALGDACQ EPEEVEIMATEL KGREAPDGMASS GC LGSSLG SLDQHQGSQETLIPPRL

SEQ ID NO: 29 5727 bp

NOV4b, ACAGGGAAGGGAAAGCACTGGGGTGGGGGAGCAGGAGAAAGCCAGATTCCCAGGGAAG

CCATGGAGCCATCCTCACCCCAGGATGAAGGCCTGAGGAAGAAACAGCCCAAGAAGCC CG124873-02 AGTTCCTGAGATTCTGCCAAGGCCACCCCGGGCCTTGTTCTGCCTGACCCTGGAGAAC DNA Sequence CCCCTGAGGAAGGCCTGCATCAGCATTGTAGAATGGAAGCCCTTCGAGACGATCATCT TGCTCACCATCTTTGCCAAT GTGTGGCCCTGGCCGTGTACCTGCCCATGCCGGAAGA TGACAACAACTCTCTGAACCTCGGCCTGGAGAAGCTGGAGTATTTCTTCCTCATTGTC TTCTCGATTGAAGCCGCCATGAAGATCATTGCCTACGGCTTCTTATTCCACCAGGACG CTTACCTGCGCAGTGGCTGGAATGTGCTGGACTTCACCATTGTCTTCCTGGGGGTCTT CACCGTGATTCTGGACAAGGTTAACGTCATCCAAAGCCACACAGCCCCCATGAGCAGC AAAGGAGCCGGCTTGGATGTCAAGGCCCTCAGAGCCTTCCGAGTGCTCAGACCCCTCC GGCTGGTGTCGGGGGTGCCTAGCCTGCAGGTGGTCCTGAACTCCATCTTCAAGGCCAT GCTCCCCCTCTTTCACATCGCCCTGCTGGTCCTCTTTATGGTCATCATCTATGCCATC ATCGGGCTGGAGCTCTTCAAGGGCAAGATGCACAAGACCTGCTACTTCATTGGTACAG ATATCGTGGCCACGGTGGAGAATGAAGAGCCATCGCCCTGCGCCAGGACGGGCTCAGG GCGCCGGTGCACCATCAATGGCAGTGAGTGCCGGGGCGGCTGGCCAGGGCCCAACCAT GGCATCACCCACTTCGACAACTTCGGCTTCTCCATGCTCACCGTGTACCAGTGCATTA CCATGGAGGGATGGACTGACGTCCTTTACTGGGTCAATGATGCCATCGGGAATGAGTG GCCCTGGATCTATTTTGTCACCCTCATTTTGCTGGGATCCTTCTTCATCCTCAACCTG GTGCTGGGTGTCCTGAGTGGGGAATTCACCAAGGAGCGGGAGAAGGCCAAGTCCAGGG GAACCTTCCAGAAGCTCCGGGAGAAGCAGCAACTAGATGAGGACCTTCGGGGCTACAT GAGCTGGATCACGCAGGGCGAGGTCATGGATGTTGAGGACTTCAGAGAAGGAAAACTG TCTTTGGATGAAGGTGGCTCTGACACAGAGAGCCTGTATGAAATTGCAGGCTTGAACA AAATCATCCAGTTCATCCGACATTGGAGGCAGTGGAACCGCATCTTTCGCTGGAAGTG CCATGACATCGTGAAGTCCAAGGTCTTCTATTGGCTGGTGATTCTCATCGTTGCCCTC AACACCCTGTCTATCGCCTCAGAGCACCACAACCAGCCTCTCTGGCTGACCCGTTTGC AAGACATTGCCAACCGGGTGCTGCTGTCCCTCTTCACCACTGAGATGCTGATGAAGAT GTACGGGCTGGGCCTGCGCCAGTACTTCATGTCTATCTTCAACCGCTTCGACTGCTTC GTGGTGTGCAGCGGTATCCTGGAGATCCTGCTGGTGGAGTCGGGCGCCATGACACCCC TGGGCATCTCCGTGCTCCGCTGCATCCGCCTCCTGAGGATCTTCAAGATCACCAAATA TTGGACGTCGCTGAGCAACCTGGTGGCATCCCTGCTCAACTCCATCCGCTCCATCGCC TCCCTGCTGCTGCTGCTCTTCCTCTTCATCGTCATCTTCGCCCTCCTGGGCATGCAGC TCTTTGGGGGGAGGTATGACTTTGAAGACACAGAAGTACGGCGCAGCAACTTTGACAA CTTTCCCCAAGCCCTCATCAGCGTCTTCCAGGTACTGACAGGGGAAGACTGGACCTCA ATGATGTACAATGGGATCATGGCCTACGGCGGGCCGTCCTACCCTGGCATGCTTGTGT GCATTTACTTCATCATCCTTTTCGTCTGTGGCAACTACATCCTGCTCAATGTCTTCCT GGCCATTGCCGTGGACAACCTGGCCGAGGCGGAGAGCCTGACTTCTGCCCAGAAGGCC AAGGCTGAGGAGAAAAAACGCAGGAAGATGTCCAAGGGTCTCCCAGACAAGTCAGAAG AGGAGAAGTCAACGATGGCCAAGAAGCTGGAGCAGAAACCCAAGGGTGAGGGCATCCC CACCACTGCCAAGCTGAAAATCGATGAGTTTGAATCTAATGTCAATGAGGTGAAGGAT CCCTACCCCTCAGCCGACTTCCCAGGGGATGACGAGGAAGATGAGCCTGAGATCCCGC TGAGCCCCCGACCACGTCCCCTGGCTGAGCTGCAGCTGAAAGAGAAGGCCGTGCCCAT TCCAGAAGCCAGCTCCTTCTTCATCTTCAGCCCCACCAATAAGATCCGTGTCCTGTGT CACCGCATCGTCAATGCCACCTGGTTTACCAACTTCATCCTGCTCTTCATCCTGCTCA GCAGCGCTGCACTGGCTGCGGAAGACCCCATCCGGGCTGATTCCATGAGAAATCAGAT CCTTAAACACTTTGACATCGGGTTCACCTCTGTCTTCACTGTGGAGATTGTCCTCAAG ATGACGACCTACGGAGCCTTCCTGCACAAGGGTTCCTTCTGCCGCAATTACTTCAACA TGCTGGACCTGCTGGTGGTGGCCGTGTCCCTCATCTCCATGGGACTTGAGTCCAGTGC CATCTCCGTGGTGAAGATCCTGAGGGTGCTGAGGGTGCTCCGACCACTCAGAGCCATC AACAGAGCCAAGGGGTTGAAGTGCATGTTCGTGGCCATCAGCACCATCGGGAΛCATCG TGCTGGTCACTACCCTCCTACAGTTCATGTTTGCCTGCATCGGCGTCCAGCTCTTCAA GGGGAAGTTCTTCAGGTGCACCGACTTGTCCAAGATGACAGAGGAGGAGTGCAGGGGC TACTACTACGTGTACAAGGACGGGGACCCCATGCAGATAGAGCTGCGTCACCGCGAGT GGGTACACAGCGACTTCCACTTCGACAATGTGCTCTCAGCCATGATGTCCCTCTTCAC GGTCTCCACCTTCGAGGGATGGCCTCAGCTGCTGTACAAGGCCATAGACTCCAATGCG GAGGACGTGGGTCCCATCTACAACAACCGTGTGGAGATGGCCATCTTCTTCATCATCT ACATCATCCTCATTGCCTTCTTCATGATGAACATCTTTGTGGGCTTCGTCATTGTCAC CTTCCAGGAGCAGGGAGAGACTGAGTACAAGAACTGTGAGCTGGACAAGAACCAGCGC CAATGTGTACAGTATGCCCTGAAGGCCCGCCCACTGAGGTGCTACATTCCCAAAAACC CATACCAGTACCAGGTGTGGTACATTGTCACCTCCTCCTACTTTGAATACCTGATGTT TGCCCTCATCATGCTCAACACCATCTGCCTCGGCATGCAGCACTACAACCAGTCGGAG CAGATGAACCACATCTCAGACATCCTCAATGTGGCCTTCACTATCATCTTCACCCTGG AGATGATCCTCAAGCTCATGGCCTTCAAGGCCAGGGGCTACTTTGGAGACCCCTGGAA TGTGTTTGACTTCCTGATTGTCATTGGCAGCATCATTGATGTCATCCTCAGTGAGATC GACACTTTCCTGGCCTCCAGCGGGGGACTGTATTGCCTGGGTGGAGGCTGCGGGAACG TTGACCCAGATGAGAGTGCCCGCATCTCCAGCGCCTTCTTCCGCCTGTTCCGTGTCAT GAGGCTGATCAAGCTGCTGAGCCGGGCAGAAGGAGTGCGAACCCTCCTGTGGACGTTC ATCAAGTCCTTCCAGGCCCTACCCTACGTGGCTCTGCTCATCGTCATGCTCTTCTTCA TCTACGCTGTCATCGGCATGCAGATGTTTGGGAAGATCGCCTTGGTGGATGGGACCCA AATAAACCGGAACAACAACTTCCAGACCTTCCCACAAGCTGTGCTACTGCTCTTCAGG CACGCGTGTGCAACAGGTGAGGCCTGGCAGGAGATCCTACTGGCCTGCAGCTATGGGA AGCTGTGTGACCCAGAGTCGGACTATGCCCCAGGGGAGGAGTACACATGTGGCACCAA CTTTGCATACTACTACTTCATCAGCTTCTACATGCTCTGTGCCTTCCTGGTCATCAAC CTCTTTGTGGCTGTCATCATGGACAATTTTGACTACCTCACCCGGGACTGGTCCATCC TGGGCCCTCATCACCTGGATGAGTTCAAGGCCATCTGGGCAGAGTATGACCCAGAGGC TAAGGGGAGAATCAAACACCTGGACGTGGTGACCCTGCTGAGAAGGATTCAGCCCCCT CTGGGCTTTGGGAAGTTCTGCCCACATCGGGTAGCTTGTAAGCGGCTGGTGGGCATGA ACATGCCCCTGAACAGCGACGGCACAGTCACCTTCAATGCCACACTCTTTGCCCTGGT CGGCACGGCACTCAAGATCAAGACGGAAGGTAACTTTGAGCAGGCCAACGAGGAGCTG AGGGCCATCATCAAGAAGATCTGGAAGAGAACCAGCATGAAGCTCTTGGACCAGGTCA TGCCTCCAATAGGAGATGATGAGGTGACAGTGGGGAAGTTCTACGCCACATTCCTGAT CCAGGAGGACTTCCGGAAGTTCATGAAACGCCAAGAGGAGTATTATGGCTATCGGCCC AAGAAGGACAT GTACAGATCCAGGCAGGGCTGCGGACCATTGAGGAAGAGGCAGCCC CCGAGATCTGTCGCACGGTCTCAGGAGACCTGGCTGCTGAGGAGGAGCTGGAGAGAGC CATGGTGGAGGCTGCGATGGAGGAGGGAATATTCCGTGTCCACTATGAAAGGGAGTTC CCAGAAGAGACAGAGACGCCTGCTACCAGAGGACGAGCCCTTGGCCAACCCTGCAGGG TCCTGGGACCCCACAGCAAACCCTGTGTGGAGATGCTGAAGGGACTGCTGACCCAGAG GGCAATGCCCAGAGGCCAGGCACCTCCTGCCCCCTGCCAGTGCCCCAGGGTGGAGTCC TCCATGCCTGAGGACAGAAAGAGCTCCACACCAGGGTCTCTTCATGAGGAGACACCCC ACAGCAGGAGCACCAGGGAGAATACTTCCAGGTGCTCAGCACCAGCTACAGCCCTGCT GATCCAAAAGGCTCTGGTTCGAGGGGGCCTGGGCACCTTGGCAGCTGATGCAAACTTC ATCATGGCAACAGGCCAGGCCCTGGCAGATGCCTGCCAAATGGAACCAGAGGAAGTGG AGATCATGGCAACAGAGCTACTGAAAGGACGAGAGGCCCCAGAGGGCATGGCCAGCTC CCTGGGATGCCTGAACCTCGGGTCCTCCCTGGGCAGCCTCGACCAACACCAGGGCTCC CAGGAGACCCTTATTCCTCCAAGGCTGTGATGCCCACACAGCATCAGCATGGGCTTAG

AGCTGGCATGACCAATGGGGGTGGGGAAGTTGCTGGGGTGGAGAAGGGCTAGCCCACC

GCAGCAGCCTCCCTCCCTCTCAGCAGCTAGATGCATGCCTGAGGCAGGGTGGTCAGGA

ACCACCTCAAAAAGTGCGGAGGAAGTAGCTGGACAGGCCCTGCCCCTCACCAGCAAGA GGCATGATTGGATGGAGCTTCTAATGTCATTCAAAAAGGCCTGGTCAGTGCCTGTCTG

GCCTAGGGACCTCCCACCTGCAGGACATTAAAATCTCCAGGCC

ORF Start: ATG at 61 ORF Stop: TGA at 5422 W

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

Table 4B. Comparison of NOV4a against NOV4b.

NOV4a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV4b 1..1599 1584/1601 (98%) 1..1597 1588/1601 (98%)

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

Table 4C. Protein Sequence Properties NOV4a

SignalP analysis: Cleavage site between residues 2 and 3

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

N-region: length 9; pos.chg 0; neg.σhg 3 H-region: length 2; peak value 0.00 PSG score: -4.40

GvH: von Heijne's method for signal seq. recognition

GvH score (threshold: -2.1): -8.25 possible cleavage site: between 42 and 43

>» 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 T S(s) for the threshold C .5: 18

INTEGRAL Likelihood = -8.17 Transmembrane 56 - 72

INTEGRAL Likelihood = -2.81 Transmembrane 91 - 107

INTEGRAL Likelihood = -5.26 Transmembrane 123 - 139

INTEGRAL Likelihood =-13.69 Transmembrane 199 - 215

INTEGRAL Likelihood = -9.34 Transmembrane 317 - 333

INTEGRAL Likelihood = -6.26 Transmembrane 433 - 449

INTEGRAL Likelihood = -2.66 Transmembrane 499 - 515

INTEGRAL Likelihood =-11.83 Transmembrane 559 - 575

INTEGRAL Likelihood = -7.96 Transmembrane 636 - 652

INTEGRAL Likelihood = -4.83 Transmembrane 801 - 817

INTEGRAL Likelihood = -3.08 Transmembrane 869 - 885

INTEGRAL Likelihood = -6.69 Transmembrane 931 - 947

INTEGRAL Likelihood =-12.26 Transmembrane 1041 -1057

INTEGRAL Likelihood = -0.85 Transmembrane 1120 -1136

INTEGRAL Likelihood = -3.40 Transmembrane 1155 -1171

INTEGRAL Likelihood = -6.58 Transmembrane 1184 -1200

INTEGRAL Likelihood =-11.62 Transmembrane 1270 -1286

INTEGRAL Likelihood = -9.39 Transmembrane 1364 -1380

PERIPHERAL Likelihood = 1.48 (at 522)

ALOM score: -13.69 (number of TMSs: 18)

MTOP: Prediction of membrane topology (Hartmann et al .)

Center position for calculation: 63

Charge difference: -5.0 C(-4.0) - N( 1.0)

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

»> membrane topology: type 3a

MITDISC: discrimination of mitochondrial targeting seq

R content: 0 Hyd Moment (75) : 9.65 Hyd Moment (95) : 1.83 G content: 0 D/E content: 2 S/T content: 2 Score: -6.45

NUCDISC: discrimination of nuclear localization signals pat4: KKRR (5) at 681 pat4: KRRK (5) at 682 pat4: RPKK (4) at 1547 pat7 : none bipartite: RKFMKRQEEYYGYRPKK at 1534 content of basic residues: 10.0% NLS Score: 0.90

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

ER Membrane Retention Signals : none

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

PTS2: 2nd peroxisomal targeting signal: none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

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

Leucine zipper pattern (PS00029) : *** found *** LFHQDAYLRSGWNVLDFTIVFL at 111 none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none

NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Predictio : cytoplasmic Reliability: 94.1

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

Final Results (k = 9/23):

77.8 %: endoplasmic reticulum 11.1 %: vacuolar 11.1 %: mitochondrial

» prediction for CG124873-01 is end (k=9) A search of the NOV4a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 4D.

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

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

Example 5. SIMILAR TO CHITINASE 3-LIKE 1.

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

CG138554-03 RTFIKSVPPFLRTHGFDG DLAWLYPGRGDKQHFTTLIKEMKAEFIKEAQPGKKQLL

Protein SAA SAGKVTIDSSYDIAKISQH DFISI YDFHGAWRGTTGHHSPLFRGQEDASPD RFSNTDYAVGYMLRLGAPASKLVMGIPTFGRSFTLASSETGVGAPISGPGIPGRFTKE

Sequence AGTLAYYEICDF RGATVHRILGQQVPYATKGNQWVGYDDQESVKSKVQY KDRQLAG AMV A D DDFQGSFCGQDLRFPLT VIKDALAAT

SEQ ID NO: 33 1202 bp

NOV5b, GGAAGAGGCCCTGTCTAGGTAGCTGGCACCAGGAGCCGTGGGCAAGGGAAGAGGCCAC CG138554-02 ACCCTGCCCTGCTCTGCTGCAGCCAGAATGGGTGTGAAGGCGTCTCAAACAGGCTTTG TGGTCCTGGTGCTGCTCCAGTGCTGCTCTGCATACAAACTGGTCTGCTACTACACCAG DNA Sequence CTGGTCCCAGTACCGGGAAGGCGATGGGAGCTGCTTCCCAGATGCCCTTGACCGCTTC CTCTGTACCCACATCATCTACAGCTTTGCCAATATAAGCAACGATCACATCGACACCT GGGAGTGGAATGATGTGACGCTCTACGGCATGCTCAACACACTCAAGAACAGATTTTC CAAGATAGCCTCCAACACCCAGAGTCGCCGGACTTTCATCAAGTCAGTACCGCTATTT CTGCGCACCCATGGCTTTGATGGGCTGGACCTTGCCTGGCTCTACCCTGGACGGAGAG ACAAACAGCATTTTACCACCCTAATCAAGGAAATGAAGGCCGAATTTATAAAGGAAGC CCAGCCAGGGAAAAAGCAGCTCCTGCTCAGCGCAGCACTGTCTGCGGGGAAGGTCACC ATTGACAGCAGCTATGACATTGCCAAGATATCCCAATACCTGGATTTCATTAGCATCA TGACCTACGATTTTCATGGAGCCTGGCGTGGGACCACAGGCCATCACAGTCCCCTGTT CCGAGGTCAGGAGGATGCAAGTCCTGACAGATTCAGCAACACTGACTATGCTGTGGGG TACATGTTGAGGCTGGGGGCTCCTGCCAGTAAGCTGGTGATGGGCATCCCCACCTTCG GGAGGAGCTTCACTCTGGCTTCTTCTGAGACTGGTGTTGGAGCCCCAATCTCAGGACC GGGAATTCCAGGCCGGTTCACCAΑGGAGGCAGGGACCCTTGCCTACTATGAGATCTGT GACTTCCTCCGCGGAGCCACAGTCCATAGAATCCTCGGCCAGCAGGTCCCCTATGCCA CCAAGGGCAACCAGTGGGTAGGATACGACGACCAGGAAAGCGTCAAAAGCAAGGTGCA GTACCTGAAGGACAGGCAGCTGGCGGGCGCCATGGTATGGGCCCTGGACCTGGATGAC TTCCAGGGCTCCTTCTGCGGCCAGGATCTGCGCTTCCCTCTCACCAATGCCATCAAGG ATGCACTCGCTGCAACGTAOCCCTCTGTTCTGCACACAGCAC

ORF Start: ATG at 86 ORF Stop: TAG at 1178

SEQ ID NO: 34 364 aa MW at 40596.7kD

NOV5b, MGVKASQTGFW V QCCSAYKLVCYYTS SQYREGDGSCFPDA DRFLCTHIIYSF CG138554-02 A ISNDHIDT EWISroVTLYGMLl^LKlrøFSKIASrWQSRRTFIKSVPLFLRTHGFDGL DLAWLYPGRRDKQHFTT I EMKAEFIKEAQPGKKQLLLSAALSAGKVTIDSSYDIAK Protein ISQYLDFISIM YDFHGA RGTTGHHSP FRGQEDASPDRFSNTDYAVGYM RLGAPA Sequence SK VMGIPTFGRSFT ASSETGVGAPISGPGIPGRFTKEAGTLAYYEICDF RGATVH RILGQQVPYATKGNQWVGYDDQESVKSKVQYLKDRQ AGAMV ALD DDFQGSFCGQD RFPL NAIKDALAAT

SEQ ID NO: 35 1741 bp

NOV5c, CTAGGTAGCTGGCACCAGGAGCCGTGGGCAAGGGAAGAGGCCACACCCTGCCCTGCTC CG138554-01 TGCTGCAGCCAGAATGGGTGTGAAGGCGTCTCAAACAGGCTTTGTGGTCCTGGTGCTG

CTCCAGTGCTGCTCTGCATACAAACTGGTCTGCTACTACACCAGCTGGTCCCAGTACC DNA Sequence GGGAAGGCGATGGGAGCTGCTTCCCAGATGCCCTTGACCGCTTCCTCTGTACCCACAT CATCTACAGCTTTGCCAATATAAGCAACGATCACATCGACACCTGGGAGTGGAATGAT GTGACGCTCTACGGCATGCTCAACACACTCAAGAACAGGAACCCCAACCTGAAGACTC TCTTGTCTGTCGGAGGATGGAACTTTGGGTCTCAAAGATTTTCCAAGATAGCCTCCAA CACCCAGAGTCGCCGGACTTTCATCAAGTCAGTACCGCCATTCCTGCGCACCCATGGC TTTGATGGGCTGGACCTTGCCTGGCTCTACCCTGGACGGAGAGACAAACAGCATTTTA CCACCCTAATCAA.GGAAATGAAGGCCGAATTTATAAAGGAAGCCCAGCCAGGGAAAAA GCAGCTCCTGCTCAGCGCAGCACTGTCTGCGGGGAAGGTCACCATTGACAGCAGCTAT GACATTGCCAAGATATCCCAACACCTGGATTTCATTAGCATCATGACCTACGATTTTC ATGGAGCCTGGCGTGGGACCACAGGCCATCACAGTCCCCTGTTCCGAGGTCAGGAGGA TGCAAGTCCTGACAGATTCAGCAACACTGACTATGCTGTGGGGTACATGTTGAGGCTG GGGGCTCCTGCCAGTAAGCTGGTGATGGGCATCCCCACCTTCGGGAGGAGCTTCACTC TGGCTTCTTCTGAGACTGGTGTTGGAGCCCCAATCTCAGGACCGGGAATTCCAGGCCG GΪTCACCAAGGAGGCAGGGACCCTTGCCTACTATGAGATCTGTGACTTCCTCCGCGGA GCCACAGTCCATAGAACCCTCGGCCAGCAGGTCCCCTATGCCACCAAGGGCAACCAGT GGGTAGGATACGACGACCAGGAAAGCGTCAAAAGCAAGGTGCAGTACCTGAAGGATAG GCAGCTGGCAGGCGCCATGGTATGGGCCCTGGACCTGGATGACTTCCAGGGCTCCTTC TGCGGCCAGGATCTGCGCTTCCCTCTCACCAATGCCATCAA.GGATGCACTCGCTGCAA CGTAGCCCTCTGTTCTGCACACAGCACGGGGGCCAAGGATGCCCCGTCCCCCTCTGGC TCCAGCTGGCCGGGAGCCTGATCACCTGCCCTGCTGAGTCCCAGGCTGAGCCTCAGTC TCCCTCCCTTGGGGCCTATGCAGAGGTCCACAACACACAGATTTGAGCTCAGCCCTGG

TGGGCAGAGAGGTAGGGATGGGGCTGTGGGGATAGTGAGGCATCGCAATGTAAGACTC GGGATTAGTACACACTTGTTGATGATTAATGGAAATGTTTACAGATCCCCAAGCCTGG CAAGGGAATTTCTTCAACTCCCTGCCCCCTAGCCCTCCTTATCAAAGGACACCATTTT

GGCAAGCTCTATCACCAAGGAGCCAAACATCCTACAAGACACAGTGACCATACTAATT

ATACCCCCTGCAAAGCCAGCTTGAAACCTTCACTTAGGAACGTAATCGTGTCCCCTAT

CCTACTTCCCCTTCCTAATTCCACAGCTGCTCAATAAΆGTACAAGAGTTTAΆCAGTGT

G

ORF Start: ATG at 72 ORF Stop: TAG at 1221

SEQ ID NO: 36 383 aa MW at 42612.9kD

NOV5c, MGVKASQTGFWLVLLQCCSAYKLVCYYTS SQYREGDGSCFPDALDRFLCTHIIYSF

A ISNDHID WEV∞DVTLYGl_iN L-v_ NPNLKTLLSVGGWl\_ CGI 38554-01 RTFIKSVPPFLRTHGFDG DLAWLYPGRRDKQHFTTLIKEMKAEFIKEAQPGKKQ LL Protein SAA SAGKVTIDSSYDIAKISQHLDFISIMTYDFHGA RGTTGHHSPLFRGQEDASPD Sequence RFSNTDYAVGYMLRLGAPASKLVMGIPTFGRSFTLASSETGVGAPISGPGIPGRFTKE

AGTLAYYEICDFLRGATVHRT GQQVPYATKGNQ VGYDDQESVKSI VQYLKDRQ AG

AMV A DLDDFQGSFCGQDLRFP TNAIKDA AAT

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 5B.

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

Table 5C. Protein Sequence Properties NOV5a

SignalP analysis: Cleavage site between residues 22 and 23

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

N-region: length 4,- pos .chg 1; neg. chg 0 H-region: length 18; peak value 10.94 PSG score: 6.54

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 0.22 possible cleavage site: between 21 and 22

>» Seems to have a σleavable signal peptide (1 to 21)

ALOM: Klein et al's method for TM region allocation Init position for calculation: 22 Tentative number of TMS(s) for the threshold 0.5: number of TMS(s) .. fixed PERIPHERAL Likelihood = 4.9₃ (at 172) ALOM score: 4.9₃ (number of TMSs: 0)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 10 Charge difference: -1.0 C( 1.0) - N( 2.0) N >= C: N-terminal side will be inside

MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75) : 4.48 Hyd Moment (95) : 6.22 G content: 2 D/E content: 1 S/T content: 6 Score: -₃.44

NUCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite: none content of basic residues: 10.2% NLS Score: -0.47

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

ER Membrane Retention Signals : none

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

PTS2: 2nd peroxisomal targeting signal: none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : MGVKASQ

Tyrosines in the tail: none

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

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

Final Results (k = 9/23):

33.3 %: extracellular, including cell wall

22.2 %: mitochondrial

22.2 %: vacuolar

22.2 %: endoplasmic reticulum W

» prediction for CG138554-03 is exc (k=9)

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

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

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

Example 6. Calcium channel alpha- 1A subunit

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: 37 7665 bp

NOV6a, CGCAGCGTAACCCGGAGCCCTTTGCTCTTTGCAGAATGGCCCGCTTCGGAGACGAGAT CG144744-01 GCCGGCCCGCTACGGGGGAGGAGGCTCCGGGGCAGCCGCCGGGGTGGTCGTGGGCAGC

GGAGGCGGGCGAGGAGCCGGGGGCAGCCGGCAGGGCGGGCAGCCCGGGGCGCAAAGGA DNA Sequence TGTACAAGCAGTCAATGGCGCAGAGAGCGCGGACCATGGCACTCTACAACCCCATCCC

CGTCCGACAGAACTGCCTCACGGTTAACCGGTCTCTCTTCCTCTTCAGCGAAGACAAC

GTGGTGAGAAAATACGCCAAAAAGATCACCGAATGGCCTCCCTTTGAATATATGATTT

TAGCCACCATCATAGCGAATTGCATCGTCCTCGCACTGGAGCAGCATCTGCCTGATGA

TGACAAGACCCCGATGTCTGAACGGCTGGATGACACAGAACCATACTTCATTGGAATT

TTTTGTTTCGAGGCTGGAATTAAAATCATTGCCCTTGGGTTTGCCTTCCACAAAGGCT

CCTACTTGAGGAATGGCTGGAATGTCATGGACTTTGTGGTGGTGCTAACGGGCATCTT

GGCGACAGTTGGGACGGAGTTTGACCTACGGACGCTGAGGGCAGTTCGAGTGCTGCGG

CCGCTCAAGCTGGTGTCTGGAATCCCAAGTTTACAAGTCGTCCTGAAGTCGATCATGA

AGGCGATGATCCCTTTGCTGCAGATCGGCCTCCTCCTATTTTTTGCAATCCTTATTTT

TGCAATCATAGGGTTAGAATTTTATATGGGAAAATTTCATACCACCTGCTTTGAAGAG

GGGACAGATGACATTCAGGGTGAGTCTCCGGCTCCATGTGGGACAGAAGAGCCCGCCC

GCACCTGCCCCAATGGGACCAAATGTCAGCCCTACTGGGAAGGGCCCAACAACGGGAT

CACTCAGTTCGACAACATCCTGTTTGCAGTGCTGACTGTTTTCCAGTGCATAACCATG

GAAGGGTGGACTGATCTCCTCTACAATAGCAACGATGCCTCAGGGAACACTTGGAACT

GGTTGTACTTCATCCCCCTCATCATCATCGGCTCCTTTTTTATGCTGAACCTTGTGCT

GGGTGTGCTGTCAGGGGAGTTTGCCAAAGAAAGGGAACGGGTGGAGAACCGGCGGGCT

TTTCTGAAGCTGAGGCGGCAACAACAGATTGAACGTGAGCTCAATGGGTACATGGAAT

GGATCTCAAAAGCAGAAGAGGTGATCCTCGCCGAGGATGAAACTGACGGGGAGCAGAG

GCATCCCTTTGATGGAGCTCTGCGGAGAACCACCATAAAGAAAAGCAAGACAGATTTG

CTCAACCCCGAAGAGGCTGAGGATCAGCTGGCTGATATAGCCTCTGTGGGTTCTCCCT

TCGCCCGAGCCAGCATTAAAAGTACCAAGCTGGAGAACTCGACCTTTTTTCACAAAAA

GGAGAGGAGGATGCGTTTCTACATCCGCCGCATGGTCAAAACTCAGGCCTTCTACTGG

ACTGTACTCAGTTTGGTAGCTCTCAACACGCTGTGTGTTGCTATTGTTCACTACAACC

AGCCCGAGTGGCTCTCCGACTTCCTTTACTATGCAGAATTCATTTTCTTAGGACTCTT

TATGTCCGAAATGTTTATAAAAATGTACGGGCTTGGGACGCGGCCTTACTTCCACTCT

TCCTTCAACTGCTTTGACTGTGGGGTTATCATTGGGAGCATCTTCGAGGTCATCTGGG

CTGTCATAAAACCTGGCACATCCTTTGGAATCAGCGTGTTACGAGCCCTCAGGTTATT

GCGTATTTTCAAAGTCACAAAGTACTGGGCATCTCTCAGAAACCTGGTCGTCTCTCTC

CTCAACTCCATGAAGTCCATCATCAGCCTGTTGTTTCTCCTTTTCCTGTTCATTGTCG

TCTTCGCCCTTTTGGGAATGCAACTCT CGGCGGCCAGTTTAATTTCGATGAAGGGAC

GCCTCCCACCAACTTCGATACTTTTCCAGCAGCAAGAATGACGGTGTTTCAGAGCCTG

ACGGGCGAAGACTGGAACGAGGTCATGTACGACGGGATCAAGTCTCAGGGGGGCGTGC

AGGGCGGCATGGTGTTCTCCATCTATTTCATTGTACTGACGCTCTTTGGGAACTACAC

CCTCCTGAATGTGTTCTTGGCCATCGCTGTGGACAATCTGGCCAACGTTTTGGAGCTC

ACCAAGGACGAGCAAGAGGAAGAAGAAGCAGCGAACCAGAAACTTGCCCTACAGAAAG

CCAAGGAGGTGGCAGAAGTGAGTCCTCTGTCCGCGGCCAACATGTCTATAGCTGTGAA

AGAGCAACAGAAGAATCAAAAGCCAGCCAAGTCCGTGTGGGAGCAGCGGACCAGTGAG

ATGCGAAAGCAGAACTTGCTGGCCAGCCGGGAGGCCCTGTATAACGAAATGGACCCGG

ACGAGCGCTGGAAGGCTGCCTACACGCGGCACCTGCGGCCAGACATGAAGACGCACTT

GGACCGGCCGCTGGTGGTGGACCCGCAGGAGAACCGCAACAACAACACCAACAAGAGC

CGGGCGGCCGAGCCCACCGTGGACCAGCGCCTCGGCCAGCAGCGCGCCGAGGACTTCC

TCAGGAAACAGGCCCGCTACCACGATCGGGCCCGGGACCCCAGCGGCTCGGCGGGCCT

GGACGCACGGAGGCCCTGGGCGGGAAGCCAGGAGGCCGAGCTGAGCCGGGAGGACCCC

TACGGCCGCGAGTCGGACCACCACGCCCGGGAGGGCAGCCTGGAGCAACCCGGGTTCT

GGGACGGCGAGGCCGAGCGAGGCAAGGCCGGGGACCCCCACCGGAGGCACGTGCACCG

GCAGGGGGGCAGCAGGGAGAGCCGCAGCGGGTCCCCGCGCACGGGCGCGGACGGGGAG

CATCGACGTCATCGCGCGCACCGCAGGCCCGGGGAGGAGGGTCCGGAGGACAAGGCGG

AGCGGAGGGCGCGGCACCGCGAGGGCAGCCGGCCGGCCCGGGGCGGCGAGGGCGAGGG

CGAGGGCCCCGACGGGGGCGAGCGCAGGAGAAGGCACCGGCATGGCGCTCCAGCCACG

TACGAGGGGGACGCGCGGAGGGAGGACAAGGAGCGGAGGCATCGGAGGAGGAAAGAGA

ACCAGGGCTCCGGGGTCCCTGTGTCGGGCCCCAACCTGTCAACCACCCGGCCAATCCA

GCAGGACCTGGGCCGCCAAGACCCACCCCTGGCAGAGGATATTGACAACATGAAGAAC

AACAAGCTGGCCACCGCGGAGTCGGCCGCTCCCCACGGCAGCCTTGGCCACGCCGGCC

TGCCCCAGAGCCCAGCCAAGATGGGAAACAGCACCGACCCCGGCCCCATGCTGGCCAT

CCCTGCCATGGCCACCAACCCCCAGAACGCCGCCAGCCGCCGGACGCCCAACAACCCG

GGGAACCCATCCAATCCCGGCCCCCCCAAGACCCCCGAGAATAGCCTTATCGTCACCA

ACCCCAGCGGCACCCAGACCAATTCAGCTAAGACTGCCAGGAAACCCGACCACACCAC

AGTGGACATCCCCCCAGCCTGCCCACCCCCCCTCAACCACACCGTCGTACAAGTGAAC AAAAACGCCAACCCAGACCCACTGCCAAAAAAAGAGGAAGAGAAGAAGGAGGAGGAGG AAGACGACCGTGGGGAAGACGGCCCTAAGCCAATGCCTCCCTATAGCTCCATGTTCAT CCTGTCCACGACCAACCCCCTTCGCCGCCTGTGCCATTACATCCTGAACCTGCGCTAC TTTGAGATGTGCATCCTCATGGTCATTGCCATGAGCAGCATCGCCCTGGCCGCCGAGG ACCCTGTGCAGCCCAACGCACCTCGGAACAACGTGCTGCGATACTTTGACTACGTTTT TACAGGCGTCTTTACCTTTGAGATGGTGATCAAGATGATTGACCTGGGGCTCGTCCTG CATCAGGGTGCCTACTTCCGTGACCTCTGGAATATTCTCGACTTCATAGTGGTCAGTG GGGCCCTGGTAGCCTTTGCCTTCACTGGCAATAGCAAAGGAAAAGACATCAACACGAT TAAATCCCTCCGAGTCCTCCGGGTGCTACGACCTCTTAAAACCATCAAGCGGCTGCCA AAGCTCAAGGCTGTGTTTGACTGTGTGGTGAACTCACTOTAAAAACGTCTTCAACATCC TCATCGTCTACATGCTATTCATGTTCATCTTCGCCGTGGTGGCTGTGCAGCTCTTCAA GGGGAAATTCTTCCACTGCACTGACGAGTCCAAAGAGTTTGAGAAAGATTGTCGAGGC AAATACCTCCTCTACGAGAAGAATGAGGTGAAGGCGCGAGACCGGGAGTGGAAGAAGT ATGAATTCCATTACGACAATGTGCTGTGGGCTCTGCTGACCCTCTTCACCGTGTCCAC GGGAGAAGGCTGGCCACAGGTCCTCAAGCATTCGGTGGACGCCACCTTTGAGAACCAG GGCCCCAGCCCCGGGTACCGCATGGAGATGTCCATTTTCTACGTCGTCTACTTTGTGG TGTTCCCCTTCTTCTTTGTCAATATCTTTGTGGCCTTGATCATCATCACCTTCCAGGA GCAAGGGGACAAGATGATGGAGGAATACAGCCTGGAGAAAAATGAGAGGGCCTGCATT GATTTCGCCATCAGCGCCAAGCCGCTGACCCGACACATGCCGCAGAACAAGCAGAGCT TCCAGTACCGCATGTGGCAGTTCGTGGTGTCTCCGCCTTTCGAGTACACGATCATGGC CATGATCGCCCTCAACACCATCGTGCTTATGATGAAGTTCTATGGGGCTTCTGTTGCT TATGAAAATGCCCTGCGGGTGTTCAACATCGTCTTCACCTCCCTCTTCTCTCTGGAAT GTGTGCTGAAAGTCATGGCTTTTGGGATTCTGAATTATTTCCGCGATGCCTGGAACAT CTTCGACTTTGTGACTGTTCTGGGCAGCATCACCGATATCCTCGTGACTGAGTTTGGG AATAACTTCATCAACCTGAGCTTTCTCCGCCTCTTCCGAGCTGCCCGGCTCATCAAAC TTCTCCGTCAGGGTTACACCATCCGCATTCTTCTCTGGACCTTTGTGCAGTCCTTCAA GGCCCTGCCTTATGTCTGTCTGCTGATCGCCATGCTCTTCTTCATCTATGCCATCATT GGGATGCAGGTGTTTGGTAACATTGGCATCGACGTGGAGGACGAGGACAGTGATGAAG ATGAGTTCCAAATCACTGAGCACAATAACTTCCGGACCTTCTTCCAGGCCCTCATGCT TCTCTTCCGGAGTGCTACCGGGGAAGCTTGGCACAACATCATGCTTTCCTGCCTCAGC GGGAAACCGTGTGATAAGAACTCTGGCATCCTGACTCGAGAGTGTGGCAATGAATTTG

CTTATTTTTACTTTGTTTCCTTCATCTTCCTCTGCTCGTTTCTGATGCTGAATCTCTT

TGTCGCCGTCATCATGGACAACTTTGAGTACCTCACCCGAGACTCCTCCATCCTGGGC

CCCCACCACCTGGATGAGTACGTGCGTGTCTGGGCCGAGTATGACCCCGCAGCTTGCG

GTCGGATTCATTATAAGGATATGTACAGTTTATTACGAGTAATGTCTCCGCCCCTCGG

CTTAGGGAAGAAGTGTCCGCCAGGAGTGGCTTACAAGCGGCTTCTGCGGATGGACCTG

CCCGTCGCAGATGACAACACCGTCCACTTCAATTCCACCCTCATGGCTCTGATCCGCA

CAGCCCTGGACATCAAGATTGCCAAGGGAGGAGCCGACAAACAGCAGATGGACGCTGA

GCTGCGGAAGGAGATGATGGCGATTTGGCCCAATCTGTCCCAGAAGACGCTAGACCTG

CTGGTCACACCTCACAAGTCCACGGACCTCACCGTGGGGAAGATCTACGCAGCCATGA

TGATCATGGAGTACTACCGGCAGAGCAAGGCCAAGAAGCTGCAGGCCATGCGCGAGGA

GCAGGACCGGACACCCCTCATGTTCCAGCGCATGGAGCCCCCGTCCCCAACGCAGGAA

GGGGGACCTGGCCAGAACGCCCTCCCCTCCACCCAGCTGGACCCAGGAGGAGCCCTGA

TGGCTCACGAAAGCGGCCTCAAGGAGAGCCCGTCCTGGGTGACCCAGCGTGCCCAGGA GATGTTCCAGAAGACGGGCACATGGAGTCCGGAACAAGGCCCCCCTACCGACATGCCC

AACAGCCAGCCTAACTCTCAGTCCGTGGAGATGCGAGAGATGGGCAGAGATGGCTACT CCGACAGCGAGCACTACCTCCCCATGGAAGGCCAGGGCCGGGCTGCCTCCATGCCCCG CCTCCCTGCAGAGAACCAGAGGAGAAGGGGCCGGCCACGTGGGAATAACCTCAGTACC ATCTCAGACACCAGCCCCATGAAGCGTTCAGCCTCCGTGCTGGGCCCCAAGGCCCGAC GCCTGGACGATTACTCGCTGGAGCGGGTCCCGCCCGAGGAGAACCAGCGGCACCACCA GCGGCGCCGCGACCGCAGCCACCGCGCCTCTGAGCGCTCCCTGGGCCGCTACACCGAT GTGGACACAGGCTTGGGGACAGACCTGAGCATGACCACCCAATCCGGGGACCTGCCGT CGAAGGAGCGGGACCAGGAGCGGGGCCGGCCCAAGGATCGGAAGCATCGACAGCACCA CCACCACCACCACCACCACCACCATCCCCCGCCCCCCGACAAGGACCGCTATGCCCAG GAACGGCCGGACCACGGCCGGGCACGGGCTCGGGACCAGCGCTGGTCCCGCTCGCCCA GCGAGGGCCGAGAGCACATGGCGCACCGGCAGGGCAGTAGTTCCGTAAGTGGAAGCCC AGCCCCCTCAACATCTGGTACCAGCACTTCTCGGCGGGGCCGCCGCCAGCTCCCCCAG ACCCCCTCCACCCCCCGGCCACACGTGTCCTATTCCCCTGTGATCCGTAAGGCCGGCG GCTCGGGGCCCCCGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA GCAGCAGCAGCAGCAGCAGCAGCAGCAGGCGGTGGCCAGGCCGGGCCGGGCGGCCACC AGCGGCCCTCGGAGGTACCCAGGCCCCACGGCCGAGCCTCTGGCCGGAGATCGGCCGC CCACGGGGGGCCACAGCAGCGGCCGCTCGCCCAGGATGGAGAGGCGGGTCCCAGGCCC GGCCCGGAGCGAGTCCCCCAGGGCCTGTCGACACGGCGGGGCCCGGTGGCCGGCATCT GGCCCGCACGTGTCCGAGGGGCCCCCGGGTCCCCGGCACCATGGCTACTACCGGGGCT CCGACTACGACGAGGCCGATGGCCCGGGCAGCGGGGGCGGCGAGGAGGCCATGGCCGG GGCCTACGACGCGCCACCCCCCGTACGACACGCGTCCTCGGGCGCCACCGGGCGCTCG CCCAGGACTCCCCGGGCCTCGGGCCCGGCCTGCGCCTCGCCTTCTCGGCACGGCCGGC GACTCCCCAACGGCTACTACCCGGCGCACGGACTGGCCAGGCCCCGCGGGCCGGGCTC CAGGAAGGGCCTGCACGAACCCTACAGCGAGAGTGACGATGATTGGTGCTAAGCCCGG

GCGAGGTGGCGCCCGCCCGGCCCCCCACGCACCCCACGCACACACCCCACCCGAGGAG

CCGCGCAGA

ORF Start: ATG at 36 ORF Stop: TAA at 7590

SEQ ID NO: 38 2518 aa MW at 283905.2kD

NOV6a, MARFGDEMPARYGGGGSGAAAGVVVGSGGGRGAGGSRQGGQPGAQRMYKQSMAQRART CG144744-01 ALY^PIPVllQNCLTVISIRS F FSEDNv KYAKKITE PPFEYMILATIIANCIVLA LEQH PDDDKTPMSER DDTEPYFIGIFCFEAGIKIIALGFAFHKGSY RNG VMDF Protein V A^LTGIIJ- VGTEFDLRTLRAVRVLRPLKLVSGIPS QVVLKSIMKAMIP QIGLL Sequence LFFAI IFAIIGLEFYMGKFHTTCFEEGTDDIQGESPAPCGTEEPARTCPNGTKCQPY EGP NGITQFDNILFAVLTVFQC ITMEGWTD YNSIVTOASGIITWNWLYFI PLI I IGS FFMLl^VLGVLSGEFAKERERVElv RAFLKLRRQQQIERELNGYMEWISKAEEVILAE DETDGEQRHPFDGALRRTTIKKSKTDLLNPEEAEDQLADIASVGSPFARASIKSTKLE NSTFFHK ERRIrøFYIRRMvXTQAFY -VLSLVAL LCVAIVHYNQPEWLSDFLYYA EFIFLGLFMSEMF IKMYGLGTRPYFHS SFNCFDCGVI IGS IFEVIWAVIKPGTSFGI S VX.RALRLLRIFKΛ^KY ASLR LVVSLLNSMKSIISL FLLFLFIVVFALLGMQLFGG QFlSIFDEGTPPllvTFDTFPAARMTVFQSLTGEDWNEV-^DGIKSQGGVQGGMVFSIYFIV LTLFG YTLLNv^LAIAVDNLANVLELTKDEQEEEEAANQKLALQKAKEVAEVSPLSA J_MSIAV EQQK QKPAKSV EQRTSE RKQ1SΠJLASREALYNEMDPDERWKAAYTRHL

RPDMKTHLDRPLVVDPQE _IKΠVTΓ KSRAAEPTVDQRLGQQRAEDFLRKQARYHDRAR DPSGSAGLDARRP AGSQEAELSREDPYGRESDHHAREGSLEQPGFWDGEAERGKAGD PHRRHVΉRQGGSRESRSGSPRTGADGEHRRHRAHRRPGEEGPED AERRARHREGSRP ARGGEGEGEGPDGGERRRRHRHGAPATYEGDARRED ERRHRRRKENQGSGVPVSGPN LSTTRPIQQDLGRQDPPLAEDIDI__ NNKLATAESAAPHGSIIGHAGLPQSPAKMGNST DPGPMLAIPA_ATNPQNAASRRTPNNPGNPSNPGPPKTPENSLIVTNPSGTQ SAKT AR PDHTOVDIPPACPPPLRØITVVQVNKNANPDPLPKKEEEK EEEEDDRGEDGPKPM PPYSSMFILSTTNPLRRLCHYILNLRYFEMCILIWIA SSIALAAEDPVQPNAPRLVΠW LRYFDYVFTGVFTFEMVIKMIDLGLVLHQGAYFRDLW ILDFIWSGALVAFAFTGNS KGKDI IKSLRVLRVLRPLKTIKRLPKLKAVFDCVVNSLKNVTNILIVYMLFMFIFA VVAVQLFKGKFFHCTDESKEFE-_CRGKYLLYEKI_VKARDRE KKYEFHYDNV_.WAL LTLFTVSTGEG PQVLKHSVDATFENQGPS PGYRMEMS IF YWYFWF PFFFVNIFVA LIIITFQEQGDKJ MEEYSLEKNERACIDFAISAKPLTRHMPQNKQSFQYRM QFVVSP PFEYTI__IAL IVL__ FYGASVAYENALRVFNIVFTSLFSLECVLK^Λ__?GILN

YFRDAW IFDFV VIiGSi ?DILV EFGrøN^INLSFLRIiFRAARLIKLLRQGYTIRILL TFVQSFKALPYVCLLIAMLFFIYAIIGMQVFGNIGIDVEDEDSDEDEFQITEHNTR TFFQAL_^LFRSATGEAH IILLSCLSGKPCDKNSGILTRECG3VIEFAYFYFVSFIFLC SFLL___FVAVII_)FEYLTRDSSILGPHHLDEYVΕ.VAEYDPAACGRIHYD-_SLL RVMSPPLGLGKKCPPGVAYKRLLRMDLPVADDKΓΓVHFNSTLALIRTALDIKIAKGGA DKQQMDAELRKEI_IAIWPNLSQKTLDLLVTPHKSTDLTVGKIYAA1_MIMEYYRQSKAK KLQAMREEQDRTPLMFQRMEPPSPTQEGGPGQNALPSTQLDPGGALMAHESGL ESPS VTQRAQEFQKTGT SPEQGPPTD PNSQPNSQSVEMREMGRDGYSDSEHYLPMEGQ GPJ_SMPRLPAENQPIRGRPRG1 LSTISDTSP_SRSASV_,GPKARRLDDYSLERVPP EENQRHHQRRRDRSHRASERSLGRYTDVDTGLGTDLSMTTQSGDLPSKERDQERGRP DRKHRQHHHHHHHHHHPPPPDKDRYAQERPDHGRARARDQRWSRSPSEGREHMAHRQG SSSVSGSPAPSTSGTSTSRRGRRQLPQTPSTPRPHVSYSPVIRKAGGSGPPQQQQQQQ QQQQQQQQQQQQQQQQQAVARPGRAATSGPRRYPGPTAEPLAGDRPPTGGHSSGRSPR MERRVPGPARSESPRACRHGGARPASGPHVSEGPPGPRHHGYYRGSDYDEADGPGSG GGEEAMAGAYDAPPPVRHASSGATGRSPRTPRASGPACASPSRHGRRLPNGYYPAHGL ARPRGPGSRKGLHEPYSESDDDC

SEQIDNO: 39 7847bp j

NOV6b, CGGCGGCGTCTTCCGCATCGTTCGCCGCAGCGTAACCCGGAGCCCTTTGCTCTTTGCA

CG144744-02 GAATGGCCCGCTTCGGAGACGAGATGCCGGCCCGCTACGGGGGAGGAGGCTCCGGGGC AGCCGCCGGGGTGGTCGTGGGCAGCGGAGGCGGGCGAGGAGCCGGGGGCAGCCGGCAG DNA Sequence GGCGGGCAGCCCGGGGCGCAAAGGATGTACAAGCAGTCAΆTGGCGCAGAGAGCGCGGA CCATGGCACTCTACAACCCCATCCCCGTCCGACAGAACTGCCTCACGGTTAACCGGTC TCTCTTCCTCTTCAGCGAAGACAACGTGGTGAGAAAATACGCCAAAAAGATCACCGAA TGTCCCTTTGAATATATGATTTTAGCCACCATCATAGCGAATTGCATCGTCCTCGCAC TGGAGCAGCATCTGCCTGATGATGACAAGACCCCGATGTCTGAACGGCTGGATGACAC AGAACCATACTTCATTGGAATTTTTTGTTTCGAGGCTGGAATTAAAATCATTGCCCTT GGGTTTGCCTTCCACAAAGGCTCCTACTTGAGGAATGGCTGGAATGTCATGGACTTTG TGGTGGTGCTAACGGGCATCTTGGCGACAGTTGGGACGGAGTTTGACCTACGGACGCT GAGGGCAGTTCGAGTGCTGCGGCCGCTCAAGCTGGTGTCTGGAATCCCAAGTTTACAA GTCGTCCTGAAGTCGATCATGAAGGCGATGATCCCTTTGCTGCAGATCGGCCTCCTCC TATTTTTTGCAATCCTTATTTTTGCAATCATAGGGTTAGAATTTTATATGGGAAAATT TCATACCACCTGCTTTGAAGAGGGGACAGATGACATTCAGGGTGAGTCTCCGGCTCCA TGTGGGACAGAAGAGCCCGCCCGCACCTGCCCCAATGGGACCAAATGTCAGCCCTACT GGGAAGGGCCCAACAACGGGATCACTCAGTTCGACAACATCCTGTTTGCAGTGCTGAC TGTTTTCCAGTGCATAACCATGGAAGGGTGGACTGATCTCCTCTACAATAGCAACGAT GCCTCAGGGAACACTTGGAACTGGTTGTACTTCATCCCCCTCATCATCATCGGCTCCT TTTTATGCTGAACCTTGTGCTGGGTGTGCTGTCAGGGGAGTTTGCCAAΑGAAAGGGA ACGGGTGGAGAACCGGCGGGCTTTTCTGAAGCTGAGGCGGCAACAACAGATTGAACGT GAGCTCAATGGGTACATGGAGTGGATCTCAAAAGCAGAAGAGGTGATCCTCGCCGAGG ATGAAACTGACGGGGAGCAGAGGCATCCCTTTGATGCTCTGCGGAGAACCACCATAAA GAAAAGCAAGACAGATTTGCTCAACCCCGAAGAGGCTGAGGATCAGCTGGCTGATATA GCCTCTGTGGGTTCTCCCTTCGCCCGAGCCAGCATTAAAAGTGCCAAGCTGGAGAACT CGACCTTTTTTCACAAAAAGGAGAGGAGGATGCGTTTCTACATCCGCCGCATGGTCAA AACTCAGGCCT CTACTGGACTGTACTCAGTTTGGTAGCTCTCAACACGCTGTGTGTT GCTATTGTTCACTACAACCAGCCCGAGTGGCTCTCCGACTTCCTTTACTATGCAGAAT TCATTTTCTTAGGACTCTTTATGTCCGAAATGTTTATAAAAATGTACGGGCTTGGGAC GCGGCCTTACTTCCACTCTTCCTTCAACTGCTTTGACTGTGGGGTTATCATTGGGAGC ATCTTCGAGGTCATCTGGGCTGTCATAAAACCTGGCACATCCTTTGGAATCAGCGTGT TACGAGCCCTCAGGTTATTGCGTATTTTCAAAGTCACAAAGTACTGGGCATCTCTCAG AAACCTGGTCGTCTCTCTCCTCAACTCCATGAAGTCCATCATCAGCCTGTTGTTTCTC CTTTTCCTGTTCATTGTCGTCTTCGCCCTTTTGGGAATGCAACTCTTCGGCGGCCAGT TTAATTTCGATGAAGGGACTCCTCCCACCAACTTCGATACTTTTCCAGCAGCAATAAT GACGGTGTTTCAGATCCTGACGGGCGAAGACTGGAACGAGGTCATGTACGACGGGATC AAGTCTCAGGGGGGCGTGCAGGGCGGCATGGTGTTCTCCATCTATTTCATTGTACTGA CGCTCTTTGGGAACTACACCCTCCTGAATGTGTTCTTGGCCATCGCTGTGGACAATCT GGCCAACGCCCAGGAGCTCACCAAGGACGAGCAAGAGGAAGAAGAAGCAGCGAACCAG AAACTTGCCCTACAGAAAGCCAAGGAGGTGGCAGAAGTGAGTCCTCTGTCCGCGGCCA ACATGTCTATAGCTGTGAAAGAGCAACAGAAGAATCAAAAGCCAGCCAAGTCCGTGTG GGAGCAGCGGACCAGTGAGATGCGAAAGCAGAACTTGCTGGCCAGCCGGGAGGCCCTG TATAACGAAATGGACCCGGACGAGCGCTGGAAGGCTGCCTACACGCGGCACCTGCGGC CAGACATGAAGACGCACTTGGACCGGCCGCTGGTGGTGGACCCGCAGGAGAACCGCAA CAACAACACCAACAAGAGCCGGGCGGCCGAGCCCACCGTGGACCAGCGCCTCGGCCAG CAGCGCGCCGAGGACTTCCTCAGGAAACAGGCCCGCTACCACGATCGGGCCCGGGACC CCAGCGGCTCGGCGGGCCTGGACGCACGGAGGCCCTGGGCGGGAAGCCAGGAGGCCGA GCTGAGCCGGGAGGGACCCTACGGCCGCGAGTCGGACCACCACGCCCGGGAGGGCAGC CTGGAGCAACCCGGGTTCTGGGAGGGCGAGGCCGAGCGAGGCAAGGCCGGGGACCCCC ACCGGAGGCACGTGCACCGGCAGGGGGGCAGCAGGGAGAGCCGCAGCGGGTCCCCGCG CACGGGCGCGGACGGGGAGCATCGACGTCATCGCGCGCACCGCAGGCCCGGGGAGGAG GGTCCGGAGGACAAGGCGGAGCGGAGGGCGCGGCACCGCGAGGGCAGCCGGCCGGCCC GGGGCGGCGAGGGCGAGGGCGAGGGCCCCGACGGGGGCGAGCGCAGGAGAAGGCACCG GCATGGCGCTCCAGCCACGTACGAGGGGGACGCGCGGAGGGAGGACAAGGAGCGGAGG CATCGGAGGAGGAAAGAGAACCAGGGCTCCGGGGTCCCTGTGTCGGGCCCCAACC GT CAACCACCCGGCCAATCCAGCAGGACCTGGGCCGCCAAGACCCACCCCTGGCAGAGGA TATTGACAACATGAAGAACAACAAGCTGGCCACCGCGGAGTCGGCCGCTCCCCACGGC AGCCTTGGCCACGCCGGCCTGCCCCAGAGCCCAGCCAAGATGGGAAACAGCACCGACC CCGGCCCCATGCTGGCCATCCCTGCCATGGCCACCAACCCCCAGAACGCCGCCAGCCG CCGGACGCCCAACAACCCGGGGAACCCATCCAATCCCGGCCCCCCCAAGACCCCCGAG AATAGCCTTATCGTCACCAACCCCAGCGGCACCCAGACCAATTCAGCTAAGACTGCCA GGAAACCCGACCACACCACAGTGGACATCCCCCCAGCCTGCCCACCCCCCCTCAACCA CACCGTCGTACAAGTGAACAAAAACGCCAACCCAGACCCACTGCCAAAAAAAGAGGAA GAGAAGAAGGAGGAGGAGGAAGACGACCGTGGGGAAGACGGCCCTAAGCCAATGCCTC CCTATAGCTCCATGTTCATCCTGTCCACGACCAACCCCCTTCGCCGCCTGTGCCATTA CATCCTGAACCTGCGCTACTTTGAGATGTGCATCCTCATGGTCATTGCCATGAGCAGC ATCGCCCTGGCCGCCGAGGACCCTGTGCAGCCCAACGCACCTCGGAACAACGTGCTGC GATACTTTGACTACGTTTTTACAGGCGTCTTTACCTTTGAGATGGTGATCAAGATGAT TGACCTGGGGCTCGTCCTGCATCAGGGTGCCTACTTCCGTGACCTCTGGAATATTCTC GACTTCATAGTGGTCAGTGGGGCCCTGGTAGCCTTTGCCTTCACTGGCAATAGCAAAG GAAAAGACATCAACACGATTAAATCCCTCCGAGTCCTCCGGGTGCTACGACCTCTTAA AACCATCAAGCGGCTGCCAAAGCTCAAGGCTGTGTTTGACTGTGTGGTGAACTCACTT AAAAACGTCTTCAACATCCTCATCGTCTACATGCTATTCATGTTCATCTTCGCCGTGG TGGCTGTGCAGCTCTTCAAGGGGAAATTCTTCCACTGCACTGACGAGTCCAAAGAGTT TGAGAAAGATTGTCGAGGCAAATACCTCCTCTACGAGAAGAATGAGGTGAAGGCGCGA GACCGGGAGTGGAAGAAGTATGAATTCCATTACGACAATGTGCTGTGGGCTCTGCTGA CCCTCTTCACCGTGTCCACGGGAGAAGGCTGGCCACAGGTCCTCAAGCATTCGGTGGA CGCCACCTTTGAGAACCAGGGCCCCAGCCCCGGGTACCGCATGGAGATGTCCATTTTC TACGTCGTCTACTTTGTGGTGTTCCCCTTCTTCTTTGTCAATATCTT GTGGCCTTGA TCATCATCACCTTCCAGGAGCAAGGGGACAAGATGATGGAGGAATACAGCCTGGAGAA AAATGAGAGGGCCTGCATTGATTTCGCCATCAGCGCCAAGCCGCTGACCCGACACATG CCGCAGAACAAGCAGAGCTTCCAGTACCGCATGTGGCAGTTCGTGGTGTCTCCGCCTT TCGAGTACACGATCATGGCCATGATCGCCCTCAACACCATCGTGCTTATGATGAAGTT CTATGGGGCTTCTGTTGCTTATGAAAATGCCCTGCGGGTGTTCAACATCGTCTTCACC TCCCTCTTCTCTCTGGAATGTGTGCTGAAAGTCATGGCTTTTGGGATTCTGAATTATT TCCGCGATGCCTGGAACATCTTCGACTTTGTGACTGTTCTGGGCAGCATCACCGATAT CCTCGTGACTGAGTTTGGGAATAACTTCATCAACCTGAGCTTTCTCCGCCTCTTCCGA GCTGCCCGGCTCATCAAACTTCTCCGTCAGGGTTACACCATCCGCATTCTTCTCTGGA CCTTTGTGCAGTCCTTCAAGGCCCTGCCTTATGTCTGTCTGCTGATCGCCATGCTCTT CTTCATCTATGCCATCATTGGGATGCAGGTGTTTGGTAACATTGGCATCGACGTGGAG GACGAGGACAGTGATGAAGATGAGTTCCAAATCACTGAGCACAATAACTTCCGGACCT TCTTCCAGGCCCTCATGCTTCTCTTCCGGAGTGCCACCGGGGAAGCTTGGCACAACAT CATGCTTTCCTGCCTCAGCGGGAAACCGTGTGATAAGAACTCTGGCATCCTGACTCGA GAGTGTGGCAATGAAT TGCTTATTTTTACTTTGTTTCCTTCATCTTCCTCTGCTCGT TTCTGATGCTGAATCTCTTTGTCGCCGTCATCATGGACAACTTTGAGTACCTCACCCG AGACTCCTCCATCCTGGGCCCCCACCACCTGGATGAGTACGTGCGTGTCTGGGCCGAG TATGACCCCGCAGCTTGCGGTCGGATTCATTATAAGGATATGTACAGTTTATTACGAG TAATATCTCCCCCTCTCGGCTTAGGCAAGAAATGTCCTCATAGGGTTGCTTGCAAGCG GCTTCTGCGGATGGACCTGCCCGTCGCAGATGACAACACCGTCCACTTCAATTCCACC CTCATGGCTCTGATCCGCACAGCCCTGGACATCAAGATTGCCAAGGGAGGAGCCGACA AACAGCAGATGGACGCTGAGCTGCGGAAGGAGATGATGGCGATTTGGCCCAATCTGTC CCAGAAGACGCTAGACCTGCTGGTCACACCTCACAAGTCCACGGACCTCACCGTGGGG AAGATCTACGCAGCCATGATGATCATGGAGTACTACCGGCAGAGCAAGGCCAAGAAGC TGCAGGCCATGCGCGAGGAGCAGGACCGGACACCCCTCATGTTCCAGCGCATGGAGCC CCCGTCCCCAACGCAGGAAGGGGGACCTGGCCAGAACGCCCTCCCCTCCACCCAGCTG GACCCAGGAGGAGCCCTGATGGCTCACGAAAGCGGCCTCAAGGAGAGCCCGTCCTGGG TGACCCAGCGTGCCCAGGAGATGTTCCAGAAGACGGGCACATGGAGTCCGGGACAAGG CCCCCCTACCGACATGCCCAACAGCCAGCCTAACTCTCAGTCCGTGGAGATGCGAGAG ATGGGCAGAGATGGCTACTCCGACAGCGAGCACTACCTCCCCATGGAAGGCCAGGGCC GGGCTGCCTCCATGCCCCGCCTCCCTGCAGAGAACCAGACCATCTCAGACACCAGCCC CATGAAGCGTTCAGCCTCCGTGCTGGGCCCCAAGGCCCGACGCCTGGACGATTACTCG CTGGAGCGGGTCCCGCCCGAGGAGAACCAGCGGCACCACCAGCGGCGCCGCGACCGCA GCCACCGCGCCTCTGAGCGCTCCCTGGGCCGCTACACCGATGTGGACACAGGCTTGGG GACAGACCTGAGCATGACCACCCAATCCGGGGACCTGCCGTCGAAGGAGCGGGACCAG GAGCGGGGCCGGCCCAAGGATCGGAAGCATCGACAGCACCACCACCACCACCACCACC ACCACCATCCCCCGCCCCCCGACAAGGACCGCTATGCCCAGGAACGGCCGGACCACGG CCGGGCACGGGCTCGGGACCAGCGCTGGTCCCGCTCGCCCAGCGAGGGCCGAGAGCAC ATGGCGCACCGGCAGGGCAGTAGTTCCGTAAGTGGAAGCCCAGCCCCCTCAACATCTG GTACCAGCACTCCGCGGCGGGGCCGCCGCCAGCTCCCCCAGACCCCCTCCACCCCCCG GCCACACGTGTCCTATTCCCCTGTGATCCGTAAGGCCGGCGGCTCGGGGCCCCCGCAG CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGGCGGTGGCCAGGCCGGGCCGGG CGGCCACCAGCGGCCCTCGGAGGTACCCAGGCCCCACGGCCGAGCCTCTGGCCGGAGA TCGGCCGCCCACGGGGGGCCACAGCAGCGGCCGCTCGCCCAGGATGGAGAGGCGGGTC CCAGGCCCGGCCCGGAGCGAGTCCCCCAGGGCCTGTCGACACGGCGGGGCCCGGTGGC CGGCATCTGGCCCGCACGTGTCCGAGGGGCCCCCGGGTCCCCGGCACCATGGCTACTA CCGGGGCTCCGACTACGACGAGGCCGATGGCCCGGGCAGCGGGGGCGGCGAGGAGGCC ATGGCCGGGGCCTACGACGCGCCACCCCCCGTACGACACGCGTCCTCGGGCGCCACCG GGCGCTCGCCCAGGACTCCCCGGGCCTCGGGCCCGGCCTGCGCCTCGCCTTCTCGGCA CGGCCGGCGACTCCCCAACGGCTACTACCCGGCGCACGGACTGGCCAGGCCCCGCGGG CCGGGCTCCAGGAAGGGCCTGCACGAACCCTACAGCGAGAGTGACGATGATTGGTGCT AAGCCCGGGCGAGGTGGCGCCCGCCCGGCCCCCCACGCACCCCACGCACACACCCCAC

CCGAGGAGCCGCGCAGAGGCCGCGGGGGCCCAGCACAGAGGGCCCGGGAGAGGGCCAG

CCGGGAGACCCCAGACTCTGGAGAGGCCAGGGCTGGGCCACAAGGGTGTCCCGCAGAG ACCCTCGGCCAAAAGAGACCCTCCTGGGCAGCCACGGCGCCCCCCAACCAGCCCCGAT CCCCCCACCCACGACAGGGGCTCTCGGGTGGGAGGCAGGGAGCAGACAAACCACACAG CCAAGGGATTTGAATGT

ORF Start: ATG at 61 ORF Stop: TAA at 7540

SEQ ID NO: 40 2493 aa MW at 280732.8kD

NOV6b, ARFGDEMPARYGGGGSGAAAGVVVGSGGGRGAGGSRQGGQPGAQRMY QSMAQRART

MALYNPIPVRQNCLTVNRSLFLFSEDNVΛ7RKYAKKITECPFEYMILATIIANCIVLAL CG144744-02 EQHLPDDDKTPMSERLDDTEPYFIGIFCFEAGIKIIALGFAFHKGSYLRNGWNVMDFV Protein VVLTGILATVGTEFDLRTLRAVRVLRPLKLVSGIPSLQVVLKSIMKAMIPLLQIGLLL Sequence FFAILIFAIIGLEFYMGKFHTTCFΞEGTDDIQGESPAPCGTEEPARTCPNGTKCQPYW

EGPIWGITQFDNILFAVL_VFQCITI_:GVWDLLYNSI_ASGNTW_^

FML LVLGVLSGEFAKERERVE RRAFLKLRRQQQIERELNGYMEWISKAEEVILAED

ETDGEQRHPFDALRRTTIKK^KTDLLNPΞEAEDQLADIASVGSPFARASIKSAKLENS

TFFHKKERRMRFYIRRMV TQAFYW VLSLVALL^LCVAIVHYNQPEWLSDFLYYAEF

IFLGLFMSEMFIKMYGLGTRPYFHSSFNCFDCGVIIGSIFEVI AVIKPGTSFGISVL

RALRLLRIFKV KYWASLRNLVVSLLNSMKSIISLLFLLFLFIVVFALLGMQLFGGQF

NFDEGTPPTNFDTFPAAIMTVTQILTGEDWNEVMYDGIKSQGGVQGGMVFSIYFIVLT

LFGISΓ_TLI_^VFLAIAVDNLANAQELTKDEQEEEEAA QKLALQKAKEVAEVSPLSAAN

MSIAV EQQK_JQKPA SVWΞQRTSEI_RKQI_LASREALY E DPDERWKAAYTRHLRP

DJVKTHLDRPLVVOPQEIvπyπsπsrT KSPAAEPTVDQRLGQQR^

SGSAGLDARRPWAGSQEAELSREGPYGRESDHHAREGSLEQPGFWEGEAERGKAGDPH

RRHVΗRQGGSRESRSGSPRTGADGEHRRffi^AHRRPGEEGPEDKAERRARHREGSRPAR

GGEGEGEGPDGGERI___RHGAPATYEGDARREDKERRHRRRKENQGSGVPVSGP LS

TTRPIQQDLGRQDPPLAEDIDlSMKJSπKLATAESAAPHGSLGHAGLPQSPAKMGNSTDP

GPMLAIPA_T_PQNAASRRTP NPGNPSNPGPPKTPENSLIV__IPSGTQTNSAKTAR

KPDHTTVDIPPACPPPLNHT^A/QV K A PDPLPKKEEEKKEEEEDDRGEDGPKPMPP

YSSl^ILSTTNPLR_a_CHYI_lV_RYFEMCILWIAMSSI

YFDYVFTGVFTFΞMVIKMIDLGLVLHQGAYFRDLWNILDFIVVSGALVAFAFTGNSKG

KDIN IKSLRVLRVLRPLKTIKIILPKLKAVFDCV^

AVQLFKGKFFHCTDESKEFEKDCRGKYLLYEKWEVKARDREWKKYEFHYDNVLWALLT

LF VSTGEGWPQVLKHSVDATFΞNQGPSPGYRMEMSIFYVVYFVVFPFFFVTIIFVALI

IITFQEQGDKJ^EEYSLEK1_RACIDFAISAKPLTRHMPQNKQSFQYRMWQFVVSPPF

EYTI__IALNTIVLMMKFYGASVAYENALRVFNIVFTSLFSLECVLKVMAFGIL YF

RDAWNIFDFV VIGSITDILV EFG NFI LSFLRLFRAARLIKLLRQGYTIRILLWT

FVQSFKALPYΛ^CLLIAMLFFIYAIIGMQVFGNIGIDVEDEDSDEDEFQITEHN FRTF

FQALMLLFRSATGEAWHNIMLSCLSGKPCDKNSGILTRECGNEFAYFYFVSFIFLCSF

LMLNLFVAVIMDNFEYLTRDSSILGPHHLDEYVRV AEYDPAACGRIHYKDMYSLLRV

ISPPLGLGKKCPHRVACKRLLRMDLPVADDl\PrVHFNSTL.__IRTAL.DIKIAKGGADK

QQMDAELRKEM_I PNLSQ TLDLLVTPHKSTDL VGKIYAAMI_3YYRQSKA KL

QAMREEQDRTPLMFQRMEPPSPTQEGGPGQNALPSTQLDPGGALMAHESGLKESPS V

TQRAQEMFQKTGTWSPGQGPPTDMPNSQPNSQSVEMREMGRDGYSDSΞHYLPMEGQGR

AASMPRLPAENQTISDTSPMKRSASVLGPKARRLDDYSLERVPPEENQRHHQRRRDRS

HRASERSLGRYTDVDTGLGTDLSMTTQSGDLPSKERDQERGRPKDRKHRQHHHHHHHH

HHPPPPDKDRYAQERPDHGRARARDQRWSRSPSEGREHMAHRQGSSSVSGSPAPSTSG

TSTPRRGRRQLPQTPSTPRPHVSYSPVIRKAGGSGPPQQQQQQQQQQQQQAVARPGRA

ATSGPRRYPGPTAEPLAGDRPPTGGHSSGRSPRMERRVPGPARSESPRACRHGGARWP

ASGPHVSEGPPGPRHHGYYRGSDYDEADGPGSGGGEEAMAGAYDAPPPVRHASSGATG

RSPRTPRASGPACASPSRHGRRLPNGYYPAHGLARPRGPGSRKGLHEPYSESDDDWC

SEQ ID NO: 41

NOV6c, CGGCGGCGTCTTCCGCATCGTTCGCCGCAGCGTAACCCGGAGCCCTTTGCTCTTTGCA GAATGGCCCGCTTCGGAGACGAGATGCCGGCCCGCTACGGGGGAGGAGGCTCCGGGGC

CG144744-03 AGCCGCCGGGGTGGTCGTGGGCAGCGGAGGCGGGCGAGGAGCCGGGGGCAGCCGGCAG DNA Sequence GGCGGGCAGCCCGGGGCGCAAAGGATGTACAAGCAGTCAATGGCGCAGAGAGCGCGGA CCATGGCACTCTACAACCCCATCCCCGTCCGACAGAACTGCCTCACGGTTAACCGGTC TCTCTTCCTCTTCAGCGAAGACAACGTGGTGAGAAAATACGCCAAAAAGATCACCGAA TGTCCCTTTGAATATATGATTTTAGCCACCATCATAGCGAATTGCATCGTCCTCGCAC TGGAGCAGCATCTGCCTGATGATGACAAGACCCCGATGTCTGAACGGCTGGATGACAC AGAACCATACTTCATTGGAATTTTTTGTTTCGAGGCTGGAATTAAAATCATTGCCCTT GGGTTTGCCTTCCACAAAGGCTCCTACTTGAGGAATGGCTGGAATGTCATGGACTTTG TGGTGGTGCTAACGGGCATCTTGGCGACAGTTGGGACGGAGTTTGACCTACGGACGCT GAGGGCAGTTCGAGTGCTGCGGCCGCTCAAGCTGGTGTCTGGAATCCCAAGTTTACAA GTCGTCCTGAAGTCGATCATGAAGGCGATGATCCCTTTGCTGCAGATCGGCCTCCTCC TATTTTTTGCAATCCTTATTTTTGCAATCATAGGGTTAGAATTTTATATGGGAAAATT TCATACCACCTGCTTTGAAGAGGGGACAGATGACATTCAGGGTGAGTCTCCGGCTCCA TGTGGGACAGAAGAGCCCGCCCGCACCTGCCCCAATGGGACCAAATGTCAGCCCTACT GGGAAGGGCCCAACAACGGGATCACTCAGTTCGACAACATCCTGTTTGCAGTGCTGAC TGTTTTCCAGTGCATAACCATGGAAGGGTGGACTGATCTCCTCTACAATAGCAACGAT GCCTCAGGGAACACTTGGAACTGGTTGTACTTCATCCCCCTCATCATCATCGGCTCCT TTTTTATGCTGAACCTTGTGCTGGGTGTGCTGTCAGGGGAGTTTGCCAAAGAAAGGGA ACGGGTGGAGAACCGGCGGGCTTTTCTGAAGCTGAGGCGGCAACAACAGATTGAACGT GAGCTCAATGGGTACATGGAGTGGATCTCAAAAGCAGAAGAGGTGATCCTCGCCGAGG ATGAAACTGACGGGGAGCAGAGGCATCCCTTTGATGCTCTGCGGAGAACCACCATAAA GAAAAGCAAGACAGATTTGCTCAACCCCGAAGAGGCTGAGGATCAGCTGGCTGATATA GCCTCTGTGGGTTCTCCCTTCGCCCGAGCCAGCATTAAAAGTGCCAAGCTGGAGAACT CGACCTTTTTTCACAAAAAGGAGAGGAGGATGCGTTTCTACATCCGCCGCATGGTCAA AACTCAGGCCTTCTACTGGACTGTACTCAGTTTGGTAGCTCTCAACACGCTGTGTGTT GCTAT GTTCACTACAACCAGCCCGAGTGGCTCTCCGACTTCCTTTACTATGCAGAAT TCATTTTCTTAGGACTCTTTATGTCCGAAATGTTTATAAAAATGTACGGGCTTGGGAC GCGGCCTTACTTCCACTCTTCCTTCAACTGCTTTGACTGTGGGGTTATCATTGGGAGC ATCTTCGAGGTCATCTGGGCTGTCATAAAACCTGGCACATCCTTTGGAATCAGCGTGT TACGAGCCCTCAGGTTATTGCGTATTTTCAAAGTCACAAAGTACTGGGCATCTCTCAG AAACCTGGTCGTCTCTCTCCTCAACTCCATGAAGTCCATCATCAGCCTGTTGTTTCTC CTTTTCCTGTTCATTGTCGTCTTCGCCCTTTTGGGAATGCAACTCTTCGGCGGCCAGT TTAATTTCGATGAAGGGACTCCTCCCACCAACTTCGATACTTTTCCAGCAGCAATAAT GACGGTGTTTCAGATCCTGACGGGCGAAGACTGGAACGAGGTCATGTACGACGGGATC AAGTCTCAGGGGGGCGTGCAGGGCGGCATGGTGTTCTCCATCTATTTCATTGTACTGA CGCTCTTTGGGAACTACACCCTCCTGAATGTGTTCTTGGCCATCGCTGTGGACAATCT GGCCAACGCCCAGGAGCTCACCAAGGACGAGCAAGAGGAAGAAGAAGCAGCGAACCAG AAACTTGCCCTACAGAAAGCCAAGGAGGTGGCAGAAGTGAGTCCTCTGTCCGCGGCCA ACATGTCTATAGCTGTGAAAGAGCAACAGAAGAATCAAAAGCCAGCCAAGTCCGTGTG GGAGCAGCGGACCAGTGAGATGCGAAAGCAGAACTTGCTGGCCAGCCGGGAGGCCCTG TATAACGAAATGGACCCGGACGAGCGCTGGAAGGCTGCCTACACGCGGCACCTGCGGC CAGACATGAAGACGCACTTGGACCGGCCGCTGGTGGTGGACCCGCAGGAGAACCGCAA CAACAACACCAACAAGAGCCGGGCGGCCGAGCCCACCGTGGACCAGCGCCTCGGCCAG CAGCGCGCCGAGGACTTCCTCAGGAAACAGGCCCGCTACCACGATCGGGCCCGGGACC CCAGCGGCTCGGCGGGCCTGGACGCACGGAGGCCCTGGGCGGGAAGCCAGGAGGCCGA GCTGAGCCGGGAGGGACCCTACGGCCGCGAGTCGGACCACCACGCCCGGGAGGGCAGC CTGGAGCAACCCGGGTTCTGGGAGGGCGAGGCCGAGCGAGGCAAGGCCGGGGACCCCC ACCGGAGGCACGTGCACCGGCAGGGGGGCAGCAGGGAGAGCCGCAGCGGGTCCCCGCG CACGGGCGCGGACGGGGAGCATCGACGTCATCGCGCGCACCGCAGGCCCGGGGAGGAG GGTCCGGAGGACAAGGCGGAGCGGAGGGCGCGGCACCGCGAGGGCAGCCGGCCGGCCC GGGGCGGCGAGGGCGAGGGCGAGGGCCCCGACGGGGGCGAGCGCAGGAGAAGGCACCG GCATGGCGCTCCAGCCACGTACGAGGGGGACGCGCGGAGGGAGGACAAGGAGCGGAGG CATCGGAGGAGGAAAGAGAACCAGGGCTCCGGGGTCCCTGTGTCGGGCCCCAACCTGT CAACCACCCGGCCAATCCAGCAGGACCTGGGCCGCCAAGACCCACCCCTGGCAGAGGA TATTGACAACATGAAGAACAACAAGCTGGCCACCGCGGAGTCGGCCGCTCCCCACGGC AGCCTTGGCCACGCCGGCCTGCCCCAGAGCCCAGCCAAGATGGGAAACAGCACCGACC CCGGCCCCATGCTGGCCATCCCTGCCATGGCCACCAACCCCCAGAACGCCGCCAGCCG CCGGACGCCCAACAACCCGGGGAACCCATCCAATCCCGGCCCCCCCAAGACCCCCGAG AATAGCCTTATCGTCACCAACCCCAGCGGCACCCAGACCAATTCAGCTAAGACTGCCA GGAAACCCGACCACACCACAGTGGACATCCCCCCAGCCTGCCCACCCCCCCTCAACCA CACCGTCGTACAAGTGAACAAAAACGCCAACCCAGACCCACTGCCAAAAAAAGAGGAA GAGAAGAAGGAGGAGGAGGAAGACGACCGTGGGGAAGACGGCCCTAAGCCAATGCCTC CCTATAGCTCCATGTTCATCCTGTCCACGACCAA.CCCCCTTCGCCGCCTGTGCCATTA CATCCTGAACCTGCGCTACTTTGAGATGTGCATCCTCATGGTCATTGCCATGAGCAGC ATCGCCCTGGCCGCCGAGGACCCTGTGCAGCCCAACGCACCTCGGAACAACGTGCTGC GATACTTTGACTACGTTTTTACAGGCGTCTTTACCTTTGAGATGGTGATCAAGATGAT TGACCTGGGGCTCGTCCTGCATCAGGGTGCCTACTTCCGTGACCTCTGGAATATTCTC GACTTCATAGTGGTCAGTGGGGCCCTGGTAGCCTTTGCCTTCACTGGCAATAGCAAAG GAAAAGACATCAACACGATTAAATCCCTCCGAGTCCTCCGGGTGCTACGACCTCTTAA AACCATCAAGCGGCTGCCAAAGCTCAAGGCTGTGTTTGACTGTGTGGTGAACTCACTT AAAAACGTCTTCAACATCCTCATCGTCTACATGCTATTCATGTTCATCTTCGCCGTGG TGGCTGTGCAGCTCTTCAAGGGGAAATTCTTCCACTGCACTGACGAGTCCAAAGAGTT TGAGAAAGATTGTCGAGGCAAATACCTCCTCTACGAGAAGAATGAGGTGAAGGCGCGA GACCGGGAGTGGAAGAAGTATGAATTCCATTACGACAATGTGCTGTGGGCTCTGCTGA CCCTCTTCACCGTGTCCACGGGAGAAGGCTGGCCACAGGTCCTCAAGCATTCGGTGGAI CGCCACCTTTGAGAACCAGGGCCCCAGCCCCGGGTACCGCATGGAGATGTCCATTTTC TACGTCGTCTACTTTGTGGTGTTCCCCTTCTTCTTTGTCAATATCTTTGTGGCCTTGA TCATCATCACCTTCCAGGAGCAAGGGGACAAGATGATGGAGGAATACAGCCTGGAGAA AAATGAGAGGGCCTGCATTGATTTCGCCATCAGCGCCAAGCCGCTGACCCGACACATG CCGCAGAACAAGCAGAGCTTCCAGTACCGCATGTGGCAGTTCGTGGTGTCTCCGCCTT TCGAGTACACGATCATGGCCATGATCGCCCTCAACACCATCGTGCTTATGATGAAGTT CTATGGGGCTTCTGTTGCTTATGAAAATGCCCTGCGGGTGTTCAACATCGTCTTCACC TCCCTCTTCTCTCTGGAATGTGTGCTGAAAGTCATGGCTTTTGGGATTCTGAATTATT TCCGCGATGCCTGGAACATCTTCGACTTTGTGACTGTTCTGGGCAGCATCACCGATAT CCTCGTGACTGAGTTTGGGAATAACTTCATCAACCTGAGCTTTCTCCGCCTCTTCCGA GCTGCCCGGCTCATCAAACTTCTCCGTCAGGGTTACACCATCCGCATTCTTCTCTGGA CCTTTGTGCAGTCCTTCAAGGCCCTGCCTTATGTCTGTCTGCTGATCGCCATGCTCTT CTTCATCTATGCCATCATTGGGATGCAGGTGTTTGGTAACATTGGCATCGACGTGGAG GACGAGGACAGTGATGAAGATGAGTTCCAAATCACTGAGCACAATAACTTCCGGACCT TCTTCCAGGCCCTCATGCTTCTCTTCCGGAGTGCCACCGGGGAAGCTTGGCACAACAT CATGCTTTCCTGCCTCAGCGGGAAACCGTGTGATAAGAACTCTGGCATCCTGACTCGA GAGTGTGGCAATGAATTTGCTTATTTTTACTTTGTTTCCTTCATCTTCCTCTGCTCGT TTCTGATGCTGAATCTCTTTGTCGCCGTCATCATGGACAACTTTGAGTACCTCACCCG AGACTCCTCCATCCTGGGCCCCCACCACCTGGATGAGTACGTGCGTGTCTGGGCCGAG TATGACCCCGCAGCTTGGGGCCGCATGCCTTACCTGGACATGTATCAGATGCTGAGAC ACATGTCTCCGCCCCTGGGTCTGGGGAAGAAGTGTCCGGCCAGAGTGGCTTACAAGCG GCTTCTGCGGATGGACCTGCCCGTCGCAGATGACAACACCGTCCACTTCAATTCCACC CTCATGGCTCTGATCCGCACAGCCCTGGACATCAAGATTGCCAAGGGAGGAGCCGACA AACAGCAGATGGACGCTGAGCTGCGGAAGGAGATGATGGCGAT TGGCCCAATCTGTC CCAGAAGACGCTAGACCTGCTGGTCACACCTCACAAGTCCACGGACCTCACCGTGGGG AAGATCTACGCAGCCATGATGATCATGGAGTACTACCGGCAGAGCAAGGCCAAGAAGC TGCAGGCCATGCGCGAGGAGCAGGACCGGACACCCCTCATGTTCCAGCGCATGGAGCC CCCGTCCCCAACGCAGGAAGGGGGACCTGGCCAGAACGCCCTCCCCTCCACCCAGCTG GACCCAGGAGGAGCCCTGATGGCTCACGAAAGCGGCCTCAAGGAGAGCCCGTCCTGGG TGACCCAGCGTGCCCAGGAGATGTTCCAGAAGACGGGCACATGGAGTCCGGAACAAGG CCCCCCTACCGACATGCCCAACAGCCAGCCTAACTCTCAGTCCGTGGAGATGCGAGAG ATGGGCAGAGATGGCTACTCCGACAGCGAGCACTACCTCCCCATGGAAGGCCAGGGCC GGGCTGCCTCCATGCCCCGCCTCCCTGCAGAGAACCAGACCATCTCAGACACCAGCCC CATGAAGCGTTCAGCCTCCGTGCTGGGCCCCAAGGCCCGACGCCTGGACGATTACTCG CTGGAGCGGGTCCCGCCCGAGGAGAACCAGCGGCACCACCAGCGGCGCCGCGACCGCA GCCACCGCGCCTCTGAGCGCTCCCTGGGCCGCTACACCGATGTGGACACAGGCTTGGG GACAGACCTGAGCATGACCACCCAATCCGGGGACCTGCCGTCGAAGGAGCGGGACCAG GAGCGGGGCCGGCCCAAGGATCGGAAGCATCGACAGCACCACCACCACCACCACCACC ACCACCATCCCCCGCCCCCCGACAAGGACCGCTATGCCCAGGAACGGCCGGACCACGG CCGGGCACGGGCTCGGGACCAGCGCTGGTCCCGCTCGCCCAGCGAGGGCCGAGAGCAC ATGGCGCACCGGCAGGGCAGTAGTTCCGTAAGTGGAAGCCCAGCCCCCTCAACATCTG GTACCAGCACTCCGCGGCGGGGCCGCCGCCAGCTCCCCCAGACCCCCTCCACCCCCCG GCCACACGTGTCCTATTCCCCTGTGATCCGTAAGGCCGGCGGCTCGGGGCCCCCGCAG CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGGCGGTGGCCAGGCCGGGCCGGG CGGCCACCAGCGGCCCTCGGAGGTACCCAGGCCCCACGGCCGAGCCTCTGGCCGGAGA TCGGCCGCCCACGGGGGGCCACAGCAGCGGCCGCTCGCCCAGGATGGAGAGGCGGGTC CCAGGCCCGGCCCGGAGCGAGTCCCCCAGGGCCTGTCGACACGGCGGGGCCCGGTGGC CGGCATCTGGCCCGCACGTGTCCGAGGGGCCCCCGGGTCCCCGGCACCATGGCTACTA CCGGGGCTCCGACTACGACGAGGCCGATGGCCCGGGCAGCGGGGGCGGCGAGGAGGCC ATGGCCGGGGCCTACGACGCGCCACCCCCCGTACGACACGCGTCCTCGGGCGCCACCG GGCGCTCGCCCAGGACTCCCCGGGCCTCGGGCCCGGCCTGCGCCTCGCCTTCTCGGCA CGGCCGGCGACTCCCCAACGGCTACTACCCGGCGCACGGACTGGCCAGGCCCCGCGGG CCGGGCTCCAGGAAGGGCCTGCACGAACCCTACAGCGAGAGTGACGATGATTGGTGCT AAGCCCGGGCGAGGTGGCGCCCGCCCGGCCCCCCACGCACCCCACGCACACACCCCAC

ORF Start: ATG at 61 ORF Stop: TAA at 7540

SEQ ID NO: 42 2493 aa MW at 280960.1kD

NOV6c, MARFGDEMPARYGGGGSGAAAGVVVGSGGGRGAGGSRQGGQPGAQRMYKQSMAQRART __YNPIPVRQNCLTV ^SLFLFSED VVI_ YAKKITECPFEYMILATIIANCIVLAL CG144744-03 EQHLPDDDKTPMSERLDDTEPYFIGIFCFΞAGIKIIALGFAFHKGSYLR G NVMDFV Protein WLTGILATVGTEFDLRTLRAVRVLRPLKLVSGI PSLQWLKS IMKAMI PLLQIGLLL Sequence FFAILIFAIIGLEFYMGKFHTTCFEEGTDDIQGESPAPCGTEEPARTCPNGTKCQPY EGPrøGITOFDNILFAVLTWOCITrøGWTDLLY Srø^ W

FML1S_VLGVLSGEFAKERERVENRRAFLKLRRQQQIERELNGYMEWISKAEEVILAED

ETDGEQRHPFDALRRTTIKKS TDLL PEEAEDQLADIASVGSPFARASIKSAKLENS

TFFHKKERRMRJYIRRIWKTQAFYWTVLSLVAI^

IFLGLFMSEMFIK YGLGTRPYFHSSFNCFDCGVIIGSIFEVIWAVIKPGTSFGISVL

RALRLLRIFK / KYWASLRNLVVSLLNSMKSIISLLFLLFLFIVVFALLGMQLFGGQF røDEGTPPTItfFDTFPAAIMTVFQILTGEDWNE ^^

LFGNYTLL VTLAIAVDNLANAQΞLTKDEQEEEEAANQKLALQKAKEVAEVSPLSAAN MSIAV EQQKNQKPAKSV EQRTSEMRKQKΓLIASREALY STEMDPDERWKAAYTRHLRP

DMKTHLDRPLVVDPQEi ivm l^SRAAEPTVDQRLGQQRAEDFLRKQARYHDRARDP

SGSAGLDARRPWAGSQEAELSREGPYGRESDHHAREGSLEQPGFWEGEAERGKAGDPH

RRHVHRQGGSRESRSGSPRTGADGEHRRHRAHRRPGEEGPEDKAERRARHREGSRPAR

GGEGEGEGPDGGERRRRHRHGAPATYEGDARREDKERRHRRRKENQGSGVPVSGPKΓLS

TTRPIQQDLGRQDPPLAEDIDNMK N LATAESAAPHGSLGHAGLPQSPAK GNSTDP

GPMLAIPA_\TNPQNAASRJLTPMVIPGNPSNPGPPKTPENSLIVTNPSGTQTNSAKTAR

KPDHTTVDIPPACPPPLLFFI VVQVNKNANPDPLPKKEEEKKEEEEDDRGEDGPKPMPP

YSSMFILSTTNPLRRLCHYILIS-RYFEMCILIWIAMSSIALAAEDPVQPNAPRJNNVLR

YFDYVFTGVFTFEMVIKMIDLGLVLHQGAYFRDLWNILDFIVVSGALVAFAFTGNSKG

KDIOTIKSLRVLRVLRPLKTII__PI_KA^

AVQLFKGKFFHCTDESKEFEKDCRG YLLYEKNEV ARDRE KYEFHYDNVLWALLT

LFTVSTGEG PQVLKHSVDATFENQGPSPGYRMEMSIFYWYFWFPFFFVNIFVALI

IITFQEQGDK^EEYSLEK ERACIDFAISAKPLTPJ_^PQNKQSFQYRMWQFVVSPPF

EYTIMAMIALNTIVLMMKFYGASVAYENALRVFNIVFTSLFSLECVLKVMAFGILNYF

RDATORIFDFVTVLGSITDILVTEFGOSIFI LSFLRLFRAARLIKLLRQGYTIRILL T

FVQSFKALPYVCLLIAMLFFIYAIIGMQVFGNIGIDVEDEDSDEDEFQITEH FRTF

FQALMI-LFRSATGEAWHNIMLSCLSGKPCDKNSGILTRECGNEFAYFYFVSFIFLCSF

LMLNLF AVIMDNFEYLTRDSSILGPHHLDEYVRV AEYDPAAWGR PYLDMYQMLRH

MSPPLGLGKKCPARVAYKRLLRMDLPVADDI_^^

QQ1^AELRKE _\IWPIS_SQKTLDLLV PHKSTDL VGKIYAA_!1I_3YYRQSKAKKL

QAMREEQDRTPLMFQRMEPPSPTQEGGPGQNALPSTQLDPGGALMAHESGLKESPSWV

TQRAQEMFQKTGT SPEQGPPTDMPNSQPNSQSVEMRE GRDGYSDSEHYLPMEGQGR

AASMPRLPAENQTISDTSPMKRSASV-.GPKARRLDDYSLERVPPEENQRHHQRRRDRS

HRASERSLGRYTDVDTGLGTDLS TTQSGDLPSKERDQERGRPKDRKHRQHHHHHHHH

HHPPPPDKDRYAQERPDHGRARARDQRWSRSPSEGREHMAHRQGSSSVSGSPAPSTSG

TSTPRRGRRQLPQTPSTPRPHVSYSPVIRKAGGSGPPQQQQQQQQQQQQQAVARPGRA

ATSGPRRYPGPTAEPLAGDRPPTGGHSSGRSPRMERRVPGPARSESPRACRHGGAR P

ASGPHVSEGPPGPRHHGYYRGSDYDEADGPGSGGGEEAMAGAYDAPPPVRHASSGATG

RSPRTPRASGPACASPSRHGRRLPNGYYPAHGLARPRGPGSRKGLHEPYSΞSDDD C

Sequence comparison ofthe above protein sequences yields the following sequence relationships shown in Table 6B.

Table 6B. Comparison ofNOV6a against NOV6b and NOV6c.

NOV6a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV6b 1..2518 2479/2518 (98%) 1..2493 2481/2518 (98%)

NOV6c 1..2518 2475/2518 (98%) 1..2493 2478/2518 (98%)

Further analysis of the NOV6a protein yielded the following properties shown in Table 6C.

Table 6C. Protein Sequence Properties NOV6a

SignalP analysis: No Known Signal Sequence Predicted PSORT π analysis: PSG: a new signal peptide prediction method

N-region: length 11; pos.chg 2; neg.chg 2 H-region: length 19; peak value 7.65 PSG score: 3.25 <

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -6.30 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: 21

INTEGRAL Likelihood = -3 98 Transmembrane ! 101 - 117

INTEGRAL Likelihood = -3 08 Transmembrane ! 143 - 159

INTEGRAL Likelihood = -3 35 Transmembrane ! 171 - 187

INTEGRAL Likelihood =-13 69 Transmembrane ! 229 - 245

INTEGRAL Likelihood -0.43 Transmembrane ! 301 - 317

INTEGRAL Likelihood -8.70 Transmembrane ! 343 - 359

INTEGRAL Likelihood -4.83 Transmembrane L 491 - 507

INTEGRAL Likelihood -3.08 Transmembrane ! 555 - 571

INTEGRAL Likelihood -15.92 Transmembrane ! 614 - 630

INTEGRAL Likelihood -2.92 Transmembrane ! 684 - 700

INTEGRAL Likelihood -1.81 Transmembranei 705 - 721

INTEGRAL Likelihood -5.15 Transmembrane : 1246 -1262

INTEGRAL Likelihood -1.86 Transmembrane ! 1287 -1303

INTEGRAL Likelihood -5.68 Transmembrane ! 1315 -1331

INTEGRAL Likelihood -13.53 Transmembrane ! 1380 -1396

INTEGRAL Likelihood -11.89 Transmembrane : 1496 -1512

INTEGRAL Likelihood -1.91 Transmembrane : 1571 -1587

INTEGRAL Likelihood -1.97 Transmembrane ! 1603 -1619

INTEGRAL Likelihood -1.12 Transmembrane ! 1632 -1648

INTEGRAL Likelihood -10.88 Transmembrane ! 1695 -1711

INTEGRAL Likelihood -9.98 Transmembrane 1794 -1810

PERIPHERAL Likelihood 1.43 (at 525)

ALOM score: -15.92 (number of TMSs: 21)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 108 Charge difference: -4.5 C(-2.5) - N( 2.0) N >= C: N-terminal side will be inside

>» membrane topology: type 3a

MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75) : 9.93 Hyd Momen (95) : 14.91 G content: 1 D/E content: 2 S/T content: 0 Score: -5.05

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 13 ARP|GD

NϋCDISC: discrimination of nuclear localization signals pat4: RRHR (3) at 957 pat4: RRRR (5) at 1002 pat4: RRRH (3) at 1003 pat4: RRHR (3) at 1004 pat4: RRHR (3) at 1025 pat4: RHRR (3) at 1026 pat4: HRRR (3) at 1027 pat4: RRRK (5) at 1028 pat4: RKHR (3) at 2206 pat7: PKARRLD (5) at 2131 pat7: PKDRKHR (5) at 2203 bipartite: RRPGEEGPEDKAERRAR at 963 content of basic residues: 12.4% NLS Score: 2.83

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

ER Membrane Retention Signals :

XXRR-like motif in the N-terminus: ARFG

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

PTS2: 2nd peroxisomal targeting signal: none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

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

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

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

709 L 0.58

710 A 0.58

711 I 0.58

712 A 0.61

713 V 0.74

714 D 0.76

715 N 0.76

716 L 0.76

717 A 0.76

718 N 0.76

719 V 0.76

720 L 0.97

721 E 0.97

722 L 0.97

723 T 0.97

724 K 0.97

725 D 0.97

726 E 0.97

727 Q 0.97

728 E 0.97

729 E 0.97

730 E .0.97

731 E 0.97

732 A 0.97

A search of the NOV6a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 6D.

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

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

Example 7. Activin receptor-like kinase 1

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

CTGCTGCGACAGCCACCTCTGCAACCACAACGTGTCCCTGGTGCTGGAGGGCTTCATC GCCTCAGACATGACCTCCCGCAACTCGAGCACGCAGCTGTGGCTCATCACGCACTACC ACGAGCACGGCTCCCTCTACGACTTTCTGCAGAGACAGACGCTGGAGCCCCATCTGGC TCTGAGGCTAGCTGTGTCCGCGGCATGCGGCCTGGCGCACCTGCACGTGGAGATCTTC GGTACACAGGGCAAACCAGCCATTGCCCACCGCGACTTCAAGAGCCGCAATGTGCTGG TCAAGAGCAACCTGCAGTGTTGCATCGCCGACCTGGGCCTGGCTGTGATGCACTCACA GGGCAGCGATTACCTGGACATCGGCAACAACCCGAGAGTGGGCACCAAGCGGTACATG GCACCCGAGGTGCTGGACGAGCAGATCCGCACGGACTGCTTTGAGTCCTACAAGTGGA CTGACATCTGGGCCTTTGGCCTGGTGCTGTGGGAGATTGCCCGCCGGACCATCGTGAA TGGCATCGTGGAGGACTATAGACCACCCTTCTATGATGTGGTGCCCAATGACCCCAGC TTTGAGGACATGAAGAAGGTGGTGTGTGTGGATCAGCAGACCCCCACCATCCCTAACC GGCTGGCTGCAGACCCGGTCCTCTCAGGCCTAGCTCAGATGATGCGGGAGTGCTGGTA CCCAAACCCCTCTGCCCGACTCACCGCGCTGCGGATCAAGAAGACACTACAAAAAATT AGCAACAGTCCAGAGAAGCCTAAAGTGATTCAATAGCCCAGGAGCACCTGATTCCTTT CTGCCTGCAGGGGGCTGGGGG

ORF Start: ATG at 28 ORF Stop: TAG at 1078

SEQ ID NO: 46 350 aa MW at 39382.0 D

NOV7b, MTLGSPRKG M L__VTQGDPV PSRGPLVTCTCESPHCKGPTCRGA CTVV VRE EGRHPQEHRGCGJSΛHRELCRGRPTEFVN-T-CCDSHLCNmWSLVLEGFIASDMTSRNS CG151723-02 STQLWLITHYHEHGSLYDFLQRQTLEPHLALRLAVSAACGLAHLHVEIFGTQGKPAIA ^Protein HRI)FKSR1WLVKSNLQCCIADLGLAVMHSQGSDYLDIG NPRVGTKRYMAPEVLDEQI Sequence RTDCFESY-OTTDIWAFGLVLWEIARRTIVNGIVEDYRPPFYDVVP DPSFEDIO KVVC VDQQTPTI PKTRLAADPV SGL AQMMRECWYPNPSARLTALRIKKT QKI SNS PEKPKV Q

SEQ ID NO: 47 1268 bp

NOV7c, CGCCACCCGCAGAGCGGGCCCAGAGGGACCATGACCTTGGGCTCCCCCAGGAAAGGCC

TTCTGATGCTGCTGATGGCCTTGGTGACCCAGGGAGACCCTGTGAAGCCGTCTCGGGG CG151723-03 CCCGCTGGTGACCTGCACGTGTGAGAGCCCACATTGCAAGGGGCCTACCTGCCGGGGG DNA Sequence GCCTGGTGCACAGTAGTGCTGGTGCGGGAGGAGGGGAGGCACCCCCAGGAACATCGGG GCTGCGGGAACTTGCACAGGGAGCTCTGCAGGGGGCGCCCCACCGAGTTCGTCAACCA CTACTGCTGCGACAGCCACCTCTGCAACCACAACGTGTCCCTGGTGCTGGAGGGAAAA GGCCGCTATGGCGAAGTGTGGCGGGGCTTGTGGCACGGTGAGAGTGTGGCCGTCAAGA TCTTCTCCTCGAGGGATGAACAGTCCTGGTTCCGGGAGACTGAGATCTATAACACAGT ATTGCTCAGACACGACAACATCCTAGGCTGCATCGCCTCAGACATGACCTCCCGCAAC TCGAGCACGCAGCTGTGGCTCATCACGCACTACCACGAGCACGGCTCCCTCTACGACT TTCTGCAGAGACAGACGCTGGAGCCCCATCTGGCTCTGAGGCTAGCTGTGTCCGCGGC ATGCGGCCTGGCGCACCTGCACGTGGAGATCTTCGGTACACAGGGCAAACCAGCCATT GCCCACCGCGACTTCAAGAGCCGCAATGTGCTGGTCAAGAGCAACCTGCAGTGTTGCA TCGCCGACCTGGGCCTGGCTGTGATGCACTCACAGGGCAGCGATTACCTGGACATCGG CAACAACCCGAGAGTGGGCACCAAGCGGTACATGGCACCCGAGGTGCTGGACGAGCAG ATCCGCACGGACTGCTTTGAGTCCTACAAGTGGACTGACATCTGGGCCTTTGGCCTGG TGCTGTGGGAGATTGCCCGCCGGACCATCGTGAATGGCATCGTGGAGGACTATAGACC ACCCTTCTATGATGTGGTGCCCAATGACCCCAGCTTTGAGGACATGAAGAAGGTGGTG TGTGTGGATCAGCAGACCCCCACCATCCCTAACCGGCTGGCTGCAGACCCGGTCCTCT CAGGCCTAGCTCAGATGATGCGGGAGTGCTGGTACCCAAACCCCTCTGCCCGACTCAC CGCGCTGCGGATCAAGAAGACACTACAAAAAATTAGCAACAGTCCAGAGAAGCCTAAA GTGATTCAATAGCCCAGGAGCACCTGATTCCTTTCTGCCTGCAGGGGGCT

ORF Start: ATG at 31 ORF Stop: TAG at 1228

SEQ ID NO: 48 399 aa MW at 45156.4 D

NOV7c, MT GS PRKGLLM LMA VTQGDPVKPSRGPLVTCTCES PHCKGPTCRGA CTWLVRΞ EGRHPQEHRGCGI^_HRELCRGRPTEFVNHYCCDSH Cl_!WSLVLEGKGRYGEvTOlGL CG151723-03 WHGESVAVKIFSSRDEQSWFRETEIYlsπ'V RHDNILGCIASDMTSRNSSTQ LITH Protein YHEHGSLYDFLQRQTLEPH ALR AVSAACGLAH HVEIFGTQGKPAIAHRDFKSRNV Sequence LVKSNLQCCIADLGLAVlfflSQGSDYLDIGNNPRVGTKRYMAPEV DEQIRTDCFESYK WTDI AFGLVLWEIARRTIVWGIVΕDYRPPFYDVVPraDPSFEDMKKVVCVDQQTPTIP NR AADPVLSG AQ1_VIREC YPNPSARLTALRIKKTLQKISNSPEKPKVIQ Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 7B.

Table 7B. Comparison of NOV7a against NOV7b and NOV7c.

NOV7a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV7b 252..497 246/246 (100%) 105..350 246/246 (100%)

NOV7c 203..497 294/295 (99%) 105..399 294/295 (99%)

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

Table 7C. Protein Sequence Properties NO 7a

SignalP analysis: Cleavage site between residues 16 and 17

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

N-region: length 2; pos.chg 2; neg.chg 0 H-region: length 13; peak value 11.70 PSG score: 7.30

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 2.20 possible cleavage site: between 15 and 16

>» Seems to have a cleavable signal peptide (1 to 15)

ALOM: Klein et al ' s method for TM region allocation Init position for calculation: 16

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

INTEGRAL Likelihood =-11.46 Transmembrane 113 - 129 PERIPHERAL Likelihood = 0.63 (at 296) ALOM score: -11.46 (number of TMSs: 1)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 7 Charge difference: -2.0 C( 1.0) - N( 3.0) N >= C: N-terminal side will be inside

»> membrane topology: type la (cytoplasmic tail 130 to 497)

MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75) : 9.10 Hyd Moment(95) : 8.40 G content: 2 D/E content: 1 S/T content: 1 Score: -4.05

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 51 CRG|A

NϋCDISC: discrimination of nuclear localization signals pat4 : none pat : 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 :

KKXX-like motif in the C-terminus: PKVI

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

PTS2 : 2nd peroxisomal targeting signal : none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: too long tail

Dileucine motif in the tail: found LL at 171 LL at 244 checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none

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

Final Results (k = 9/23) :

44.4 %: endoplasmic reticulum

22.2 %: Golgi

11.1 %: plasma membrane

11.1 %: vesicles of secretory system

11.1 %: extracellular, including cell wall

» prediction for CG151723-01 is end (k=9) A search of the NOV7a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 7D.

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

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

Example 8. Glycerol -3-phosphate dehydrogenase 1 (soluble).

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: 49 1103 bp

NOV8a, AGCAAGAAAGTCTGCATTGTAGGCTCCGGGAACTGGGGCTCAGCCATCGCCAAGATC CG159280-05 GTGGGTGGCAATGCAGCCCAGCTGGCACAGTTTGACCCACGGGTGACCATGTGGGTAT TTGAGGAAGACATTGGAGGCAAAAAGCTGACTGAGATCATCAACACGCAGCATGAGAA DNA Sequence TGTCAAATACCTGCCAGGGCACAAGTTGCCCCCAAATGTGGTGGCTGTCCCAGATGTG GTCCAGGCTGCAGAGGATGCTGACATCCTGATCTTTGTGGTGCCCCATCAGTTCATCG GCAAGATCTGTGAC^'CAGCTCAAGGGCCATCTGAAGGCAAACGCCACTGGCATATCTCT TATTAAGGGGGTAGACGAGGGCCCCAATGGGCTGAAGCTCATCTCGGAAGTGATTGGG GAGCGCCTCGGCATCCCCATGAGTGTGCTGATGGGGGCCAACATTGCCAGCGAGGTGG CTGATGAGAAGTTCTGTGAGACAACCATTGGCTGCAAGGACCCGGCCCAGGGACAACT CCTGAAAGAGCTGATGCAGACACCAAACTTCCGTATCACAGTGGTGCAAGAGGTGGAC ACAGTAGAGATCTGTGGAGCCTTAAAGAATGTAGTGGCCGTGGGGGCTGGCTTCTGTG ATGGCCTGGGCTTTGGCGACAACACCAAGGCGGCAGTGATCCGGCTGGGACTCATGGA GATGATAGCCTTCGCCAAGCTCTTCTGCAGTGGCCCTGTGTCCTCTGCCACCTTCTTG GAGAGCTGTGGTGTTGCTGACCTGATCACTACCTGCTATGGAGGGCGGAACCGGAAAG TGGCTGAGGCCTTTGCGCGTACAGGAAAGTCCATTGAGCAGCTGGAGAAAGAGTTGCT GAATGGGCAGAAACTGCAGGGGCCCGAGACAGCCCGGGAGCTATACAGCATCCTCCAG CACAAGGGCCTGGTAGACAAGTTTCCCTTGTTCATGGCTGTGTACAAGGTGTGCTACG AGGGCCAGCCAGTGGGTGAATTCATCCACTGCCTGCAGAATCATCCAGAACATATGCA TCATCACCACCATCACTGAGCAGGTGGCGGCCGCACTCGAGCACCACCACCACCACCA C

ORF Start: at 2 ORF Stop: TGA at 1061

SEQ ID NO: 50 353 aa W at 38187.9kD

NOV8a, SKI VCIVGSGIWGSAIAKIVGGNAAQLAQFDPRV MWVFEEDIGGKK TEII TQHEN CG159280-05 VKYLPGHK PPNWAVPDWQAAEDADILIFWPHQFIGKICDQ KGHLKANATGIS

IKGVDEGPNGLK ISEVIGER GIPMSVLMGANIASEVADEKFCETTIGCKDPAQGQ Protein KELMQTPNFRITWQEVDTVEICGALKNWAVGAGF Sequence MIAFAK FCSGPVSSATF ESCGVADLITTCYGGRNRKVAEAFARTGKSIEQLEKELL

NGQK QGPETAl_LYSILQHKGLVDKFPLF]_a^/YKVCYEGQPVGEFIHCLQNΗPEHMH

HHHHH

SEQ ID NO: 51 1413 bp

NOV8b, CGGGCCGGCTCAGGCAGAGACGCGGCACCATGGCTAGCAAGAAAGTCTGCATTGTAGG CG159280-01 CTCCGGGAACTGGGGCTCAGCCATCGCCAAGATCGTGGGTGGCAATGCAGCCCAGCTC GCACAGTTTGACCCACGGGTGACCATGTGGGTATTTGAGGAAGACATTGGAGGCAAAA DNA Sequence AGCTGACTGAGATCATCAACACGCAGCATGAGAATGTCAAATACCTGCCAGGGCACAA GTTGCCCCCAAATGTGGTGGCTGTCCCAGATGTGGTCCAGGCTGCAGAGGATGCTGAC ATCCTGATCTTTGTGGTGCCCCATCAGTTCATCGGCAAGATCTGTGACCAGCTCAAGG GCCATCTGAAGGCAAACCCCACTGGCATATCTCTTATTAAGGGGGTAGACGAGGGCCC CAATGGGCTGAAGCTCATCTQGGAAGTGATTGGGGAGCGCCTCGGCATCCCCATGAGT GTGCTGATGGGGGCCAACATTGCCAGCGAGGTGGCTGATGAGAAGTTCTGTGAGACAA CCATTGGCTGCAAGGACCCGGCCCAGGGACAACTCCTGAAAGAGCTGATGCAGACACC AAACTTCCGTATCACAGTGGTGCAAGAGGTGGACACAGTAGAGATCTGTGGAGCCTTA AAGAATGTAGTGGCCGTGGGGGCTGGCjTTCTGTGATGGCCTGGGCTTTGGCGACAACA CCAAGGCGGCAGTGATCCGGCTGGGACTCATGGAGATGATAGCCTTCGCCAAGCTCTT CTGCAGTGGCCCTGTGTCCTCTGCCACCTTCTTGGAGAGCTGTGGTGT GCTGACCTG ATCACTACCTGCTATGGAGGGCGGAACCGGAAAGTGGCTGAGGCCTTTGCGCGTACAG GAAAGTCCATTGAGCAGCTGGAGAAAGAGTTGCTGAATGGGCAGAAACTGCAGGGGCC CGAGACAGCCCGGGAGCTATACAGCATCCTCCAGCACAAGGGCCTGGTAGACAAGTTT CCCTTGTTCATGGCTGTGTACAAGGTGTGCTACGAGGGCCAGCCAGTGGGTGAATTCA TCCACTGCCTGCAGAATCATCCAGAACATATGTGAGTGGGGCCAGGCCCAGGCCAGGC GTTTTTACCCCAGTGGAGACCAGCAGAAGCCTGGGGTACCTAGTCACCAGGATCTCCA GGACTCCCAGGGAGCAGAGTCTTCTCATCGTTTCACTGGAGGACAGGTGGCTATGGGG CCCAGCTACGCACCTGGAGATCCTGAACTGTCAAGCCACTGGCAGCCTCATGCCACCAl

CATTCGCCAGAAATGCAGTTGCCCTGTCCCTCTCCAGATGTGGGGCTTTCTCCATATC

CTCTGGGAGGGGTGGAATCAAGCCCCAGTGCTGCCTGCTTGGTGGCGGGGGTGATGTAI

TGTGGAGAAGGGTCGGGGCCG

ORF Start: ATG at 30 ORF Stop: TGA at 1077

SEQ ID NO: 52 349 aa MW at 37593.3kD

NOV8b, MASKK^CIVGSGN GSAIAKIVGGNAAQIAQFDPRVTMWVFEEDIGGKKLTEIINTQH ENVKYLPGHKIiPP WAVPDWQAAEDADILIFWPHQFIGKICDQLKGH KANPTGI CG159280-01 SLIKGVDEGPNG KLISEVIGERLGIPMSVLMGANIASEVADEKFCETTIGCKDPAQG Protein Q LKE MQTPNFRITVVQEVDTVEICGALKJWVAVGAGFCDG GFGDN KAAVIRLGL Sequence l ffiMIAFAKLFCSGPVSSATFLESCGVADLITTCYGGRNRKVAEAFARTGKSIEQ EKE LLNGQK QGPETARE YSILQHKGLVDKFPLFMAVYKVCYEGQPVGEFIHCLQNHPEH M i

SEQ ID NO: 53 1080 bp

NOV8c, ACGGGATCCACCATGGCTAGCAAGAAAGTCTGCATTGTAGGCTCCGGGAACTGGGGCT CAGCCATCGCCAAGATCGTGGGTGGCAATGCAGCCCAGCTGGCACAGTTTGACCCACG 258685898 GGTGACCATGTGGGTATTTGAGGAAGACATTGGAGGCAAAAAGCTGACTGAGATCATC DNA Sequence AACACGCAGCATGAGAATGTCAAATACCTGCCAGGGCACAAGTTGCCCCCAAATGTGG TGGCTGTCCCAGATGTGGTCCAGGCTGCAGAGGATGCTGACATCCTGATCT7TGTGGT GCCCCATCAGTTCATCGGCAAGATCTGTGACCAGCTCAAGGGCCATCTGAAGGCAAAC GCCACTGGCATATCTCTTATTAAGGGGGTAGACGAGGGCCCCAATGGGCTGAAGCTCA TCTCGGAAGTGATTGGGGAGCGCCTCGGCATCCCCATGAGTGTGCTGATGGGGGCCAA CATTGCCAGCGAGGTGGCTGATGAGAAGTTCTGTGAGACAACCATTGGCTGCAAGGAC CCGGCCCAGGGACAACTCCTGAAAGAGCTGATGCAGACACCAAACTTCCGTATCACAG TGGTGCAAGAGGTGGACACAGTAGAGATCTGTGGAGCCTTAAAGAATGTAGTGGCCGT GGGGGCTGGCTTCTGTGATGGCCTGGGCTTTGGCGACAACACCAAGGCGGCAGTGATC CGGCTGGGACTCATGGAGATGATAGCCTTCGCCAAGCTCTTCTGCAGTGGCCCTGTGT CCTCTGCCACCTTCTTGGAGAGCTGTGGTGTTGCTGACCTGATCACTACCTGCTATGG AGGGCGGAACCGGAAAGTGGCTGAGGCCTTTGCGCGTACAGGAAAGTCCATTGAGCAG CTGGAGAAAGAGTTGCTGAATGGGCAGAAACTGCAGGGGCCCGAGACAGCCCGGGAGC TATACAGCATCCTCCAGCACAAGGGCCTGGTAGACAAGTTTCCCTTGTTCATGGCTGT GTACAAGGTGTGCTACGAGGGCCAGCCAGTGGGTGAATTCATCCACTGCCTGCAGAAT CATCCAGAACATATGTGAGCAGGTGGCGGCCGCAAG

ORF Start: at 1 ORF Stop: TGA at 1060

" »"M^

SEQ ID NO: 54 353 aa MW at 37913.6kD

NOV8c, TGSTMASKKVCIVGSG1WGSAIAKIVGGNAAQLAQFDPRV MWVFEEDIGGKK TEII I\_QHEIWKY PGHKLPP VVAVPDVVQAAEDADILIFVVPHQFIGKICDQLKGH KA!VI 258685898 ATGISLIKGVDEGPNGLK ISEVIGERLGIPMSVLMGAWIASEVADEKFCETTIGCKD Protein PAQGQL KΞLMQTPNFRITVVQEVDTVEICGALKJWVAVGAGFCDGLGFGDNT AAVI Sequence RLGLMEMIAFAKLFCSGPVSSATF ESCGVADLITTCYGGRRKVAEAFARTGKSIEQ LEKEL NGQKLQGPETARΞLYSILQHKGLVDKFP FMAVYKVCYEGQPVGEFIHCLQN HPEHM

SEQ ID NO: 55 993 bp

NOV8d, ACGGGATCCACCATGGCTAGCAAGAAAGTCTGCATTGTAGGCTCCGGGAACTGGGGCT CAGCCATCGCCAAGATCGTGGGTGGCAATGCAGCCCAGCTGGCACAGTTTGACCCACG 258685953 GGTGACCATGTGGGTATTTGAGGAAGACATTGGAGGCAAAAAGCTGACTGAGATCATC DNA Sequence AACACGCAGCATGAGAATGTCAAATACCTGCCAGGGCACAAGTTGCCCCCAAATGTGG CTGTCCCAGATGTGGTCCAGGCTGCAGAGGATGCTGACATCCTGATCTTTGTGGTGCC CCATCAGTTCATCGACAAGATCTGTGACCAGCTCAAGGGCCATCTGAAGGCAAACGCC ACTGGCATATCTCTTATTAAGGGGGCCAACATTGCCAGCGAGGTGGCTGATGAGAAGT TCTGTGAGACAACCATTGGCTGCAAGGACCCGGCCCAGGGACAACTCCTGAAAGAGCT GATGCAGACACCAAACTTCCGTATCACAGTGGTGCAAGAGGTGGACACAGTAGAGATC TGTGGAGCCTTAAAGAATGTAGTGGCCGTGGGGGCTGGCTTCTGTGATGGCCTGGGCT TTGGCGACAACACCAAGGCGGCAGTGATCCGGCTGGGACTCATGGAGATGATAGCCTT CGCCAAGCTCTTCTGCAGTGGCCCTGTGTCCTCTGCCACCTTCTTGGAGAGCTGTGGT GTTGCTGACCTGATCACTACCTGCTATGGAGGGCGGAACCGGAAAGTGGCTGAGGCCT TTGCGCGTACAGGAAAGTCCATTGAGCAGCTGGAGAAAGAGTTGCTGAATGGGCAGAA ACTGCAGGGGCCCGAGACAGCCCGGGAGCTATACAGCATCCTCCAGCACAAGGGCCTG W 03

GTAGACAAGTTTCCCTTGTTCATGGCTGTGTACAAGGTGTGCTACGAGGGCCAGCCAG TGGGTGAATTCATCCACTGCCTGCAGAATCATCCAGAACATATGTGAGCAGGTGGCGG CCGCAAG

ORF Start: at 1 ORF Stop: TGA at 973

SEQ ID NO: 56 324 aa MW at 34966. lkD

NOV8d, TGSTI^SKK^CIVGSGIWGSAIAKIVGGNAAQLAQFDPRVTMWVFEEDIGGKKLTΞII N QHENVKY PGHKLPP VAVPDVVQAAEDADI IFVVPHQFIDKICDQLKGHLKANA 258685953 TGISLIKGANIASEVADEKFCETTIGCKDPAQGQLLKELMQTPNFRITWQEVDTVEI Protein CGALKNWAVGAGFCDGLGFGDNTKAAVIRLGL EMIAFAKLFCSGPVSSATFLESCG Sequence VADLITTC YGGRNRKVAEAFARTGKS lEQLEKELLNGQKLQGPETARELYS ILQHKGL VDKFPLFMAVYKVCYEGQPVGEFIHCLQNHPEHM

SEQ ID NO: 57 1167 bp

NOV8e, GAATCGCCTTACATGGCTAGCAAGAAAGTCTGCATTGTAGGCTCCGGGAACTGGGGCT CG159280-02 CAGCCATCGCCAAGATCGTGGGTGGCAATGCAGCCCAGCTGGCACAGTTTGACCCACG GGTGACCATGTGGGTATTTGAGGAAGACATTGGAGGCAAAAAGCTGACTGAGATCATC DNA Sequence AACACGCAGCATGAGAATGTCAAATACCTGCCAGGGCACAAGTTGCCCCCAAATGTGG TGGCTGTCCCAGATGTGGTCCAGGCTGCAGAGGATGCTGACATCCTGATCTTTGTGGT GCCCCATCAGTTCATCGGCAAGATCTGTGACCAGCTCAAGGGCCATCTGAAGGCAAAC GCCACTGGCATATCTCTTATTAAGGGGGTAGACGAGGGCCCCAATGGGCTGAAGCTCA TCTCGGAAGTGATTGGGGAGCGCCTCGGCATCCCCATGAGTGTGCTGATGGGGGCCAA CATTGCCAGCGAGGTGGCTGATGAGAAGTTCTGTGAGACAACCATTGGCTGCAAGGAC CCGGCCCAGGGACAACTCCTGAAAGAGCTGATGCAGACACCAAACTTCCGTATCACAG TGGTGCAAGAGGTGGACACAGTAGAGATCTGTGGAGCCTTAAAGAATGTAGTGGCCGT GGGGGCTGGCTTCTGTGATGGCCTGGGCTTTGGCGACAACACCAAGGCGGCAGTGATC CGGCTGGGACTCATGGAGATGATAGCCTTCGCCAAGCTCTTCTGCAGTGGCCCTGTGT CCTCTGCCACCTTCTTGGAGAGCTGTGGTGTTGCTGACCTGATCACTACCTGCTATGG AGGGCGGAACCGGAAAGTGGCTGAGGCCTTTGCGCGTACAGGAAAGTCCATTGAGCAG CTGGAGAAAGAGTTGCTGAATGGGCAGAAACTGCAGGGGCCCGAGACAGCCCGGGAGC TATACAGCATCCTCCAGCACAAGGGCCTGGTAGACAAGTTTCCCTTGTTCATGGCTGT GTACAAGGTGTGCTACGAGGGCCAGCCAGTGGGTGAATTCATTCACTGCCTGCAGAAT CATCCAGAACATATGTGAGTGGGGCCAGGGCCCAGGCCAGGCCGCTTTTTTACCCCAG

TGGAGACCAGCAGAAGCCTGGGGTACCTAGTCACCAGGATCTCCAGGACTCCAAGGCG

ATTCCAC

ORF Start: ATG at 13 ORF Stop: TGA at 1060

SEQ ID NO: 58 349 aa MW at 37567.3kD

NOV8e, MASI_VCIVGSGN GSAIAKIVGGNAAQLAQFDPRVTMWVFEEDIGGKKLTEIINTQH CG159280-02 ENV^YLPGHKLPPNVVAVPDVVQAAEDADILIFVVPHQFIGKICDQLKGHLKANATGI SLIKGVDEGPNGLKLISEVIGERLGIPMSVLMGANIASEVADEKFCETTIGCKDPAQG Protein QLLKELMQTPNFRITVVQEVT⁾TVEICGALKJWVAVGAGFCDGLGFGDNTKAAVIRLGL Sequence MEMIAFAKLFCSGPVSSATFLΞSCGVADLITTCYGGRNRKVAEAFARTGKSIEQLEKE LNGQKLQGPETARELYSILQHKGLVDKFPLFMAVYKVCYEGQPVGEFIHCLQNHPEH M

SEQ ID NO: 59 993 bp

NOV8f, ACGGGATCCACCATGGCTAGCAAGAAAGTCTGCATTGTAGGCTCCGGGAACTGGGGCT CG159280-03 CAGCCATCGCCAAGATCGTGGGTGGCAATGCAGCCCAGCTGGCACAGTTTGACCCACG GGTGACCATGTGGGTATTTGAGGAAGACATTGGAGGCAAAAAGCTGACTGAGATCATC DNA Sequence AACACGCAGCATGAGAATGTCAAATACCTGCCAGGGCACAAGTTGCCCCCAAATGTGG CTGTCCCAGATGTGGTCCAGGCTGCAGAGGATGCTGACATCCTGATCTTTGTGGTGCC CCATCAGTTCATCGACAAGATCTGTGACCAGCTCAAGGGCCATCTGAAGGCAAACGCC ACTGGCATATCTCTTATTAAGGGGGCCAACATTGCCAGCGAGGTGGCTGATGAGAAGT TCTGTGAGACAACCATTGGCTGCAAGGACCCGGCCCAGGGACAACTCCTGAAAGAGCT GATGCAGACACCAAACTTCCGTATCACAGTGGTGCAAGAGGTGGACACAGTAGAGATC TGTGGAGCCTTAAAGAATGTAGTGGCCGTGGGGGCTGGCTTCTGTGATGGCCTGGGCT TTGGCGACAACACCAAGGCGGCAGTGATCCGGCTGGGACTCATGGAGATGATAGCCTT CGCCAAGCTCTTCTGCAGTGGCCCTGTGTCCTCTGCCACCTTCTTGGAGAGCTGTGGT GTTGCTGACCTGATCACTACCTGCTATGGAGGGCGGAACCGGAAAGTGGCTGAGGCCT TTGCGCGTACAGGAAAGTCCATTGAGCAGCTGGAGAAAGAGTTGCTGAATGGGCAGAA ACTGCAGGGGCCCGAGACAGCCCGGGAGCTATACAGCATCCTCCAGCACAAGGGCCTG

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 8B.

Table 8C.

Table 8C. Protein Sequence Properties NO 8a

SignalP analysis: No Known Signal Sequence Predicted

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

N-region: length 3 ; pos .chg 2; ne . chg 0 H-region: length 14; peak value 4.25 PSG score: -0.15

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -6.03 possible cleavage site: between 17 and 18

>» Seems to have no N-terminal signal peptide

Tentative number of TMS(s) for the threshold 0.5: Number of TMS(.s) for threshold 0.5: 0 PERIPHERAL Likelihood = 1.54 (at 84) ALOM score: -0.48 (number of TMSs: 0)

MITDISC: discrimination of mitochondrial targeting seq

R content: 0 Hyd Moment (75) 67 Hyd Moment (95) 6.66 G content: D/E content: 1 S/T content: Score: -6.19

NUCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite: none content of basic residues: 9.3% NLS Score: -0.47

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

ER Membrane Retention Signals : none

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

PTS2 : 2nd peroxisomal targeting signal : none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

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

Final Results (k = 9/23) ;

47.8 %: cytoplasmic 26.1 %: mitochondrial 26.1 %: nuclear

» prediction for CG159280-05 is cyt (k=23) A search of the NOV8a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 8D.

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

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

Example 9. Serine/threonine-protein kinase receptor R2.

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

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

Table 9B. Comparison of NOV9a against NOV9b.

NOV9a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV9b 1..379 378/379 (99%) 1..379 378/379 (99%)

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

Table 9C. Protein Sequence Properties NOV9a

SignalP analysis: Cleavage site between residues 25 and 26

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

N-region: length 3; pos.chg 0; neg.chg 1 H-region: length 23; peak value 0.00 PSG score: -4.40 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 0.75 possible cleavage site: between 23 and 24

>» Seems to have no N-terminal signal peptide

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

INTEGRAL Likelihood =-15.81 Transmembrane 133 - 149 PERIPHERAL Likelihood = 2.92 (at 299) ALOM score: -15.81 (number of TMSs: 1)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 140 Charge difference: 2.0 C( 1.0) - N(-1.0) C > N: C-terminal side will be inside

>» membrane topology: type lb (cytoplasmic tail 133 to 505)

MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75) : 6.23 Hyd Moment (95) : 6.18 G content: 6 D/E content: 2 S/T content: 8 Score: -6.55

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 37 PRG|vQ

NUCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite: none content of basic residues: 10.7% NLS Score: -0.47

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

ER Membrane Retention Signals :

KKXX-like motif in the C-terminus: EDVK

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

PTS2 : 2nd peroxisomal targeting signal: none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: too long tail

Dileucine motif in the tail: found LL at 137 checking 63 PROSITE DNA binding motifs: none

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

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

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

Example 10. Proprotein convertase subtilisin/kexin type 5 precursor.

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

Table 10A. NOV10 Sequence Analysis

SEQ ID NO: 67 6201 bp

NOVlOa, GGCACGAGGCGGAGGGAGCGCTGGGAGCGAGCAAGCGAGCGTTTGGAGCCCGGGCCAG

CAGAGGGGGCGCCCGGTCGCTGCCTGTACCGCTCCCGCTGGTCATCTCCGCCGCGCTC CG169667-01 GGGGGCCCCGGGAGGAGCGAGACCGAGTCGGAGAGTCCGGGAGCCAAGCCGGGCGAAA DNA Sequence CCCAACTGCGGAGGACGCCCGCCCCACTCAGCCTCCTCCTGCGTCCGAGCCGGGGAGC

ATCGCCGAGCGCCCCACGGGCCGGAGAGCTGGGAGCACAGGTCCCGGCAGCCCCAGGG

ATGGTCTAGGAGCCGGCGTAAGGCTCGCTGCTCTGCTCCCTGCCGGGGCTAGCCGCCT

CCTGCCGATCGCCCGGGGCTGCGAGCTGCGGCGGCCCGGGGCTGCTCGCCGGGCGGCG

CAGGCCGGAGAAGTTAGTTGTGCGCGCCCTTAGTGCGCGGAACCAGCCAGCGAGCGAG

GGAGCAGCGAGGCGCCGGGACCATGGGCTGGGGGAGCCGCTGCTGCTGCCCGGGACGT

TTGGACCTGCTGTGCGTGCTGGCGCTGCTCGGGGGCTGCCTGCTCCCCGTGTGTCGGA CGCGCGTCTACACCAACCACTGGGCAGTCAAAATCGCCGGGGGCTTCCCGGAGGCCAA CCGTATCGCCAGCAAGTACGGATTCATCAACATAGGACAGATAGGGGCCCTGAAGGAC TACTACCACTTCTACCATAGCAGGACGATTAAAAGGTCAGTTATCTCGAGCAGAGGGA CCCACAGTTTCATTTCAATGGAACCAAAGGTGGAATGGATCCAACAGCAAGTGGTAAA AAAGCGGACAAAGAGGGATTATGACTTCAGTCGTGCCCAGTCTACCTATTTCAATGAT CCCAAGTGGCCCAGCATGTGGTATATGCACTGCAGTGACAATACACATCCCTGCCAGT CTGACATGAATATCGAAGGAGCCTGGAAGAGAGGCTACACGGGAAAGAACATTGTGGT CACTATCCTGGATGACGGAATTGAGAGAACCCATCCAGATCTGATGCAAAACTACGAT GCTCTGGCAAGTTGCGACGTGAATGGGAATGACTTGGACCCAATGCCTCGTTATGATG CAAGCAACGAGAACAAGCATGGGACTCGCTGTGCTGGAGAAGTGGCAGCCGCTGCAAA CAATTCGCACTGCACAGTCGGAATTGCTTTCAACGCCAAGATCGGAGGAGTGCGAATG CTGGACGGAGATGTCACGGACATGGTTGAAGCAAAATCAGTTAGCTTCAACCCCCAGC ACGTGCACATTTACAGCGCCAGCTGGGGCCCGGATGATGATGGCAAGACTGTGGACGG ACCAGCCCCCCTCACCCGGCAAGCCTTTGAAAACGGCGTTAGAATGGGGCGGAGAGGC CTCGGCTCTGTGTTTGTTTGGGCATCTGGAAATGGTGGAAGGAGCAAAGACCACTGCT CCTGTGATGGCTACACCAACAGCATCTACACCATCTCCATCAGCAGCACTGCAGAAAG CGGAAAGAAACCTTGGTACCTGGAAGAGTGTTCATCCACGCTGGCCACAACCTACAGC AGCGGGGAGTCCTACGATAAGAAAATCATCACTACAGATCTGAGGCAGCGTTGCACGG ACAACCACACTGGGACGTCAGCCTCAGCCCCCATGGCTGCAGGCATCATTGCGCTGGC CCTGGAAGCCAATCCGTTTCTGACCTGGAGAGACGTACAGCATGTTATTGTCAGGACT TCCCGTGCGGGACATTTGAACGCTAATGACTGGAAAACCAATGCTGCTGGTTTTAAGG TGAGCCATCTTTATGGATTTGGACTGATGGACGCAGAAGCCATGGTGATGGAGGCAGA GAAGTGGACCACCGTTCCCCGGCAGCACGTGTGTGTGGAGAGCACAGACCGACAAATC AAGACAATCCGCCCTAACAGTGCAGTGCGCTCCATCTACAAAGCTTCAGGCTGCTCGG ATAACCCCAACCGCCATGTCAACTACCTGGAGCACGTCGTTGTGCGCATCACCATCAC CCACCCCAGGAGAGGAGACCTGGCCATCTACCTGACCTCGCCCTCTGGAACTAGGTCT CAGCTTTTGGCCAACAGGCTATTTGATCACTCCATGGAAGGATTCAAAAACTGGGAGT TCATGACCATTCATTGCTGGGGAGAAAGAGCTGCTGGTGACTGGGTCCTTGAAGTTTA TGATACTCCCTCTCAGCTAAGGAACTTTAAGACTCCAGGTAAATTGAAAGAATGGTCT TTGGTCCTCTACGGCACCTCCGTGCAGCCATATTCACCAACCAATGAATTTCCGAAAG TGGAACGGTTCCGCTATAGCCGAGTTGAAGACCCCACAGACGACTATGGCACAGAGGA TTATGCAGGTCCCTGCGACCCTGAGTGCAGTGAGGTTGGCTGTGACGGGCCAGGACCA GACCACTGCAATGACTGTTTGCACTACTACTACAAGCTGAAAAACAATACCAGGATCT GTGTCTCCAGCTGCCCCCCTGGCCACTACCACGCCGACAAGAAGCGCTGCAGGAAGTG TGCCCCCAACTGTGAGTCCTGCTTTGGGAGCCATGGTGACCAATGCATGTCCTGCAAA TATGGATACTTTCTGAATGAAGAAACCAACAGCTGTGTTACTCACTGCCCTGATGGGT CATATCAGGATACCAAGAAAAATCTT GCCGGAAATGCAGTGAAAACTGCAAGACATG TACTGAATTCCATAACTGTACAGAATGTAGGGATGGGTTAAGCCTGCAGGGATCCCGG TGCTCTGTCTCCTGTGAAGATGGACGGTATTTCAACGGCCAGGACTGCCAGCCCTGCC ACCGCTTCTGCGCCACTTGTGCTGGGGCAGGAGCTGATGGGTGCATTAACTGCACAGA GGGCTACTTCATGGAGGATGGGAGATGCGTGCAGAGCTGTAGTATCAGCTATTACTTT GACCACTCTTCAGAGAATGGATACAAATCCTGCAAAAAATGTGATATCAGTTGTTTGA CGTGCAATGGCCCAGGATTCAAGAACTGTACAAGCTGCCCTAGTGGGTATCTCTTAGA CTTAGGAATGTGTCAAATGGGAGCCATTTGCAAGGATGGAGAATATGTTGATGAGCAT GGCCACTGCCAGACCTGTGAGGCCTCATGTGCCAAGTGCCAGGGACCAACCCAGGAAG ACTGCACTACCTGCCCCATGACAAGGATTTTTGATGATGGCCGCTGTGTTTCGAACTG CCCCTCATGGAAATTTGAATTTGAGAACCAATGCCATCCATGCCACCACACCTGCCAG AGATGCCAAGGAAGTGGCCCTACCCACTGCACCTCCTGTGGAGCAGACAACTATGGCC GAGAGCACTTCCTGTACCAGGGAGAGTGTGGAGATAGCTGCCCAGAGGGCCACTATGC CACTGAGGGGAACACCTGCCTGCCCTGCCCAGACAACTGTGAGCTTTGCCACAGCGTG CATGTCTGCACAAGATGCATGAAGGGCTACTTCATAGCGCCCACCAACCACACATGCC AGAAGTTAGAGTGTGGACAAGGTGAAGTCCAAGACCCAGACTATGAAGAATGTGTCCC TTGTGAAGAAGGATGTCTGGGATGCAGCTTGGATGATCCAGGAACATGTACATCTTGC GCTATGGGGTATTACAGGTTTGATCACCATTGTTATAAAACCTGTCCTGAGAAGACCT ACAGTGAGGAAGTGGAATGCAAGGCGTGTGATAGTAACTGTGGCAGCTGTGACCAGAA TGGGTGTTACTGGTGTGAAGAGGGCTTCTTTCTCTTAGGTGGCAGTTGTGTGAGGAAA TGTGGTCCTGGATTCTATGGTGACCAAGAAATGGGAGAATGTGAGTCCTGCCACCGAG CATGCGAAACCTGCACAGGCCCTGGTCATGACGAGTGCAGCAGCTGCCAGGAAGGACT GCAGCTGCTGCGTGGGATGTGCGTGCATGCCACCAAGACCCAGGAGGAGGGCAAATTC TGGAATGAAGCTGTGTCCACTGCAAACCTATCTGTGGTGAAGAGCCTGCTGCAGGAGC GACGAAGGTGGAAAGTTCAAATCAAAAGAGATATTTTGAGAAAACTCCAGCCTTGTCA TTCTTCTTGTAAAACCTGCAATGGATCTGCAACTCTGTGCACTTCATGTCCCAAAGGT GCATATCTTCTGGCTCAGGCCTGTGTTTCCTCCTGTCCCCAAGGCACATGGCCTTCCG TAAGGAGTGGGAGCTGCGAGAACTGTACGGAGGCCTGTGCCATCTGCTCTGGAGCCGA TCTTTGCAAAAAATGCCAGATGCAGCCGGGCCACCCTCTCTTCCTCCATGAAGGCAGG TGCTACTCCAAGTGCCCGGAGGGCTCTTATGCAGAAGACGGCATATGTGAACGCTGTA GCTCTCCTTGCAGAACATGTGAAGGAAACGCCACCAACTGCCATTCTTGTGAAGGAGG CCACGTCCTGCACCACGGAGTGTGCCAGGAAAACTGCCCCGAGAGGCACGTGGCTGTG AAGGGGGTATGCAAGCATTGCCCAGAGATGTGTCAGGACTGCATCCATGAGAAAACAT GCAAAGAGTGCACGCCTGAGTTCTTCCTGCACGATGATATGTGCCACCAGTCCTGTCC CCGTGGCTTCTATGCAGACTCGCGCCACTGTGTCCCCTGCCATAAAGACTGTCTGGAG TGCAGTGGCCCCAAAGCCGACGACTGCGAGCTCTGTCTTGAGAGTTCCTGGGTCCTCT ATGATGGACTGTGCTTGGAGGAGTGTCCAGCAGGAACCTATTATGAAAAGGAGACTAA GGAGTGCAGAGATTGCCACAAGTCCTGCTTGACCTGCTCATCATCTGGGACCTGCACC ACCTGTCAGAAAGGCCTGATCATGAACCCTCGTGGGAGCTGCATGGCCAACGAGAAGT GCTCACCCTCCGAGTACTGGGATGAGGATGCTCCCGGGTGCAAGCCCTGCCATGTTAA GTGCTTCCACTGCATGGGGCCGGCGGAGGACCAGTGTCAAACATGCCCCATGAACAGC CTTCTTCTCAACACAACCTGTGTGAAGGACTGCCCAGAGGGCTATTATGCCGATGAGG ACAGCAACCGGTGTGCCCACTGCCACAGCTCTTGCAGGACATGTGAAGGGAGACACAG CAGGCAGTGCCACTCCTGCCGACCGGGCTGGTTCCAGCTAGGAAAAGAGTGCCTGCTC CAGTGCAGGGAAGGATATTACGCAGACAACTCCACTGGCCGGTGTGAGAGGTGCAACA GGAGCTGCAAGGGGTGCCAGGGCCCACGGCCCACAGACTGCCTGTCTTGCGATAGATT TTTCTTTCTGCTCCGCTCCAAAGGAGAGTGTCATCGCTCCTGCCCAGACCATTACTAT GTAGAGCAAAGCACACAGACCTGTGAGAGATGCCATCCGACTTGTGATCAATGCAAAG GAAAAGGAGCGTTGAATTGTTTATCCTGTGTGTGGAGTTACCACCTCATGGGAGGGAT CTGCACCTCGGACTGTCTTGTGGGGGAATACAGAGTGGGAGAGGGAGAGAAG TTAAC TGTGAAAAATGCCACGAGAGCTGCATGGAATGCAAGGGACCAGGGGCCAAGAACTGCA CCTTGTGCCCTGCCAACCTGGTGCTGCACATGGACGACAGCCACTGCCTCCACTGCTG CAACACCTCTGATCCCCCCAGTGCCCAGGAGTGCTGTGACTGCCAGGACACCACGGAC GAATGCATCCTTCGAACAAGCAAGGTTAGGCCTGCAACTGAGCATTTCAAGACAGCTC TGTTCATCACCTCCTCCATGATGCTGGTGCTTCTGCTCGGGGCAGCTGTGGTAGTGTG GAAGAAATCTCGTGGCCGAGTCCAGCCAGCAGCAAAGGCCGGCTATGAAAAACTGGCC GACCCCAACAAGTCTTACTCCTCCTATAAGAGCAGCTATAGAGAGAGCACCAGCTTTG AAGAGGATCAGGTGATTGAGTACAGGGATCGGGACTATGATGAGGATGATGATGATGA CATCGTCTACATGGGCCAGGATGGCACAGTCTACCGGAAATTTAAATATGGGCTGCTG GATGACGATGACATAGATGAGCTGGAATATGATGACGAGAGTTACTCCTACTACCAGT AAACAGGCACTCCCCCACCAACACCACCATTCCACTCTCAGGCATGCCTGTGA

ORF Start: ATG at 487 ORF Stop: TAA at 6148

SEQ ID NO: 68 1887 aa MW at 210117.4kD

NOVlOa, MG GSRCCCPGR DL CVLALLGGCL PVCRTRVYTKIHWAVKIAGGFPEA RIAS YG

FINIGQIGALKDYY iFYHSRTI RSVISSRGTHSFISMEPKVΞWIQQQVVKKRTKRDY CG169667-01 DFSRAQSTYFlsroPKWPSMWY__lCSD frHPCQSD]_IIEGAWKRGYTGK IVv^ Protein ERTHPDLMQJSr_DAI_ SCDWGlTOLDPMPRYDASl Sequence IAFNA IGGVRMLDGDV DMVEA SVSFNPQHVHIYSASWGPDDDGKTVDGPAPLTRQ

AFENGVRMGRRG GSVFVWASGNGGRSKDHCSCDGYTNSIYTISISSTAESGKKPWYL

EECSSTLATTYSSGESYDKi IITTDLRORCTD]N_TGTSASAPMAAGIIA__EANPF T RDVQHVIVRTSRAGHLNA V«TNAAGFK^SH YGFG MDAEAMvT_EAEK TTVPR QHVCVESTDRQIKTIRPNSAVRSIYKASGCSDNPNR / YLEHVVVRITITHPRRGDL AIYLTSPSGTRSQL ARLFDHSMEGFKN EFMTIHC GERAAGDWVLEVYDTPSQ R NFKTPG LKEWSLVLYGTSVQPYSPTNEFPKVERFRYSRVEDPTDDYGTEDYAGPCDP ΞCSEVGCDGPGPDHCNDCLHY ΪK K N RICVSSCPPGHYHADKKRCRKCAPNCESC FGSHGDQC SCKYGYFLNEΞTNSCVTHCPDGSYQDTKKNLCRKCSENCKTCTEFH CT ECRDGLS QGSRCSVSCEDGRYFNGQDCQPCHRFCATCAGAGADGCINCTEGYFMEDG RCVQSCSISYYFDHSSENGYKSCKKCDISCLTCNGPGFKNCTSCPSGY LD GMCQMG AICKDGEYVDEHGHCQTCEASCAKCQGPTQEDCTTCPM RIFDDGRCVSNCPSWKFEF ENQCHPCHHTCQRCQGSGPTHCTSCGADNYGREHFLYQGECGDSCPEGHYATEGN CL PCPDNCE CHSVHVCTRCM GYFIAPTNHTCQK ECGQGEVQDPDYEECVPCEEGCLG CS DDPGTCTSCAMGYYRFDHHCYKTCPEKTYSEEVECKACDSNCGSCDQNGCYWCEE GFF LGGSCVRKCGPGFYGDQEMGECESCHRACETCTGPGHDECSSCQEG QLLRG C VHATKTQEEGKFVmΞAVSTANIiSVVTSLLQERRR KVQIKRDILRKLQPCHSSCKTCN GSATLCTSCPKGAY LAQACVSSCPQG PSVRSGSCENCTEACAICSGAD CKKCQM QPGHPLFLHEGRCYSKCPEGSYAEDGICERCSSPCRTCEGNATNCHSCEGGHV HHGV CQENCPERHVAVKGVCKHCPEMCQDCIHEKTCKECTPEFFLHDDMCHQSCPRGFYADS RHCVPCHKDCLECSGPKADDCELCLESSWV YDGLCLEECPAGTYYEKETKECRDCHK SCLTCSSSGTCTTCQKGLI NPRGSCMANEKCSPSEYWDEDAPGCKPCHV CFHCMGP AEDQCQTCPM SLLLNTTCVKDCPEGYYADEDSNRCAHCHSSCRTCEGRHSRQCHSCR PGWFQLGKEC QCREGYYADNSTGRCERCNRSCKGCQGPRPTDCLSCDRFFFL RSK GECHRSCPDHYYVEQSTQTCERCHPTCDQCKGKGALNC SCVWSYH MGGICTSDC V GEYRVGEGEKFNCEKCHESCMECKGPGAKVJCTLCPANLV H DDSHCLHCCNTSDPPS AQECCDCQDTTDECI RTSKΛ/RPATEHFKTALFITSSMM VLLLGAAVVVWKKSRGRV QPAAKAGYEK ADPN SYSSYKSSYRESTSFEEDQVIEYRDRDYDEDDDDDIVYMGQD G VYRKFKYGLLDDDDIDELEYDDESYSYYQ

Further analysis ofthe NO V 10a protein yielded the following properties shown in Table 10B.

Table 10B. Protein Sequence Properties NOVlOa

SignalP analysis: Cleavage site between residues 33 and 34

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

N-region: length 6 ; pos .chg 1; neg.chg 0 H-region: length 5; peak value -6.21 PSG score: -10.61

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 1.82 possible cleavage site: between 32 and 33

>» Seems to have no N-terminal signal peptide

Tentative number of TMS(s) for the threshold 0.5: 2 INTEGRAL Likelihood = -6.79 Transmembrane 13 - 29 INTEGRAL Likelihood = -7.59 Transmembrane 1774 -1790 PERIPHERAL Likelihood = 1.22 (at 384) ALOM score: -7.59 (number of TMSs: 2)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 20 Charge difference: 1.5 C( 3.5) - N( 2.0) C > N: C-terminal side will be inside

»> membrane topology: type 3b

MITDISC: discrimination of mitochondrial targeting seq R content: 4 Hyd Moment (75) : 6.39 Hyd Moment (95) : 8.11 G content: 7 D/E content: 2 S/T content: 3 Score: -5.55

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 98 SRG|TH

NϋCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite : none content of basic residues: 10.2% NLS Score: -0.47

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

ER Membrane Retention Signals : none

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

PTS2 : 2nd peroxisomal targeting signal: none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: nuclear Reliability: 94.1

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

Final Results (k = 9/23):

34.8 %: nuclear

34.8 %: endoplasmic reticulum

13.0 % : vacuolar

4.3 %: Golgi

4.3 %: mitochondrial

4.3 %: vesicles of secretory system

4.3 % : cytoplasmic

» prediction for CG169667-01 is nuc (k=23) A search of the NOVlOa protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table IOC.

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

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

Example 11. Protein tyrosine phosphatase, non-receptor type 18

The NO VI 1 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 11 A.

Table 11A. NOV11 Sequence Analysis

SEQ ID NO: 69 1499 bp

NOVlla, GAATTCGGCACGAGCGGGCTGGACCTTGCTGGCCCGCGGCGCCA GAGCCGCAGCCTG CG169754-01 GACTCGGCGCGGAGCTTCCTGGAGCGGCTGGAAGCGCGGGGCGGCCGGGAGGGGGCAG TCCTCGCCGGCGAGTTCAGCGACATCCAGGCCTGCTCGGCCGCCTGGAAGGCTGACGG DNA Sequence CGTGTGCTCCACCGTGGCCGGCAGTCGGCCAGAGAACGTGAGGAAGAACCGCTACAAA GACGTGCTGCCTTATGATCAGACGCGAGTAATCCTCTCCCTGCTCCAGGAAGAGGGAC ACAGCGACTACATTAATGGCAACTTCATCCGGGGCGTGGATGGAAGCCTGGCCTACAT TGCCACGCAAGGACCCTTGCCTCACACCCTGCTAGACTTCTGGAGACTGGTCTGGGAG TTTGGGGTCAAGGTGATCCTGATGGCCTGTCGAGAGATAGAGAATGGGCGGAAAAGGT GTGAGCGGTACTGGGCCCAGGAGCAGGAGCCACTGCAGACTGGGCTTTTCTGCATCAC TCTGATAAAGGAGAAGTGGCTGAATGAGGACATCATGCTCAGGACCCTCAAGGTCACA TTCCAGAAGGAGTCCCGTTCTGTGTACCAGCTACAGTATATGTCCTGGCCAGACCGTG GGGTCCCCAGCAGTCCTGACCACATGCTCGCCATGGTGGAGGAAGCCCGTCGCCTCCA GGGATCTGGCCCTGAACCCCTCTGTGTCCACTGCAGTGCGGGTTGTGGGCGAACAGGC GTCCTGTGCACCGTGGATTATGTGAGGCAGCTGCTCCTGACCCAGATGATCCCACCTG ACTTCAGTCTCTTTGATGTGGTCCTTAAGATGAGGAAGCAGCGGCCTGCGGCCGTGCA GACAGAGGAGCAGTACAGGTTCCTGTACCACACGGTGGCTCAGATGTTCTGCTCCACA CTCCAGAATGCCAGCCCCCACTACCAGAACATCAAAGAGAATTGTGCCCCACTCTACG ACGATGCCCTCTTCCTCCGGACTCCCCAGGCACTTCTCGCCATACCCCGCCCACCAGG AGGGGTCCTCAGGAGCATCTCTGTGCCCGGGTCCCCGGGCCACGCCATGGCTGACACC TACGCGGAGGAGCAGAAGCGCGGGGCTCCAGCGGGCGCCGGGAGTGGGACGCAGACGG GGACGGGGACGGGGGCGCGCAGCGCGGAGGAGGCGCCGCTCTACAGCAAGGTGACGCC GCGCGCCCAGCGACCCGGGGCGCACGCGGAGGACGCGAGGGGGACGCTGCCTGGCCGC GTTCCTGCTGACCAAAGTCCTGCCGGATCTGGCGCCTACGAGGACGTGGCGGGTGGAG CTCAGACCGGTGGGCTAGGTTTCAACCTGCGCATTGGGAGGCCGAAGGGTCCCCGGGA CCCGCCTGCTGAGTGGAGCCGGGTGTCTCTGGACCTC-'GACTGACACTGGGCCTGCCC

CGGTCCCTGTATGCACTGCCACAGTGCCCTGGGCCCCATGTCCACCCCT

ORF Start: ATG at 44 ORF Stop: TGA at 1430

SEQ ID NO: 70 462 aa MW at 50812.1kD jNOVlla, MSRS DSARSF ERLEARGGRΞGAVLAGEFSDIQACSAAWKADGVCSTVAGSRPENVR KNRYKDVLPYDOTRVILSL OEEGHSDYING FIRGλTDGS AYIATOGPLPHT DFW CG169754-01 RLWEFGVKVILMACRΞIENGRKRCERYWAQEQEPLQTGLFCITLIKEKWLNEDIMLR

Protein TLKVTFQKESRSVYQLQYMSWPDRGVPSSPDH AMVEEARRLQGSGPEPLCVHCSAG CGRTGVLC VDY /RQLLLTQMIPPDFS FDVVliKMRKQRPAAVQTEEQYRFLYHTVAQ

Sequence MFC STLQNAS PHYQNIKENCAPLYDDALFLRTPQALLAI PRPPGGVLRS ISVPGS PGH AMADTYAEEQKRGAPAGAGSGTQTGTGTGARSAΞΞAPLYSKVTPRAQRPGAHAEDARG TLPGRVPADQSPAGSGAYEDVAGGAQTGGLGFNLRIGRPKGPRDPPAEWTRVSLDL

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

Table 11B. Protein Sequence Properties NO Vila

SignalP analysis: No Known Signal Sequence Predicted

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

N-region: length 9; pos.chg 2 ; neg.chg 1 H-region: length 3; peak value -9.00 PSG score: -13.40

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -₂.1): -4.37 possible cleavage site: between 35 and 36

>» Seems to have no N-terminal signal peptide

Tentative number of TMS(s) for the threshold 0.5: 0 number of TMS(s) .. fixed PERIPHERAL Likelihood = 2.38 (at 115) ALOM score: .38 (number of TMSs: 0)

MITDISC: discrimination of mitochondrial targeting seq R content: 2 Hyd Moment (75) : 5.69 Hyd Moment (95) : 12.32 G content: 0 D/E content: 2 S/T content: 4 Score: -3.34

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 13 SRS|LD

NϋCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite: none content of basic residues: 11.5% NLS Score: -0.47

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

ER Membrane Retention Signals :

XXRR-like motif in the N-terminus: SRSL

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

A search of the NOVl la protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 11C.

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

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

Example 12.

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

Table 12A. NOV12 Sequence Analysis

SEQ ID NO: 71 2264 bp j

NOV12a, TTGACTGTATCGCCGGAATTCAGGCAGCGCCAGGCGGCAGGTCGGAGCCGCCGCAGC

TCCCCGAGTACAGCTGCAGCTACATGGTGTCGCGGCCGGTCTACAGCGAGCTCGCTTT CG170739-01 CCAGCAACAGCACGAGCGGCGCCTGCAGGAGCGCAAGACGCTGCGGGAGAGCCTGGCC DNA Sequence AAGTGCTGCAGTTGTTCAAGAAAGAGAGCCTTTGGTGTGCTAAAGACTCTAGTGCCCA TCTTGGAGTGGCTCCCCAAATACCGAGTCAAGGAATGGCTGCTTAGTGACGTCATTTC GGGAGTTAGTACTGGGCTAGTGGCCACGCTGCAAGGACCTTTTCCAGTGGTGAGTTTA ATGGTGGGATCTGTTGTTCTGAGCATGGCCCCCGACGAACACTTTCTCGTATCCAGCA GCAATGGAACTGTATTAAATACTACTATGATAGACACTGCAGCTAGAGATACAGCCAG AGTCCTGATTGCCAGTGCCCTGACTCTGCTGGTTGGAATTATACAGTTGATATTTGGT GGCTTGCAGATTGGATTCATAGTGAGGCACTTGGCAGATCCTTTGGTTGGTGGCTTCA CAACAGCTGCTGCCTTCCAAGTGCTGGTCTCACAGCTAAAGATTGTCCTCAATGTTTC AACCAAAAACTACAATGGAGTTCTCTCTATTATCTATACGCTGGTTGAGATTTTTCAA AATATTGGTGATACCAATCTTGCTGATTTCACTGCTGGATTGCTCACCATTGTCGTCT GTATGGCAGTTAAGGAATTAAATGATCGGTTTAGACACAAAATCCCAGTCCCTATTCC TATAGAAGTAATTGTGACGATAATTGCTACTGCCATTTCATATGGAGCCAACCTGGAA AAAAATTACAATGCTGGCATTGTTAAATCCATCCCAAGGGGGTTTTTGCCTCCTGAAC TTCCACCTGTGAGCTTGTTCTCGGAGATGCTGGCTGCATCATTTTCCATCGCTGTGGT GGCTTATGCTATTGCAGTGTCAGTAGGAAAAGTATATGCCACCAAGTATGATTACACC ATCGATGGGAACCAGGAATTCATTGCCTTTGGGATCAGCAACATCTTCTCAGGATTCT TCTCTTGTTTTGTGGCCACCACTGCTCTTTCCCGCACGGCCGTCCAGGAGAGCACTGG AGGAAAGACACAGGTTGCTGGCATCATCTCTGCTGCGATTGTGATGATCGCCATTCTT GCCCTGGGGAAGCTTCTGGAACCCTTGCAGAAGTCGGTCTTGGCAGCTGTTGTAATTG CCAACCTGAAAGGGATGTTTATGCAGCTGTGTGACATTCCTCGTCTGTGGAGACAGAA TAAGATTGATGCTGTTATCTGGGTGTTTACGTGTATAGTGTCCATCATTCTGGGGCTG GATCTCGGTTTACTAGCTGGCCTTATATTTGGACTGTTGACTGTGGTCCTGAGAGTTC AGTTTCCTTCTTGGAATGGCCTTGGAAGCATCCCTAGCACAGATATCTACAAAAGTAC CAAGAATTACAAAAACATTGAAGAACCTCAAGGAGTGAAGATTCTTAGATTTTCCAGT CCTATTTTCTATGGCAATGTCGATGGTTTTAAAAAATGTATCAAGTCCACAGTTGGAT TTGATGCCATTAGAGTATATAATAAGAGGCTGAAAGCGCTGAGGAAAATACAGAAACT AATAAAAAGTGGACAATTAAGAGCAACGAAGAATGGCATCATAAGTGATGCTGTTTCA ACAAATAATGCTTTTGAGCCCGATGAGGATATTGAAGATCTGGAGGAACTTGATATCC CAACCAAGGAAATAGAGATTCAAGTGGATTGGAACTCTGAGCTTCCAGTCAAAGTGAA CGTTCCCAAAGTGCCAATCCATAGCCTTGTGCTTGACTGTGGAGCTATATCTTTCCTG GACGTTGTTGGAGTGAGATCACTGCGGGTGATTGTCAAAGAATTCCAAAGAATTGATG TGAATGTGTATTTTGCATCACTTCAAGATTATGTGATAGAAAAGCTGGAGCAATGCGG GTTCTTTGACGACAACATTAGAAAGGACACATTCTTTTTGACGGTCCATGATGCTATA CTCTATCTACAGAACCAAGTGAAATCTCAAGAGGGTCAAGGTTCCATTTTAGAAACGA TCACTCTCATTCAGGATTGTAAAGATACCCTTGAATTAGTAGAAACAGAGCTGACGGA AGAAGAACTTGATGTCCAGGATGAGGCTATGCGTACACTTGCATCCTGACTGC GCCA AG

ORF Start: ATG at 22 ORF Stop: TGA at 2251

Further analysis of the NOVl 2a protein yielded the following properties shown in Table 12B.

Table 12B. Protein Sequence Properties NOV12a

SignalP analysis: No Known Signal Sequence Predicted

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

N-region: length 9; pos.chg 1; neg.chg 1 H-region: length 5; peak value -5.32 PSG score: -9.72

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -8.79 possible cleavage site: between 61 and 62

»> Seems to have no N-terminal signal peptide

Tentative number of TMS ( s) Eor the threshold 0 .5 : 11

INTEGRAL Likelihood = 0 47 Transmembrane 84 - 100

INTEGRAL Likelihood = -2 18 Transmembrane 103 - 119

INTEGRAL Likelihood = -8 86 Transmembrane 149 - 165

INTEGRAL Likelihood = -4 73 Transmembrane 232 - 48

INTEGRAL Likelihood = -3 93 Transmembrane 261 - 277

INTEGRAL Likelihood = -5 89 Transmembrane 312 - 328

INTEGRAL Likelihood = -1 28 Transmembrane 349 - 365

INTEGRAL Likelihood =-11 30 Transmembrane 385 - 401

INTEGRAL Likelihood = -1 22 Transmembrane 412 - 428

INTEGRAL Likelihood = -7 64 Transmembrane 450 - 466

INTEGRAL Likelihood = -1 86 Transmembrane 620 - 636

PERIPHERAL Likelihood = 0 63 (at 189 )

ALOM score -11.30 (number of TMSs : 11 )

MTOP : Prediction of membrane topology (Hartmann et al . ) Center position for calculation: 91 Charge difference: -1.5 C(-1.5) - N( 0.0) N >= C: N-terminal side will be inside

>» membrane topology: type 3a

MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75) : 5.67 Hyd Moment (95) : 8.17 G content: 2 D/E content: 2 S/T content: 1 Score: -6.60

Gavel: prediction of cleavage sites for mitochondrial preseq

A search of the NOVl 2a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 12C.

In a BLAST search of public sequence datbases, the NOV12a protein was found o have homology to the proteins shown in the BLASTP data in Table 12D.

U 03/03401

PFam analysis predicts that the NOVl 2a protein contains the domains shown in the Table 12E.

Example 13. D-GLUCURONYL C5 EPEVIERASE.

The NOVl 3 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 13 A.

Table 13A. NOV13 Sequence Analysis

SEQ ID NO: 73 2274 bp

NOV13a, GGCGCTGCTCAGGAATTTGACAAGAAACTGAAGTTTTGATTCAGATATATTTTGAATT

GAAACCAGAGATGTTCTAGAGTTTAGATTCTTTCATTTGATTAAGGTATGGTCTGAAT CG170764-01 A GCGTTGCTTGGCAGCTCGGGTCAACTATAAGACTTTGATTATTATCTGCGCACTCT DNA Sequence TCACTTTGGTCACAGTACTTTTGTGGAATAAGTGTTCCAGTGACAAAGCAATCCAGTT TCCACGGCGTTCGAGTAGTGGCTTCAGAGTGGATGGGTTTGAAAAAAGAGCAGCAGCA TCTGAGAGTAACAACTATATGAACCACGTGGCCAAACAACAGTCTGAGGAAGCATTCC CTCAGGAACAGCAGAAAGCACCCCCTGTTGTTGGGGGCTTCAATAGCAATGTGGGAAG TAAGGTGTTAGGGCTCAAATATGAAGAAATTGACTGTCTCATAAATGATGAACACACA ATTAAAGGGAGACGAGAGGGGAACGAAGTCTTTCTTCCATTCACTTGGGTTGAGAAAT ATTTTGATGTTTATGGAAAGGTGGTTCAGTATGATGGCTATGATCGGTTTGAATTCTC TCATAGCTATTCCAAAGTCTATGCACAGAGAGCCCCCTATCACCCCGATGGTGTGTTT ATGTCTTTTGAAGGCTACAATGTGGAAGTCCGAGACAGAGTCAAGTGCATAAGTGGGG TTGAAGGTGTGCCATTATCTACACAATGGGGACCTCAAGGCTATTTCTATCCAATCCA GATTGCACAGTATGGATTAAGTCATTACAGCAAGAATCTAACTGAGAAACCTCCTCAC ATAGAGGTATATGAAACAGCAGAAGACAGAGACAAAAACAAGCCTAATGACTGGACTG TGCCAAAGGGCTGCTTTATGGCGAATGTGGCTGATAAGTCTAGATTCACCAATGTCAA ACAGTTTATTGCACCAGAAACCAGTGAAGGTGTATCCTTGCAACTGGGAAACACAAAA GATTTTATTATTTCATTTGACCTCAAGTTCTTGACAAATGGAAGTGTGTCCGTGGTTC TAGAGACCACAGAAAAGAATCAGCTCTTCACTATACATTATGTCTCAAATGCTCAGCT AATTGCTTTTAAAGAAAGAGATATATACTATGGCATTGGGCCCAGAACTTCATGGAGC ACAGTTACCAGGGACCTGGTCACTGACCTCAGGAAAGGAGTGGGTCTTTCAAACACAA AAGCTGTCAAGCCAACCAAAATAATGCCCAAGAAGGTGGTTAGGTTGATTGCAAAAGG TAAGGGATTCCTCGACAACATTACCATCTCTACCACAGCCCACATGGCTGCATTTTTT GCTGCTAGTGATTGGCTAGTAAGGAΑCCAGGATGAGAAAGGTGGCTGGCCAATTATGG TGACCCGTAAGTTAGGGGAAGGGTTCAAGTCTTTAGAGCCAGGATGGTATTCTGCCAT GGCCCAAGGGCAAGCCATTTCTACATTAGTCAGGGCCTATCTGTTAACAAAAGACCAT ATATTCCTCAATTCAGCTTTAAGGGCAACAGCCCCTTATAAGTTTCTATCTGAGCAGC ATGGAGTTAAAGCTGTGTTTATGAATAAACATGACTGGTATGAAGAATATCCAACCAC ACCTAGCTCTTTTGTTTTAAATGGCTTTATGTATTCTTTAATTGGGCTGTATGACTTA AAAGAAACTGCAGGGGAAAAACTCGGAAAAGAAGCAAGGTCCTTGTATGAGCGTGGCA TGGAATCTCTTAAAGCCATGCTGCCCTTGTATGACACTGGCTCAGGAACCATCTATGA CCTCCGTCACTTCATGCTTGGCATCGCTCCTAACCTGGCTCGCTGGGACTATCATACC ACCCACATCAATCAGTTGCAGCTACTCAGTACCATTGATGAGTCCCCAATCTTCAAAG AATTTGTCAAGAGGTGGAAAAGCTACCTTAAAGGCAGCAGGGCAAAGCACAACTAGAG CTCACAACCAAAACTGCCCTTCAGCCTCTGCTGTACACAGAAACTACAGGCTCTGTCT

CAGGAGAGCATAGGCACATTTTAAAAGGTTATGTACTAGGTTTTTGTGGATTCTATCA

AAGTGATAAGTGATCCTTAAAACCAGCCTTCTAAAATAATTGCATTCCATGGGTTGGG TATTTAGAAATGTAGGTGGCATTTAGAACACAATGTTTAATCAATGGGCTGAACAAAGi

ATGCTTCACTTTGCCTTGCCCATCACCCTATACAGTTTCGCAGATAGTCTAGTCACTC

TATGTGAGAAAG

ORF Start: ATG at 117 ORF Stop: TAG at 1968

SEQ ID NO: 74 617 aa MW at 70114.4kD

NOV13a, MRCLAARVlvr_KTLIIICALFTLVTVLLW KCSSDKAIQFPRRSSSGFRVDGFE-α_ AA CG170764-01 SES KTYl_ _VAKQQSEEAFPQEQQKAPPVVGGFNSlWGSKVLGLKYEEIDC IlvroEHT IKGRPJ3Gl_VFLPFTTι<ΛtEKY^DVYG ΛtQYDGYDRFEFSHSYSIWYAQRAPYHPDGVF Protein MSFEGYKT ΕVRDRVKCISGVEGVPLSTQWGPQGYFYPIQIAQYGLSHYSKNLTEKPPH Sequence IEΛ/YETAEDRDKITOP D TVPKGCFMAIW^'ADKSRFTI KQFIAPETSEGVSLQ GNTK DFIISFD KFLTNGSVSWLETTEKNQLFTIHYVSNAQLIAFKERDIYYGIGPRTSWS TVTRDLVTD RKGVG SNTKAVKPTKIMPKKVVRLIAKGKGF DNITISTTAHMAAFF AASD LVR QDEKGGWPI VTRK GEGFKSLEPG YSAMAQGQAISTLVRAY LTKDH IFLNSALRATAPYKFESEQHGVKAVF_N7KHDWYEEYPTTPSSFVLNGFMYSLIGLYDL KETAGEK GKEARSLYERGMESLKAMLPLYDTGSGTIYD RHFMLGIAPN ARWDYHT THINQLQLLSTIDESPIF EFVKRWKSYLKGSRAKHN

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

Table 13B. Protein Sequence Properties NO 13a

SignalP analysis: Cleavage site between residues 34 and 35

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

N-region: length 11; pos.chg 3; neg.chg 0 H-region: length 18; peak value 11.63 PSG score: 7.₂3

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -3.90 possible cleavage site: between 33 and 34

>» Seems to have no N-terminal signal peptide

Tentative number of TMS(s) for the threshold 0.5: 1 Number of TMS(s) for threshold 0.5: 1 INTEGRAL Likelihood = -9.61 Transmembrane 12 - 28 PERIPHERAL Likelihood = 3.45 (at 293) ALOM score: -9.61 (number of TMSs: 1)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 19 Charge difference: -1.0 C( 3.0) - N( 4.0) N >= C: N-terminal side will be inside

>» membrane topology: type 2 (cytoplasmic tail 1 to 12)

MITDISC: discrimination of mitochondrial targeting seq R content: 2 Hyd Moment (75) : 13.46 Hyd Moment (95) : 9.77 G content: 0 D/E content: S/T content: Score: -0.22

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 58 FRV|DG

NUCDISC: discrimination of nuclear localization signals pat4: none pat7 : none bipartite: none content of basic residues : 12.5% NLS Score: -0.47

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

ER Membrane Retention Signals:

XXRR-like motif in the N-terminus: RCLA

KKXX-like motif in the C-terminus: RAKH

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

PTS2 : 2nd peroxisomal targeting signal: none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail:9

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):

39.1 %: cytoplasmic

34.8 %: mitochondrial

13.0 %: Golgi

4.3 %: extracellular, including cell wall

4.3 %: vacuolar

4.3 %: endoplasmic reticulum » prediction for CG170764-01 is cyt (k=23)

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 13C.

In a BLAST search of public sequence datbases, the NOV13a protein was found to have homology to the proteins shown in the BLASTP data in Table 13D.

PFam analysis predicts that the NOV13a protein contains the domains shown in the Table 13E.

Table 13E. Domain Analysis of NOV13a

Identities/

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

No Significant Matches Found

Example 14. Cyclin G-associated kinase

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: 75 3889 bp

NOV14a, A GTCGCTGCTGCAGTCTGCGCTCGACTTCTTGGCGGGTCCAGGCTCCCTGGGCGGTG CG170882-01 CTTCCGGCCGCGACCAGAGTGACTTCGTGGGGCAGACGGTGGAACTGGGCGAGCTGCG GCTGCGGGTGCGGCGGGTCCTGGCCGAAGGAGGGTTTGCATTTGTGTATGAAGCTCAA DNA Sequence GATGTGGGGAGTGGCAGAGAGTATGCATTAAAGAGGCTATTATCCAATGAAGAGGAAA AGAACAGAGCCATCATTCAAGAAGTTTGCTTCATGAAAAAGCTTTCCGGCCACCCGAA CATTGTCCAGTTTTGTTCTGCAGCGTCTATAGGAAAAGAGGAGTCAGACACGGGGCAG GCTGAGTTCCTCTTGCTCACAGAGCTCTGTAAAGGGCAGCTGGTGGAATTTTTGAAGA AAATGGAATCTCGAGGCCCCCTTTCGTGCGACACGGTTCTGAAGATCTTCTACCAGAC GTGCCGCGCCGTGCAGCACATGCACCGGCAGAAGCCGCCCATCATCCACAGGGACCTC AAGGTTGAGAACTTGTTGCTTAGTAACCAAGGGACCATTAAGCTGTGTGACTTTGGCA GTGCCACGACCATCTCGCACTACCCTGACTACAGCTGGAGCGCCCAGAGGCGAGCCCT GGTGGAGGAAGAGATCACGAGGAATACAACACCAATGTATAGAACACCAGAAATCATA GACTTGTATTCCAACTTCCCGATCGGCGAGAAGCAGGATATCTGGGCCCTGGGCTGCA TCTTGTACCTGCTGTGCTTCCGGCAGCACCCTTTTGAGGATGGAGCGAAACTTCGAAT AGTCAATGGGAAGTACTCGATCCCCCCGCACGACACGCAGTACACGGTCTTCCACAGC CTCATCCGCGCCATGCTGCAGGTGAACCCGGAGGAGCGGCTGTCCATCGCCGAGGTGG TGCACCAGCTGCAGGAGATCGCGGCCGCCCGCAACGTGAACCCCAAGTCTCCCATCAC AGAGCTCCTGGAGCAGAATGGAGGCTACGGGAGCGCCACACTGTCCCGAGGGCCACCC CCTCCCGTGGGCCCCGCTGGCAGTGGCTACAGTGGAGGCCTGGCGCTGGCGGAGTACG ACCAGCCGTATGGCGGCTTCCTGGACATTCTGCGGGGTGGGACAGAGCGGCTCTTCAC CAACCTCAAGGACACCTCCTCCAAGGTCATCCAGTCCGTCGCTAATTATGCAAAGGGT GACCTGGACATATCTTACATCACATCCAGAATTGCAGTGATGTCATTCCCAGCAGAAG GTGTGGAGTCAGCGCTCAAAAACAACATCGAAGATGTGCGGTTGTTCCTGGACTCCAA GCACCCAGGGCACTATGCCGTCTACAACCTGTCCCCGAGGACCTACCGGCCCTCCAGG TTCCACAACCGGGTCTCCGAGTGTGGCTGGGCAGCACGGCGGGCCCCACACCTGCACA CCCTGTACAACATCTGCAGGAACATGCACGCCTGGCTGCGGCAGGACCACAAGAACGT CTGCGTCGTGCACTGCATGGACGGGAGAGCCGCGTCTGCTGTGGCCGTCTGCTCCTTC CTGTGCTTCTGCCGTCTCTTCAGCACCGCGGAGGCCGCCGTGTACATGTTCAGCATGA AGCGCTGCCCACCAGGCATCTGGCCATCCCACAAAAGGTACATCGAGTACATGTGTGA CATGGTGGCGGAGGAGCCCATCACACCCCACAGCAAGCCCATCCTGGTGAGGGCCGTG GTCATGACACCCGTGCCGCTGTTCAGCAAGCAGAGGAGCGGCTGCAGGCCCTTCTGCG AGGTCTACGTGGGGGACGAGCGTGTGGCCAGCACCTCCCAGGAGTACGACAAGATGCG GGACTTTAAGATTGAAGATGGCAAAGCGGTGATTCCCCTGGGCGTCACGGTGCAAGGA GACGTGCTCATCGTCATCTATCACGCCCGGTCCACTCTGGGCGGCCGGCTGCAGGCCA AGATGGCATCCATGAAGATGTTCCAGATTCAGTTCCACACGGGGTTTGTGCCTCGGAA CGCCACCACTGTGAAATTTGCCAAGTATGACCTGGACGCGTGTGACATTCAAGAAAAA TACCCGGATTTATTTCAAGTGAACCTGGAAGTGGAGGTGGAGCCCAGGGACAGGCCGA GCCGGGAAGCCCCACCATGGGAGAACTCGAGCATGAGGGGGCTGAACCCCAAAATCCT GTTTTCCAGCCGGGAGGAGCAGCAAGACATTCTGCCTAAGTTTGAAGAGAAGGAGGCA GAGACTGGTGCAGAAAATGCCTCTTCCAAGGAGAGCGAGTCTGCCCTGATGGAGGACA GAGACGAGAGTGAGGTGTCAGATGAAGGGGGATCCCCGATCTCCAGCGAGGGCCAGGA ACCCAGGGCCGACCCAGAGCCCCCCGGCCTGGCAGCAGGGCTGGTGCAGCAGGACTTG GTTTTTGAGGTGGAGACACCGGCTGTGCTGCCAGAGCCTGTGCCACAGGAAGACGGGG TCGACCTCCTGGGCCTGCACTCCGAGGTGGGCGCAGGGCCAGCTGTACCCCCGCAGGC CTGCAAGGCCCCCTCCAGCAACACCGACCTGCTCAGCTGCCTCCTTGGGCCCCCTGAG GCCGCCTCCCAGGGGCCCCCGGAGGATCTGCTCAGCGAGGACCCGCTGCTCCTGGCAA GCCCGGCCCCTCCCCTGAGCGO?GCAGAGCACCCCAAGAGGAGGGCCCCCTGCCGCTGC TGACCCCΪTTGGCCCGCTTCTGCCGTCTTCAGGCAACAACTCCCAGCCCTGCTCCAAT CCTGATCTCTTCGGCGAATTTCTCAATTCGGACTCTGTGACCGTCCCACCATCCTTCC CGTCTGCCCACAGCGCTCCGCCCCCATCCTGCAGCGCCGACTTCCTGCACCTGGGGGA TCTGCCAGGAGAGCCCAGCAAGATGACAGCCTCGTCCAGCAACCCAGACCTGCTGGGA GGATGGGCTGCCTGGACCGAGACTGCAGCGTCGGCAGTGGCCCCCACGCCAGCCACAG AAGGCCCCCTCTTCTCTCCTGGAGGTCAGCCGGCCCCTTGTGGCTCTCAGGCCAGCTG GACCAAGTCTCAGAACCCGGACCCATTTGCTGACCTTGGCGACCTCAGCTCCGGCCTC CAAGGCTCACCAGCTGGATTTCCTCCTGGGGGCTTCATTCCCAAAACGGCCACCACGG CCAAAGGCAGCAGCTCCTGGCAGACAAGTCGGCCGCCAGCCCAGGGCGCCTCATGGCC CCCTCAGGCCAAGCCGCCCCCCAAAGCCTGCACACAGCCAAGGCCTAACTATGCCTCG AACTTCAGTGTGATCGGGGCGCGGGAGGAGCGGGGGGTCCGCGCACCCAGCTTTGCTC AAAAGCCAAAAGTCTCTGAGAACGACTTTGAAGATCTGTTGTCCAATCAAGGCTTCTC CTCCAGGTCTGACAAGAAAGGGCCAAAGACCATTGCAGAGATGAGGAAGCAGGACCTG GCTAAAGACACGGACCCACTCAAGCTGAAGCTCCTGGACTGGAT GAGGGCAAGGAGC GGAACATCCGGGCCCTGCTGTCCACGCTGCACACAGTGCTGTGGGACGGGGAGAGCCG CTGGACGCCCGTGGGCATGGCCGACCTGGTGGCTCCGGAGCAAGTGAAGAAGCACTAT CGCCGCGCGGTGCTGGCCGTGCACCCCGACAAGGCTGCGGGGCAGCCGTACGAGCAGC ACGCCAAGATGATCTTCATGGAGCTGAATGACGCCTGGTCGGAGTTTGAGAACCAGGG CTCCCGGCCCCTCTTCTGAGGCCGCAGTGGTGGTGGCTGCGCACACAGCTCCACAGGT

TGGGAGCCGTCGTGGGACCTGGGTCCCCACCGTGAGGACCCCGTGGGCGACAGCAGGT

GTG

ORF Start: ATG at 1 ORF Stop: TGA at 3787

SEQ ID NO: 76 1262 aa MW at 138077.5kD

NOV14a, MS LQSALDFLAGPGSLGGASGRDQSDFVGQTVELGELRLRVRRVLAEGGFAFVYEAQ DVGSGREYALKR LSNEEEKNRAIIQEVCFMKKLSGHPNIVQFCSAASIGKEESDTGQ CG170882-01 AEF L TELCKGQ VEFLKKMESRGPLSCDTVLKIFYQTCRAVQHMHRQKPPIIHRDL Protein KVEN LSNQGTIKLCDFGSATTISHYPDYSWSAQRIτ___JVEEEITRNTTPl(r_RTPΞII Sequence D YSNFPIGEKQDIWALGCILYLLCFRQHPFEDGAK RIV GKYSIPPHDTQYTVFHS IRAMLQVNPEER SIAEVVΗQLQEIAAAR VNPKSPITEL EQNGGYGSATLSRGPP PPVGPAGSGYSGG ALAEYDQPYGGF DILRGGTERLFTNLKDTSSKV'IQSVANYAKG DLDISYITSRIAVMSFPAEGVESALKNNIEDVRLF DSKHPGHYAVY LSPRTYRPSR FHNRVSECGWAARRAPHLHT Y ICRMHAW RQDHKNVCVVHCMDGRAASAVAVCSF LCFCRLFSTAEAA\T FS_N_lCPPGIWPSHKRYIEYMCDMVAEEPITPHSKPILVRAV VM PVPLFSKQRSGCRPFCFΛT-rVG ERVASTSQEYDKMRDFKIEDGKAVIPLGVTVQG DV IVIYHARSTLGGRLQAKi Sl_KMFQIQFHTGFVPRNATTVKFAKYDLDACDIQEK YPDLFQVNLEVEVEPRDRPSREAPPWENSSMRG NPKILFSSREEQQDI PKFEEKEA ETGAENASSKESESAMEDRDESEVSDΞGGSPISSEGQEPRADPEPPGLAAGVQQDL VFEΕTPAVLPEPVPQEDGVDLLGLHSEVGAGPAVPPQACKAPSSN DL SC LGPPE AASQGPPEDLLSEDPLLLASPAPPLSVQSTPRGGPPAAADPFGP LPSSGNNSQPCSN PDLFGEFL-ISDSVTVPPSFPSAHSAPPPSCSADF HLGDLPGEPSK TASSSNPDL G GWAAWTETAASAVAPTPATEGPLFSPGGQPAPCGSQASWTKSQNPDPFADLGD SSGL QGSPAGFPPGGFIPKTATTAKGSSSWQTSRPPAQGASWPPQAKPPP ACTQPRPNYAS NFSVIGAREERGVRAPSFAQKPKVSENDFEDLLSNQGFSSRSDKKGP TIAEMRKQD AKDTDPLKiKL DWIEGKERNIRALLST H VLWDGESR TPVGMADVAPEQVKKHY P^V AVHPDKAAGQPYEQHAK IFMELNDAWSEFENQGSRPLF

SEQ ID NO: 77 3759 bp

NOV14b, ATGTCGCTGCTGCAGTCTGCGCTCGACTTCTTGGCGGGTCCAGGCTCCCTGGGCGGTG CTTCCGGCCGCGACCAGAGTGACTTCGTGGGGCAGACGGTGGAACTGGGCGAGCTGCG CG170882-02 GCTGCGGGTGCGGCGGGTCCTGGCCGAAGGAGGGTTTGCAT TGTGTATGAAGCTCAA DNA Sequence GATGTGGGGAGTGGCAGAGAGTATGCATTAAAGGTTGAGAACTTGTTGCTTAGTAACC AAGGGACCATTAAGCTGTGTGACTTTGGCAGTGCCACGACCATCTCGCACTACCCTGA CTACAGCTGGAGCGCCCAGAGGCGAGCCCTGGTGGAGGAAGAGATCACGAGGAATACA ACACCAATGTATAGAACACCAGAAATCATAGACTTGTATTCCAACTTCCCGATCGGCG AGAAGCAGGATATCTGGGCCCTGGGCTGCATCTTGTACCTGCTGTGCTTCCGGCAGCA CCCTTTTGAGGATGGAGCGAAACTTCGAATAGTCAATGGGAAGTACTCGATCCCCCCG CACGACACGCAGTACACGGTCTTCCACAGCCTCATCCGCGCCATGCTGCAGGTGAACC CGGAGGAGCGGCTGTCCATCGCCGAGGTGGTGCACCAGCTGCAGGAGATCGCGGCCGC CCGCAACGTGAACCCCAAGTCTCCCATCACAGAGCTCCTGGAGCAGAATGGAGGCTAC GGGAGCGCCACACTGTCCCGAGGGCCACCCCCTCCCGTGGGCCCCGCTGGCAGTGGCT ACAGTGGAGGCCTGGCGCTGGCGGAGTACGACCAGCCGTATGGCGGCTTCCTGGACAT TCTGCGGGGTGGGACAGAGCGGCTCTTCACCAACCTCAAGGACACCTCCTCCAAGGTC ATCCAGTCCGTCGCTAATTATGCAAAGGGTGACCTGGACATATCTTACATCACATCCA GAATTGCAGTGATGTCATTCCCAGCAGAAGGTGTGGAGTCAGCGCTCAAAAACAACAT CGAAGATGTGCGGTTGTTCCTGGACTCCAAGCACCCAGGGCACTATGCCGTCTACAAC CTGTCCCCGAGGACCTACCGGCCCTCCAGGTTCCACAACCGGGTCTCCGAGTGTGGCT GGGCAGCACGGCGGGCCCCACACCTGCACACCCTGTACAACATCTGCAGGAACATGCA CGCCTGGCTGCGGCAGGACCACAAGAACGTCTGCGTCGTGCACTGCATGGACGGGAGA GCCGCGTCTGCTGTGGCCGTCTGCTCCTTCCTGTGCTTCTGCCGTCTCTTCAGCACCG CGGAGGCCGCCGTGTACATGTTCAGCATGAAGCGCTGCCCACCAGGCATCTGGCCATC CCACAAAAGGTACATCGAGTACATGTGTGACATGGTGGCGGAGGAGCCCATCACACCC CACAGCAAGCCCATCCTGGTGAGGGCCGTGGTCATGACACCCGTGCCGCTGTTCAGCA AGCAGAGGAGCGGCTGCAGGCCCTTCTGCGAGGTCTACGTGGGGGACGAGCGTGTGGC CAGCACCTCCCAGGAGTACGACAAGATGCGGGACTTTAAGATTGAAGATGGCAAAGCG GTGATTCCCCTGGGCGTCACGGTGCAAGGAGACGTGCTCATCGTCATCTATCACGCCC

NOV14c, ATGTCGCTGCTGCAGTCTGCGCTCGACTTCTTGGCGGGTCCAGGCTCCCTGGGCGGTG

CTTCCGGCCGCGACCAGAGTGACTTCGTGGGGCAGACGGTGGAACTGGGCGAGCTGCG CG170882-03 GGTGCGGGTGCGGCGGGTCCTGGCCGAAGGAGGGTTTGCATTTGTGTATGAAGCTCAA DNA Sequence GATGTGGGGAGTGGCAGAGAGTATGCATTAAAGGGCAGCTGGTGGAATTTTTGAAGAA

AATGGAATCTCGAGGCCCCCTTTCGTGCGACACGGTTCTGAAGATCTTCTACCAGACG TGCCGCGCCGTGCAGCACATGCACCGGCAGAAGCCGCCCATCATCCACAGGGACTTCA AGGTTGAGAACTTGTTGCTTAGTAACCAAGGGACCATTAAGCTGTGTGACTTTGGCAG TGCCACGACCATCTCGCACTACCCTGACTACAGCTGGAGCGCCCAGAGGCGAGCCCTG GTGGAGGAAGAGATCACGAGGAATACAACACCAATGTATAGAACACCAGAAATCATAG ACTTGTATTCCAACTTCCCGATCGGCGAGAAGCAGGATATCTGGGCCCTGGGCTGCAT CTTGTACCTGCTGTGCTTCCGGCAGCACCCTTTTGAGGATGGAGCGAAACTTCGAATA GTCAATGGGAAGTACTCGATCCCCCCGCACGACACGCAGTACACGGTCTTCCACAGCC TCATCCGCGCCATGCTGCAGGTGAACCCGGAGGAGCGGCTGTCCATCGCCGAGGTGGT GCACCAGCTGCAGGAGATCGCGGCCGCCCGCAACGTGAACCCCAAGTCTCCCATCACA GAGCTCCTGGAGCAGAATGGAGGCTACGGGAGCGCCACACTGTCCCGAGGGCCACCCC CTCCCGTGGGCCCCGCTGGCAGTGGCTACAGTGGAGGCCTGGCGCTGGCGGAGTACGA CCAGCCGTATGGCGGCTTCCTGGACATTCTGCGGGGTGGGACAGAGCGGCTCTTCACC AACCTCAAGGACACCTCCTCCAAGGTCATCCAGTCCGTCGCTAATTATGCAAAGGGTG ACCTGGACATATCTTACATCACATCCAGAATTGCAGTGATGTCATTCCCAGCAGAAGG TGTGGAGTCAGCGCTCAAAAACAACATCGAAGATGTGCGGTTGTTCCTGGACTCCAAG CACCCAGGGCACTATGCCGTCTACAACCTGTCCCCGAGGACCTACCGGCCCTCCAGGT TCCACAACCGGGTCTCCGAGTGTGGCTGGGCAGCACGGCGGGCCCCACACCTGCACAC CCTGTACAACATCTGCAGGAACATGCACGCCTGGCTGCGGCAGGACCACAAGAACGTC TGCGTCGTGCACTGCATGGACGGGAGAGCCGCGTCTGCTGTGGCCGTCTGCTCCTTCC TGTGCTTCTGCCGTCTCTTCAGCACCGCGGAGGCCGCCGTGTACATGTTCAGCATGAA GCGCTGCCCACCAGGCATCTGGCCATCCCACAAAAGGTACATCGAGTACATGTGTGAC ATGGTGGCGGAGGAGCCCATCACACCCCACAGCAAGCCCATCCTGGTGAGGGCCGTGG TCATGACACCCGTGCCGCTGTTCAGCAAGCAGAGGAGCGGCTGCAGGCCCTTCTGCGA GGTCTACGTGGGGGACGAGCGTGTGGCCAGCACCTCCCAGGAGTACGACAAGATGCGG GACTTTAAGATTGAAGATGGCAAAGCGGTGATTCCCCTGGGCGTCACGGTGCAAGGAG ACGTGCTCATCGTCATCTATCACGCCCGGTCCACTCTGGGCGGCCGGCTGCAGGCCAA GATGGCATCCATGAAGATGTTCCAGATTCAGTTCCACACGGGGTTTGTGCCTCGGAAC GCCACCACTGTGAAATTTGCCAAGTATGACCTGGACGCGTGTGACATTCAAGAAAAAT ACCCGGATTTATTTCAAGTGAACCTGGAAGTGGAGGTGGAGCCCAGGGACAGGCCGAG CCGGGAAGCCCCACCATGGGAGAACTCGAGCATGAGGGGGCTGAACCCCAAAATCCTG TTTTCCAGCCGGGAGGAGCAGCAAGACATTCTGTCTAAGTTTGGGAAGCCGGAGCTTC CCCGGCAGCCTGGCTCCACGGCTCAGTATGATGCTGGGGCAGGGTCCCCGGAAGCCGA ACCCACAGACTCTGACTCACCGCCAAGCAGCAGCGCGGACGCCAGTCGCTTCCTGCAC ACGCTGGACTGGCAGGAAGAGAAGGAGGCAGAGACTGGTGCAGAAAATGCCTCTTCCA AGGAGAGCGAGTCTGCCCTGATGGAGGACAGAGACGAGAGTGAGGTGTCAGATGAAGG GGGATCCCCGATCTCCAGCGAGGGCCAGGAACCCAGGGCCGACCCAGAGCCCCCCGGC CTGGCAGCAGGGCTGGTGCAGCAGGACTTGGTTTTTGAGGTGGAGACACCGGCTGTGC TGCCAGAGCCTGTGCCACAGGAAGACGGGGTCGACCTCCTGGGCCTGCACTCCGAGGT GGGCGCAGGGCCAGCTGTACCCCCGCAGGCCTGCAAGGCCCCCTCCAGCAACACCGAC CTGCTCAGCTGCCTCCTTGGGCCCCCTGAGGCCGCCTCCCAGGGGCCCCCGGAGGATC TGCTCAGCGAGGACCCGCTGCTCCTGGCAAGCCCGGCCCCTCCCCTGAGCGTGCAGAG CACCCCAAGAGGAGGGCCCCCTGCCGCTGCTGACCCCTTTGGCCCGCTTCTGCCGTCT TCAGGCAACAACTCCCAGCCCTGCTCCAATCCTGATCTCTTCGGCGAATTTC?CAATT CGGACTCTGTGACCGTCCCACCATCCTTCCCGTCTGCCCACAGCGCTCCGCCCCCATC CTGCAGCGCCGACTTCCTGCACCTGGGGGATCTGCCAGGAGAGCCCAGCAAGATGACA GCCTCGTCCAGCAACCCAGACCTGCTGGGAGGATGGGCTGCCTGGACCGAGACTGCAG CGTCGGCAGTGGCCCCCACGCCAGCCACAGAAGGCCCCCTCTTCTCTCCTGGAGGTCA GCCGGCCCCTTGTGGCTCTCAGGCCAGCTGGACCAAGTCTCAGAACCCGGACCCATTT GCTGACCTTGGCGACCTCAGCTCCGGCCTCCAAGGCTCACCAGCTGGATT CCTCCTG GGGGCTTCATTCCCAAAACGGCCACCACGGCCAAAGGCAGCAGCTCCTGGCAGACAAG TCGGCCGCCAGCCCAGGGCGCCTCATGGCCCCCTCAGGCCAAGCCGCCCCCCAAAGCC TGCACACAGCCAAGGCCTAACTATGCCTCGAACTTCAGTGTGATCGGGGCGCGGGAGG AGCGGGGGGTCCGCGCACCCAGCTTTGCTCAAAAGCCAAAAGTCTCTGAGAACGACTT TGAAGATCTGTTGTCCAATCAAGGCTTCTCCTCCAGGTCTGACAAGAAAGGGCCAAAG ACCATTGCAGAGATGAGGAAGCAGGACCTGGCTAAAGACACGGACCCACTCAAGCTGA AGCTCCTGGACTGGATTGAGGGCAAGGAGCGGAACATCCGGGCCCTGCTGTCCACGCT GCACACAGTGCTGTGGGACGGGGAGAGCCGCTGGACGCCCGTGGGCATGGCCGACCTG GTGGCTCCGGAGCAAGTGAAGAAGCACTATCGCCGCGCGGTGCTGGCCGTGCACCCCG ACAAGGCTGCGGGGCAGCCGTACGAGCAGCACGCCAAGATGATCTTCATGGAGCTGAA TGACGCCTGGTCGGAGTTTGAGAACCAGGGCTCCCGGCCCCTCTTCTGAGGCCGCAGT GGTGGTGGCTGCGCACACAGCTCCACAGGTTGGGAGCCGTCGTGGGACCTGGGTCCCC

ACCGTGAGGACCCCGTGGGCGACAGCAGGTGTGGCCAGGGTGGGGCTCCGAGCCCCGG

GTCACCGCCCGCC

ORF Start: ATG at 234 ORF Stop: TGA at 3759

SEQ ID NO: 80 1175 aa MW at 12837 l.OkD

NOV14c, ESRGPLSCDTV KIFYQTCRAVQHMHRQKPPIIHRDFKVENL SNQGTIKLCDFGS CG170882-03 ATTISHYPDYS SAQRRALVEEEITR TTPMYRTPEIIDLYS FPIGΞKQDIWALGCI LY LCFRQHPFEDGAKLRIV GKYSIPPHDTQYTVFHS IRAMLQVNPEERLSIAEW Protein HQLQEIAAARNVNPKSPITE LEQNGGYGSATLSRGPPPPVGPAGSGYSGGLALAEYD Sequence QPYGGFLDI RGGTERLFT LKDTSSK/IQSVANYAKGD DISYITSRIAVMSFPAEG VESALKNNIEDVRLF DSKHPGIIYAVY LSPRTYRPSRFHi VSECG AARRAPHLHT LYNICR1S_^ LRQDHK1WCVVHCMDGRAASAVAVCSFLCFCR FSTAEAAVYMFS_ RCPPGIWPSHKRYIEYMCDMVAEEPITPHSKPII.VRAWMTPVP FSKQRSGCRPFCE VYVGDERVASTSQEYDKMRDFKIEDGKAVIPLGVTVQGDVLIVIYHARSTLGGRLQAK MASM MFQIQFHTGFVPRNATTVKFAKYD DACDIQEKYPDLFQVNLEVEVEPRDRPS RΞAPP ENSSMRG NPKILFSSREEQQDILSKFGKPELPRQPGSTAQYDAGAGSPEAE PTDSDSPPSSSADASRF HT D QEEKEAETGAENASSKESESALMEDRDESEVSDEG GSPISSEGQEPRADPEPPG AAG VQQDLVFEVETPAVLPEPVPQEDGVD G HSEV GAGPAVPPQACKAPSSN D LSCLLGPPEAASQGPPEDLLSEDPLLLASPAPP SVQS TPRGGPPAAADPFGPLLPSSGNNSQPCSNPDLFGEFLNSDSV VPPSFPSAHSAPPPS CS DF HLGDLPGEPSKMTASSSNPD GGWAA TETAASAVAPTPATEGPLFSPGGQ PAPCGSQASWTKSQNPDPFADLGD SSGLQGSPAGFPPGGFIPKTATTAKGSSSWQTS RPPAQGASWPPQAKPPPKACTQPRPNYASNFSVIGAREERGVRAPSFAQKPKVSENDF EDL SNQGFSSRSD KGPKTIAEMRKQDLAKDTDP KLKLLDWIEGKERNIRALLST HTV TODGESR TPVGI__LVAPEQV KHYRRAVLAVHPDKAAGQPYEQHAKMIF ELN DA SEFENQGSRP F

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

Table 14B. Comparison of NOV14a against NOV14b and NOV14c.

NOV14a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV14b 174..1262 1085/1138 (95%) 68..1205 1086/1138 (95%)

NOV14c 137..1262 1121/1175 (95%) 1..1175 1122/1175 (95%)

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

Table 14C. Protein Sequence Properties NO 14a

SignalP analysis: No Known Signal Sequence Predicted

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

N-region: length 9; pos.chg 0; neg.chg 1 H-region: length 13; peak value 0.00 PSG score: -4.40

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -1.03 possible cleavage site: between 22 and 23

>» Seems to have no N-terminal signal peptide

Tentative number of TMS(s) for the threshold 0.5: 2 INTEGRAL Likelihood = -3.24 Transmembrane 513 - 529 INTEGRAL Likelihood = -3.66 Transmembrane 628 - 644 PERIPHERAL Likelihood = 3.29 (at 572) ALOM score: -3.66 (number of TMSs: 2)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 520 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: 1 Hyd Moment (75) : 4.11 Hyd Moment (95) : 3.68 G content: 5 D/E content: 2 S/T content: 4 Score: -7.87

Gavel: prediction of cleavage sites for mitochondrial preseq R-3 motif at 67 GREY|A

NϋCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite: none content of basic residues: 10.0% 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: found ILPK at 745

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

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

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

Final Results (k = 9/23) :

34.8 %: nuclear

26.1 %: mitochondrial

26.1 %: endoplasmic reticulum

4.3 %: plasma membrane

4.3 %: cytoplasmic

4.3 %: peroxisomal

» prediction for CG170882-01 is nuc (k=23) A search of the NOV14a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 14D.

In a BLAST search of public sequence datbases, the NOV14a protein was found o have homology to the proteins shown in the BLASTP data in Table 14E.

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

Example 15. malic enzyme.

The NOVl 5 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 15 A.

Table 15A. NOV15 Sequence Analysis

SEQ ID NO: 81 1374 bp

NOV15a, CGGAAGGAGAGGACCGAGGTCTGCCAAGGACCATGGGTGCCGCGCTGGGGACAGGCAC CG171205-01 GCGGCTGGCTCCCTGGCCGGGCCGGGCCTGCGGCGCCCTCCCGCGCTGGACACCCACC GCGCCCGCCCAAGGCTGCCACTCCAAGCTTGGCCCGGCGCGCCCTGTGCCCCTGAAGA DNA Sequence AGCGCGGATACGATGTCACCAGGAACCCTCATCTCAACAAGGGGATGGCCTTTACCCT TGAAGAAAGGCTGCAGCTTGGAATCCACGGCCTAATCCCGCCCTGCTTTCTGAGCCAG GACGTCCAGCTCCTCCGAATCATGAGATATTACGAGCGGCAGCAGAGTGACCTGGACA AGTACATCATTCTCATGACACTCCAAGACCGGAACGAGAAGCTCTTCTACCGAGTGCT GACTTCGGATGTGGAGAAGTTCATGCCAATCGTGTACACGCCTACCGTGGGGCTGGCC TGTCAGCACTATGGCCTGACTTTCCGCAGGCCCCGTGGACTGTTCATCACCATTCATG ACAAAGGTCATCTTGCAACAATGCTGAATTCTTGGCCAGAAGACAATATTAAGGCCGT GGTGGTGACTGATGGGGAGCGCATCCTGGGCCTGGGAGACCTGGGCTGCTACGGCATG GGCATCCCTGTGGGCAAGCTGGCCCTGTACACGGCATGCGGAGGGGTGAACCCGCAGC AGTGCCTCCCTGTGCTGCTGGACGTCGGCACCAACAATGAGGAGCTGCTCAGAGACCC TCTGTACATCGGCCTGAAACACCAGCGCGTGCACGGGAAGGCATACGATGACTTGCTG GATGAGTTCATGCAGGCTGTGACAGACAAGTTTGGAATAAATTGCCTCATCCAATTTG AAGACTTCGCCAATGCCAATGCCTTCCGCCTGCTCAACAAATACCGTAACAAGTACTG CATGTTCAATGATGACATCCAAGGCACAGCCTCCGTTGCTGTGGCAGGGATCTTGGCT GCTCTGCGAATCACCAACAACAAGCTTTCCAATCACGTGTTTGTTTTCCAAGGTGCAG GCGAGGCAGCTATGGGCATTGCCCACCTCCTTGTCATGGCCCTAGAGAAAGAAGGTGT ACCGAAGGCAGAGGCCACAAGAAAGATCTGGATGGTGGACTCTAAAGGGCTCATTGTC AAGGGGAGGAGCCACCTGAACCATGAAAAGGAGATGTTTGCCCAAGACCATCCTGAAG TCAACTCCCTGGAGGAGGTGGTGAGGCTGGTGAAGCCCACAGCCATCATAGGGCCGAG GGATTTTTGCCAGTGGAAGTCCTTTTAAGAGTGTGACTCTGGAAGATGGCAAGACCTT

CATTCCTGGGCAGGGAAACAATGCTTACGTGTTCCCCGGG

ORF Start: ATG at 33 ORF Stop: TAA at 1302 SEQ ID NO: 82 423 aa MW at47227.4kD

NOV15a, MGAALGTGTR APWPGRACGALPRWTPTAPAQGCHSK GPARPVPLKFRGYDvTRNPH GMAFT EΞRLQLGIHGLIPPCFLSQDVQLLRIMRYYERQQSDLDKYII MTLQDR CG171205-01 NEKLFYRV TSDVEKFMPIVYTPTVGLACQHYG TFRRPRG FITIHDKGH ATMLNS Protein PEDNIKAVVVTDGΞRILG GDLGCYGMGIPVGKLALYTACGGV PQQCLPVLLDVGT Sequence MSTEΞL RDPLYIGLKHQRVHGKAYDDLLDEFMQAV DKFGINCLIQFEDFAAAFRL NKYRIvπYCMFlvTODIQGTASV^^

VMA EKEGVP_\EATRKIWMVDSKGLIVKGRSHNHEKEMFAODHPEVNSLEEVVRLV IKPTAI IGPRDFCQ KSF

Further analysis of the NOVl 5a protein yielded the following properties shown in Table 15B.

Table 15B. Protein Sequence Properties NO 15a

SignalP analysis: No Known Signal Sequence Predicted

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

N-region: length 10; pos.chg 1; neg.chg 0 H-region: length 6; peak value -1.99 PSG score: -6.39

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -6.89 possible cleavage site: between 20 and 21

>» Seems to have no N-terminal signal peptide

ALOM: Klein et al's method for IM 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 = 1.01 (at 336) ALOM score: 0.21 (number of TMSs: 0)

MITDISC: discrimination of mitochondrial targeting seq R content: 5 ^'Hyd Moment (75) : 1.33 Hyd Momen (95) : 2.04 G content: 8 D/E content: 1 S/T content: 5 Score: -3.87

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 65 TRN|PH

NUCDISC: discrimination of nuclear localization signals pat4: RRPR (4) at 153 pat7: PVPLKKR (3) at 43 pat7: PLKKRGY (5) at 45 bipartite: none content of basic residues: 11.6% NLS Score: 0.47

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

ER Membrane Retention Signals:

KKXX-like motif in the C-terminus: QWKS

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 : MGAALGT

Prenylation motif: none

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 15C.

In a BLAST search of public sequence datbases, the NOV15a protein was foundo have homology to the proteins shown in the BLASTP data in Table 15D.

Table 15D. Public BLASTP Results for NOV15a

Identities/

NOV15a

Protein Similarities

Residues/ Expect

Accession Protein/Organism Length for the

Match Value

Number Matched

Residues

Portion

Q16798 NADP-dependent malic enzyme, 1..413 412/413 (99%) 0.0 mitochondrial precursor (EC 1..413 412/413 (99%) 1.1.1.40) (NADP-ME) (Malic enzyme 3) - Homo sapiens (Human), 604 aa.

Q8TBJ0 Malic enzyme 3, NADP(+ 1..413 410/413 (99%) 0.0 dependent, mitochondrial - Homo 1..413 410/413 (99%) sapiens (Human), 604 aa.

BAC27751 Adult male olfactory brain cDNA, 1..413 386/413 (93%) 0.0 RIKEN full-length enriched library, 1..413 393/413 (94%) clone:6430411I17 product:NADP- DEPENDENT MALIC ENZYME, MITOCHONDRIAL PRECURSOR (EC 1.1.1.40) (NADP-ME) homolog - Mus musculus (Mouse), 604 aa.

P40927 NADP-dependent malic enzyme 48..413 282/366 (77%) e-172 (EC 1.1.1.40) (NADP-ME) - 2..367 331/366 (90%) Columba livia (Domestic pigeon), 557 aa.

Q90XC0 Malic enzyme (EC 1.1.1.40) - 48..413 280/366 (76%) e-171 Gallus gallus (Chicken), 557 aa. 2..367 329/366 (89%)

PFam analysis predicts that the NOV15a protein contains the domains shown in the Table 15E.

Example 16. L-LACTATE DEHYDROGENASE

The NOVl 6 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 16 A.

Table 16A. NOV16 Sequence Analysis

SEQ ED NO: 83 1550 bp

NOV16a, GGGACCTAACTGTGGAAGAACTAAGGAATGTAAATGTATTTTTCCCACATTTCAAATA

T CCATGGACACCTATGTTTTTAAAGATAGTTCTCAGAAAGACCTGCTGAATTTTACT CG171793-01 GGCACAATTCCTGTGATGTATCAGGGTAATACATATAACATACCAATTCGTTTCTGGA DNA Sequence TTTTGGATTCTCACCCTTTCGCTCCCCCTATTTGCTTCTTGAAGCCAACTGCAAATAT GGGAATCTTAGTCGGAAAACATGTGGATGCTCAAGGCAGAATATATTTGCCCTATCTC CAAAACTGGAGCCATCCTAAATCTGTCATTGTTGGATTAATTAAAGAAATGATTGCCA AGTTTCAAGAGGAACTTCCCATGTATTCTCTATCATCATCTGATGAGGCACGGCAGGT AGACTTGCTAGCCTATATTGCAAAAATCACTGAAGGTGTTTCAGATACAAATTCAAAG AGCTGGGCAAATCATGAGAATAAAACAGTCAATAAAATTACTGTGGTTGGAGGTGGAG AACTCGGTATTGCCTGCACATTAGCAATTTCAGCAAAGGGTATTGCAGACAGGCTTGT CCTCTTAGACCTCTCAGAAGGGACTAAAGGAGCCACGATGGACCTTGAAATCTTCAAC CTTCCTAATGTGGAGATCAGCAAAGATTTGTCTGCCTCTGCTCATTCCAAGGTGGTGA TCTTCACAGTCAACTCTTTGGGTAGTTCTCAGTCGTACCTTGATGTGGTACAGAGCAA TGTGGATATGTTCAGAGCCCTTGTCCCAGCTCTGGGACATTATAGTCAACACAGTGTC CTGCTCGTTGCATCTCAACCAGTGGAAATCATGACCTATGTAACATGGAAACTGAGTA CATTTCCTGCAAATCGAGTGATCGGAATTGGATGTAATCTGGATTCACAGAGATTACA GTATATTATTACAAATGTTT GAAGGCACAGACTTCAGGCAAAGAAGTATGGGTTATT GGCGAGCAAGGAGAAGACAAAGTGCTCACATGGAGTGGCCAAGAAGAAGTAGTGAGTC ATACCTCTCAAGTGCAGCTGTCCAACAGAGCCATGGAACTGCTAAGAGTAAAAGGTCA AAGATCCTGGTCTGTTGGACTATCAGTAGCTGACATGGTTGACAGTATTGTAAACAAT AAGAAGAAAGTCCATTCTGTATCAGCTTTAGCAAAGGGATATTATGATATAAATAGTG AAGTGTTTTTAAGTTTGCCTTGCATCCTTGGAACCAATGGAGTATCTGAAGTTATCAA AACCACACTGAAAGAAGATACAGTTACTGAGAAACTCCAAAGCAGTGCATCCTCAATC CACAGTCTCCAACAACAGTTAAAACTTTGATTCTCAAATGCAATTTGAGAGGCTGGAC

TTCTACCTAAAGGGAAAAGTCTTTAATTTTACCTATATA'TGGNTTGAGGATTTCTGTA

TCCTGCTACTTACNTTTACAAACTGCCTGGTTAAGTAAGGGGTTCCTGATTAGCTTT

GNGAGGTAAANCCCTAGGGGTTTTCCAGGGGGGGGAAATTAA

ORF Stop: TGA at 1362

ISEQ ID NO: 84 433 aa JMW at 47583.1kD

NOV16a, MDTY tFKDSSQKDLLNFTGTIPVMYQGlsπ'Y_^IPIRF ILDSHPFAPPICFLKPTANMG

ILVGKHVDAQGRIYLPYLQNWSHPKSVIVGLIKEMIAKFQEELPMYSLSSSDEARQVD CG171793-01 rAYIAKITEGVSDT SKSWATsraE KO KI VVGGGELGIACTLAISAKGIADRLV Protein LDLSEGTKGATMDLEIF Plvr^ISKXlLSASAHSKVVIFTVNS GSSQSY DVVQSNV Sequence DMFRALVPA GHYSQHSVL VASQPVEIMTYVT KLSTFPANRVIGIGC LDSQR QY

IIT1WLKAQTSGKEVWVIGEQGEDKVLTWSGQEEVVSHTSQVQ SNRAMEL RV GQR

SWS VGLSVAD]_VOS IVNIKIO ^

TLKEDTVTEKLQSSASSIHS QQQLK Further analysis of the NOVl 6a protein yielded the following properties shown in Table 16B.

Table 16B. Protein Sequence Properties NOVl 6a

SignalP analysis: No Known Signal Sequence Predicted

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

N-region: length 8; pos.chg 1; neg.chg 2 H-region: length 3; peak value 0.00 PSG score: -4.40

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -10.52 possible cleavage site: between 47 and 48

>» Seems to have no N-terminal signal peptide

Tentative number of TMS(s) for the threshold 0.5: 1 Number of TMS(s) for threshold 0.5: 0 PERIPHERAL Likelihood = 2.38 (at 388) ALOM score: -1.17 (number of TMSs: 0)

MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75) : 12.96 Hyd Moment (95) : 5.03 G content: 0 D/E content: 2 S/T content: 1 Score: -5.92

NCTCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite: none content of basic residues : 8.8% NLS Score: -0.47

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

ER Membrane Retention Signals :

KKXX-like motif in the C-terminus : QQLK

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

PTS2: 2nd peroxisomal targeting signal: none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

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

AAU23670 Novel human enzyme 213..433 220/221 (99%) e-120 polypeptide #756 - Homo 1..221 220/221 (99%) sapiens, 221 aa. [WO200155301-A2, 02-AUG- 2001]

ABG21596 Novel human diagnostic protein 109..432 133/350 (38%) le-60 #21587 - Homo sapiens, 354 aa. 9..352 205/350 (58%) [WO200175067-A2, 11-OCT- 2001]

In a BLAST search of public sequence datbases, the NOV16a protein was found to have homology to the proteins shown in the BLASTP data in Table 16D.

PFam analysis predicts that the NOVl 6a protein contains the domains shown in the Table 16E.

Example 17. EPHA4, Ephrin type-A receptor 4

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

Table 17A. NOV17 Sequence Analysis

SEQ ID NO: 85 3136 bp

NOV17a, AAGCGGCAGGAGCAGCGTTGGCACCGGCGAACCATGGCTGGGATTTTCTATTTCGCCC CG172979-01 TATTTTCGTGTCTCTTCGGGATTTGCGACGCTGTCACAGGTTCCAGGGTATACCCCGC GAATGAAGTTACCTTATTGGATTCCAGATCTGTTCAGGGAGAACTTGGGTGGATAGCA DNA Sequence AGCCCTCTGGAAGGAGGGTGGGAGGAAGTGAGTATCATGGATGAAAAAAATACACCAA TCCGAACCTACCAAGTGTGCAATGTGATGGAACCCAGCCAGAATAACTGGCTACGAAC TGATTGGATCACCCGAGAAGGGGCTCAGAGGGTGTATATTGAGATTAAATTCACCTTG AGGGACTGCAATAGTCTTCCGGGCGTCATGGGGACTTGCAAGGAGACGTTTAACCTGT ACTACTATGAATCAGACAACGACAAAGAGCGTTTCATCAGAGAGAACCAGTTTGTCAA AATTGACACCATTGCTGCTGATGAGAGCTTCACCCAAGTGGACATTGGTGACAGAATC ATGAAGCTGAACACCGAGATCCGGGATGTAGGGCCATTAAGCAAAAAGGGGTTTTACC TGGCTTTTCAGGATGTGGGGGCCTGCATCGCCCTGGTATCAGTCCGTGTGTTCTATAA AAAGTGTCCACTCACAGTCCGCAATCTGGCCCAGTTTCCTGACACCATCACAGGGGCT GATACGTCTTCCCTGGTGGAAGTTCGAGGCTCCTGTGTCAACAACTCAGAAGAGAAAG ATGTGCCAAAAATGTACTGTGGGGCAGATGGTGAATGGCTGGTACCCATTGGCAACTG CCTATGCAACGCTGGGCATGAGGAGCGGAGCGGAGAATGCCAAGCTTGCAAAATTGGA TATTACAAGGCTCTCTCCACGGATGCCACCTGTGCCAAGTGCCCACCCCACAGCTACT CTGTCTGGGAAGGAGCCACCTCGTGCACCTGTGACCGAGGCTTTTTCAGAGCTGACAA CGATGCTGCCTCTATGCCCTGCACCCGTCCACCATCTGCTCCCCTGAACTTGATTTCA AATGTCAACGAGACATCTGTGAACTTGGAATGGAGTAGCCCTCAGAATACAGGTGGCC GCCAGGACATTTCCTATAATGTGGTATGCAAGAAATGTGGAGCTGGTGACCCCAGCAA GTGCCGACCCTGTGGAAGTGGGGTCCACTACACCCCACAGCAGAATGGCTTGAAGACC ACCAAAGTCTCCATCACTGACCTCCTAGCTCATACCAATTACACCTTTGAAATCTGGG CTGTGAATGGAGTGTCCAAATATAACCCTAACCCAGACCAATCAGTTTCTGTCACTGT GACCACCAACCAAGCAGCACCATCATCCATTGCTTTGGTCCAGGCTAAAGAAGTCACA AGATACAGTGTGGCACTGGCTTGGCTGGAACCAGATCGGCCCAATGGGGTAATCCTGG AATATGAAGTCAAGTATTATGAGAAGGATCAGAATGAGCGAAGCTATCGTATAGTTCG GACAGCTGCCAGGAACACAGATATCAAAGGCCTGAACCCTCTCACTTCCTATGTTTTC CACGTGCGAGCCAGGACAGCAGCTGGCTATGGAGACTTCAGTGAGCCCTTGGAGGTTA CAACCAACACAGTGCCTTCCCGGATCATTGGAGATGGGGCTAACTCCACAGTCCTTCT GGTCTCTGTCTCGGGCAGTGTGGTGCTGGTGGTAATTCTCATTGCAGCTTTTGTCATC AGCCGGAGACGGAGTAAATACAGTAAAGCCAAACAAGAAGCGGATGAAGAGAAACATT TGAATCAAGGTGTAAGAACATATGTGGACCCCTTTACGTACGAAGATCCCAACCAAGC AGTGCGAGAGTTTGCCAAAGAAATTGACGCATCCTGCATTAAGATTGAAAAAGTTATA GGAGTTGGTGAATTTGGTGAGGTATGCAGTGGGCGTCTCAAAGTGCCTGGCAAGAGAG AGATCTGTGTGGCTATCAAGACTCTGAAAGCTGGTTATACAGACAAACAGAGGAGAGA CTTCCTGAGTGAGGCCAGCATCATGGGACAGTTTGACCATCCGAACATCATTCACTTG GAAGGCGTGGTCACTAAATGTAAACCAGTAATGATCATAACAGAGTACATGGAGAATG GCTCCTTGGATGCATTCCTCAGGAAAAATGATGGCAGATTTACAGTCATTCAGCTGGT GGGCATGCTTCGTGGCATTGGGTCTGGGATGAAGTATTTATCTGATATGAGCTATGTG CTGATTTTGGCATGTCCCGAGTGCTTGAGGATGATCCGGAAGCAGCTTACACCACCAG GGGTGGCAAGATTCCTATCCGGTGGACTGCGCCAGAAGCAATTGCCTATCGTAAATTC ACATCAGCAAGTGATGTATGGAGCTATGGAATCGTTATGTGGGAAGTGATGTCGTACG GGGAGAGGCCCTATTGGGATATGTCCAATCAAGATGTGATTAAAGCCATTGAGGAAGG CTATCGGTTACCCCTCCCAATGGACTGCCCCATTGCGCTCCACCAGCTGATGCTAGAC TGCTGGCAGAAGGAGAGGAGCGACAGGCCTAAATTTGGGCAGATTGTCAACATGTTGG ACAAACTCATCCGCAACCCCAACAGCTTGAAGAGGACAGGGACGGAGAGCTCCAGACC TAACACTGCCTTGTTGGATCCAAGCTCCCCTGAATTCTCTGCTGTGGTATCAGTGGGC GATTGGCTCCAGGCCATTAAAATGGACCGGTATAAGGATAACTTCACAGCTGCTGGTT ATACCACACTAGAGGCTGTGGTGCACGTGAACCAGGAGTA&GTACTCAACGATGTAAC ACGAAAGGGGACCTGGCAAGAATTGGTATCACAGCCATCACGCACCAGAATAAGATTT

TGAGCAGTGTCCAGGCAATGCGAACCCAAATGCAGCAGATGCACGGCAGAATGGTTCC

CGTCTGAGCCAGTACTGAATAAACTCAAAACTCTTGAAATTAGTTTACCTCATCCATG

CACTTTAATTGAAGAACTGCACTTTTTTTACTTCGTCTTCGCCCTCTGAAATTAAAGA

AATT

ORF Start: ATG at 34 ORF Stop: TAA at 2881

SEQ ID NO: 86 949 aa MW at 105730.9kD

NOV17a, _ GIFYFA FSC FGICDAVTGSRVYPANEV LDSRSVQGELG IASPLEGGWEEVS CG172979-01 IMDEKNTPIRTYQVOSIVMEPSQlv^^

TCKETFNLYYΥESDI_KERFIRENQFVKIDTIAADESFTQ\7DIGDRI_SLNTEIRDVG Protein PLSKKGFYLAFQDVGACIALVSVRVFYKKCPL VRNLAQFPDTITGADTSSLVEVRGS Sequence CVNNSEE DVPKMYCGADGEWLVPIGNCLCNAGHEERSGECQACKIGYYKALSTDATC AKC PPHS YS VWEGATSCTCDRGFFRADNDAASMPCTRPPS P LI SNVNETSVNLEW S SPQN GGRQDI SYNWCKKCGAGDPSKCRPCGSGVHYTPQQNGLKTTKVS ITDL AH TISΓ_TFEI AVNGVSKY PNPDQSVSV V TNQAAPSSIALVQAKΞVTRYSVA AW EP DRPNGVI EYEVKYYEKDQNERSYRIVRTAARN DIKGLNPLTSYVFHVRARTAAGYG DFSEPLEVTTN VPSRIIGDGA S VL VSVSGSVV VVILIAAFVISRRRSKYSKAK QEADEEKH NQGVRTYVDPFTYEDPNQAVREFAKΞIDASCIKIEKVIGVGEFGEVCSG R KVPGKREICVAI TLKAGYTDKQRRDFLSEASIMGQFDHPNIIH EGW KCKPVM IITEYMENGS DAF RKXVTDGRF_VIQ VG_-,RGIGSGMKY SDMSYVHRDLAAR ILV NSN VCKVSDFGMSRV EDDPEAAYTTRGGKIPIRWTAPEAIAYRKFTSASDV SYGI VMWΞVMSYGERPY DMSNQDVIKAIEEGYRLP PMDCPIALHQLMLDCWQKERSDRPK FGQIVLVM DKLIRNPNSL RTGTESSRPNTALLDPSSPEFSAVVSVGD LQAIKMDRY KD FTAAGYTTLEAWHVNQE

SEQ ID NO: 87 2913 bp

NOV17b, AAGCGGCAGGAGCAGCGTTGGCACCGGCGAACCATGGCTGGGATTTTCTATTTCGCCC CG172979-02 TATTTTCGTGTCTCTTCGGGATTTGCGACGCTGTCACAGGTTCCAGGGTATACCCCGC GAATGAAGTTACCTTATTGGATTCCAGATCTGTTCAGGGAGAACTTGGGTGGATAGCA DNA Sequence AGCCCTCTGGAAGGAGGGTGGGAGGAAGTGAGTATCATGGATGAAAAAAATACACCAA TCCGAACCTACCAAGTGTGCAATGTGATGGAACCCAGCCAGAATAACTGGCTACGAAC TGATTGGATCACCCGAGAAGGGGCTCAGAGGGTGTATATTGAGATTAAATTCACCTTG AGGGACTGCAATAGTCTTCCGGGCGTCATGGGGACTTGCAAGGAGACGTTTAACCTGT ACTACTATGAATCAGACAACGACAAAGAGCGTTTCATCAGAGAGAACCAGTTTGTCAA AATTGACACCATTGCTGCTGATGAGAGCTTCACCCAAGTGGACATTGGTGACAGAATC ATGAAGCTGAACACCGAGATCCGGGATGTAGGGCCATTAAGCAAAAAGGGGTTTTACC TGGCTTTTCAGGATGTGGGGGCCTGCATCGCCCTGGTATCAGTCCGTGTGTTCTATAA AAAGTGTCCACTCACAGTCCGCAATCTGGCCCAGTTTCCTGACACCATCACAGGGGCT GATACGTCTTCCCTGGTGGAAGTTCGAGGCTCCTGTGTCAACAACTCAGAAGAGAAAG ATGTGCCAAAAATGTACTGTGGGGCAGATGGTGAATGGCTGGTACCCATTGGCAACTG CCTATGCAACGCTGGGCATGAGGAGCGGAGCGGAGAATGCCAAGCTTGCAAAATTGGA TATTACAAGGCTCTCTCCACGGATGCCACCTGTGCCAAGTGCCCACCCCACAGCTACT CTGTCTGGGAAGGAGCCACCTCGTGCACCTGTGACCGAGGCTTTTTCAGAGCTGACAA CGATGCTGCCTCTATGCCCTGCACCCGTCCACCATCTGCTCCCCTGAACTTGATTTCA AATGTCAACGAGACATCTGTGAACTTGGAATGGAGTAGCCCTCAGAATACAGGTGGCC GCCAGGACATTTCCTATAATGTGGTATGCAAGAAATGTGGAGCTGGTGACCCCAGCAA GTGCCGACCCTGTGGAAGTGGGGTCCACTACACCCCACAGCAGAATGGCTTGAAGACC ACCAAAGTCTCCATCACTGACCTCCTAGCTCATACCAATTACACCTTTGAAATCTGGG CTGTGAATGGAGTGTCCAAATATAACCCTAACCCAGACCAATCAGTTTCTGTCACTGT GACCACCAACCAAGCAGCACCATCATCCATTGCTTTGGTCCAGGCTAAAGAAGTCACA AGATAdAGTGTGGCACTGGCTTGGCTGGAACCAGATCGGCCCAATGGGGTAATCCTGG AATATGAAGTCAAGTATTATGAGAAGGATCAGAATGAGCGAAGCTATCGTATAGTTCG GACAGCTGCCAGGAACACAGATATCAAAGGCCTGAACCCTCTCACTTCCTATGTTTTC CACGTGCGAGCCAGGACAGCAGCTGGCTATGGAGACTTCAGTGAGCCCTTGGAGGTTA CAACCAACACAGTGCCTTCCCGGATCATTGGAGATGGGGCTAACTCCACAGTCCTTCT GGTCTCTGTCTCGGGCAGTGTGGTGCTGGTGGTAATTCTCATTGCAGCTTTTGTCATC AGCCGGAGACGGAGTAAATACAGTAAAGCCAAACAAGAAGCGGATGAAGAGAAACATT TGAATCAAGGTGTAAGAACATATGTGGACCCCTTTACGTACGAAGATCCCAACCAAGC AGTGCGAGAGTTTGCCAAAGAAATTGACGCATCCTGCATTAAGATTGAAAAAGTTATA GGAGTTGGTGAATTTGGTGAGGTATGCAGTGGGCGTCTCAAAGTGCCTGGCAAGAGAG AGATCTGTGTGGCTATCAAGACTCTGAAAGCTGGTTATACAGACAAACAGAGGAGAGA CΪTCCTGAGTGAGGCCAGCATCATGGGACAGTTTGACCATCCGAACATCATTCACTTG GAAGGCGTGGTCACTAAATGTAAACCAGTAATGATCATAACAGAGTACATGGAGAATG GCTCCTTGGATGCATTCCTCAGGAAAAATGATGGCAGATTTACAGTCATTCAGCTGGT GGGCATGCTTCGTGGCATTGGGTCTGGGATGAAGTATTTATCTGATATGAGCTATGTG CATCGTGATCTGGCCGCACGGAACATCCTGGTGAACAGCAACTTGGTCTGCAAAGTGT CTGATTTTGGCATGTCCCGAGTGCTTGAGGATGATCCGGAAGCAGCTTACACCACCAG GGGTGGCAAGATTCCTATCCGGTGGACTGCGCCAGAAGCAATTGCCTATCGTAAATTC ACATCAGCAAGTGATGTATGGAGCTATGGAATCGTTATGTGGGAAGTGATGTCGTACG GGGAGAGGCCCTATTGGGATATGTCCAATCAAGATACCTAACACTGCCTTGTTGGATC

CAGCCTCCCCTGAATTCTCTGCTGTGGTATCAGTGGGCGATTGGCTCCAGGCCATTAA

AATGGACCGGTATAAGGATAACTTCACAGCTGCTGGTTATACCACACTAGAGGCTGTG

GTGCACGTGAACCAGGAGGACCTGGCAAGAATTGGTATCACAGCCATCACACACCAGA ATAAGATTTTGAGCAGTGTCCAGGCAATGCGAACCCAAATGCAGCAGATGCACGGCAG

AATGGTTCCCGTCTGAGCCAGTACTGAATAAACTCAAAACTCTTGAAATTAGTTTACC

TCATCCATGCACTTTAATTGAAGAACTGCACTTTTTTTACTTCGTCTTCGCCCTCTGAl

1AATTAAAGAAATG

ORF Start: ATG at 34 ORF Stop: TAA at 2533

SEQ ID NO: 88 833 aa MW at 92619.0kD

NOV17b, MAGIFYFA FSC FGICDAV GSRVYPA EVTLLDSRSVQGE G IASPLEGG EEVS CG172979-02 I3_>EKOTPIRTYQVCNVMEPSQN1W^

TCKETFI_YYYESDM3KERFIRENQFVKIDTIAADESFTQVDIGDRIM LN EIRDVG Protein P SKKGFYLAFQDVGACIALVSVRVFYKKCP TVRN AQFPDTITGADTSSLVEVRGS Sequence CVNNSEEKOVPKMYCGADGEWLVPIGNCLCNAGHEERSGECQACKIGYYKALSTDATC

A CPPHSYSVWEGATSCTCDRGFFRADNDAASMPCTRPPSAP lV_IS vTffiTSVN EW

SSPQNTGGRQDISYWCKKCGAGDPSKCRPCGSGVHYTPQQNGLKTTKVSITD LAH

TKπfTFEI AV GVSKYNPNPDQSVSV V TNQAAPSSIALVQAKEV RYSVALA EP

DRPNGVILEYEVKYYEKIJQNERSYRIVRTAARl^DIKG NPLTSYVFHVRARTAAGYG

DFSEPLEVTTNTVPSRIIGDGANSTVL VSVSGSVVLVVILIAAFVISRRRSKYSKAK

QEADEEKH NQGVRTYVDPFTYEDPNQAVREFAKEIDASCIKIEKVIGVGEFGEVCSG

R KVPGKREICVAIKT KAGYTDKQRRDFLSEASIMGQFDHPNIIHLEGWTKCKPVM

IITEY ENGS DAFLRKNDGRFTVIQ VGMLRGIGSGMKY SDMSYVHRD AARNILV

NSNLVCKVSDFGMSRV EDDPEAAYTTRGG IPIR TAPEAIAYRKFTSASDV SYGI

VMWEVMSYGERPY DMSNQDT

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

Table 17B. Comparison of NOV17a against NOV17b.

NOV17a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV17b 1..832 832/832 (100%) 1..832 832/832 (100%)

Further analysis ofthe NOVl 7a protein yielded the following properties shown in Table 17C.

Table 17C. Protein Sequence Properties NOV17a SignalP analysis: Cleavage site between residues 20 and 21

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

N-region: length 0; pos.chg 0; neg.chg 0 H-region: length 17; peak value 10.94 PSG score: 6.54

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 0.18 possible cleavage site: between 19 and 20

>» Seems to have a cleavable signal peptide (1 to 19)

INTEGRAL Likelihood =-12.68 Transmembrane 548 - 564 PERIPHERAL Likelihood = 2.81 (at 682) ALOM score: -12.68 (number of TMSs: 1)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 9 Charge difference: -2.0 C(-1.0) - N( 1.0) N >= C: N-terminal side will be inside

»> membrane topology: type la (cytoplasmic tail 565 to 949)

MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75) : 0.36 Hyd Moment (95) : 3.21 G content: 2 D/E content: 1 S/T content: 1 Score: -6.78

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

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

ER Membrane Retention Signals: none

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

PTS2: 2nd peroxisomal targeting signal: none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

A search of the NOV17a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 17D.

In a BLAST search of public sequence datbases, the NOV17a protein was found o have homology to the proteins shown in the BLASTP data in Table 17E.

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

Example 18. Discoidin domain receptor 2.

The NOVl 8 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 18 A.

Table 18A. NOV18 Sequence Analysis

SEQ ID NO: 89 2812 bp

NOV18a, CTGCACCCGTTGATACTCCAGTTCCAACACCATCTTCTGAGATGATCCTGATTCCCAG

AATGCTCTTGGTGCTGTTCCTGCTGCTGCCTATCTTGAGTTCTGCAAAAGCTCAGGTT CG173488-01 AATCCAGGGTCAAGATGGCAATCAGGGACACTATGACATGGACTACAGTCAAAAGATC DNA Sequence CAGGAATGCCTGCCTTGGACAGCTAGATCTTCTCCCCAGGAGGTGCCTGAGGGAGACA TACACACAGGTGACAGTCACAAAGGCTATATGCCGCTATCCTCTGGGCATGTCAGGAG GCCAGATTCCAGATGAGGACATCACAGCTTCCAGTCAGTGGTCAGAGTCCACAGCTGC CAAATATGGAAGGCTGGACTCAGAAGAAGGGGATGGAGCCTGGTGCCCTGAGATTCCA GTGGAACCTGATGACCTGAAGGAGTTTCTGCAGATTGACTTGCACACCCTCCATTTTA TCACTCTGGTGGGGACCCAGGGGCGCCATGCAGGAGGTCATGGCATCGAGTTTGCCCC CATGTACAAGATCAATTACAGTCGGGATGGCACTCGCTGGATCTCTTGGCGGAACCGT CATGGGAAACAGGTGCTGGATGGAAATAGTAACCCCTATGACATTTTCCTAAAGGACT TGGAGCCGCCCATTGTAGCCAGATTTGTCCGGTTCATTCCAGTCACCGACCACTCCAT GAATGTGTGTATGAGAGTGGAGCTTTACGGCTGTGTCTGGCTAGATGGCTTGGTGTCT TACAATGCTCCAGCTGGGCAGCAGTTTGTACTCCCTGGAGGTTCCATCATTTATCTGA ATGATTCTGTCTATGATGGAGCTGTTGGATACAGCATGACAGAAGGGCTAGGCCAATT GACCGATGGTGTGTCTGGCCTGGACGATTTCACCCAGACCCATGAATACCACGTGTGG CCCGGCTATGACTATGTGGGCTGGCGGAACGAGAGTGCCACCAATGGCTACATTGAGA TCATGTTTGAATTTGACCGCATCAGGAATTTCACTACCATGAAGGTCCACTGCAACAA CATGTTTGCTAAAGGTGTGAAGATCTTTAAGGAGGTACAGTGCTACTTCCGCTCTGAA GCCAGTGAGTGGGAACCTAATGCCATTTCCTTCCCCCTTGTCCTGGATGACGTCAACC CCAGTGCTCGGTTTGTCACGGTGCCTCTCCACCACCGAATGGCCAGTGCCATCAAGTG TCAATACCATTTTGCAGATACCTGGATGATGTTCAGTGAGATCACCTTCCAATCAGAT GCTGCAATGTACAACAACTCTGAAGCCCTGCCCACCTCTCCTATGGCACCCACAACCT ATGATCCAATGCTTAAAGTTGATGACAGCAACACTCGGATCCTGATTGGCTGCTTGGT GGCCATCATCTTTATCCTCCTGGCCATCATTGTCATCATCCTCTGGAGGCAGTTCTGG CAGAAAATGCTGGAGAAGGCTTCTCGGAGGATGCTGGATGATGAAATGACAGTCAGCC TTTCCCTGCCAAGTGATTCTAGCATGTTCAACAATAACCGCTCCTCATCACCTAGTGA ACAAGGGTCCAACTCGACTTACGATCGCATCTTTCCCCTTCGCCCTGACTACCAGGAG CCATCCAGGCTGATACGAAAACTCCCAGAATTTGCTCCAGGGGAGGAGGAGTCAGGCT GCAGCGGTGTTGTGAAGCCAGTCCAGCCCAGTGGCCCTGAGGGGGTGCCCCACTATGC AGAGGCTGACATAGTGAACCTCCAAGGAGTGACAGGAGGCAACACATACTCAGTGCCT GCCGTCACCATGGACCTGCTCTCAGGAAAAGATGTGGCTGTGGAGGAGTTCCCCAGGA AACTCGTAACTTTCAAAGAGAAGCTGGGAGAAGGACAGTTTGGGGAGGTTCATCTCTG TGAAGTGGAGGGAATGGAAAAATTCAAAGACAAAGATTTTGCCCTAGATGTCAGTGCC AACCAGCCTGTCCTGGTGGCTGTGAAAATGCTCCGAGCAGATGCCAACAAGAATGCCA GGAATGATTTTCTTAAGGAGATAAAGATCATGTCTCGGCTCAAGGACCCAfiACATCAT CCATCTATTATCTGTGTGTATCACTGATGACCCTCTCTGTATGATCACTGAATACATG GAGAATGGAGATCTCAATCAGTTTCTTTCCCGCCACGAGCCCCCTAATTCTTCCTCCA GCGATGTACGCACTGTCAGTTACACCAATCTGAAGTTTATGGCTACCCAAATTGCCTC TGGCATGAAGTACCTTTCCTCTCTTAATTTTGTTCACCGAGATCTGGCCACACGAAAC TGTTTAGTGGGTAAGAACTACACAATCAAGATAGCTGACTTTGGAATGAGCAGGAACC TGTACAGTGGTGACTATTACCGGATCCAGGGCCGGGCAGTGCTCCCTATCCGCTGGAT GTCTTGGGAGAGTATCTTGCTGGGCAAGTTCACTACAGCAAGTGATGTGTGGGCCTTT GGGGTTACTTTGTGGGAGACTTTCACCTTTTGTCAAGAACAGCCCTATTCCCAGCTGT CAGATGAACAGGTTATTGAGAATACTGGAGAGTTCTTCCGAGACCAAGGGAGGCAGAC TTACCTCCCTCAACCAGCCATTTGTCCTGACTCTGTGTATAAGCTGATGCTCAGCTGC TGGAGAAGAGATACGAAGAACCGTCCCTCATTCCAAGAAATCCACCTTCTGCTCCTTC AACAAGGCGACGAGTGATGCTGTCAGTGCCTGGCCATGTTCCTACGGCTCAGGTCCTC

CCTACAAGACCTACCACTCACCCATGCC

ORF Start: ATG at 131 ORF Stop: TGA at 2741

SEQ ID NO: 90 870 aa MW at 98674. lkD

NOV18a, _^IRDTMTWT VTRSI_IACLGQ DLLPRRC RETYTQVTV KAICRYPLGMSGGQIPD

EDITASSQ SESTAAKYGRLDSEEGDGAWCPEIPVEPDDLKEFLQIDLHT HFIT VG CG173488-01 TQGRHAGGHGIEFAPMYKI YSRDGTRWIS RNRHGKQVLDGNSNPYDIF KD EPPI Protein VARFVRFIPVTDHS NVCMRVELYGCV DGLVSYAPAGQQFVLPGGS11YL DSVY Sequence DGAVGYSMTEGLGQLTDGVSGLDDFTQTHEYHV PGYDYVGWRNESATNGYIEIMFEF

DRIRNFTTMKΛ CNIMFAKGVKIFKEVQCYFRSEASEWEPNAISFPLV DDV^

VTVPLHHR_SAIKCQYHFADTWI FSEITFQSDAAinr]MSEALPTSPMAPTTYDPl^ VDDSNTRILIGC VAIIFIL AIIVII WRQFWQKMLEKASRRMLDDEMTVS S PS

DSSMFNHNRSSSPSEQGSNSTYDRIFPLRPDYQEPSR IRKLPEFAPGEEESGCSGVV

KPVQPSGPEGVPHYAEADIV LQGV GGISπ'YSVPAvTMD SGKDVAVEEFPRK LTF

KEKLGEGQFGEVΗLCEVEGMEKFKDKDFALDVSANQPVLVAVKI^RADAKNARNDFL

KEIKI SRLKDPNIIHLLSVCITDDPLCMITEYMENGDLNQF SRHEPPNSSSSDVRT

VSYTNLKF_\TQIASG_Y SS NFVHRDLATRNCLVGKNYTIKIADFGMSRNLYSGD

YYRIQGRAVLPIRWMS ESILLGKFTTASDVWAFGVTLWETFTFCQEQPYSQ SDEQV lENTGEFFRDQGRQTYLPQPAICPDSVYKLM SCWRRDTKNRPSFQEIH QQGDE

SEQ ID NO: 91 12648 bp

NOV18b, CCGTTGATACTCCAGTTCCAACACCATCTTCTGAGATGATCCTGATTCCCAGAATGCT CTTGGTGCTGTTCCTGCTGCTGCCTATCTTGAGTTCTGCAAAAGCTCAGGTTAATCCA CG173488-02 GCTATATGCCGCTATCCTCTGGGCATGTCAGGAGGCCAGATTCCAGATGAGGACATCA DNA Sequence CAGCTTCCAGTCAGTGGTCAGAGTCCACAGCTGCCAAATATGGAAGGCTGGACTCAGA AGAAGGGGATGGAGCCTGGTGCCCTGAGATTCCAGTGGAACCTGATGACCTGAAGGAG TTTCTGCAGATTGACTTGCACACCCTCCATTTTATCACTCTGGTGGGGACCCAGGGGC GCCATGCAGGAGGTCATGGCATCGAGTTTGCCCCCATGTACAAGATCAATTACAGTCG GGATGGCACTCGCTGGATCTCTTGGCGGAACCGTCATGGGAAACAGGTGCTGGATGGA AATAGTAACCCCTATGACATTTTCCTAAAGGACTTGGAGCCGCCCATTGTAGCCAGAT TTGTCCGGTTCATTCCAGTCACCGACCACTCCATGAATGTGTGTATGAGAGTGGAGCT TTACGGCTGTGTCTGGCTAGATGGCTTGGTGTCTTACAATGCTCCAGCTGGGCAGCAG TTTGTACTCCCTGGAGGTTCCATCATTTATCTGAATGATTCTGTCTATGATGGAGCTG TTGGATACAGCATGACAGAAGGGCTAGGCCAATTGACCGATGGTGTGTCTGGCCTGGA CGATTTCACCCAGACCCATGAATACCACGTGTGGCCCGGCTATGACTATGTGGGCTGG CGGAACGAGAGTGCCACCAATGGCTACATTGAGATCATGTTTGAATTTGACCGCATCA GGAATTTCACTACCATGAAGGTCCACTGCAACAACATGTTTGCTAAAGGTGTGAAGAT CTTTAAGGAGGTACAGTGCTACTTCCGCTCTGAAGCCAGTGAGTGGGAACCTAATGCC ATTTCCTTCCCCCTTGTCCTGGATGACGTCAACCCCAGTGCTCGGTTTGTCACGGTGC CTCTCCACCACCGAATGGCCAGTGCCATCAAGTGTCAATACCATTTTGCAGATACCTG GATGATGTTCAGTGAGATCACCTTCCAATCAGATGCTGCAATGTACAACAACTCTGAA GCCCTGCCCACCTCTCCTATGGCACCCACAACCTATGATCCAATGCTTAAAGTTGATG

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

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

Table 18C. Protein Sequence Properties NOV18a SignalP analysis: No Known Signal Sequence Predicted

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

N-region: length 5; pos.chg 1; neg.chg 1 H-region: length 7; peak value 2.0₃ PSG score: -2.-8

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -9.49 possible cleavage site: between ₃6 and 37

»> Seems to have no N-terminal signal peptide

INTEGRAL Likelihood =-19.69 Transmembrane 419 - 435 PERIPHERAL Likelihood = 3.34 (at 191) ALOM score: -19.69 (number of TMSs: 1)

MTOP: prediction of membrane topology (Hartmann et al.) Center position for calculation: 426 Charge difference: 5.0 C( 4.0) - N(-1.0) C > N: C-terminal side will be inside

>» membrane topology: type lb (cytoplasmic tail 419 to 870)

MITDISC: discrimination of mitochondrial targeting seq R content: 3 Hyd Moment (75) : 12.74 Hyd Moment (95) : 8.67 G content: 1 D/E content: 2 S/T content: 5 Score: -2.06

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 26 SRN|AC

NϋCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite: none content of basic residues: 9.7% NLS Score: -0.47

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

ER Membrane Retention Signals :

XXRR-like motif in the N-terminus: AIRD

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

PTS2 : 2nd peroxisomal targeting signal : none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: too long tail

Dileucine motif in the tail: found LL at 427 LL at 56₂ LL at 577 LL at 655 LL at 775 LL at 862 LL at 863 checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none

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

Final Results (k = 9/23):

34.8 %: nuclear 26.1 % : cyoplasmic 21.7 %: mitochondrial

4.3 % : vacuolar

4.3 %: vesicles of secretory system

4.3 %: endoplasmic reticulum

4.3 % : peroxisomal

» prediction for CG173488-01 is nuc (k=23)

A search ofthe NOVl 8a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 18D.

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

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

Example 19. Novel Pyrin domain containing protein.

The NOVl 9 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 19 A.

Further analysis ofthe NOVl 9a protein yielded the following properties shown in Table 19B.

Table 19B. Protein Sequence Properties NOV19a

SignalP analysis: Cleavage site between residues 11 and 12

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

N-region: length 11; pos.chg 4; neg.chg 1 H-region: length 7; peak value 2.55 PSG score: -1.85

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -7.30 possible cleavage site: between 40 and 41

>» Seems to have no N-terminal signal peptide

ALOM: Klein et al's method for TM region allocation Init position for calculation: 1 Tentative number of TMS(s) for the threshold 0, 5: 4

INTEGRAL Likelihood = -7.06 Transmembrane 578 - 594

INTEGRAL Likelihood = 0.32 Transmembrane 640 - 656

INTEGRAL Likelihood = -9.98 Transmembrane 659 675

INTEGRAL Likelihood = -0.16 Transmembrane 729 745

PERIPHERAL Likelihood = 0.85 (at 606)

ALOM score: -9.98 (number of TMSs: 4)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 585 Charge difference: -1.5 C(-0.5) - N( 1.0) N >= C: N-terminal side will be inside

>» membrane topology: type 3a

MITDISC: discrimination of mitochondrial targeting seq R content: 2 Hyd Moment (75) : 3.65 Hyd Moment (95) : 6.10 G content: 0 D/E content: 2 S/T content: 1 Score: -5.26

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 17 ARK|PR

NUCDISC: discrimination of nuclear localization signals pat4: RKPR (4) at 7 pat7 : none bipartite: none content of basic residues: 11.3% NLS Score: -0.22

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 : none

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

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

Final Results (k = 9/23) :

47.8 % : endoplasmic reticulum 34.8 %: mitochondrial 13.0 % : nuclear 4.3 %: vesicles of secretory system

» prediction for CG173939-01 is end (k=23)

A search of the NOVl 9a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 19C.

Table 19C. Geneseq Results for NOV19a

In a BLAST search of public sequence datbases, the NOV19a protein was found o have homology to the proteins shown in the BLASTP data in Table 19D.

PFam analysis predicts that the NOVl 9a protein contains the domains shown in the Table 19E.

Example 20.

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

Table 20A. NOV20 Sequence Analysis

SEQ ID NO: 95 3188 bp

NOV20a, GGAGCCGCAGTGAGCACCATGGAGCTGGCGGCCTTGTGCCGCTGGGGGCTCCTCCTCG

CCCTCTTGCCCCCCGGAGCCGCGAGCACCCAAGTGTGCACCGGCACAGACATGAAGCT CG174189-01 GCGGCTCCCTGCCAGTCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGC DNA Sequence TGCCAGGTGGTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCCAGCCTGT CCTTCCTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGT GAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGAC AACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCA CAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGAGATCTT GAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTTTG TGGAAGGACATCTTCCACAAGAACAACCAGCTGGCTCTCACACTGATAGACACCAACC GCTCTCGGGCCTGCCACCCCTGTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGA GAGTTCTGAGGATTGTCAGAGCCTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCGC TGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTGCCGGCTGCACGG GCCCCAAGCACTCTGACTGCCTGGCCTGCCTCCACTTCAACCACAGTGGCATCTGTGA GCTGCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGCCCAAT CCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTACAACTACC TTTCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACAACCAAGAGGTGAC AGCAGAGGATGGAACACAGCGGTGTGAGAAGTGCAGCAAGCCCTGTGCCCGAGTGTGC TATGGTCTGGGCATGGAGCACTTGCGAGAGGTGAGGGCAGTTACCAGTGCCAATATCC AGGAGTTTGCTGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAGAGCTT TGATGGGGACCCAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAAGTGTTT GAGACTCTGGAAGAGATCACAGGTTACCTATACATCTCAGCATGGCCGGACAGCCTGC CTGACCTCAGCGTCTTCCAGAACCTGCAAGTAATCCGGGGACGAATTCTGCACAATGG CGCCTACTCGCTGACCCTGCAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTCACTG AGGGAACTGGGCAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTCGTGC ACACGGTGCCCTGGGACCAGCTCTTTCGGAACCCGCACCAAGCTCTGCTCCACACTGC CAACCGGCCAGAGGACGAGTGTGTGGGCGAGGGCCTGGCCTGCCACCAGCTGTGCGCC CGAGGGCACTGCTGGGGTCCAGGGCCCACCCAGTGTGTCAACTGCAGCCAGTTCCTTC

TGCACGGGCCCCAAGCACTCTGACTGCCTGGCCTGCCTCCACTTCAACCACAGTGGCA TCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCAT GCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTAC AACTACCTTTCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACAACCAAG AGGTGACAGCAGAGGATGGAACACAGCGGTGTGAGAAGTGCAGCAAGCCCTGTGCCCG AGTGTGCTATGGTCTGGGCATGGAGCACTTGCGAGAGGTGAGGGCAGTTACCAGTGCC TTCAGCCCAGCCTTCGACAACCTCTATTACTGGGACCAGGACCCACCAGAGCGGGGGG CTCCACCCAGCACCTTCAAAGGGACACCTACGGCAGAGAACCCAGAGTACATGGGTCT GGACGTGCCAGTGTGACTGCAGCCAAGCTAATTCCGG

ORF Start: ATG at 26 ORF Stop: TGA at 1232

SEQ ID NO: 98 402 aa MW at 44063.7kD

NOV20b, ELAALCR GLL ALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQWQG NLELTYLPTNASLSFLQDIQEVQGYVLIAH QVRQVPLQRLRIVRGTQLFED YALAV CG174189-03 LDNGDPLNNTTPVTGASPGGLRELQLRS TEI KGGVLIQRNPQ CYQDTILWKDIFH Protein KNNQLA T IDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLP Sequence TDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPAVTYNTDTFESMPNPEGRYT FGASCVTACPYNYLSTDVGSCTLVCP HNQEVTAEDGTQRCEKCSKPCARVCYG GME HLREVRAVTSAFSPAFD LYYWDQDPPERGAPPSTFKGTPTAENPEYMGLDVPV

SEQ ID NO: 99 2261 bp

NOV20c, GCCGCAGTGAGCACCATGGAGCTGGCGGCCTTGTGCCGCTGGGGGCTCCTCCTCGCCC CG174189-02 TCTTGCCCCCCGGAGCCGCGAGCACCCAAGTGTGCACCGGCACAGACATGAAGCTGCG GCTCCCTGCCAGTCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGC DNA Sequence CAGGTGGTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCCAGCCTGTCCT TCCTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGAG GCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGACAAC TATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAG GGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGAGATCTTGAA AGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTTTGTGG AAGGACATCTTCCACAAGAACAACCAGCTGGCTCTCACACTGATAGACACCAACCGCT CTCGGGCCTGCCACCCCTGTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAG TTCTGAGGATTGTCAGAGCCTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCGCTGC AAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTGCCGGCTGCACGGGCC CCAAGCACTCTGACTGCCTGGCCTGCCTCCACTTCAACCACAGTGGCATCTGTGAGCT GCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGCCCAATCCC GAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTACAACTACCTTT CTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACAACCAAGAGGTGACAGC AGAGGATGGAACACAGCGGTGTGAGAAGTGCAGCAAGCCCTGTGCCCGAGTGTGCTAT GGTCTGGGCATGGAGCACTTGCGAGAGGTGAGGGCAGTTACCAGTGCCAATATCCAGG AGTTTGCTGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAGAGCTTTGA TGGGGACCCAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAAGTGTTTGAG ACTCTGGAAGAGATCACAGGTTACCTATACATCTCAGCATGGCCGGACAGCCTGCCTG ACCTCAGCGTCTTCCAGAACCTGCAAGTAATCCGGGGACGAATTCTGCACAATGGCGC CTACTCGCTGACCCTGCAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTCACTGAGG GAACTGGGCAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTCGTGCACA CGGTGCCCTGGGACCAGCTCTTTCGGAACCCGCACCAAGCTCTGCTCCACACTGCCAA CCGGCCAGAGGACGAGTGTGTGGGCGAGGGCCTGGCCTGCCACCAGCTGTGCGCCCGA GGGCACTGCTGGGGTCCAGGGCCCACCCAGTGTGTCAACTGCAGCCAGTTCCTTCGGG GCCAGGAGTGCGTGGAGGAATGCCGAGTACTGCAGGGGCTCCCCAGGGAGTATGTGAA TGCCAGGCACTGTTTGCCGTGCCACCCTGAGTGTCAGCCCCAGAATGGCTCAGTGACC TGTTTTGGACCGGAGGCTGACCAGTGTGTGGCCTGTGCCCACTATAAGGACCCTCCCT TCTGCGTGGCCCGCTGCCCCAGCGGTGTGAAACCTGACCTCTCCTACATGCCCATCTG GAAGTTTCCAGATGAGGAGGGCGCATGCCAGCCTTGCCCCATCAACTGCACCCACTCC TGTGTGGACCTGGATGACAAGGGCTGCCCCGCCGAGCAGAGAGCCAGCCCTCTGACGT CCATCGTCTCTGCGGTGGTTGGCATTCTGCTGGTCGTGGTCTTGGGGGTGGTCTTTGG GATCCTCATCAAGCGACGGCAGCAGAAGATCCGGAAGTACACGATGCGGAGACTGCTG CAGGAAACGGAGCTGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGC AGATGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCGC TTTTGGCACAGTCTACAAGGGCATCTGGATCCCTTAGGGAGTGTCTAAGAACAAAAG

ORF Start: ATG at 16 ORF Stop: TAG at 2239

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 20B.

Table 20B. Comparison of NOV20a against NOV20b and NOV20c.

NOV20a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV20b 1..359 359/359 (100%) 1..359 359/359 (100%)

NOV20c 1..741 741/741 (100%) 1..741 741/741 (100%)

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

Table 20C. Protein Sequence Properties NOV20a

SignalP analysis: Cleavage site between residues 23 and 24

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

N-region: length 8; pos .chg 1; neg.chg 1 H-region: length 21; peak value 9.66 PSG score: 5.26

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 2.88 possible cleavage site: between 21 and 22

>» Seems to have a cleavable signal peptide (1 to 21)

ALOM: Klein et al's method for TM region allocation Init position for calculation: 2₂

INTEGRAL Likelihood =-15.18 Transmembrane 659 - 675 PERIPHERAL Likelihood = 0.90 (at 945) ALOM score: -15.18 (number of TMSs: 1)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 10 Charge difference: -1.5 C(-0.5) - N( 1.0) N >= C: N-terminal side will be inside >» membrane topology: type la (cytoplasmic tail 676 to 1042)

MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75) : 8.47 Hyd Moment (95) : 3.65 G content: 3 D/E content: 2 S/T content: 4 Score: -6.32

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 18 CR |GL

NUCDISC: discrimination of nuclear localization signals pat4: none pat7 : none bipartite: none content of basic residues: 9.1% NLS Score: -0.47

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

ER Membrane Retention Signals : none

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

PTS2 : 2nd peroxisomal targeting signal: none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: too long tail

Dileucine motif in the tail: found LL at 690 LL at 785 LL at 806 LL at 8₂₂ LL at 869 LL at 934 LL at 1008 checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none

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

Final Results (k = 9/23):

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

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

Table 20E. Public BLASTP Results for NOV20a

PFam analysis predicts that the NOV20a protein contains the domains shown in the Table 20F.

Example 21. RTK TIEl splice variant.

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

Table 21A. NOV21 Sequence Analysis

SEQ ID NO: 101 1277 bp

NOV21a, CGCTCGTCCTGGCTGGCCTGGGTCGGCCTCTGGAGTATGGTCTGGCGGGTGCCCCCTT TCTTGCTCCCCATCCTCTTCTTGGCTTCTCATGTGGGCGCGGCGGTGGACCTGACGCT CG174799-01 GCTGGCCAACCTGCGGCTCACGGACCCCCAGCGCTTCTTCCTGACTTGCGTGTCTGGG DNA Sequence GAGGCCGGGGCGGGGAGGGGCTCGGACGCCTGGGGCCCGCCCCTGCTGCTGGAGAAGG ACGACCGTATCGTGCGCACCCpGCCCGGGCCACCCGTCCTAGAGACTGACCCAGCTTT TGCTCGAGAGCATGGGACAGCCTCTACCCTTAGCTCCCGGCAGCTGCTGCGTTTCGCC AGTGATGCGGCCAATGGCATGCAGTACCTGAGTGAGAAGCAGTTCATCCACAGGGACC TGGCTGCCCGGAATGTGCTGGTCGGAGAGAACCTAGCCTCCAAGATTGCAGACTTCGG CCTTTCTCGGGGAGAGGAGGTTTATGTGAAGAAGACGATGGGGCGTCTCCCTGTGCGC TGGATGGCCATTGAGTCCCTGAACTACAGTGTCTATACCACCAAGAGTGATGTCTGGT CCTTTGGAGTCCTTCTTTGGGAGATAGTGAGCCTTGGAGGTACACCCTACTGTGGCAT GACCTGTGCCGAGCTCTATGAAAAGCTGCCCCAGGGCTACCGCATGGAGCAGCCTCGA AACTGTGACGATGAAGTGTACGAGCTGATGCGTCAGTGCTGGCGGGACCGTCCCTATG AGCGACCCCCCTTTGCCCAGATTGCGCTACAGCTAGGCCGCATGCTGGAAGCCAGGAA GGCCTATGTGAACATGTCGCTGTTTGAGAACTTCACTTACGCGGGCATTGATGCCACA GCTGAGGAGGCCTGAGCTGCCATCCAGCCAGAACGTGGCTCTGCTGGCCGGAGCAAAC TCTGCTGTCTAACCTGTGACCAGTCTGACCCTTACAGCCTCTGACTTAAGCTGCCTCA

AGGAATTTTTTTAACTTAAGGGAGAAAAAAAGGGATCTGGGGATGGGGTGGGCTTAGG GGAACTGGGTTCCCATGCTTTGTAGGTGTCTCATAGCTATCCTGGGCATCCTTCTTTC TAGTTCAGCTGCCCCACAGGTGTGTTTCCCATCCCACTGCTCCCCCAACACAAACCCC

CACTCCAGCTCCTTCGCTTAAGCCAGCACTCACACCACTAACATGCCCTGTTCAGCTA CTCCCACTCCCGGCCTGTCATTCAGAAAAAAATAAATGTTCTAATAAGCTCCAAAAAA A

ORF Start: ATG at 37 ORF Stop: TGA at 883

SEQ ID NO: 102 MW at 31678.9kD

|NOV21a, iMvT^røVPPF LPILF ASHVGAAVD TLLAT^RLTDPQRFFLTCVSGEAGAGRGSDA G! CG174799-01 PPLLLEKDDRIVRTPPGPPV ETDPAFAREHGTAST SSRQ LRFASDAANGMQYLSE

Protein KQFIHRDLAARVLVGEN ASKIADFGLSRGEEVYVKKTMGR PVR iLAIES NYSVY TTKSDVWSFGV LWEIVSLGGTPYCGMTCAELYEK PQGYRMEQPRNCDDEVy/E MRQ

Sequence CWRDRPYERPPFAQIALQLGRM ΞARKAYvTl SLFE FTYAGIDATAEEA

SEQ ID NO: 103 3140 bp

NOV21b, CGCTCGTCCTGGCTGGCCTGGGTCGGCCTCTGGAGTATGGTCTGGCGGGTGCCCCCTT CG174799-02 TCTTGCTCCCCATCCTCTTCTTGGCTTCTCATGTGGGCGCGGCGGTGGACCTGACGCT GCTGGCCAACCTGCGGCTCACGGACCCCCAGCGCTTCTTCCTGACTTGCGTGTCTGGG DNA Sequence GAGGCCGGGGCGGGGAGGGGCTCGGACGCCTGGGGCCCGCCCCTGCTGCTGGAGAAGG ACGACCGTATCGTGCGCACCCCGCCCGGGCCACCCCTGCGCCTGGCGCGCAACGGTTC GCACCAGGTCACGCTTCGCGGCTTCTCCAAGCCCTCGGACCTCGTGGGCGTCTTCTCC TGCGTGGGCGGTGCTGGGGCGCGGCGCACGCGCGTCATCTACGTGCACAACAGCCCTG GAGCCCACCTGCTTCCAGACAAGGTCACACACACTGTGAACAAAGGTGACACCGCTGT ACTTTCTGCACGTGTGCACAAGGAGAAGCAGACAGACGTGATCTGGAAGAGCAACGGA TCCTACTTCTACACCCTGGACTGGCATGAAGCCCAGGATGGGCGGTTCCTGCTGCAGC TCCCAAATGTGCAGCCACCATCGAGCGGCATCTACAGTGCCACTTACCTGGAAGCCAG CCCCCTGGGCAGCGCCTTCTTTCGGCTCATCGTGCGGGGTTGTGGGGCTGGGCGCTGG GGGCCAGGCTGTACCAAGGAGTGCCCAGGTTGCCTACATGGAGGTGTCTGCCACGACC ATGACGGCGAATGTGTATGCCCCCCTGGCTTCACTGGCACCCGCTGTGAACAGGCCTG CAGAGAGGGCCGTTTTGGGCAGAGCTGCCAGGAGCAGTGCCCAGGCATATCAGGCTGC CGGGGCCTCACCTTCTGCCTCCCAGACCCCTATGGCTGCTCTTGTGGATCTGGCTGGA GAGGAAGCCAGTGCCAAGAAGCTTGTGCCCCTGGTCATTTTGGGGCTGATTGCCGACT CCAGTGCCAGTGTCAGAATGGTGGCACTTGTGACCGGTTCAGTGGTTGTGTCTGCCCC TCTGGGTGGCATGGAGTGCACTGTGAGAAGTCAGACCGGATCCCCCAGATCCTCAACA TGGCCTCAGAACTGGAGTTCAACTTAGAGACGATGCCCCGGATCAACTGTGCAGCTGC AGGGAACCCCTTCCCCGTGCGGGGCAGCATAGAGCTACGCAAGCCAGACGGCACTGTG CTCCTGTCCACCAAGGCCATTGTGGAGCCAGAGAAGACCACAGCTGAGTTCGAGGTGC CCCGCTTGGTTCTTGCGGACAGTGGGTTCTGGGAGTGCCGTGTGTCCACATCTGGCGG CCAAGACAGCCGGCGCTTCAAGGTCAATGTGAAAGTGCCCCCCGTGCCCCTGGCTGCA CCTCGGCTCCTGACCAAGCAGAGCCGCCAGCTTGTGGTCTCCCCGCTGGTCTCGTTCT CTGGGGATGGACCCATCTCCACTGTCCGCCTGCACTACCGGCCCCAGGACAGTACCAT GGACTGGTCGACCATTGTGGTGGACCCCAGTGAGAACGTGACGTTAATGAACCTGAGG CCAAAGACAGGATACAGTGTTCGTGTGCAGCTGAGCCGGCCAGGGGAAGGAGGAGAGG GGGCCTGGGGGCCTCCCACCCTCATGACCACAGACTGTCCTGAGCCTTTGTTGCAGCC GTGGTTGGAGGGCTGGCATGTGGAAGGCACTGACCGGCTGCGAGTGAGCTGGTCCTTG CCCTTGGTGCCCGGGCCACTGGTGGGCGACGGTTTCCTGCTGCGCCTGTGGGACGGGA CACGGGGGCAGGAGCGGCGGGAGAACGTCTCATCCCCCCAGGCCCGCACTGCCCTCCT GACGGGACTCACGCCTGGCACCCACTACCAGCTGGATGTGCAGCTCTACCACTGCACC CTCCTGGGCCCGGCCTCGCCCCCTGCACACGTGCTTCTGCCCCCCAGTGGGCCTCCAG CCCCCCGACACCTCCACGCCCAGGCCCTCTCAGACTCCGAGATCCAGCTGACATGGAA GCACCCGGAGGCTCTGCCTGGGCCAATATCCAAGTACGTTGTGGAGGTGCAGGTGGCT GGGGGTGCAGGAGACCCACTGTGGATAGACGTGGACAGGCCTGAGGAGACAAGCACCA TCATCCGTGGCCTCAACGCCAGCACGCGCTACCTCTTCCGCATGCGGGCCAGCATTCA GGGGCTCGGGGACTGGAGCAACACAGTAGAAGAGTCCACCCTGGGCAACGGGCTGCAG GCTGAGGGCCCAGTCCAAGAGAGCCGGGCAGCTGAAGAGGGCCTGGATCAGCAGCTGA TCCTGGCGGTGGTGGGCTCCGTGTCTGCCACCTGCCTCACCATCCTGGCCGCCCTTTT AACCCTGGTGTGCATCCGCAGAAGCTGCCTGCATCGGAGACGCACCTTCACCTACCAG TCAGGCTCGGGCGAGGAGACCATCCTGCAGTTCAGCTCAGGGACCTTGACACTTACCC GGGGCATGACCTGTGCCGAGCTCTATGAAAAGCTGCCCCAGGGCTACCGCATGGAGCA GCCTCGAAACTGTGACGATGAAGTGTACGAGCTGATGCGTCAGTGCTGGCGGGACCGT CCCTATGAGCGACCCCCCTTTGCCCAGATTGCGCTACAGCTAGGCCGCATGCTGGAAG CCAGGAAGGCCTATGTGAACATGTCGCTGTTTGAGAACTTCACTTACGCGGGCATTGA TGCCACAGCTGAGGAGGCCTGAGCTGCCATCCAGCCAGAACGTGGCTCTGCTGGCCGG

AGCAAACTCTGCTGTCTAACCTGTGACCAGTCTGACCCTTACAGCCTCTGACTTAAGC

TGCCTCAAGGAATTTTTTTAACTTAAGGGAGAAAAAAAGGGATCTGGGGATGGGGTGG

GCTTAGGGGAACTGGGTTCCCATGCTTTGTAGGTGTCTCATAGCTATCCTGGGCATCC

TTCTTTCTAGTTCAGCTGCCCCACAGGTGTGTTTCCCATCCCACTGCTCCCCCAACAC

AAACCCCCACTCCAGCTCCTTCGCTTAAGCCAGCACTCACACCACTAACATGCCCTGTI

ITCAGCTACTCCCACTCCCGGCCTGTCATTCAGAAAAAAΆTAAATGTTCTAATAAGCTC

CAAAAAAA

ORF Start: ATG at 37 ORF Stop: TGA at 2746

Sequence SYPVLEWEDITFEDLIGEG1_GQVIRAMIKJDG KMNAAIKMLKEYASENDHRDFAGE LEVLCKLGHHPNIINLLGACKiπiGYLYIAIEYAPYG LDF RKSRV ETDPAFAREH GTAST SSRQLLRFASDAANGMQYLSEKQFIHRD_ARNV VGENLASKIADFG SRG EEVYVKKTMGRliPVRWMAIES NYSVYTTKSDV SFGVL EIVS GGTPYCGMTCAE YEKLPQGYFJdEQPRNCDDEVYE MRQC RDRPYERPPFAQIALQLGRMLEAR AYVN MS FENFTYAGIDATAEEA

Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 21B.

Table 21B. Comparison of NOV21a against NOV21b and NOV21c.

NOV21a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV21b 200..282 83/83 (100%) 821..903 83/83 (100%)

NOV21c 78..282 205/205 (100%) 337..541 205/205 (100%)

Further analysis of the NOV21a protein yielded the following properties shown in Table 21C.

Table 21C. Protein Sequence Properties NOV21a

SignalP analysis: Cleavage site between residues 22 and 23

PSORTH analysis: PSG: a new signal peptide prediction method

N-region: length 4; pos.chg 1; neg.chg 0 H-region: length 19; peak value 11.25 PSG score: 6.85

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 0.86 possible cleavage site: between 21 and 22

>» Seems to have a cleavable signal peptide (1 to 21)

ALOM: Klein et al ' s method for TM region allocation Init position for calculation: 22

Tentative number of TMS(s) for the threshold 0.5: 0 number of TMS(s) .. fixed PERIPHERAL Likelihood = 2.33 (at 180) ALOM score: 2.33 (number of TMSs: 0)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 10 Charge difference: -1.5 C( 0.5) - N( 2.0) N >= C: N-terminal side will be inside

MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75) : 7.17 Hyd Moment (95) : 6.43 G content: 1 D/E content: 1 S/T content: 1 Score: -4.07

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 14 RV|PP

NUCDISC: discrimination of nuclear localization signals pat4: none pat7 : none bipartite: none content of basic residues : 10.6%

NLS Score: -0.47

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

ER Membrane Retention Signals:

XXRR-like motif in the N-terminus: V RV none

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

PTS2 : 2nd peroxisomal targeting signal : none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

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

Final Results (k = 9/23):

33.3 %: extracellular, including cell wall 33.3 %: endoplasmic reticulum 22.2 %: mitochondrial 11.1 %: vacuolar

» prediction for CG174799-01 is exc (k=9) A search of the NOV21a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 21D.

In a BLAST search of public sequence datbases, the NOV21 a protein was found to have homology to the proteins shown in the BLASTP data in Table 21E.

PFam analysis predicts that the NOV21a protein contains the domains shown in the Table 2 IF.

Example 22.

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

Table 22A. NOV22 Sequence Analysis SEQ ID NO: 107 1432 bp

NOV22a, CACCGGATCCACCA GGAACTAATTTCCCCAACAGTGATTATAATCCTGGGTTGCCTT

GCTCTGTTCTTACTCCTTCAGCGGAAGAATTTGCGTAGACCCCCGTGCATCAAGGGCT CG175105-03 GGATTCCTTGGATTGGAGTTGGATTTGAGTTTGGGAAAGCCCCTCTAGAATTTATAGA DNA Sequence GAAAGCAAGAATCAAGTATGGACCAATATTTACAGTCTTTGCTATGGGAAACCGAATG ACCTTTGTTACTGAAGAAGAAGGAATTAATGTGTTTCTAAAATCCAAAAAAGTAGATT TTGAACTAGCAGTGCAAAATATCGTTTATCGTACAGCATCAATTCCAAAGAATGTCTT TTTAGCACTGCATGAAAAACTCTATATTATGTTGAAAGGGAAAATGGGGACTGTCAAT CTCCATCAGTTTACTGGGCAACTGACTGAAGAATTACATGAACAACTGGAGAATTTAG GCACTCATGGGACAATGGACCTGAACAACTTAGTAAGACATCTCCTTTATCCAGTCAC AGTGAATATGCTCTTTAATAAAAGTTTGTTTTCCACAAACAAGAAAAAAATCAAGGAG TTCCATCAGTATTTCCAAGTTTATGATGAAGATTTTGAGTATGGGTCCCAGTTGCCAG AGTGTCTTCTAAGAAACTGGTCAAAATCCAAAAAGTGGTTCCTGGAACTGTTTGAGAA AAACATTCCAGATATAAAAGCATGTAAATCTGCAAAAGATAATTCCATGACATTATTG CAAGCTACGCTGGATATTGTAGAGACGGAAACAAGTAAGGAAAACTCACCCAATTATG GGCTCTTACTGCTTTGGGCTTCTCTGTCTAATGCTGTTCCTGTTGCATTTTGGACACT TGCATACGTCCTTTCTCATCCTGATATCCACAAGGCCATTATGGAAGGCATATCTTCT GTGTTTGGCAAAGCAGGCAAAGATAAGATTAAAGTGTCTGAGGATGACCTGGAGAATC TCCTTCTAATTAAATGGTGTGTTTTGGAAACCATTCGTTTAAAAGCTCCTGGTGTCAT TACTAGAAAAGTGGTGAAGCCTGTGGAAATTTTGAATTACATCATTCCTTCTGGTGAC TTGTTGATGTTGTCTCCATTTTGGCTGCATAGAAATCCAAAGTATTTTCCTGAGCCTG AATTGTTCAAACCTGAACGTTGGAAAAAGGCAAATTTAGAGAAGCACTCTTTCTTGGA CTGCTTCATGGCATTTGGAAGCGGGAAGTTCCAGTGTCCTGCAAGGTGGTTTGCTCTG TTAGAGGTTCAGATGTGTATTATTTTAATACTTTATAAATATGACTGTAGTCTTCTGG ACCCATTACCCAAACAGAGTTATCTCCATTTGGTGGGTGTCCCCCAGCCGGAAGGGCA ATGCCGAATTGAATATAAACAAAGAATATAGGTCGACGGC

ORF Start: ATG at 14 ORF Stop: TAG at 1421

SEQ ID NO: 108 469 aa MW at 54115.2kD

NOV22a, MELISPTVIIILGC ALFL QRKNLRRPPCIKGWIPWIGVGFEFG AP EFIEKARI

KYGPIFTWAMGNIrøTFVTEEEGIIWF K^ CG175105-03 EK YIMLKGKMGTVrø__QFTGQ_TEELHEQ EN^ Protein Fl^SLFSTI^KKIKEFHQYFQVYDEDFEYGSQ PECL RN SKSKKWFLELFEK IPD Sequence IKACKSAKDNSMTL QATLDIVETETSKENSPNYGL LLWAS SNAVPVAFWTLAYV

SHPDIHKAIMEGISSVFGKAGKDKIKVSEDD ENLLLIKWCVLΞTIRLKAPGV TRKV

VKPVEILNYIIPSGDLLMLSPF LHRNPKYFPEPELFKPERWKKANLEKHSF DCFMA

FGSGKFQCPAR FA EVQMCIILILYKYDCSLLDPLPKQSY H VGVPQPEGQCRIE

YKQRI

SEQ ID NO: 109 1414 bp j^'

NOV22b, ACTGTTTCACACTTTTCTGCTTCTGGAAGGTGCTGGACAAAAACATGGAACTAATTTC CG175105-01 CCCAACAGTGATTATAATCCTGGGTTGCCTTGCTCTGTTCTTACTCCTTCAGCGGAAG AATTTGCGTAGACCCCCGTGCATCAAGGGCTGGATTCCTTGGATTGGAGTTGGATTTG DNA Sequence AGTTTGGGAAAGCCCCTCTAGAATTTATAGAGAAAGCAAGAATCAAGTATGGACCAAT ATTTACAGTCTTTGCTATGGGAAACCGAATGACCTTTGTTACTGAAGAAGAAGGAATT AATGTGTTTCTAAAATCCAAAAAAGTAGATTTTGAACTAGCAGTGCAAAATATCGTTT ATCGTACAGGGAAAATGGGGACTGTCAATCTCCATCAGTTTACTGGGCAACTGACTGA AGAATTACATGAACAACTGGAGAATTTAGGCACTCATGGGACAATGGACCTGAACAAC TTAGTAAGACATCTCCTTTATCCAGTCACAGTGAATATGCTCTTTAATAAAAGTTTGT TTTCCACAAACAAGAAAAAAATCAAGGAGTTCCATCAGTATTTTCAAGTTTATGATGA AGATTTTGAGTATGGGTCCCAGTTGCCAGAGTGTCTTCTAAGAAACTGGTCAAAATCC AAAAAGTGGTTCCTGGAACTGTTTGAGAAAAACATTCCAGATATAAAAGCATGTAAAT CTGCAAAAGATAATTCCATGACATTATTGCAAGCTACGCTGGATATTGTAGAGACGGA AACAAGTAAGGAAAACTCACCCAATTATGGGCTCTTACTGCTTTGGGCTTCTCTGTCT AATGCTGTTCCTGTTGCATTTTGGACACTTGCATACGTCCTTTCTCGTCCTGATATCC ACAAGGCCATTATGGAAGGCATATCTTCTGTGTTTGGCAAAGCAGGCAAAGATAAGAT TAAAGTGTCTGAGGATGACCTGGAGAATCTCCTTCTAATTAAATGGTGTGTTTTGGAA ACCATTCGTTTAAAAGCTCCTGGTGTCATTACTAGAAAAGTGGTGAAGCCTGTGGAAA TTTTGAATTACATCATTCCTTCTGGTGACTTGTTGATGTTGTCTCCATTTTGGCTGCA TAGAAATCCAAAGTATTTTCCTGAGCCTGAATTGTTCAAACCTGAACGTTGGAAAAAG GCAAATTTAGAGAAGCACTCTTTCTTGGACTGCTTCATGGCATTTGGAAGCGGGAAGT TCCAGTGTCCTGCAAGGTGGTTTGCTCTGTTAGAGGTTCAGATGTGTATTATTTTAAT ACTTTATAAATATGACTGTAGTCTTCTGGACCCATTACCCAAACAGAGTTATCTCCAT TTGGTGGGTGTCCCCCAGCCGGAAGGGCAATGCCGAATTGAATATAAACAAAGAATAT GACATCTGTTGGGCCTCACAAG

ORF Start: ATG at 45 ORF Stop: TGA at 1392

SEQ ID NO: 110 449 aa MW at 51823.4kD

NOV22b, MELI SPTVI I ILGC A FLLLQRKNLRRPPC IKGWI P IGVGFEFGKAP EF IEKARI

KYGPIFTWAMGNRMTFVTEEEGINWLKSK^ CG175105-01 GQLTEELHEQLEI^GTHGTMDLKII^^ Protein QVYDEDFEYGSQLPECLLRIWSKSKKWFLΞ FEKNIPDIKACKSAKDNSMT QATLD Sequence IVETETSKENSP Γ_GLL LWASLSNAVPVAF TLAYVLSRPDIHKAIMEGISSVFGKA

GKDKIKVSEDDLEIS_ IKMCVLETIR KAPGVITRKΛA PVEI ]SNRIIPSGDLLMLS

PFWLHRNPKYFPEPELFKPERWKKAN EKHSFLDCFMAFGSGKFQCPAR FALLEVQM

CIILILYKYDCSL DPLPKQSYLHLVGVPQPEGQCRIEYKQRI

SEQ ID NO: 111 2183 bp

NOV22c, GGGGGAAGTGGAGGTGGGAGGGAGCGACAATGGAAAAATCACCTGAAAACTGGGACAG

AGGAAGGAAGCTACAGTTACGAΆGGAGAGCTGCAAΆAGTTGCAGCAGAAΆGGTTGGGA CG175105-02 GTCCCGACAGGTTCCGTAGCCCACAGAAAAGAAGCAAGGGACGGCAGGACTGTTTCAC DNA Sequence ACTTTTCTGCTTCTGGAAGGTGCTGGACAAAAACATGGAACTAATTTCCCCAACAGTG ATTATAATCCTGGGTTGCCTTGCTCTGTTCTTACTCCTTCAGCCGAAGAATTTGCGTA GACCCCCGTGCATCAAGGGCTGGATTCCTTGGATTGGAGTTGGATTTGAGTTTGGGAA AGCCCCTCTAGAATTTATAGAGAAAGCAAGAATCAAGTATGGACCAATATTTACAGTC TTTGCTATGGGAAACCGAΆTGACCTTTGTTACTGAAGAAGAAGGAATTAATGTGTTTC TAAAATCCAAAAAAGTAGATTTTGAACTAGCAGTGCAAAATATCGTTTATCGTACAGC ATCAATTCCAAAGAATGTCTTTTTAGCACTGCATGAAAAACTCTATATTATGTTGAAA GGGAAAATGGGGACTGTCAATCTCCATCAGTTTACTGGGCAACTGACTGAAGAATTAC ATGAACAACTGGAGAATTTAGGCACTCATGGGACAATGGACCTGAACAACTTAGTAAG ACATCTCCTTTATCCAGTCACAGTGAATATGCTCTTTAATAAAAGTTTGTTTTCCACA AACAAGAAAAAAATCAAGGAGTTCCATCAGTATTTTCAAGTTTATGATGAAGATTTTG AGTATGGGTCCCAGTTGCCAGAGTGTCTTCTAAGAAACTGGTCAAAATCCAAAAAGTG GTTCCTGGAACTGTTTGAGAAAAACATTCCAGATATAAAAGCATGTAAATCTGCAAAA GATAATTCCATGACATTATTGCAAGCTACGCTGGATATTGTAGAGACGGAAACAAGTA AGGAAAACTCACCCAATTATGGGCTCTTACTGCTTTGGGCTTCTCTGTCTAATGCTGT TCCTGTTGCATTTTGGACACTTGCACACGTCCTTTCTCATCCTGATATCCACAAGGCC ATTATGGAAGGCATATCTTCTGTGTTTGGCAAAGCAGGCAAAGATAAGATTAAAGTGT CTGAGGATGACCTGGAGAATCTCCTTCTAATTAAATGGTGTGTTTTGGAAACCATTCG TTTAAAAGCTCCTGGTGTCATTACTAGAAAAGTGGTGAAGCCTGTGGAAATTTTGAAT TACATCATTCCTTCTGGTGACTTGTTGATGTTGTCTCCATTTTGGCTGCATAGAAATC CAAAGTATTTTCCTGAGCCTGAATTGTTCAAACCTGAACGTTGGAAAAAGGCAAATTT AGAGAAGCACTCTTTCTTGGACTGCTTCATGGCATTTGGAAGCGGGAAGTTCCAGTGT CCTGCAAGGTGGTTTGCTCTGTTAGAGGTTCAGATGTGTATTATTTTAATACTTTATA AATATGACTGTAGTCTTCTGGACCCATTACCCAAACAGAGTTATCTCCATTTGGTGGG TGTCCCCCAGCCGGAAGGGCAATGCCGAATTGAATATAAACAAAGAATATGACATCTG

TTGGGCCTCACAAGGACCAGGGCCTTCTGGAGGAGTGGCACTACCCCACCTGGCAGCA

CCTAGACCTGAGCTCTACAAAAACACACTGCTTCACTTTGTTTTAGGACTTAGTTCAA

GAACACATTCAAATGGTGCATGTGTTTGGTATCTTCAACAGTAGACCAAGAATCTAAC ATCACTCTCAGTAATATAGAGACCGGAATACATGGTTTATAGGAAATGATCAAATGAT CCAAAAAAACTCCACATTTTTTAAGAAGTTGGAATTTGATTTCATGCATAACTGTATT

AAAACATTAAATAGAAATAATGTCATTTGAATGAAAATCTTATCACATTAAATTCACT

GTGAAGGCAGCATACTTAAATTTTTATTTTGAAAAGTCTAAAAGGCTTAGATTTTTAA

AATTTAATAATTATTTCTACAAATTTTCTATTTTTCTTGAGGTGATTCTCAACTAGCA

ATTGGAACTCCTAGGCTCTATTAACATAATTCTTTATTGTAAACGTATCTAATGCTAA

AAGTAATAAAATGGTAGTTTTCTGAGACCTGCGAAAA.

ORF Start: ATG at 209 ORF Stop: TGA at 1616

SEQ ID NO: 112 469 aa MW at 54030. lkD

NOV22c, MELI S PTVI I ILGCLALFLLLQPKNLRRPPCIKG I P IGVGFEFGKAPLΞFIEKARI

KYGPIFTVFAMGKTRMTFVTEEEGI VFLKSKKVDFELAVQNIVYRTASIPK1WFLALH CG175105-02 EKLYI__KGK GTWLHQFTGQLTEELHEQL Protein FI^SLFSTMKKKIKEFHQYFQVYDΞDFEYGSQLPECLLRN SKSK WFLELFEKNIPD Sequence IKACKSAKDNSMTLLQATLDIVETETSKE SPNYGLLLLWASLSNAVPVAF TLAHVL SHPDIHKAIMEGISSVFGKAGKDKIKVSEDDLENLLLIKWCVLETIRLKAPGVITRKV V^PVEILl^IIPSGDLLMLSPF LHRNP YFPEPELFKPER KKANLEKHSFLDCFMA FGSGKFQCPARWFALLEVQMCIILILYKYDCSIiLDPLPKQSYLHLVGVPQPEGQCRIE YKQRI

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

Table 22B. Comparison of NOV22a against NOV22b and NOV22c.

NOV22a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV22b 1..469 448/469 (95%) 1..449 448/469 (95%)

NOV22c 1..469 467/469 (99%) 1..469 468/469 (99%)

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

Table 22C. Protein Sequence Properties NOV22a

SignalP analysis: Cleavage site between residues 27 and 28

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

N-region: length 2; pos.chg 0; neg.chg 1 H-region: length 20; peak value 0.00 PSG score: -4.40

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -1.50 possible cleavage site: between 22 and 23

»> Seems to have no N-terminal signal peptide

Tentative number of TMS(s) for the threshold 0.5: 3 INTEGRAL Likelihood = -9.50 Transmembrane 3 - 19 INTEGRAL Likelihood = -0.37 Transmembrane 267 - 283 INTEGRAL Likelihood = -2.34 Transmembrane 416 - 432 PERIPHERAL Likelihood = 3.71 (at 354) ALOM score: -9.50 (number of TMSs: 3)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 10 Charge difference: 5.0 C( 5.0) - N( 0.0) C > N: C-terminal side will be inside

>» membrane topology: type 3b

MITDISC: discrimination of mitochondrial targeting seq R content: 3 Hyd Moment (75) : 4.91 Hyd Moment (95) : 7.70 G content: 4 D/E content: 2 S/T content: 2 Score: -5.59

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 38 RRP|PC NϋCDISC: discrimination of nuclear localization signals pat4 : none p t7: PERWKKA (4) at 387 bipartite: none content of basic residues: 12.4% NLS Score: -0.13

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

ER Membrane Retention Signals:

KKXX-like motif in the C-terminus: YKQR

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

PTS2: 2nd peroxisomal targeting signal: none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

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

Final Results (k = 9/23) :

44.4 %: endoplasmic reticulum

22.2 %: vacuolar

11.1 %: mitochondrial

11.1 %: Golgi

11.1 %: cytoplasmic

» prediction for CG175105-03 is end (k=9)

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

NOV22a Identities/

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

Residues Region

AAE05173 Human drug metabolising enzyme 1..469 454/469 (96%) 0.0 (DME-4) protein - Homo sapiens, 1..469 456/469 (96%) 469 aa. [WO200151638-A2, 19- JUL-2001]

ABG23259 Novel human diagnostic protein 57..244 185/188 (98%) e-106 #23250 - Homo sapiens, 682 aa. 17..204 187/188 (99%) [WO200175067-A2, 11-OCT- 2001]

ABP00222 Human ORFX protein sequence 315..441 104/127 (81%) le-60 SEQ ID NO:426 - Homo sapiens, 2..128 120/127 (93%) 128 aa. [WO200192523-A2, 06- DEC-2001]

AAY36206 Human secreted protein #78 - 1..90 90/90 (100%) le-46 Homo sapiens, 92 aa. 1..90 90/90 (100%) [WO9925825-A2, 27-MAY-1999]

ABB57286 Mouse ischaemic condition related 1..468 133/503 (26%) 4e-44 protein sequence SEQ ID NO:806 7..503 241/503 (47%) - Mus musculus, 507 aa. [WO200188188-A2, 22-NOV- 2001]

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

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

Example 23.

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

Table 23A. NOV23 Sequence Analysis

SEQ ID NO: 113 1423 bp

NOV23a, GCCACGGCAGAGAGGATGAGAGCGTGGATCCCTGGGTGGGTTGGGCGGCCGCACGGGG CG175257-01 GTGCCGAGGCGTCTGGGGGCCTGCGCTTCGGGGCGAGCGCAGCGCAAGGCTGGCGCGC GCGCATGGAGGATGCTCACTGCACTTGGCTTTCGTTACCTGGTCTGCCCCCGGGCTGG DNA Sequence GCCTTGTTTGCCGTCCTCGACGGCCACGGTGGGGCTCGAGCTGCCCGCTTCGGTGCAC GCCATTTGCCAGGCCATGTGCTCCAGGAGCTGGGCCCGGAGCCTAGCGAGCCCGAGGG CGTGCGCGAGGCGCTGCGCCGAGCCTTCTTGAGCGCCGACGAGCGCCTGCGCTCCCTC TGGCCCCGCGTGGAAACGGGCGGCTGCACGGCCGTAGTGTTGCTGGTCTCCCCGCGGG TTCTGTACCTGGCGCACTGCGGTGACTCCCGCGCGGTGCTGAGCCGCGCTGGCGCCGT GGCCTTCAGCACAGAGGACCACCGGCCCCTTCGACCCCGGGAACGCGAGCGCATCCAC GCCGCTGGCGGCACCATCCGCCGCCGACGCGTCGAGGGCTCTCTGGCCGTGTCGCGAG CGTTGGGCGACTTTACCTACAAGGAGGCTCCGGGGAGGCCCCCCGAGCTACAGCTCGT TTCTGCGGAGCCAGAGGTGGCCGCACTGGCACGCCAGGCTGAGGACGAGTTCATGCTC CTGGCCTCTGATGGCGTCTGGGACACTGTGTCTGGTGCTTGCCTGGCGGGACTGGTGG CTTCACGCCTCCGCTTGGGCCTGGCCCCAGAGCTTCTCTGCGCGCAGCTGTTGGACAC GTGTCTGCTCCACCGGCTTCAGGGCAGCCTGGACAACATGACCTGCATCCTGGTCTGC TTCCCTGGGGCCCCTAGGCCTTCTGAGGAGGCGATCAGGAGGGAGCTAGCACTGGACG CAGCCCTGGGCTGCAGAATCGCTGGTGAGCAGACTCTGGGGTCTGCTCAGAAGCCCCC CAGCCTGAACACAGTTTTCAGGACTCTGGCCTCAGAGGACATCCCAGATTTACCTCCT GGGGGAGGGCTGGACTGCAAGGCCACTGTCATTGCTGAAGTTTATTCTCAGATCTGCC AGGTCTCAGAAGAGTGCGGAGAGAAGGGGCAGGATGGGGCTGGGAAGTCCAACCCCAC GCATTTGGGCTCAGCCTTGGACATGGAGGCCTGACAGCTGTTGTCCTTTGGGGATCCT TTGCTTCTCTGGGGCCTCAACAGAACTAAAGAAGAAAACCGACCCTTTCCCCAACTAC

ATGTACCAGCGGAAGGAAGGAAGGCCAATGTAGGAACCCAAAATGCTTATTTCTTCTT CTCTTACTTCCCTCTCACAGAAAAGTCTTACGAATGGGGAAATTCCACCAACATCCAG

ACCAAAAAGAAAAAAGCCCAAATCGAAAAAA

ORF Start: ATG at 16 ORF Stop: TGA at 1192

SEQ ID NO: 114 392 aa MW at 41721.9kD

NOV23a, M^WIPGWGRPHGGAEASGGLRFGASAAQGWRARMEDAHCTWLSLPGLPPGWALFAV LDGHGGARAARFGARH PGIf_,QE_GPEPSEPEGVREALRRAFLSADER_RS_WPRVE CG175257-01 TGGCTAWL VSPRVLYLAHCGDSRAVLSRAGAVAFSTEDHRPLRPRERERIHAAGGT Protein IRRRRVEGSLAVSRA GDFTYKEAPGRPPΞLQLVSAEPEVAA ARQAEDEFMLLASDG Sequence VTTOTVSGACLAG VASRLRLGLAPEL CAQL DTCLLHRLQGS DNMTCILVCFPGAP RPSEEAIRRE ALDAA GCRIAGEQTLGSAQ PPSL TVFRTLASEDIPDLPPGGGLD CKATVIAEVYSQICQVSEECGEKGQDGAGKSNPTHLGSAL.DMEA

Further analysis of the NOV23a protein yielded the following properties shown in Table 23B.

Table 23B. Protein Sequence Properties NOV23a

SignalP analysis: No Known Signal Sequence Predicted

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

N-region: length 11; pos.chg 2; neg.chg 0 H-region: length 5; peak value -2.92 PSG score: -7.32

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -2.86 possible cleavage site: between 54 and 55

»> Seems to have no N-terminal signal peptide

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 252) ALOM score: -1.01 (number of TMSs: 0)

MITDISC: discrimination of mitochondrial targeting seq R content: 2 Hyd Moment (75) : 7.32 Hyd Moment (95) : 8.82 G content: 4 D/E content: 1 S/T content: 0 Score: -4.59

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 45 ARM|ED

NϋCDISC: discrimination of nuclear localization signals pat4: RRRR (5) at 176 pat7 : none bipartite: none content of basic residues: 10.7% NLS Score: -0.16

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

ER Membrane Retention Signals:

XXRR-like motif in the N-terminus: RA I

SKL: peroxisomal targeting signal in the C-terminus: none PTS2 : 2nd peroxisomal targeting signal : none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

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

Final Results (k = 9/23):

43.5 % : cytoplasmic

34.8 % : mitochondrial

17.4 %: nuclear

4.3 % : peroxisomal

» prediction for CG175257-01 is cyt (k=23)

A search of the NOV23a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 23C.

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

PFam analysis predicts that the NOV23a protein contains the domains shown in the Table 23E.

Table 23E. Domain Analysis of NOV23a

Identities/

Pfam Domain NOV23a Match Region J Similarities Expect Value for the Matched Region

PP2C 22..279 112/304 (37%) l.le-72 197/304 (65%)

Example 24. ADAMTS-12 precursor

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

SEQ ID NO: 115 3528 bp

NOV24a, AGTGTGACGGCGCCCTGCTGCCTCCGCTCGTCCTGCCCGTGCTGCTGCTGCTGGTTT GGGGACTGGACCCGGGCACAGCTGTCGGCGACGCGGCGGCCGACGTGGAGGTGGTGCT CG175427-01 CCCGTGGCGGGTGCGCCCCGACGACGTGCACCTGCCGCCGCTGCCCGCAGCCCCCGGG DNA Sequence CCCCGACGGCGGCGACGCCCCCGCACGCCCCCAGCCGCCCCGCGCGCCCGGCCCGGAG AGCGCGCCCTGCTGCTGCACCTGCCGGCCTTCGGGCGCGACCTGTACCTTCAGCTGCG CCGCGACCTGCGCTTCCTGTCCCGAGGCTTCGAGGTGGAGGAGGCGGGCGCGGCCCGG CGCCGCGGCCGCCCCGCCGAGCTGTGCTTCTACTCGGGCCGTGTGCTCGGCCACCCCG GCTCCCTCGTCTCGCTCAGCGCCTGCGGCGCCGCCGGCGGCCTGGTTGGCCTCATTCA GCTTGGGCAGGAGCAGGTGCTAATCCAGCCCCTCAACAACTCCCAGGGCCCATTCAGT GGACGAGAACATCTGATCAGGCGCAAATGGTCCTTGACCCCCAGCCCTTCTGCTGAGG CCCAGAGACCTGAGCAGCTCTGCAAGGTTCTAACAGAAAAGAAGAAGCCGACGTGGGG CAGGCCTTTGCGGGACTGGCGGGAGCGGAGGAACGCTATCCGGCTCACCAGCGAGCAC ACGGTGGAGACCCTGGTGGTGGCCGACGCCGACATGGTGCAGTACCACGGGGCCGAGG CTGCCCAGAGGTTCATCCTGACCGTCATGAACATGGTATACAATATGTTTCAGCACCA GAGCCTGGGGATTAAAATTAACATTCAAGTGACCAAGCTTGTCCTGCTACGACAACGT CCCGCTAAGTTGTCCATTGGGCACCATGGTGAGCGGTCCCTGGAGAGCTTCGTCCACT GGCAGAACGAGGAGTATGGAGGAGCGCGATACCTCGGCAATAACCAGGTTCCCGGCGG GAAGGACGACCCGCCCCTGGTGGATGCTGCTGTGTTTGTGACCAGGACAGATTTCTGT GTACACAAAGATGAACCGTGTGACACTGTTGGAATTGCTTACTTAGGAGGTGTGTGCA GTGCTAAGAGGAAGTGTGTGCTTGCCGAAGACAATGGTCTCAATTTGGCCTTTACCAT CGCCCATGAGCTGGGCCACAACTTGGGCATGAACCACGACGATGACCACTCATCTTGC GCTGGCAGGTCCCACATCATGTCAGGAGAGTGGGTGAAAGGCCGGAACCCAAGTGACC TCTCTTGGTCCTCCTGCAGCCGAGATGACCTTGAAAACTTCCTCAAGTCAAAAGTCAG CACCTGCTTGCTAGTCACGGACCCCAGAAGCCAGCACACAGTACGCCTCCCGCACAAG CTGCCGGGCATGCACTACAGTGCCAACGAGCAGTGCCAGATCCTGTTTGGCATGAATG CCACCTTCTGCAGAAACATGGAGCATCTAATGTGTGCTGGACTGTGGTGCCTGGTAGA AGGAGACACATCCTGCAAGACCAAGCTGGACCCTCCCCTGGATGGCACCGAGTGTGGG GCAGACAAGTGGTGCCGCGCGGGGGAGTGCGTGAGCAAGACGCCCATCCCGGAGCATG TGGACGGAGACTGGAGCCCGTGGGGCGCCTGGAGCATGTGCAGCCGAACATGTGGGAC GGGAGCCCGCTTCCGGCAGAGGAAATGTGACAACCCCCCCCCTGGGCCTGGAGGCACA CACTGCCCGGGTGCCAGTGTAGAACATGCGGTCTGCGAGAACCTGCCCTGCCCCAAGG GTCTGCCCAGCTTCCGGGACCAGCAGTGCCAGGCACACGACCGGCTGAGCCCCAAGAA GAAAGGCCTGCTGACAGCCGTGGTGGTTGACGATAAGCCATGTGAACTCTACTGCTCG CCCCTCGGGAAGGAGTCCCCACTGCTGGTGGCCGACAGGGTCCTGGACGGTACACCCT GCGGGCCCTACGAGACTGATCTCTGCGTGCACGGCAAGTGCCAGAAAATCGGCTGTGA CGGCATCATCGGGTCTGCAGCCAAAGAGGACAGATGCGGGGTCTGCAGCGGGGACGGC AAGACCTGCCACTTGGTGAAGGGCGACTTCAGCCACGCCCGGGGGACAGGTTATATCG AAGCTGCCGTCATTCCTGCTGGAGCTCGGAGGATCCGTGTGGTGGAGGATAAACCTGC CCACAGCTTTCTGGCTCTCAAAGACTCGGGTAAGGGGTCCATCAACAGTGACTGGAAG ATAGAGCTCCCCGGAGAGTTCCAGATTGCAGGCACAACTGTTCGCTATGTGAGAAGGG GGCTGTGGGAGAAGATCTCTGCCAAGGGACCAACCAAACTACCGCTGCACTTGATGGT GTTGTTATTTCACGACCAAGATTATGGAATTCATTATGAATACACTGTTCCTGTAAAC CGCACTGCGGAAAATCAAAGCGAACCAGAAAAACCGCAGGACTCTTTGTTCATCTGGA CCCACAGCGGCTGGGAAGGGTGCAGTGTGCAGTGCGGCGGAGGGGAGCGCAGAACCAT CGTTTCGTGTACACGGATTGTCAACAAGACCACAACTCTGGTGAACGACAGTGACTGC CCTCAAGCAAGCCGCCCAGAGCCCCAGGTCCGAAGGTGCAACTTGCACCCCTGCCAGT CACGGTGGGTGGCAGGCCCGTGGAGCCCCTGCTCGGCGACCTGTGAGAAAGGCTTCCA GCACCGGGAGGTGACCTGCGTGTACCAGCTGCAGAACGGCACACACGTCGCTACGCGG CCCCTCTACTGCCCGGGCCCCCGGCCGGCGGCAGTGCAGAGCTGTGAAGGCCAGGACT GCCTGTCCATCTGGGAGGCGTCTGAGTGGTCACAGTGCTCTGCCAGCTGTGGTAAAGG GGTGTGGAAACGGACCGTGGCGTGCACCAACTCACAAGGGAAATGCGACGCATCCACG AGGCCGAGAGCCGAGGAGGCCTGCGAGGACTACTCAGGCTGCTACGAGTGGAAAACTG GGGACTGGTCTCAGTGCTCGTCGACCTGCGGGAAGGGCCTGCAGTCCCGGGTGGTGCA GTGCATGCACAAGGTCACAGGGCGCCACGGCAGCGAGTGCCCCGCCCTCTCGAAGCCT GCCCCCTACAGACAGTGCTACCAGGAGGTCTGCAACGACAGGATCAACGCCAACACCA TCACCTCCCCCCGCCTTGCTGCTCTGACCTACAAATGCACACGAGACCAGTGGACGGT ATATTGCCGGGTCATCCGAGAAAAGAACCTCTGCCAGGACATGCGGTGGTACCAGCGC TGCTGCCAGACCTGCAGGGACTTCTATGCAAACAAGATGCGCCAGCCACCGCCGAGCT CGTGACACGCAGTCCCAΑGGGTCGCTCAAAGCTCAGACTCAGG-CTGAAAGCCACCCA CCCGCAAGCCTACCAGCCTTGTGGCCACACCCCCACCCGGCTGCCACAAGAATCCAAC

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

Table 24B. Protein Sequence Properties NOV24a

SignalP analysis: Cleavage site between residues 28 and 29

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

N-region: length 3; pos.chg 0; neg.chg 1 H-region: length 19; peak value 0.00 PSG score: -4.40

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 0.56 possible cleavage site: between 21 and 22

>» Seems to have no N-terminal signal peptide

Tentative number of TMS(s) for the threshold 0.5: INTEGRAL Likelihood = -7.32 Transmembrane 6 22 INTEGRAL Likelihood = -3.13 Transmembrane 138 154 PERIPHERAL Likelihood = 5.30 (at 271) ALOM score: -7.32 (number of TMSs: 2)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 13 Charge difference: -4.0 C(-4.0) - N( 0.0) N >= C: N-terminal side will be inside

>» membrane topology: type 3a

MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75) : 7.80 Hyd Momen (95) : 5.89 G content: 2 D/E content: 2 S/T content: 0 Score: -7.79 Gavel: prediction of cleavage sites for mitochondrial preseq cleavage site motif not found

NϋCDISC: discrimination of nuclear localization signals pat4: PRRR (4) at 59 pat4: RRRR (5) at 60 pat4: RRRR (5) at 61 pat4: RRRP (4) at 62 pat4: RRPR (4) at 63 pat4: KKKP (4) at 207 pat4: PKKK (4) at 617 pat7: PGPRRRR (5) at 57 pat7: PRRRRRP (5) at 59 pat7: PKKKGLL (5) at 617 bipartite: KKKPT GRPLRDWRERR at 207 bipartite: KKPTWGRPLRDWRERRN at 208 content of basic residues: 12.3% NLS Score: 3.69

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

ER Membrane Retention Signals: none

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

PTS2 : 2nd peroxisomal targeting signal : none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

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

Zinc finger, C2H2 type, domain (PS00028) : *** found *** CSSTCGKGLQSRWQCMHKVTGRH at 1011 checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none

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

Final Results (k = 9/23) :

34.8 %: nuclear

30.4 %: endoplasmic reticulum

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

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

W

PFam analysis predicts that the NOV24a protein contains the domains shown in the Table 24E.

Example 25. TGFbRII splice variant.

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

Table 25A. NOV25 Sequence Analysis

SEQ ID NO: 117 1727 bp

NOV25a, GTTGGCGAGGAGTTTCCTGTTTCCCCCGCAGCGCTGAGTTGAAGTTGAGTGAGTCACT

CGCGCGCACGGAGCGACGACACCCCCGCGCGTGCACCCGCTCGGGACAGGAGCCGGAC CG175516-01 TCCTGTGCAGCTTCCCTCGGCCGCCGGGGGCCTCCCCGCGCCTCGCCGGCCTCCAGGC DNA Sequence CCCTCCTGGCTGGCGAGCGGGCGCCACATCTGGCCCGCACATCTGCGCTGCCGGCCCG

GCGCGGGGTCCGGAGAGGGCGCGGCGCGGAGCGCAGCCAGGGGTCCGGGAAGGCGCCG TCCGTGCGCTGGGGGCTCGGTCTATGACGAGCAGCGGGGTCTGCCATGGGTCGGGGGC TGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGTGGACGCGTATCGCCAGCACGAT CCCACCGCACGTTCAGAAGGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATA TTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCA TCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAGCTGAGTTCAACCTGGGAAAC CGGCAAGACGCGGAAGCTCATGGAGTTCAGCGAGCACTGTGCCATCATCCTGGAAGAT GACCGCTCTGACATCAGCTCCACGTGTGCCAACAACATCAACCACAACACAGAGCTGC TGCCCATTGAGCTGGACACCCTGGTGGGGAAAGGTCGCTTTGCTGAGGTCTATAAGGC CAAGCTGAAGCAGAACACTTCAGAGCAGTTTGAGACAGTGGCAGTCAAGATCTTTCCC TATGAGGAGTATGCCTCTTGGAAGACAGAGAAGGACATCTTCTCAGACATCAATCTGA AGCATGAGAACATACTCCAGTTCCTGACGGCTGAGGAGCGGAAGACGGAGTTGGGGAA ACAATACTGGCTGATCACCGCCTTCCACGCCAAGGGCAACCTACAGGAGTACCTGACG

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

Table 25B. Protein Sequence Properties NOV25a

SignalP analysis: Cleavage site between residues 24 and 25

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

N-region: length 7; pos.chg 2; neg.chg 0 H-region: length 11; peak value 6.69 PSG score: 2.29

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -1.94 possible cleavage site : between 51 and 52 '

>» Seems to have a cleavable signal peptide (1 to 51)

ALOM: Klein et al's method for _Λ region allocation Init position for calculation: 52

INTEGRAL Likelihood = -3.82 Transmembrane 52 - 68 PERIPHERAL Likelihood = 4.77 (at 264) ALOM score: -3.82 (number of TMSs: 1)

MTOP : Prediction of membrane topology (Hartmann et al . ) Center position for calculation: 25 Charge difference: -3.0 C(-0.5) - N( 2.5) N >= C: N-terminal side will be inside

>» membrane topology: type la (cytoplasmic tail 69 to 446)

MITDISC: discrimination of mitochondrial targeting seq R content: 3 Hyd Moment (75) : 13.49 Hyd Moment (95) : 15.91 G content: 3 D/E content: 1 S/T content: 3 Score: -0.61 Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 29 TRl|AS

NUCDISC: discrimination of nuclear localization signals pat4 : none pat7 : none bipartite: none content of basic residues: 11.4% NLS Score: -0.47

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

ER Membrane Retention Signals:

XXRR-like motif in the N-terminus: GRGL

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

PTS2 : 2nd peroxisomal targeting signal : none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: too long tail

Dileucine motif in the tail: found LL at 120 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) :

55.6 %: endoplasmic reticulum

22.2 %: Golgi

11.1 %: plasma membrane

11.1 %: extracellular, including cell wall

» prediction for CG175516-01 is end (k=9) A search of the NOV25a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 25C.

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

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

Example 26.

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

Table 26A. NOV26 Sequence Analysis SEQ ID NO: 119 2218 bp

NOV26a, TCCAAGCAGAGCCTCGGCGTGCCCCCAGGACCGGTAAAGTTCCTCTCGCCAGCCGCAT

CCATGCTTCTGGCGCGGATGAACCCGCAGGTGCAGCCCGAGAACAACGGGGCGGACAC CG175700-01 GGGTCCAGAGCAGCCCCTTCGGGCGCGCAAAACTGCGGAGCTGCTGGTGGTGAAGGAG DNA Sequence CGCAACGGCGTCCAGTGCCTGCTGGCGCCCCGCGACGGCGACGCGCAGCCCCGGGAGA CCTGGGGCAAGAAGATCGACTTCCTGCTGTCCGTAGTCGGCTTCGCAGTGGACCTGGC CAACGTGTGGCGCTTCCCCTACCTCTGCTACAAGAACGGCGGCGGTGCCTTCTTGATC CCGTACACACTGTTCCTTATCATCGCGGGGATGCCCCTGTTCTACATGGAGCTGGCTC TGGGACAGTACAACCGGGAGGGGGCTGCCACCGTTTGGAAAATCTGCCCATTCTTCAA AGGCGTTGGCTATGCTGTCATCCTGATCGCCCTGTACGTTGGCTTCTACTACAACGTC ATCATCGCCTGGTCACTCTACTACCTCTTCTCCTCCTTCACCCTCAACCTGCCCTGGA CCGACTGTGGCCACACCTGGAACAGCCCCAACTGTACCGACCCCAAGCTCCTCAATGG CTCCGTGCTTGGCAACCACACCAAGTACTCCAAGTACAAGTTCACGCCGGCAGCCGAG TTTTATGAGCGTGGTGTCCTGCACCTTCACGAGAGCAGCGGGATTCATGACATCGGCC TGCCCCAGTGGCAGCTCTTGCTCTGTCTGATGGTCGTCGTCATCGTCTTGTATTTTAG CCTCTGGAAAGGGGTGAAGACATCAGGAAAGGTATGGATTGATGCCGCAACTCAGATA TTTTTTTCCTTGGGGGCTGGATTTGGAGTATTGATTGCATTTGCCAGTTACAACAAAT TTGACAACAACTGTTACAGGGATGCCCTGCTGACCAGCAGCATCAACTGTATCACCAG CT CGTCTCTGGGTTCGCCATCTTCTCCATCCTTGGTTACATGGCCCATGAACACAAG GTCAACATTGAGGATGTGGCCACAGAAGGAGCTGGCCTAGTGTTCATCCTGTATCCAG AGGCCATT CTACCCTGTCTGGATCTACATTCTGGGCTGTTGTGTTTTTCGTCATGCT CCTGGCGCTGGGCCTTGACAGCTCAATGGGAGGCATGGAGGCTGTCATCACAGGCCTG GCAGATGACTTCCAGGTCCTGAAGCGACACCGGAAACTCTTCACATTTGGCGTCACCT TCAGCACTTTCCTTCTCGCCCTGTTCTGCATAACCAAGGGTGGAATTTACGTCTTGAC CCTCCTGGACACCTTTGCTGCGGGCACCTCCATCCTTTTTGCTGTCCTCATGGAAGCC ATCGGAGTTTCCTGGTTTTATGGAGTGGACAGGTTCAGCAACGACATCCAGCAGATGA TGGGGTTCAGGCCGGGTCTATACTGGAGACTGTGCTGGAAGTTCGTCAGTCCTGCCTT CCTCCTGTTCGTGGTTGTGGTCAGCATCATCAACTTCAAGCCACTCACCTACGACGAC TACATCTTCCCGCCCTGGGCCAACTGGGTGGGGTGGGGCATCGCCCTGTCCTCCATGG TCCTGGTGCCCATCTACGTCATCTATAAGTTCCTCAGCACGCAGGGCTCTCTT GGGA GAGACTGGCCTATGGCATCACGCCAGAGAACGAGCACCACCTGGTGGCTCAGAGGGAC ATCAGACAGTTCCAGTTGCAACACTGGCTGGCCATCTGAGCCTGCCTGGAGGAGAAGG

AGGAACCCCCATGCCAATGTCCAGGTCACAGGCATCCGCTGCGCTCCCACCTCGGACA

CCATCTTGGGATTCCTCCCCTGGAAGTTGTCCTTTCTGATCCTCTCTTCTTTTCCCAT iTTACAAATGATTTCGTGACTGTAGTTTTTGTTCACCTTCTGTGCATCTGGCCTGGGGG

CTGTTAGCTCAGAGGAGAGGAGCAAACAGGAAAATGACTTCTGTTCTGTCCCCGCTGT

TTTGGGGGAAGTCTCTCCCACTTTGGGATCCTGCTGAAGCTAGGTTCATGAGGTCGGA

AATCCCCACCACATTTGCCTAGACTTTGGGGCACAGGAGTTCTTAGTCCACCAAATCA

GAGAGAGGATGGGCTTTTGATCAGATACCCCTCCCAAAAAAAAAAAAAAATCTATGAC GGCTAAATGCCCGA

ORF Start: ATG at 61 ORF Stop: TGA at 1777

SEQ ID NO: 120 572 aa MW at 64315.4kD

NOV26a, M LARM PQVQPΞN GADTGPEQP RAR TAELLVVKERNGVQCLLAPRDGDAQPRET CG175700-01 GKKIDFL SVVGFAVDLAN TNFRFPYLCYKNGGGAFLIPYT FLIIAGMP FYMELA GQYI EGAATV KICPFFKGVGYAVI IALY /GFYYIWIIA SLYY FSSFTLNLPWT Protein DCGHTWNSPNCTDP L NGSVLG HTKYSKYKFTPAAEFYERGVLHLHESSGIHDIGL Sequence PQWQLLLC VVVIVLYFSLWKGVKTSGK^ IDAATQIFFSLGAGFGVLIAFASYNKF DLVLNCYI-A LTSSINCITSFVSGFAIFSILGYI HEH VNIEDVATEGAGLVFILYPE AISTLSGSTFWAVVTFVM I__^GLDSSMGGMEAVITGLADDFQVLKRHR LFTFGV F STF LA FCITKGGIYV TLLDTFAAGTSILFAVL_Ϊ:AIGVSWFYGVDRFSNDIQQMM GFRPGLY RLCWKFVS PAF LFWWS I INFKP TYDDYIFPPWANWVGWGI A S SMV VPIYVIYKF STQGS WERLAYGITPENEHH VAQRDIRQFQLQHWLAI

Further analysis of the NOV26a protein yielded the following properties shown in Table 26B.

Table 26B. Protein Sequence Properties NOV26a

SignalP analysis: No Known Signal Sequence Predicted PSORT II analysis: PSG: a new signal peptide prediction method

N-region: length 5; pos.chg 1; neg.chg 0 H-region: length 7; peak value -8.16 PSG score: -12.56

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -4.46 possible cleavage site: between 53 and 54

>» 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: 11

INTEGRAL Likelihood = -1.75 Transmembrane 63 - 79

INTEGRAL Likelihood = -4.41 Transmembrane 94 - 110

INTEGRAL Likelihood = -5.26 Transmembrane 140 - 156

INTEGRAL Likelihood =-11.36 Transmembrane 237 - 253

INTEGRAL Likelihood -4.09 Transmembrane 270 - 286

INTEGRAL Likelihood -2.55 Transmembrane 304 - 320

INTEGRAL Likelihood -6.37 Transmembrane 356 - 372

INTEGRAL Likelihood -4.35 Transmembrane 400 - 416

INTEGRAL Likelihood -2.23 Transmembrane 431 - 447

INTEGRAL Likelihood -7.54 Transmembrane 478 - 494

INTEGRAL Likelihood -2.28 Transmembrane 513 - 529

PERIPHERAL Likelihood 2.70 (at 334)

ALOM score: -11.36 (number of TMSs: 11)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 70 Charge difference: 2.0 C( 1.0) - N(-1.0) C > N: C-terminal side will be inside

>» membrane topology: type 3b

MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75) : 5.17 Hyd Moment (95) : 4.41 G content: 1 D/E content: 2 S/T content: 0 Score: -6.94

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 15 ARM|NP

NUCDISC: discrimination of nuclear localization signals pat4: KRHR (3) at 394 pat4: RHRK (3) at 395 pat7: PLRARKT (4) at 24 bipartite: none content of basic residues: 7.0% NLS Score: 0.25

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

ER Membrane Retention Signals:

XXRR-like motif in the N-terminus: LLAR

SKL: peroxisomal targeting signal in the C-terminus : none PTS2 : 2nd peroxisomal targeting signal : none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif: type 1 : none type 2 : none

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

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

Leucine zipper pattern (PS00029) : *** found *** LIPYTLFLIIAGMPLFYMELAL at 95 none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none

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

Final Results (k = 9/23) :

88.9 %: endoplasmic reticulum 11.1 %: mitochondrial

» prediction for CG175700-01 is end (k=9)

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

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

PFam analysis predicts that the NOV26a protein contains the domains shown in the Table 26E.

Example 27. Canalicular multispecific organic anion transporter 1.

The NOV27 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 27 A. Table 27A. NOV27 Sequence Analysis

SEQ ID NO: 121 ;4596 bp

NOV27a, GAAGAAACAACACAATCATATTAATAGAAGAGTCTTCGTTCCAGACGCAGTCCAGGAA CG175869-01 TCATGCTGGAGAAGTTCTGCAACTCTAC TTTTGGAATTCCTCATTCCTGGACAGTCC

GGAGGCAGACCTGCCACTTTGTTTTGAGCAAACTGTTCTGGTGTGGATTCCCT GGGC DNA Sequence TTCCTATGGCTCCTGGCCCCCTGGCAGCTTCTCCACGTGTATAAATCCAGGACCAAGA

GATCCTCTACCACCAAACTCTATCTTGCTAAGCAGGTATTCGTTGG TTTCTTCTTAT

TCTAGCAGCCATAGAGCTGGCCCTTGTACTCACAGAAGACTCTGGACAAGCCACAGTC

CCTGCTGTTCGATATACCAATCCAAGCCTCTACCTAGGCACATGGCTCCTGGTTTTGC

TGATCCAATACAGCAGACAATGGTGTGTACAGAAAAACTCCTGGTTCCTGTCCCTATT

CTGGATTCTCTCGATACTCTGTGGCACTTTCCAATTTCAGACTCTGATCCGGACACTC

TTACAGGGTGACAATTCTAATCTAGCCTACTCCTGCCTGTTCTTCATCTCCTACGGAT

TCCAGATCCTGATCCTGATCTTTTCAGCATTTTCAGAAAATAATGAGTCATCAAATAA

TCCATCATCCATAGCTTCATTCCTGAGTAGCATTACCTACAGCTGGTATGACAGCATC

ATTCTGAAAGGCTACAAGCGTCCTCTGACACTCGAGGATGTCTGGGAAGTTGATGAAG

AGATGAAAACCAAGACATTAGTGAGCAAGTTTGAAACGCACATGAAGAGAGAGCTGCA

GAAAGCCAGGCGGGCACTCCAGAGACGGCAGGAGAAGAGCTCCCAGCAGAACTCTGGA

GCCAGGCTGCCTGGCTTGAACAAGAATCAGAGTCAAAGCCAAGATGCCCTTGTCCTGG

AAGATGTTGAAAAGAAAAAAAAGAAGTCTGGGACCAAAAAAGATGTTCCAAAATCCTG

GTTGATGAAGGCTCTGTTCAAAACTTTCTACATGGTGCTCCTGAAATCAT CCTACTG

AAGCTAGTGAATGACATCTTCACGTTTGTGAGTCCTCAGCTGCTGAAATTGCTGATCT

CCTTTGCAAGTGACCGTGACACATATTTGTGGAΪTGGATATCTCTGTGCAATCCTCTT

ATTCACTGCGGCTCTCATTCAGTCTTTCTGCCTTCAGTGTTATTTCCAACTGTGCTTC

AAGCTGGGTGTAAAAGTACGGACAGCTATCATGGCTTCTGTATATAAGAAGGCATTGA

CCCTATCCAACTTGGCCAGGAAGGAGTACACCGTTGGAGAAACAGTGAACCTGATGTC

TGTGGATGCCCAGAAGCTCATGGATGTGACCAACTTCATGCACATGCTGTGGTCAAGT

GTTCTACAGATTGTCTTATCTATCTTCTTCCTATGGAGAGAGTTGGGACCCTCAGTCT

TAGCAGGTGTTGGGGTGATGGTGCTTGTAATCCCAATTAATGCGATACTGTCCACCAA

GAGTAAGACCATTCAGGTCAAAAATATGAAGAATAAAGACAAACGTTTAAAGATCATG

AATGAGATTCTTAGTGGAATCAAGATCCTGAAATATTTTGCCTGGGAACCTTCATTCA

GAGACCAAGTACAAAACCTCCGGAAGAAAGAGCTCAAGAACCTGCTGGCCTTTAGTCA

ACTACAGTGTGTAGTAATATTCGTCTTCCAGTTAACTCCAGTCCTGGTATCTGTGGTC

ACATTTTCTGTTTATGTCCTGGTGGATAGCAACAATATTTTGGATGCACAAAAGGCCT

TCACCTCCATTACCCTCTTCAATATCCTGCGCTTTCCCCTGAGCATGCTTCCCATGAT

GATCTCCTCCATGCTCCAGGCCAGTGTTTCCACAGAGCGGCTAGAGAAGTACTTGGGA

GGGGATGACTTGGACACATCTGCCATTCGACATGACTGCAATTTTGACAAAGCCATGC

AGTTTTCTGAGGCCTCCTTTACCTGGGAACATGAT CGGAAGCCACAGTCCGAGATGT

GAACCTGGACATTATGGCAGGCCAACTTGTGGCTGTGATAGGCCCTGTCGGCTCTGGG

AAATCCTCCTTGATATCAGCCATGCTGGGAGAAATGGAAAATGTCCACGGGCACATCA

CCATCAAGGGCACCACTGCCTATGTCCCACAGCAGTCCTGGATTCAGAATGGCACCAT

AAAGGACAACATCCTTTTTGGAACAGAGTTTAATGAAAAGAGGTACCAGCAAGTACTG

GAGGCCTGTGCTCTCCTCCCAGACTTGGAAATGCTGCCTGGAGGAGATTTGGCTGAGA

TTGGAGAGAAGGGTATAAATCTTAGTGGGGGTCAGAAGCAGCGGATCAGCCTGGCCAG

AGCTACCTACCAAAATTTAGACATCTATCTTCTAGATGACCCCCTGTCTGCAGTGGAT

GCTCATGTAGGAAAACATATTTTTAATAAGGTCTTGGGCCCCAATGGCCTGTTGAAAG

GCAAGACTCGACTCTTGGTTACACATAGCATGCACTTTCTTCCTCAAGTGGATGAGAT

TGTAGTTCTGGGGAATGGAACAATTGTAGAGAAAGGATCCTACAGTGCTCTCCTGGCC

AAAAAAGGAGAGTTTGCTAAGAATCTGAAGACATTTCTAAGACATACAGGCCCTGAAG

AGGAAGCCACAGTCCATGA GGCAGTGAAGAAGAAGACGATGACTATGGGCTGATATC

CAGTGTGGAAGAGATCCCCGAAGATGCAGCCTCCATAACCATGAGAAGAGAGAACAGC

TTTCGTCGAACACTTAGCCGCAGTTCTAGGTCCAATGGCAGGCATCTGAAGTCCCTGA

GAAACTCCTTGAAAACTCGGAATGTGAATAGCCTGAAGGAAGACGAAGAACTAGTGAA

AGGACAAAAACTAATTAAGAAGGAATTCATAGAAACTGGAAAGGTGAAGTTCTCCATC

TACCTGGAGTACCTACAAGCAATAGGATTGTTTTCGATATTCTTCATCATCCTTGCGT

TTGTGATGAATTCTGTGGCTTTTATTGGATCCAACCTCTGGCTCAGTGCTTGGACCAG

TGACTCTAAAATCΪTCAATAGCACCGACTATCCAGCATCTCAGAGGGACATGAGAGTT

GGAGTCTACGGAGCTCTGGGATTAGCCCAAGGTATATTTGTGTTCATAGCACATTTCT

GGAGTGCCTTTGGTTTCGTCCATGCATCAAATATCTTGCACAAGCAACTGCTGAACAA

TATCCTTCGAGCACCTATGAGATTTTTTGACACAACACCCACAGGCCGGATTGTGAAC

AGGTTTGCCGGCGATATTTCCACAGTGGATGACACCCTGCCTCAGTCCTTGCGCAGCT

GGATTACATGCTTCCTGGGGATAATCAGCACCCTTGTCATGATCTGCATGGCCACTCC

TGTCTTCACCATCATCGTCATTCCTCTTGGCATTATTTATGTATCTGTTCAGATGT T

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

Table 27B. Protein Sequence Properties NOV27a

SignalP analysis: Cleavage site between residues 51 and 52

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

N-region: length 4; pos.chg 1; neg.chg 1 H~region: length 12; peak value 5.06 PSG score: 0.66 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -5.39 possible cleavage site: between 45 and 46

>» Seems to have no N-terminal signal peptide

Tentative number of TMS(s) J Eor the threshold 0 1.5: 14

INTEGRAL Likelihood = -1 .38 Transmembrane 30 - 46

INTEGRAL Likelihood =- •11 .04 Transmembrane 70 - 86

INTEGRAL Likelihood = -2 .07 Transmembrane 131 - 147

INTEGRAL Likelihood = -4 .99 Transmembrane 165 - 181

INTEGRAL Likelihood = -0 .22 Transmembrane 334 - 350

INTEGRAL Likelihood = -6 .90 Transmembrane 357 - 373

INTEGRAL Likelihood = -2 .92 Transmembrane 440 - 456

INTEGRAL Likelihood = -7 .86 Transmembrane 463 - 479

INTEGRAL Likelihood = -8 .55 Transmembrane 544 - 560

INTEGRAL Likelihood = 0 .47 Transmembrane 657 - 673

INTEGRAL Likelihood = -8 .92 Transmembrane 971 - 987

INTEGRAL Likelihood = -2 .71 Transmembrane 1024 -1040

INTEGRAL Likelihood = -7, .80 Transmembrane 1112 -1128

INTEGRAL Likelihood = -3 .24 Transmembrane 1213 -1229

PERIPHERAL Likelihood = 1 .27 (at 588)

ALOM score: -11.04 (number of TMSs: 14)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 37 Charge difference: 9.5 C( 5.5) - N(-4.0) 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: 0 Hyd Moment (75) : 10.88 Hyd Moment (95) : 12.51 G content: 0 D/E content: 2 S/T content: 4 Score: -4.43

NUCDISC: discrimination of nuclear localization signals pat4: KKKK (5) at 294 pat4: KKKK (5) at 295 pat7 : none bipartite: KRELQKARRALQRRQEK at 247 content of basic residues: 10.4% NLS Score: 0.65

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

ER Membrane Retention Signals:

KKXX-like motif in the C-terminus: NSTK

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

Tyrosines in the tail: none

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

Leucine zipper pattern (PS00029) : *** found *** LSIGQRQLLCLGRALLRKSKIL at 1384 none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none

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

Final Results (k = 9/23) :

77.8 %: endoplasmic reticulum 11.1 %: mitochondrial 11.1 %: vacuolar

» prediction for CG175869-01 is end (k=9)

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

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

PFam analysis predicts that the NOV27a protein contains the domains shown in the Table 27E.

Example 28. Phospholipid transporting ATPase Class II type 9A.

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

Table 28A. NOV28 Sequence Analysis

SEQ ID NO: 123 7466 bp

NOV28a, GCGAGCGGGCGCAGCGCGGAGCTCGGGCCCATGGTGCGCCCGTGTCCGTCGGTCGGGC

CGCGCGGGCGCCTCCGCGCGTGGCCCGGCGCTCGCGAGCTCGCCCCCTCGCTGCGGGC CG175900-01 CCGGCCCGCCCGCTGCCGCCGCCTCCTCCCCCGGGGCGGCGCGGCGCCGGCGGGCGGC DNA Sequence GGCGCGGAGGCCGGACCGGGCGGCGGCCCAGGCGGCGCGGGGGGCGCGGCGGCCAAGG CGGGCGGCGCCGCCGACATGACGGACAACATCCCGCTGCAGCCGGTGCGCCAGAAGAA GCGGATGGACAGCAGGCCCCGCGCCGGGTGCTGCGAGTGGCTGAGATGCTGCGGTGGA GGGGAGGCCAGGCCCCGCACTGTCTGGCTGGGGCACCCCGAGAAGAGAGACCAGAGGT ATCCTCGGAATGTCATCAACAATCAGAAGTACAATTTCTTCACCTTTCTTCCTGGGGT GCTGTTCAACCAGTTCAAATACTTTTTCAACCTCTATTTCT ACTTCTTGCCTGCTCT CAGTTTGTTCCCGAAATGAGACTTGGTGCACTCTATACCTACTGGGTTCCCCTGGGCT TCGTGCTGGCCGTCACTGTCATCCGTGAGGCGGTGGAGGAGATCCGATGCTACGTGCG GGACAAGGAAGTCAACTCCCAGGTCTACAGCCGGCTCACAGCACGAGGTACTGTTGTG GGTGTTGTTCTTTACACTGGCAGAGAACTCCGGAGTGTCATGAATACCTCAAATCCCC GAAGTAAGATCGGCCTGTTCGACTTGGAAGTGAACTGCCTCACCAAGATCCTCTTTGG TGCCCTGGTGGTGGTCTCGCTGGTCATGGTTGCCCTTCAGCACTTTGCAGGCCGTTGG TACCTGCAGATCATCCGCTTCCTCCTCTTGTTTTCCAACATCATCCCCATTAGTTTGC GCGTGAACCTGGACATGGGCAAGATCGTGTACAGCTGGGTGATTCGAAGGGACTCAAA AATCCCCGGGACCGTGGTTCGCTCCAGCACGATTCCTGAGCAGCTGGGCAGGATTTCG TACTTACTCACAGACAAGACAGGCACTC TACCCAGAACGAGATGATTTTCAAACGGC TCCATCTCGGAACAGTAGCCTACGGCCTCGACTCAATGGACGAAGTACAAAGCCACAT TTTCAGCATTTACACCCAGCAATCCCAGGACCCACCGGCTCAGAAGGGCCCAACGCTC ACCACTAAGGTCCGGCGGACCATGAGCAGCCGCGTGCACGAAGCCGTGAAGGCCATCG CGCTCTGCCACAACGTGACTCCCGTGTATGAGTCCAACGGTGTGACTGATCAGGCTGA GGCCGAGAAGCAGTACGAAGACTCCTGCCGCGTATACCAGGCATCCAGCCCCGATGAG GTGGCCCTGGTACAGTGGACGGAAAGTGTGGGCTTAACCCTGGTGGGCCGAGACCAGT CTTCCATGCAGCTGAGGACCCCTGGCGACCAGATCCTGAACTTCACCATCCTACAGAT CTTCCCTTTCACCTATGAAAGCAAACGTATGGGCATCATCGTGCGGGATGAATCAACT GGAGAAATTACGTTTTACATGAAGGGAGCAGATGTGGTCATGGCTGGCAT GTGCAGT ACAATGACTGGTTGGAGGAAGAGTGTGGCAACATGGCCCGAGAAGGGCTGCGGGTGCT CGTGGTGGCAAAGAAGTCTCTTGCAGAGGAGCAGTATCAGGACTTTGAAGCCCGCTAC GTCCAGGCCAAGCTGAGTGTGCACGACCGCTCCCTCAAAGTGGCCACGGTGATCGAGA GCCTGGAGATGGAGATGGAACTGCTGTGCCTGACGGGCGTGGAGGACCAGCTGCAGGC AGATGTGCGGCCCACGCTGGAGACCCTGAGGAATGCTGGCATCAAGGTTTGGATGCTG ACAGGGGACAAGCTGGAGACAGCTACGTGCACAGCGAAGAATGCACATCTGGTGACCA GAAACCAAGACATCCACGTTTTTCGGCTGGTGACCAACCGCGGGGAGGCTCACCTCGA GCTGAACGCCTTCCGCAGGAAGCATGATTGTGCCCTGGTCATCTCGGGAGACTCCCTG GAGGTTTGCCTCAAGTACTATGAGTACGAGTTCATGGAGCTGGCCTGCCAGTGCCCGG CCGTAGTCTGCTGCCGATGTGCCCCCACCCAGAAGGCCCAGATCGTGCGCCTGCTTCA GGAGCGCACGGGCAAGCTCACCTGTGCAGTAGGGGACGGAGGCAATGACGTCAGCATG ATTCAGGAATCTGACTGCGGCGTGGGAGTGGAAGGAAAGGAAGGAAAACAGGCTTCGT TGGCTGCAGACTTCTCCATCACTCAATTTAAGCATCTTGGCCGGTTGCTTATGGTGCA TGGCCGGAACAGCTACAAGCGGTCAGCCGCCCTCAGCCAGTTCGTGATTCACAGGAGC CTCTGTATCAGCACCATGCAGGCTGTCTTTTCCTCCGTGTTTTACTTTGCCTCCGTCC CTCTCTATCAAGGATTCCTCATCATTGGGTACTCCACAATTTACACCATGTTTCCTGT GT TTCTCTGGTCCTGGACAAAGATGTCAAATCGGAAGTTGCCATGCTGTATCCTGAG CTCTACAAGGATCTTCTCAAGGGACGGCCGTTGTCCTACAAGACATTCTTAATATGGG TTTTGATTAGCATCTATCAAGGGAGCACCATCATGTACGGGGCGCTGCTGCTGTTTGA GTCGGAGTTCGTGCACATCGTGGCCATCTCCTTCACCTCGCTGATCCTCACCGAGCTG CTCATGGTGGCGCTGACCATCCAGACCTGGCACTGGCTCATGACAGTGGCGGAGCTGC TCAGCCTGGCCTGCTACATCGCCTCCCTGGTGTTCTTACACGAGTTCATCGATGTGTA CTTCATCGCCACCTTGTCATTCTTGTGGAAAGTCTCCGTCATCACTCTGGTCAGCTGC CTCCCCCTCTATGTCCTCAAGTACCTGCGAAGACGGTTCTCTCCCCCCAGCTACTCAA AGCTCACATCATAGGCCGTGCGTTCGCTGGAGGGGGCCCTGGTCTTGGCGCTTCCCTG ATGGACAGAGCTCAAGTTCCATTTATATTAACCGCCACCTGTGGATTTTGCAGTAATT GCTAACACATGCAGTTTTAATGGGAAGCGGCTCTGCGCCTAAACGGAGTCCTAACGCT

GCATCAACGGGAGGGAGGGTCCTGAAAGAGACCCATCTGGGCCTGTCTGAACCCCTCG TTCTTCATGTTTAGGTGAATATGAATATGTTAAAGCTGGTGGCTCAGCTGGGAGATTT ATATGGGTCACTGTGCGAGCTTCCTTATGACTTGAATTTTGTTGTCACATGATAAAAG

TTTCTGTGTAGCTGAAGGTTGTAGAAGGCTTGTGTGTGTGTGTGTGTGTGTGTGTGTG TGTGTGTGTGTGTT TTAAAGAGTCCTAATGTGTATGTACTCTTTATGTCTTTCTTGC TCTTACAAAGAGGTGTCAGAAAAATAGAAAGCTC GGTGTCGGTTTGGGAGGAAAAG

ACAGTGACATTTGGTAAAAAGTTATCCACACAATAATCTCCATTCGGAAATGCTCAGT

ATCGTCTCCAGCCAGCCCTGCTTATCCAGGTTACACTGGATTCCTGGGATCGTAACCA

GTAAATGAGAGGAGAGGGAGAGAGAGTGTCCTAAGTCCAATCTGTTATCCTTGATCTG

ATTCAGCATCCATAGTGTGTGAGTTAACTTCACCTGCCACCTCGTAAAAGAATTTCAG AGGTGTGATCCCGCTTTATTGGGACCTGGTAACAATCACAAAGCCAGTGGCTGTTTGA GAAGGACCTCAGACATTTTCAGCAGAGTTGTTTTAGCAGGAAACGTGCCACTGAATGG

CCCCTAAATGTGTCGACAGTGTGATAAGAGACTCAACTAATTCTTTAGGCAACATGGC AGATGTGACTCAGATCCTCCAAGACCAAAGCGGAAAGGTCAGGGGGCTGGGACTCTTC TCTTCCATAGAAGCCTGTTTCCTGTTAGGAGGCATAATGGAAGATGACCCCACAAAGG

CAGAGGCATCTTTCGGAACAACACTGGTGGCAGCTTTCAGAACAAGGAACCCCTGGTG

IGGAGGACGCCCAAGCTACAGCGTTGGGATCTGGGATCTGTTCCACTGCCGGCAGATTT

CAAGGGGAACTTGCTGAAAGGCAGCCAGTGGTGAAGATTTCTCCCCTCCCAGGATGGA

CTACATGCCGGCATGTTTCTTATAAAGCTGTGGCTGCTTGTTTCAGAGGAAGGGAGTT TGCAGTCGCGGGACGTGGTAGAGCAAGGCATTCTTGGGTTTTCAAGTTGCTTCTTGCA

GAAGCCACATATGCATGCCATAAGGGTTAAGTTGGTGGATCTTTAAGAGCCAAGTGTG

GTTGAGATCTTGGATTTGCGTTTACTTCTTGATGAATACATATCCT CAAACCCTCTG

CCTGGCGCTACTTCTGTGTGCTTCAGAGAGGTACATCACAGCCTGGT TCTGATGCCT

ACTAACTCCTGCTCTTGGAGAGCTGGAGACACGAGGATCAGATAGTCCCTTGCCTTTG

GAGCACTCTTGATAAGCTTTTGTATTTTGTGT GTCCTTTTAAAATGTTCTAGAATGA

CTTTACGTTGCAGGTACTGGTTAATTGGCTGTTGACACCACATCTATTTTGTCTTATG

ATTCTGCAGTTTTGCAGTACTTTTCTCTATCTGATTCAGCCAT TCTGCCAGAGGGAA

AAGGTCGGCAGAAAAGATGTATTGAGTGAATAGTTAAGGATAGGATCTTTGTCCAAAA

ATTTCAGAAAGATTGAGCAAATCTGACGTATTCATTGAGTGAGTTTCTGTGTTTTCAA lAGGTGGAGGAGAAATTTGTGCTGGAAGTTTTTAAGCCTCCGTTTTCTTGGAAATCAGT

CTGTAACACTGGCAAGTCTTAAGATAGTCCCGTTTAGACTTTGCAGATGCTGAACCTG

IGCTCTGTAACGCTGGGAAGTCTTAAGATAGTCCTGTTTAGACTTTGCAAACCCTGTAC

CTGGCTTTGCTCGGAGATTCGGGATGCTGGCTCCTGCAGGCAGGGCGTGTGGGAGCCT

CGTCAGAAAGTTTTAGAGGTTTCCAGCAGAAGCAGAATGAAGATGGTCTCCCTGGCCT

TTTCCTTAATTCTCAATTTTGAT GAGGTGCACAAGTTGACTTTTAAAGCCAACGCTT

AAGATACTGATTGACATCTTCAAGGGAGAATGCTCCCAGGAGGGGCTGAAGAAGCCAT

AGTTGGAAGTGGAAGGTACTCGTCAGTGTTCTCCACAAACCTTTTTACTCTGTTGTCT

CAGCCGCACTGGGGCGGAGGCGGTCAAGGGTGAGAAGTACCGACACTCAAGTGCAAAC

TGCCACGTCGTTGGCCCATCCCATCAGTGGGCAGCTGGCTGACGCCATTCACTGGACG GTCCCTGAACACCTAGGAATGCACACACCGTGCTTCTCAGACACTGGAGACGCAAAGG

CAGGAGGATGCAGTCCGGTGAGAGGACACGATCTTTACCTGCACAATCAGACTGTAAG

CCCAGCAGAGAACCCCAGGGGCGCCTGGGTACTTCTCGGAGGTCATCTTAGTTGTGGT

GGGGAAGACAAAGAAATAAGCAAACAAGAAACTAGAGTTACTATACAAGAAACTCTCC

TGAGTTTGTAAACCTTAAGCATAAGGATTCAGTTGACCTTTTTCTTGGTTCATCAATC

TGGAAAGAACTTACATAAAGCGCCATTGACACTGTCACCTGGGAGCTCCATGGGCCGT AAGTCTTTGACAGCCAATTTAATTTGAGGTCAGAGGGCCTTGAGGTACACAGTCAGCA CTGTTTGAACACTTTTCCTGAAAGCAAAACTCACAGCTCCCTGCGCCCTCTGACAACA

CTAGCTATTTCTGCCAGAGTAAGAACTTCTATTACTATTTTATTATTGTTCATATGTC T TTGATGATGGTTGTGTGACAGGGGGAAGCAGGATCTATTTGGTTTCTTCCCCCTCC CCCCACCCCTTCCTTTTTGTCTCTCT TTTTTTTCTCTAAGAAAATCACCAGACTAGT

TTTTCCATCTTGAGTAATTTCTTATGTGGGACAGTTTTGATCCTCATTTTGAAAGCAT GCGTGCGCACATGTGTGTTGCCTGTGGTGCCAGGTGAGACAGGTGGCACTAACTCCAG CTGCTTGGAAGGCATCCCAAGGGCGCATCTTAAAGTTGGAGCAGACCTCCCTTTTCCA

GCCCCTGGGGCCATTAGACCACGTGCTGGAACTAGCATTGTAAAATTCCCATCCCAGT TCCACTCCCCTGAAGTGAAACCCTTTTTT TTTGTGACAGTAAATCTTAAAAATCATT GTCTCTTTATGAACATTTCCTCAGTTTCTTCTCTGCTGAAAATGTAAGCCATGCTACT

TTTTAATGTATTTTGAATTTTGTGCTCATTGGAAATTGATATGCTAATGCCTCCCCCA CCCCCCGCCAGACTTTTCTTTTTATACTTTGTCTTGTT TTACTGGGGTAGGCTGGGC ATGCGTGCGTGCCTTTAGGGCAGCATTTTAAACCTTTGCCAAAATTGCAAATGGGACA

TGTACATTCTTCTGCTCCATCCTACTTAAACACCTATCAGCTATTTTTATCTTTAACC TTTTCTGTATGTTTGAAGTGTGTGGGGGGTGTGTGTGTGTGTGAAAGAGCGAGAGAAT GATGTCATCTAAAGTTTTTTGAAGAATTATTTGGTTTTCATTGCATTAAAATTCTATC ACTCCCAGCTT GTTTTCATTTAAAAAAATATACAAAGAGCTTTGTAAATACAACACA TTTTATTTCTCCCCCTTCTTTTAATGTACAGCTTTTTTGCCACTTATATATACTTAAAJ

ATATTCCCATGAATTATGTCCAGTTCTTCTTGGAAAAAAAT TGGTTTTGAATGAACC

TGCAAAGCATCCTGCAGCGTGAGCAGCTCCTCCACCTGGAGCTCCGAAGCATCTTCTC

AGGCCAAAGCGGCATTACCCGTGAATCTGTCTTCTCCGCCACAGCATGGTTTGAGGCGi

CAGTCTGTTAATATAGCTGGGCCATGTCAGTGACTGTTGTGTTTGTGGGGTCAGGTGGl

GGGGCATGGTATTTGCAAAAAAAACAAATTATGGCTAATTTATTATTTTGTTGCAGTG

GGGTTAACTGTAAACTCATGTAAGAGTCTGTGATTTCCTCATTGGTTGATCTCTCTCT

CTGTAATCCTCATTGCAAATTTTCACCAGGACAGCGTTTT--'GATTAGAGGGGAGCTCi

TGGCACAGTATGCTTTAATTTAGCAGGAACTTCCAGATGATTTAAATTCTCGATGCTGl

TGATGACACACATATGATCTTTCGTGTTTCTGAGCGACTCTACTTTCATTGTTTGCCAl

GCGTGGCTCGTTGCTGTTGCCCAATAAAGCTTGTGTACGTTC

ORF Start: ATG at 31 ORF Stop: TAG at 3028

SEQ ID NO: 124 999 aa MW at ll2020.0kD

NOV28a, MVRPCPSVGPRGRLRA PGARELAPSLRARPARCRR LPRGGAAPAGGGAEAGPGGGP GGAGGAAAKAGGAADMTDNIP QPVRQKKRMDSRPRAGCCEW RCCGGGEARPRTVWL CG175900-01 GHPEKJRDQRYPR VI NQKYWFTFLPGVLFNQFKyFF LYFIilj ACSQFVPEMRLGA Protein LYTY VPLGFVLATVIREAVEEIRCYVRDKEVNSQVYSRLTARGTVVGVVLYTGREL Sequence RSVMNTSNPRSKIGLFDLEVNCLTKILFGALVVVSLVMVALQHFAGR YLQIIRFL L FSNIIPISLRVNLDMGKIVYS VIRRDSKIPG WRSSTIPEQLGRISY DKTGT TQNEMIFKR H GTVAYG DSMDEVQSHIFSIYTQQSQDPPAQKGPTLTTKVRRTMSS RVHEAVKAIALCH V PVYESNGVTDQAEAEKQYEDSCRVYQASSPDEVALVQWTESV GLTLVGRDQSSMQ RTPGDQILNFTILQIFPFTYESKR GIIVRDΞSTGEITFY KGA DV/MAGIVQYrø3 LEEECGl\ AREGLRVLVVAKKSLAEEQYQDFEARYVQAKLSVHDR SLKVAVIESLEMEMELLCLTGVEDQLQADVRPTLETLRNAGIKVWMLTGDKLETATC TA NAHLV R QDIHvTRLV RGEAHLE NAFRRKHDCA VISGDSLEVCLKYYEYE FME ACQCPAWCCRCAPTQKAQIVR LQERTGKLTCAVGDGG DVSMIQESDCGVGV EGKEGKQASLAADFSITQFKΗ GRLL VHGRNSY RSAA SQFVIHRSLCIST QAVF SSVFYFASVPLYQGF IIGYSTIYTMFPVFSLV DKDVKSEVAMLYPELY D KGRP LSYKTF IWWilSIYQGSTIMYGAL FESEFVHIVAISFTSLILTE LMVALTIQT rWLMTVAEL SLACYIASLVFLHEFIDVYFIATLSFL KVSVITLVSC P YVLKY R RRFSPPSYSK-TS

Further analysis of the NOV28a protein yielded the following properties shown in Table 28B.

Table 28B. Protein Sequence Properties NOV28a

SignalP analysis: No Known Signal Sequence Predicted

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

N-region: length 11; pos.chg 2 ; neg.chg 0 H-region: length 1; peak value -8.64 PSG score: -13.04

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -₂.1): -6.80 possible cleavage site: between 34 and 35

>» Seems to have no N-terminal signal peptide

ALOM: Klein et al's method for TM region allocation nit position for calculation: 1 Tentative number of TMS(s) for

INTEGRAL Likelihood = -1 86 Transmembrane 175 - 191

INTEGRAL Likelihood = -9 24 Transmembrane 258 - 274

INTEGRAL Likelihood = -0 90 Transmembrane ₂82 - 298

INTEGRAL Likelihood = -5 41 Transmembrane 905 - 921

INTEGRAL Likelihood = -4 94 Transmembrane 934 - 950

INTEGRAL Likelihood = -3 03 Transmembrane 958 - 974

PERIPHERAL Likelihood = 0 53 (at 815)

ALOM score: -9. 4 (number of TMSs: 6) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 182 Charge difference: -1.0 C(-1.0) - N( 0.0) N >= C: N-terminal side will be inside

>» membrane topology: type 3a

MITDISC: discrimination of mitochondrial targeting seq R content: 5 Hyd Moment (75) : 7.20 Hyd Moment (95) : 9.64 G content: 3 D/E content: 1 S/T content: 1 Score: -1.00

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 50 PRG|GA

NϋCDISC: discrimination of nuclear localization signals pat4: RRKH (3) at 672 pat7: PARCRRL (4) at 31 pat7: PVRQKKR (4) at 82 bipartite: none content of basic residues: 11.0% NLS Score: 0.40

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

ER Membrane Retention Signals:

XXRR-like motif in the N-terminus: VRPC

KKXX-like motif in the C-terminus: SKLT

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

PTS2 : 2nd peroxisomal targeting signal : none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

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

66.7 % : endoplasmic reticulum

22.2 % : nuclear

11.1 %: mitochondrial

» prediction for CG175900-01 is end (k=9)

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

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

PFam analysis predicts that the NOV28a protein contains the domains shown in the Table 28E.

Example 29.

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

Table 29A. NOV29 Sequence Analysis

SEQ ID NO: 125 2599 bp

NOV29a, CCAGCAGTTGTAATTAGCACCCCGGGTGTCAGCCAGAAACCAACAAACAGCCAAATCC;

CTGCAGCCCCGCCCAGCCTATCCACCGGCGGGGGACCGATTAACCATTAACCCCCACC CG176069-01 CCTCCCCGGCAGAGCCTCCACCCCTTCACAGAGGCTAGGCCAAGACTCCCAGCAGATC DNA Sequence TTCCCAGAGGACGGTTTGAAAGGAAGGCAGAGAGGGCACTGGGAGGAGGCAGTGGGAG

GGCGGAGGGCGGGGGCCTTCGGGGTGGGCGCCCAGGGTAGGGCAGGTGGCCGCGGCGT

GGAGGCAGGGAGAATGCGACTCTCCAAAACCCTCGTCGACATGGACATGGCCGACTAC AGTGCTGCACTGGACCCAGCCTACACCACCCTGGAATTTGAGAATGTGCAGGTGTTGA CGATGGGCAATGGCCCTTCCAGTCCTCACTGCCTCACAGTGGCTCTGCTTGGCGCTTG GCACAGTGACATGATGATTTTGTTGCCGCTGCGTCTCGCCAGATTGAGGCATCCCCTC CGACATCACTGGAGCATATCTGGAGGGGTGGACAGTTCTCCACAGGGAGACACGTCCC CATCAGAAGGCACCAACCTCAACGCGCCCAACAGCCTGGGTGTCAGCGCCCTGTGTGC CATCTGCGGGGACCGGGCCACGGGCAAACACTACGGTGCCTCGAGCTGTGACGGCTGC AAGGGCTTCTTCCGGAGGAGCGTGCGGAAGAACCACATGTACTCCTGCAGATTTAGCC GGCAGTGCGTGGTGGACAAAGACAAGAGGAACCAGTGCCGCTACTGCAGGCTCAAGAA ATGCTTCCGGGCTGGCATGAAGAAGGAAGCCGTCCAGAATGAGCGGGACCGGATCAGC ACTCGAAGGTCAAGCTATGAGGACAGCAGCCTGCCCTCCATCAATGCGCTCCTGCAGG CGGAGGTCCTGTCCCGACAGATCACCTCCCCCGTCTCCGGGATCAACGGCGACATTCG GGCGAAGAAGATTGCCAGCATCGCAGATGTGTGTGAGTCCATGAAGGAGCAGCTGCTG GTTCTCGTTGAGTGGGCCAAGTACATCCCAGCTTTCTGCGAGCTCCCCCTGGACGACC AGGTGGCCCTGCTCAGAGCCCATGCTGGCGAGCACCTGCTGCTCGGAGCCACCAAGAG ATCCATGGTGTTCAAGGACGTGCTGCTCCTAGGCAATGACTACATTGTCCCTCGGCAC TGCCCGGAGCTGGCGGAGATGAGCCGGGTGTCCATACGCATCCTTGACGAGCTGG GC TGCCCTTCCAGGAGCTGCAGATCGATGACAATGAGTATGCCTACCTCAAAGCCATCAT CTTCTTTGACCCAGATGCCAAGGGGCTGAGCGATCCAGGGAAGATCAAGCGGCTGCGT TCCCAGGTGCAGGTGAGCTTGGAGGACTACATCAACGACCGCCAGTATGACTCGCGTG GCCGCTTTGGAGAGCTGCTGCTGCTGCTGCCCACCTTGCAGAGCATCACCTGGCAGAT GATCGAGCAGATCCAG CATCAAGCTCTTCGGCATGGCCAAGATTGACAACCTGT G CAGGAGATGCTGCTGGGAGGTCCGTGCCAAGCCCAGGAGGGGCGGGGTTGGAGTGGGG ACTCCCCAGGAGACAGGCCTCACACAGTGAGCTCACCCCTCAGCTCCTTGGCTTCCCC ACTGTGCCGCTTTGGGCAAGTTGCTTAACCTGTCTGTGCCTCAGTTTCCTCACCAGAA

AAATGGGAACAAGGCAATGGTCTATTTGTTCAGGCACCGAGAACCTAGCACGTGCCAG

TCACTGTTCTAAGTGCTGGCAA-TCAGCAAAGAACAAGATCTTTGCCCTCGGGGAGGC

TGTGTGTGTGTGAGTATGTATGGATGCGTGGATATCTGTGTATATGCCCGTATGTGCG TGCATGTGTATATAAAGCCTCACATTTTATGATTTTGAAATAAACAGGTAATATGAGG TCCCCCAGCGATGCACCCCATGCCCACCACCCCCTGCACCCTCACCTGATGCAGGAAC

ATATGGGAACCAACGTCATCGTTGCCAACACAATGCCCACTCACCTCAGCAACGGACA GATGTGTGAGTGGCCCCGACCCAGGGGACAGGCAGCCACCCCTGAGACCCCACAGCCC TCACCGCCAGGTGGCTCAGGGTCTGAGCCCTATAAGCTCCTGCCGGGAGCCGTCGCCA

CAATCGTCAAGCCCCTCTCTGCCATCCCCCAGCCGACCATCACCAAGCAGGAAGTTAT CTAGCAAGCCGCTGGGGCTTGGGGGCTCCACTGGCTCCCCCCAGCCCCCTAAGAGAGC ACCTGGTGATCACGTGGTCACGGCAAAGGAAGACGTGATGCCAGGACCAGTCCCAGAG

CAGGAATGGGAAGGATGAAGGGCCCGAGAACATGGCCTAAGGCACATCCCACTGCACC CTGACGCCCTGCTCTGATAACAAGACTTTGACTTGGGGAGACCCTCTACTGCCTTGGA

CAACT TCTCATGTTGAAGCCACTGCCTTCACCTTCACCTTCATCCATGTCCAACCCC CGACTTCATCCCAAAGGACAGCCGCCTGGAGATGACTTGAGCCTTAC

ORF Start: ATG at 304 ORF Stop: TAA at 1708

SEQ ID NO: 126 468 aa MW at 52166.3kD

NOV29a, iyRLSKTLVD] ro__YSAALDPAYTTLEFE ntQVLTMGNGPSSPHCLTVA GAWHSDM

MILLPLRIARLRHPLRHHWSISGGVDSSPQGDTSPSEGTNLNAPNSLGVSA CAICGD

CG176069-01 RATGKHYGASSCDGCKGFFRRSVRKNHMYSCI^

Protein G K EAVQNERDRISTI^SSYEDSSLPSINALLQAEV SRQITSPVSGINGDIRAKKI

ASIADVCESMKEO LVLVEWA YIPAFCE P DDOVALLRAHAGEHLLLGATKRSMVF Sequence KDV LLGNDYIVPRHCPELAEMSRVSIRILDE VLPFQΞLQIDDNEYAYLKAIIFFDP DAKGLSDPGKIKR RSQVQVSLEDYINDRQYDSRGRFGELL LLPTLQSITWQMIEQI QFIKLFGMAKIDN LQEML GGPCQAQEGRG SGDSPGDRPHTVSSPLSSLASP CRF GQVA

Further analysis ofthe NO V29a protein yielded the following properties shown in Table 29B.

Table 29B. Protein Sequence Properties NOV29a

SignalP analysis: Cleavage site between residues 68 and 69

PSORT π PSG: a new signal peptide prediction method

N-region: length 11; pos.chg 2; neg.chg 2 analysis: H-region: length 2; peak value 0.89 PSG score: -3.51

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -7.17 possible cleavage site: between 55 and 56

>» Seems to have no N-terminal signal peptide

Tentative number of TMS(s) for the threshold 0.5: 0 number of TMS (s) .. fixed PERIPHERAL Likelihood = 0.69 (at 46) ALOM score: 0.69 (number of TMSs: 0)

MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment (75) : 4.40 Hyd Moment (95) : 10.64 G content: 0 D/E content: 2 S/T content: 2 Score: -5.24

Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 12 MRL|SK

NUCDISC: discrimination of nuclear localization signals pat : none pat7: PGKIKRL (4) at 356 bipartite: none content of basic residues : 12.0% NLS Score: -0.13

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

ER Membrane Retention Signals :

XXRR-like motif in the N-terminus: RLSK

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

Tyrosines in the tail: none

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

Nuclear hormones receptors DNA-binding region signature (PS00031) : *** found ***

CAICGDRATGKHYGASSCDGCKGFPRR at 111 checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none

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

Final Results (k = 9/23):

82.6 %: nuclear

8.7 %: mitochondrial

4.3 %: cytoplasmic

4.3 %: peroxisomal

» prediction for CG176069-01 is nuc (k=23)

A search of the NOV29a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 29C.

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

PFam analysis predicts that the NOV29a protein contains the domains shown in the Table 29E.

Example 30.

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

Table 30A. NOV30 Sequence Analysis SEQ ID NO: 127 992 bp

NOV30a, CTGTCTTTTGTTTCTCTTGCATGCAAGGCCCCATACTGTGGATCATGGCAAATCTGAG CCAGCCCTCCGAATTTGTCCTCTTGGGCTTCTCCTCCTTTGGTGAGCTGCAGGCCCTT CG50303-01 CTGTATGGCCCCTTCCTCATGCTTTATCTTCTCGCCTTCATGGGAAACACCATCATCA DNA Sequence TAGTTATGGTCATAGCTGACACCCACCTACATACACCCATGTACTTCTTCCTGGGCAA TTTTTCCCTGCTGGAGATCTTGGTAACCATGACTGCAGTGCCCAGGATGCTCTCAGAC CTGCTGGTCCCCCACAAAGTCATTACCTTCACTGGCTGCATGGTCCAGTTCTACTTCC ACTTTTCCCTGGGGTCCACCTCCTTCCTCATCCTGACAGACATGGCCCTTGATCGCTT TGTGGCCATCTGCCACCCACTGCGCTATGGCACTCTGATGAGCCGGGCTATGTGTGTC CAGCTGGCTGGGGCTGCCTGGGCAGCTCCTTTCCTAGCCATGGTACCCACTGTCCTCT CCCGAGCTCATCTTGATTACTGCCATGGCGACGTCATCAACCACTTCTTCTGTGACAA TGAACCTCTCCTGCAGTTGTCATGCTCTGACACTCGCCTGTTGGAATTCTGGGACTTT CTGATGGCCTTGACCTTTGTCCTCAGCTCCTTCCTGGTGACCCTCATCTCCTATGGCT ACATAGTGACCACTGTGCTGCGGATCCCCTCTGCCAGCAGCTGCCAGAAGGCTTTCTC CACTTGCGGGTCTCACCTCACACTGGTCTTCATCGGCTACAGTAGTACCATCTTTCTG TATGTCAGGCCTGGCAAAGCTCACTCTGTGCAAGTCAGGAAGGTCGTGGCCTTGGTGA CTTCAGTTCTCACCCCCTTTCTCAATCCCTTTATCCTTACCTTCTGCAATCAGACAGT TAAAACAGTGCTACAGGGGCAGATGTAGAGGCTGAAAGGCCTTTGCAAGGCACAATGA TGAGCC

ORF Start: ATG at 21 ORF Stop: TAG at 954

SEQ ID NO: 128 311 aa MW at 34714.8kD

NOV30a, MQGPILWIMANLSQPSEFV GFSSFGELQALLYGPFLM Y LAFMGNTIIIV VIAD CG50303-01 THLHTPi^FFLGNFS LEILV MTAVPRMLSD LVPHKVITFTGCMVQFYFHFS GST SFLILTDMALDRFVAICHP RYGTL SRAMCVQLAGAA AAPFLAMVPTVLSRAH DY Protein CHGDVINHFFCDI_PLLQ SCSDTRLLEFWDFLMATFVLSSF V LISYGYIVTTVL Sequence RIPSASSCQKAFSTCGSHLTLVFIGYSSTIFLYVRPGKAHSVQVRKWAV SV TPF LNPFI TFCNQTVKTVLQGQM

SEQ ID NO: 129 992 bp

NOV30b, CTGTCTTTTGTTTCTCTTGCATGCAAGGCCCCATACTGTGGATCATGGCAAATCTGAG CG50303-02 CCAGCCCTCCGAATTTGTCCTCTTGGGCTTCTCCTCCTTTGGTGAGCTGCAGGCCCTT CTGTATGGCCCCTTCCTCATGCTTTATCTTCTCGCCTTCATGGGAAACACCATCATCA DNA Sequence TAGTTATGGTCATAGCTGACACCCACCTACATACACCCATGTACTTCTTCCTGGGCAA TTTTTCCCTGCTGGAGATCTTGGTAACCATGACTGCAGTGCCCAGGATGCTCTCAGAC CTGTTGGTCCCCCACAAAGTCATTACCTTCACTGGCTGCATGGTCCAGTTCTACTTCC ACTTTTCCCTGGGGTCCACCTCCTTCCTCATCCTGACAGACATGGCCCTTGATCGCTT TGTGGCCATCTGCCACCCACTGCGCTATGGCACTCTGATGAGCCGGGCTATGTGTGTC CAGCTGGCTGGGGCTGCCTGGGCAGCTCCTTTCCTAGCCATGGTACCCACTGTCCTCT CCCGAGCTCATCTTGATTACTGCCATGGCGACGTCATTAACCACTTCTTCTGTGACAA TGAACCTCTCCTGCAGTTGTCATGCTCTGACACTCGCCTGTTGGAATTCTGGGACTTT CTGATGGTCTTGACCTTTGTCCTCAGCTCCTTCCTGGTGACCCTCATCTCCTATGGCT ACATAGTGACCACTGTGCTGCGGATCCCCTCTGCCAGCAGCTGCCAGAAGGCTTTCTC CACTTGCGGGTCTCACCTCACACTGGTCTTCATCGGCTACAGTAGTACCATCTTTCTG TATGTCAGGCCTGGCAAAGCTCACTCTGTGCAAGTCAGGAAGGTCGTGGCCTTGGTGA CTTCAGTTCTCACCCCCTTTCTCAATCCCTTTATCCTTACCTTCTGCAATCAGACAGT TAAAACAGTGCTACAGGGGCAGATGTAGAGGCTGAAAGGCCTTTGCAAGGCACAATGA TGAGCC

ORF Start: ATG at 21 ORF Stop: TAG at 954

SEQ ID NO: 130 J311 aa [MW at 34742.9kD

NOV30b, MQGPI I_^L_SQPSEFVL GFSSFGELQALLYGPFLK_Y LAFMGΪSΠ^LIIIVMVIAD THLHTPMYFFLG1^S LEILVT1_AVPRM SDL VPHKVITFTGC VQFYFHFSLGST CG50303-02 SF I TDMA DRFVAICHP RYGT MSFJ—CVQLAGAAWAAPFL-^MVPTVLSRAHLDY Protein CHGDVINHFFCDNEPLLQLSCSI ΓRLLEFWDFLIWLTF^SSF VTLISYGYIVTTVL Sequence RIPSASSCQI__^,STCGSHLT VFIGYSSTIFLYVRPGKAHSVQVRKVVALVTSVLTPF LNPFI TFCNQTVKTVLQGQM

SEQ ID NO: 131 964 bp

NOV30c, GGCCCCATACTGTGGATCATGGCAAATCTGAGCCAGCCCTCCGAATTTGTCCTCTTGG GCTTCTCCTCCTTTGGTGAGCTGCAGGCCCTTCTGTATGGCCCCTTCCTCATGCTTTA CG50303-03 r r f2rr^,'wr_ !r3-_aΛ _rp_ a r_ _πτ»τ'_ ;α P_ -f_θ f;-p_r^,r'P_r^ι

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

Table 30B. Comparison of NOV30a against NOV30b and NOV30c.

NOV30a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV30b 1..311 310/311 (99%) 1..311 310/311 (99%)

NOV30c 9..311 303/303 (100%) 1..303 303/303 (100%)

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

Table 30C. Protein Sequence Properties NOV30a

SignalP analysis: Cleavage site between residues 58 and 59

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

N-region: length 0; pos.chg 0; neg.chg 0 H-region: length 16; peak value 8.74 PSG score: 4.34

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -2.13 possible cleavage site: between 31 and 32

>» 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.

INTEGRAL Likelihood = -7 .11 TTrraannssmmembrane 40 56

INTEGRAL Likelihood = -0 .48 TTrraannssmmembrane 68 - 84

INTEGRAL Likelihood = -0. .37 TTrraannssmmembrane 147 - 163

INTEGRAL Likelihood = -7 , .48 TTrraannssmmembrane 206 - 222

INTEGRAL Likelihood = -2. .28 TTrraannssmmembrane 279 - 295

PERIPHERAL Likelihood = 0 , .90 (at 117)

ALOM score: -7.48 (number of TMSs: 5)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 47 Charge difference: 2.0 C( 0.0) - N(-2.0) 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: 0 Hyd Moment (75) : 2.02 Hyd Moment (95) : 3.18 G content: 1 D/E content: 1 S/T content: 2 Score: -5.78

N0CDISC: discrimination of nuclear localization signals pa 4 : none pat7 : none bipartite: none content of basic residues: 4.5%

NLS Score: -0.47

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

ER Membrane Retention Signals : none

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

PTS2: 2nd peroxisomal targeting signal: none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

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

Leucine zipper pattern (PS00029) : *** found *** LSCSDTRLLEFWDFLMALTFVL at 193 none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none

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

Final Results (k = 9/23):

44.4 %: endoplasmic reticulum

22.2 %: vacuolar

11.1 %: Golgi

11.1 %: vesicles of secretory system

11.1 %: mitochondrial

» prediction for CG50303-01 is end (k=9)

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

In a BLAST search of public sequence datbases, the NOV30a protein was found to have homology to the proteins shown in the BLASTP data in Table 30E.

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

Example 31.

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

DNA Sequence GGAGCTCACTCACACAGGAATCCCAGCCCATCTACGAGGGGATGGTGGGAAAAGCAAA CCAACTTTCTTCAGTAAGAATGACCCTTCTGAGCAATGGTAGGTCTCTGGCCCACCAC AGAGGCAGGCATAGCCAAGGCCAGAGCCAGGGCCCGGGCAGCGGGGACACGAACCGTT CCGACTGCTGCCCGCAGCCGCCGCCGCCGCCCAGGTGCGAGCTCTTGCATGTGGCCAT CGTGTGTGCGGGGCATAACTCCAGCCGAGACGTCATCACCCTGGTGAAGTCCATGCTC TTCTACAGGAAAAATCCACTGCACCTCCACTTGGTGACTGACGCCGTGGCCAGAAACA TCCTGGAGACGCTCTTCCACACATGGATGGTCTATCGATCCCCTGTCAGCTTTTATCA TGCCGACCAGCTCAAGCCCCAGGTCTCCTGTATCCCCAACTTTCACTACTCCGGCCTC TATGGGCTAATGAAGCTGGTGCTGCCCAATGCCTTGCCTGCTGAGCTGGCCCGCGTCA TTGTCCTGGACACGGATGTCACCTTCGCCTCTGACATCTCGGAGCTCTGGGCCCTCTT TGCTCACTTTTCTGACACGCAGGCGATCGGTCTTGTGGAGAACCAGAGTGACTGGTAC CTGGGCAACCTCTGGAAGAACCACAGGCCCTGGCCTGCCTTGGGCCGGGGATTTAACA CAGGTGTGATCCTGCTGCGGCTGGACCGGCTCCGGCAGGCTGGCTGGGAGCAGATGTG GAGGCTGACAGCCAGGCGGGAGCTCCTTAGCCTGCCTGCCACCTCACTGGCTGACCAG GACATCTTCAACGCTGTGATCAAGGAGCACCCGGGGCTAGTGCAGCGTCTGCCTTGTG TCTGGAATGTGCAGCTGTCAGATCACACACTGGCCGAGCGCTGCTACTCTGAGGCGTC TGACCTCAAGGTGATCCACTGGAACTCACCAAAGAAGCTTCGGGTGAAGAACAAGCAT GTGGAATTCTTCCGCAATTTCTACCTGACCTTCCTGGAGTACGATGGGAACCTGCTGC GGAGAGAGCTCTTTGTGTGCCCCAGCCAGCCCCCACCTGGTGCTGAGCAGTTGCAGCA GGCCCTGGCACAACTGGACGAGGAAGACCCCTGCTTTGAGϊTCCGGCAGCAGCAGCTC ACTGTGCACCGTGTGCATGTCACTTTCCTGCCCCATGAACCGCCACCCCCCCGGCCTC ACGATGTCACCCTTGTGGCCCAGCTGTCCATGGACCGGCTGCAGATGTTGGAAGCCCT GTGCAGGCACTGGCCTGGCCCCATGAGCCTGGCCTTGTACCTGACAGACGCAGAAGCT CAGCAGTTCCTGCATTTCGTCGAGGCCACACCAGTGGGTGTTGCCCGGCACGACGTGG CCTACCATGTGGTGTACCGGGGGGCGCCCCTATACCCCGTCAACCAGCTTCGCAACGT GGCCTTGGCCCAGGCCCTCACGCCTTACGTCTTCCTCAGTGACATTGACTTCCTGCCT GCCTATTCTCTCTACGACTACCTCAGGGCCTCCATTGAGCAGCTGGGGCTGGGCAGCC GGCGCAAGGCAGCACTGGTGGTGCCGGCATTCGAGACCCTGCGCTACCGCTTCAGCTT CCCCCATTCCAAGGTGGAGCTGTTGGCCTTGCTGGATGCGGGCACTCTCTACACCTTC AGGTACCACGAGTGGCCCCGAGGCCACGCACCCACAGACTATGCCCGCTGGCGGGAGG CTCAGGCCCCGTACCGTGTGCAATGGGCGGCCAACTATGAACCCTACGTGGTGGTGCC ACGAGACTGTCCCCGCTATGATCCTCGC TGTGGGCTTCGGCTGGAACAAAGTGGCC CACATTGTGGAGCTGGATGCCCAGGAATATGAGCTCCTGGTGCTGCCCGAGGCCTTCA CCATCCATCTGCCCCACGCTCCAAGCCTGGACATCTCCCGCTTCCGCTCCAGCCCCAC CTATCGTGACTGCCTCCAGGCCCTCAAGGACGAATTCCACCAGGACTTGTCCCGCCAC CATGGGGCTGCTGCCCTCAAATACCTCCCAGCCCTGCAGCAGCCCCAGAGCCCTGCCC GAGGCTGAGGCTGGGCCGGCGCTGCCCCTCATCTTAGCATTGGGCAGACACCAGGGCA

ACCTGCCCTCCGCCATCCCTGCTATT AAATTATTTAAGGTCTCTGGGAAGGGCTGGG

GCAGAGCATCTGTGGGGTGGGGTCTTCCCCTTGCTGCTATTGTATGGCTGGGGACTGG

TCTCTCTCTGCCCCAGCCAGTTTGGGGCTGGTTCCCCCATCTTGAATTGTTTATCCCT TTOTCATAATTAAAGTTTTAAAACATCAAAAAAAAAA^

ORF Start: ATG at 54 ORF Stop: TGA at 2268

SEQ ID NO: 138 738 aa MW at 84233.5kD

NOV31c, MKS SLFCLFFLTF VLPSSGFLLGRQQS SVNFHIRTTRGSSLTQESQPIYEGMVGK ANQ SSVRMT LSNGRSLAHHRGRHSQGQSQGPGSGDTNRSDCCPQPPPPPRCELLHV CG50595-04 AIVCAGHNSSRDVITLVKS_^FYRKNPLHLHLV DAVARNI ET FHT MVYRSPVSF Protein YHADQLKPQVSCIPNFHYSGLYG LV PNALPAE ARVIV DTDV FASDISELWA Sequence LFAHFSDTQAIGLVENQSDWYLGNLWKNHRPWPALGRGF TGVILLR DRLRQAGWEQ I_W^ TARRE LSLPATSLADQDIFNAVIKEHPG VQR PCVWNVQLSDHTLAERCYSE ASDLVIHVrøSPKK RV JπHVEFFRNFY TF EYDG LLRRE FVCPSQPPPGAEQ QQA AQLDΞEDPCFEFRQQQLTVHRVHVTFLPHEPPPPRPHDVT VAQ SMDRLQMLE ALCRHWPGPMS ALYLTDAEAQQFLHFVEATPVGVARHDVAYrlVVYRGAPLYPVNQLR NVALAQALTPYVFLSDIDFLPAYSLYDY RASIEQLG GSRRKAALWPAFET RYRF SFPHSKVE LALLDAGTLYTFRYHEWPRGHAPTDYARWREAQAPYRVQWAANYEPYW VPRDCPRYDPRFVGFG NKVAHIVELDAQEYE VLPEAFTIH PHAPS DISRFRSS PTYRDC QALKDEFHQDLSRHHGAAALKYLPALQQPQSPARG

SEQ ID NO: 139 3042 bp

NOV31d, AGCGGGCGAGCGCAGGGCCCAGCCCGGGAGCCGCTGGAGCAGGGCCTGCGATGGAGCC

TGCAGCCCCGGGTCGCGTCCCTCCCTGAGCGCCCCCGTCGGCGGCCATGCTGCCCCGA CG50595-07 GGGCGCCCCCGGGCGCTGGGGGCCGCCGCGCTGTTGCTGCTGCTGCTGCTGCTCGGAT DNA Sequence TCCTCCTGTTCGGTGGGGACCTGGGGTGTGAGCGCCGCAAGCCTGGCGGGCGAGCGGG GGCCCCGGGATGCTTCCCCGGCCCGCTCATGCCACGTGTCCCCCCAGACGGGAGGCTG CGGAGAGCCGCCGCCCTCGACGGAGACCCGGGGGCCGGCCCCGGGGACCACAACCGCT CCGACTGCGGCCCGCAGCCGCCGCCGCCGCCCAAGTGCGAGCTCTTGCATGTGGCCAT CGTGTGTGCGGGGCATAACTCCAGCCGAGACGTCATCACCCTGGTGAAGTCCATGCTC TTCTACAGGAAAAATCCACTGCACCTCCACTTGGTGACTGACGCCGTGGCCAGAAACA TCCTGGAGACGCTCTTCCACACATGGATGGTGCCTGCTGTCCGTGTCAGCTTTTATCA TGCCGACCAGCTCAAGCCACAGGTCTCCTGGATCCCCAACAAGCACTACTCCGGCCTC TATGGGCTAATGAAGCTGGTGCTGCCCAGTGCCTTGCCTGCTGAGCTGGCCCGCGTCA TTGTCCTGGACACGGATGTCACCTTCGCCTCTGACATCTCGGAGCTCTGGGCCCTCTT TGCTCACTTTTCTGGTGAGCAGGCGATCGGTCTTGTGGAGAACCAGAGTGACTGGTAC CTGGGCAACCTCTGGAAGAACCACAGGCCCTGGCCTGCCTTGGGCCGGGGATTTAACA CAGGTGTGATCCTGCTGCGGCTGGACCGGCTCCGGCAGGCTGGCTGGGAGCAGATGTG GAGGCTGACAGCCAGGCGGGAGCTCCTTAGCCTGCCTGCCACCTCACTGGCTGACCAG GACATCTTCAACGCTGTGATCAAGGAGCACCCGGGGCTAGTGCAGCGTCTGCCTTGTG TCTGGAATGTGCAGCTGTCAGATCACACACTGGCCGAGCGCTGCTACTCTGAGGCGTC TGACCTCAAGGTGATCCACTGGAACTCACCAAAGAAGCTTCGGGTGAAGAACAAGCAT GTGGAATTCTTCCGCAATTTCTACCTGACCTTCCTGGAGTACGATGGGAACCTGCTGC GGAGAGAGCTCTTTGTGTGCCCCAGCCAGCCCCCACCTGGTGCTGAGCAGTTGCAGCA GGCCCTGGCACAACTGGACGAGGAAGACCCCTGCTTTGAGTTCCGGCAGCAGCAGCTC ACTGTGCACCGTGTGCATGTCACTTTCCTGCCCCATGAACCGCCACCCCCCCGGCCTC ACGATGTCACCCTTGTGGCCCAGCTGTCCATGGACCGGCTGCAGATGTTGGAAGCCCT GTGCAGGCACTGGCCTGGCCCCATGAGCCTGGCCTTGTACCTGACAGACGCAGAAGCT CAGCAGTTCCTGCATTTCGTCGAGGCCTCACCAGTGCTTGCTGCCCGGCAGGACGTGG CCTACCATGTGGTGTACCGTGAGGGGCCCCTATACCCCGTCAACCAGCTTCGCAACGT GGCCTTGGCCCAGGCCCTCACGCCTTACGTCTTCCTCAGTGACATTGACTTCCTGCCT GCCTATTCTCTCTACGACTACCTCAGGGCCTCCATTGAGCAGCTGGGGCTGGGCAGCC GGCGCAAGGCAGCACTGGTGGTGCCGGCATTCGAGACCCTGCGCTACCGCTTCAGCTT CCCCCATTCCAAGGTGGAGCTGTTGGCCTTGCTGGATGCGGGCACTCTCTACACCTTC AGGTACCACGAGTGGCCCCGAGGCCACGCACCCACAGACTATGCCCGCTGGCGGGAGG CTCAGGCCCCGTACCGTGTGCAATGGGCGGCCAACTATGAACCCTACGTGGTGGTGCC ACGAGACTGTCCCCGCTATGATCCTCGCTTTGTGGGCTTCGGCTGGAACAAAGTGGCC CACATTGTGGAGCTGGATGCCCAGGAATATGAGCTCCTGGTGCTGCCCGAGGCCTTCA CCATCCATCTGCCCCACGCTCCAAGCCTGGACATCTCCCGCTTCCGCTCCAGCCCCAC CTATCGTGACTGCCTCCAGGCCCTCAAGGACGAA CCACCAGGACTTGTCCCGCCAC CATGGGGCTGCTGCCCTCAAATACCTCCCAGCCCTGCAGCAGCCCCAGAGCCCTGCCC GAGGCTGAGGCTGGGCCGGCGCTGCCCCTCATCTTAGCATTGGGCAGACACCAGGGCA

ACCTGCCCTCCGCCATCCCTGCTATTTAAATTATTTAAGGTCTCTGGGAAGGGCTGGG

GCAGAGCATCTGTGGGGTGGGGTCTTCCCCTTGCTGCTATTGTATGGCTGGGGACTGG

TCTCTCTCTGCCCCAGCCAGTTTGGGGCTGGTTCCCCCATCTTGAATTGTTTATCCCT

TTTTCATAATTAAAGTTTTAAAACATCAAAAAAAAAACAAAAAACAAAAAAAAAAAAA

CAAAAAAAAAAAAAAAAAAAAAAAACAACAAAAAAAAAAAAAAGGGGGCCCGCCAAAA lAAAAGCACCCCACCGAGGGGAGCACAACAAGATGAGGAGCACGCACACTCGGGAAACC

AAGGCTGGCCCCACAAGAGCGGGGACGGAAAATAAGAACACAGAGAGCGCAGAGGCAAl

GTGTATATAACAGGGCAGCGGAGGGGGAAAAAACCGCCGGCCCGGGGCACCAACTCGG

CGCAGAGAGCCGCCCCCCAACACAGACTGGCGGGCGAGGGAACCCACACAGGGACGCAl

CACACCACCACCCCCCAAAAACACTAGAGGGAGACACGCCCACAGGCGCCACCACCAAl

CACCGACAGCAGCCCCAGCGGCGGCAGCAAACACGCGCGGCAAACACACACACGACCG;

IGCACAGCAGCCGACCCGCGGCCCCACGAACGCACGCCCGACACACGCGGACGGAGAAC

CACGCAGCGACCATCTCACCCCAGGG

ORF Start: ATG at 105 ORF Stop: TGA at 2268

SEQ ID NO: 140 721 aa MW at 81755. lkD

NOV31d, MLPRGRPRA GAAA LLL LLGF IiFGGDLGCΞRRKPGGRAGAPGCFPGP PRVPP

DGR RRAAA DGDPGAGPGDHNRSDCGPQPPPPPKCELLHVAIVCAGHNSSRDVITLV CG50595-07 KSM FYRK PLHLHLVTDAVARNILETLFHTVMVPAVRVSFYHADQLKPQVSWIPN H Protein YSG YGLMKLVLPSA PAE ARVIV DTDVTFASDISE A FAHFSGEQAIG VENQ Sequence SDVfYLGrøTOmRPWPALGRGFN GVIL^

LADQDIFNAVIKEHPG VQRLPCTfflSlVQLSDHT AERCYSEASDLKVIrlϊmSPKKLR

KHKHVEFFRNFY TFLEYDGN LRRE FVCPSQPPPGAEQLQQALAQLDEEDPCFEFR

QQQ TVHRVHV F PHEPPPPRPHDVT VAQLSMDRLQMLEA CRH PGPMSLAYLT

DAEAQQFLHFVEASPVLAARQDVAYHVVYREGPLYPVNQ RNVALAQALTPYVFLSDI

DF PAYSLYDY RASIEQLGLGSRRKAALWPAFET RYRFSFPHSKVΞIi A DAGT

LYTFRYHE PRGHAPTDYAR REAOAPYRVO AANYEPYVWPRDCPRYDPRFVGFGW NKVAHIVE DAQEYELLVLPEAFTIHLPHAPS DISRFRSSPTYRDC QALKDEFHQD SRHHGAAALKYLPALQQPQSPARG

SEQ ID NO: 141 2459 bp

NOV31e, AAGCAAACAACTTCAACTTTCCATGAGTGCTAGCCAGCCCTGGGAGGCTCCGCTGCCA

GAAGGAACGTCAAGGGGCCTGCGATGGAGCCTGCAGCCCCGGGTCGCGTCCCTCCCTG CG50595-06 AGCGCCCCCGTCGGCGGCCATGCTGCCCCGAGGGCGCCCCCGGGCGCTGGGGGCCGCC DNA Sequence GCGCTGTTGCTGCTGCTGCTGCTGCTCGGATTCCTCCTGTTCGACGGGAGGCTGCGGA GAGCCGCCGCCCTCGACGGAGACCCGGGGGCCGGCCCCGGGGACCACAACCGCTCCGA CTGCGGCCCGCAGCCGCCGCCGCCGCCCAAGTGCGAGCTCTTGCATGTGGCCATCGTG TGTGCGGGGCATAACTCCAGCCGAGACGTCATCACCCTGGTGAAGTCCATGCTCTTCT ACAGGAAAAATCCACTGCACCTCCACTTGGTGACTGACGCCGTGGCCAGAAACATCCT GGAGACGCTCTTCCACACATGGATGGTGCCTGCTGTCCGTGTCAGCTTTTATCATGCC GACCAGCTCAAGCCCCAGGTCTCCTGGATCCCCAACAAGCACTACTCCGGCCTCTATG GGCTAATGAAGCTGGTGCTGCCCAGTGCCTTGCCTGCTGAGCTGGCCCGCGTCATTGT CCTGGACACGGATGTCACCTTCGCCTCTGACATCTCGGAGCTCTGGGCCCTCTTTGCT CACTTTTCTGACACGCAGGCGATCGGTCTTGTGGAGAACCAGAGT ACTGGTACCTGG GCAACCTCTGGAAGAACCACAGGCCCTGGCCTGCCTTGGGCCGGGGATTTAACACAGG TGTGATCCTGCTGCGGCTGGACCGGCTCCGGCAGGCTGGCTGGGAGCAGATGTGGAGG CTGACAGCCAGGCGGGAGCTCCTTAGCCTGCCTGCCACCTCACTGGCTGACCAGGACA TCTTCAACGCTGTGATCAAGGAGCACCCGGGGCTAGTGCAGCGTCTGCCTTGTGTCTG GAATGTGCAGCTGTCAGATCACACACTGGCCGAGCGCTGCTACTCTGAGGCGTCTGAC CTCAAGGTGATCCACTGGAACTCACCAAAGAAGCTTCGGGTGAAGAACAAGCATGTGG AATTCTTCCGCAATTTCTACCTGACCTTCCTGGAGTACGATGGGAACCTGCTGCGGAG AGAGCTCTTTGTGTGCCCCAGCCAGCCCCCACCTGGTGCTGAGCAGTTGCAGCAGGCC CTGGCACAACTGGACGAGGAAGACCCCTGCTTTGAGTTCCGGCAGCAGCAGCTCACTG TGCACCGTGTGCATGTCAC TCCTGCCCCATGAACCGCCACCCCCCCGGCCTCACGA TGTCACCCTTGTGGCCCAGCTGTCCATGGACCGGCTGCAGATGTTGGAAGCCCTGTGC AGGCACTGGCCTGGCCCCATGAGCCTGGCCTTGTACCTGACAGACGCAGAAGCTCAGC AGTTCCTGCATTTCGTCGAGGCCTCACCAGTGCTTGCTGCCCGGCAGGACGTGGCCTA CCATGTGGTGTACCGTGAGGGGCCCCTATACCCCGTCAACCAGCTTCGCAACGTGGCC TTGGCCCAGGCCCTCACGCCTTACGTCTTCCTCAGTGACATTGACTTCCTGCCTGCCT ATTCTCTCTACGACTACCTCAGGGCCTCCATTGAGCAGCTGGGGCTGGGCAGCCGGCG CAAGGCAGCACTGGTGGTGCCGGCAT TGAGACCCTGCGCTACTGCTTCAGCTTCCCC CATTCCAAGGTGGAGCTGTTGGCCTTGCTGGATGCGGGCACTCTCTACACCTTCAGGT ACCACGAGTGGCCCCGAGGCCACGCACCCACAGACTATGCCCGCTGGCGGGAGGCTCA GGCCCCGTACCGTGTGCAATGGGCGGCCAACTATGAACCCTACGTGGTGGTGCCACGA GACTGTCCCCGCTATGATCCTCGCTTTGTGGGCTTCGGCTGGAACAAAGTGGCCCACA TTGTGGAGCTGGATGCCCAGGAATATGAGCTCCTGGTGCTGCCCGAGGCCTTCACCAT CCATCTGCCCCACGCTCCAAGCCTGGACATCTCCCGCTTCCGCTCCAGCCCCACCTAT CGTGACTGCCTCCAGGCCCTCAAGGACGAATTCCACCAGGACTTGTCCCGCCACCATG GGGCTGCTGCCCTCAAATACCTCCCAGCCCTGCAGCAGCCCCAGAGCCCTGCCCGAGG CTGAGGCTGGGCCGGCGCTGCCCCTCATCTTAGCATTGGGCAGACACCAGGGCAACCT

GCCCTCCGCCATCCCTGCTATTTAAATTATTTAAGGTCTCTGGGAAGGGCTGGGGCAG

AGCATCTGTGGGGTGGGGTCTTCCCCTTGCTGCTATTGTATGGCTGGGGACTGGTCTC TCTCTGCCCCAGCCAGTTTGGGGCTGGTTCCCCCATCTTGAATTGTTTATCCCTTTTT CATAATTAAAGTTTTAAAACATC

ORF Start: ATG at 136 ORF Stop: TGA at 2206

SEQ ID NO: 142 690 aa MW at 78646.5kD

NOV31e, MLPRGRPRALGAAALLLLLLL GFLLFDGRLRRAAALDGDPGAGPGDHNRSDCGPQPP PPPKCE LRIVAIVCAGHNSSRDVIT VKS_L,FYRKNP HLHLV DAVAR ILETLFHT CG50595-06 WL^PAVRVSFYHADQLKPQVSWIPNKHYSGLYGLRØSLVLPSALPAEIARVIVLDTDV Protein FASDISELWALFAHFSI ΓQAIGLVENQSD Y GN WKNHRP PALGRGF TGVI_LR Sequence DRLRQAG EQM RLTARRELLS PATS ADQDIFNAVIKEHPGLVQPJJPCVWNVQLSD

HTLAERCYSEASDLIWIHM^SPKK RVKLΠΑWEFFRNFYLTFLEYDGNLLRRELFVCP SQPPPGAEQLQQALAQ DEEDPCFEFRQQQ TVHRVHVTFLPHEPPPPRPHDV LVAQ LS_DR QMLEALCRHWPGPMSLA YLTDAEAQQFLHFVEASPVLIAARQDVAYHVVΥRE GPLYPVT ΓQ RNVALAQA TPYVF SDIDF PAYSLYDYLRASIEQLGLGSRRKAALVV PAFETLRYCFSFPHSK /EL AL DAGTLYTFRYHE PRGHAPTDYAR REAQAPYRVQ WAANYΞPYVVVPRDCPRYDPRFVGFGWNKVAHIVE DAQEYELLVLPΞAFTIHLPHAP SLDISRFRSSPTYRDCLQALKDΞFHQDLSRHHGAAA KYLPA QQPQSPARG SEQ ID NO: 143 2105 bp

NOV31f, CATCTAGGCCACCATGGCCACCATGCTGCCCCGAGGGCGCCCCCGGGCGCTGGGGGCC GCCGCGCTGTTGCTGCTGCTGCTGCTGCTCGGATTCCTCCTGTTCGACGGGAGGCTGC 306448537 GGAGAGCCGCCGCCCTCGACGGAGACCCGGGGGCCGGCCCCGGGGACCACAACCGCTC DNA Sequence CGACTGCGGCCCGCAGCCGCCGCCGCCGCCCAAGTGCGAGCTCTTGCATGTGGCCATC GTGTGTGCGGGGCATAACTCCAGCCGAGACGTCATCACCCTGGTGAAGTCCATGCTCT TCTACAGGAAAAATCCACTGCACCTCCACTTGGTGACTGACGCCGTGGCCAGAAACAT CCTGGAGACGCTCTTCCACACATGGATGGTGCCTGCTGTCCGTGTCAGCTTTTATCAT GCCGACCAGCTCAAGCCCCAGGTCTCCTGGATCCCCAACAAGCACTACTCCGGCCTCT ATGGGCTAATGAAGCTGGTGCTGCCCAGTGCCTTGCCTGCTGAGCTGGCCCGCGTCAT TGTCCTGGACACGGATGTCACCTTCGCCTCTGACATCTCGGAGCTCTGGGCCCTCTTT GCTCACTTTTCTGACACGCAGGCGATCGGTCTTGTGGAGAACCAGAGTGACTGGTACC TGGGCAACCTCTGGAAGAACCACAGGCCCTGGCCTGCCTTGGGCCGGGGATTTAACAC AGGTGTGATCCTGCTGCGGCTGGACCGGCTCCGGCAGGCTGGCTGGGAGCAGATGTGG AGGCTGACAGCCAGGCGGGAGCTCCTTAGCCTGCCTGCCACCTCACTGGCTGACCAGG ACATCTTCAACGCTGTGATCAAGGAGCACCCGGGGCTAGTGCAGCGTCTGCCTTGTGT CTGGAATGTGCAGCTGTCAGATCACACACTGGCCGAGCGCTGCTACTCTGAGGCGTCT GACCTCAAGGTGATCCACTGGAACTCACCAAAGAAGCTTCGGGTGAAGAACAAGCATG TGGAATTCTTCCGCAATTTCTACCTGACCTTCCTGGAGTACGATGGGAACCTGCTGCG GAGAGAGCTCTTTGTGTGCCCCAGCCAGCCCCCACCTGGTGCTGAGCAGTTGCAGCAG GCCCTGGCACAACTGGACGAGGAAGACCCCTGCTTTGAGTTCCGGCAGCAGCAGCTCA CTGTGCACCGTGTGCATGTCACTTTCCTGCCCCATGAACCGCCACCCCCCCGGCCTCA CGATGTCACCCTTGTGGCCCAGCTGTCCATGGACCGGCTGCAGATGTTGGAAGCCCTG TGCAGGCACTGGCCTGGCCCCATGAGCCTGGCCTTGTACCTGACAGACGCAGAAGCTC AGCAGTTCCTGCATTTCGTCGAGGCCTCACCAGTGCTTGCTGCCCGGCAGGACGTGGC CTACCATGTGGTGTACCGTGAGGGGCCCCTATACCCCGTCAACCAGCTTCGCAACGTG GCCTTGGCCCAGGCCCTCACGCCTTACGTCTTCCTCAGTGACATTGACTTCCTGCCTG CCTATTCTCTCTACGACTACCTCAGGGCCTCCATTGAGCAGCTGGGGCTGGGCAGCCG GCGCAAGGCAGCACTGGTGGTGCCGGCATTTGAGACCCTGCGCTACCGC TCAGCTTC CCCCATTCCAAGGTGGAGCTGTTGGCCTTGCTGGATGCGGGCACTCTCTACACCTTCA GGTACCACGAGTGGCCCCGAGGCCACGCACCCACAGACTATGCCCGCTGGCGGGAGGC TCAGGCCCCGTACCGTGTGCAATGGGCGGCCAACTATGAACCCTACGTGGTGGTGCCA CGAGACTGTCCCCGCTATGATCCTCGCTTTGTGGGCTTCGGCTGGAACAAAGTGGCCC ACATTGTGGAGCTGGATGCCCAGGAATATGAGCTCCTGGTGCTGCCCGAGGCCTTCAC CATCCATCTGCCCCACGCTCCAAGCCTGGACATCTCCCGCTTCCGCTCCAGCCCCACC TATCGTGACTGCCTCCAGGCCCTCAAGGACGAATTCCACCAGGACTTGTCCCGCCACC ATGGGGCTGCTGCCCTCAAATACCTCCCAGCCCTGCAGCAGCCCCAGAGCCCTGCCCG AGGCTGAACGCGTGATC

ORF Start: at 8 ORF Stop: TGA at 2093

SEQ ID NO: 144 695 aa MW at 79175.1kD

NOV31f, ATMATMLPRGRPRALGAAAL L LLLLGF LFDGRLRRAAALDGDPGAGPGDHNRSDC GPQPPPPPKCE LHVAIVCAGHNSSRDVITLVKSMLFYRKNP HLH VTDAVARNILE 306448537 TLFHTVMVPAVRVSFYHADQLKPQVS IPI^HYSGLYGLMKiVLPSALPAELARVIVL Protein DTDVTFASDISEL ALFAHFSDTQAIG VENQSDVreLGNLWIOSraRP PALGRGF GV Sequence ILLR DRLRQAGWEQM RLTARREL SLPATSLADQDIFNAVIKEHPG VQRLPCVWN VQ SDHTIAERCYSEASDLKVIHWNSPKKiRVI<_ΛαiVEFFRl_ YLTF EYDGNLLRRE FVCPSQPPPGAEQ QQALAQ DEEDPCFEFRQQQLTVHRVHVTF PHEPPPPRPHDV TLVAQ SMDRLQMLEALCRHWPGPMSLALY TDAEAQQFLHFVEASPVLAARQDVAYH WYREGPLYPV QLRNVALAQALTPYVF SDIDF PAYSLYDY RASIEQLG GSRRK AALWPAFETLRYRFSFPHSKVE LALLDAGTLYTFRYHE PRGHAPTDYAR REAQA PY lVQWAAlvrYEPYVVVPRDCPRYDPRFVGFGVrøK tAHIVELDAQEYE VLPEAFTIH LPHAPSLDISRFRSSPTYRDCLQALKDEFHQDLSRHHGAAALKY PALQQPQSPARG

SEQ ID NO: 145 2351 bp

NOV31g, TAAAAATACAAAAAATTAGCCGGGCGTAGTGGCGGGCGCCTGTAGTCCCAGCTACT G GGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCAGAGCTTGCAGTGAGCCGAG CG50595-01 ATCCCGCCACTGCACTCCAGCCTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAA DNA Sequence AAAAAGAACATCCTGAGCCGGGCGTGGAAAAGCTCTTTGCAGATGGCGCTTCCATCTC TGCGCCCCTCGGGGTGGGGGCTGTCCCATGTTGCTCCTGCTGGGGCCTCTCAGGCTTC CTCTTTGCCCACCCAAAAGGAAAAATCCACTGCACCTCCACTTGGTGACTGACGCCGT GGCCAGAAACATCCTGGAGACGCTCTTCCACACATGGATGGTGCCTGCTATCGATCCC C GTCAGCTTTTATCATGCCGACCAGCTCAAGCCCCAGGTCTCCTGGATCCCCAACA AGCACTACTCCGGCCTCTATGGGCTAATGAAGCTGGTGCTGCCCAATGCCTTGCCTGC TGAGCTGGCCCGCGTCATTGTCCTGGACACGGATGTCACCTTCGCCTCTGACATCTCG GAGCTCTGGGCCCTCTTTGCTCACTTTTCTGACACGCAGGCGATCGGTCTTGTGGAGA ACCAGAGTGACTGGTACCTGGGCAACCTCTGGAAGAACCACAGGCCCTGGCCTGCCTT GGGCCGGGGATTTAACACAGGTGTGATCCTGCTGCGGCTGGACCGGCTCCGGCAGGCT GGCTGGGAGCAGATGTGGAGGCTGACAGCCAGGCGGGAGCTCCTTAGCCTGCCTGCCA CCTCACTGGCTGACCAGGACATCTTCAACGCTGTGATCAAGGAGCACCCGGGGCTAGT GCAGCGTCTGCCTTGTGTCTGGAATGTGCAGCTGTCAGATCACACACTGGCCGAGCGC TGCTACTCTGAGGCGTCTGACCTCAAGGTGATCCACTGGAACTCACCAAAGAAGCTTC GGGTGAAGAACAAGCATGTGGAATTCTTCCGCAATTTCTACCTGACCTTCCTGGAGTA CGATGGGAACCTGCTGCGGAGAGAGCTCTTTGTGTGCCCCAGCCAGCCCCCACCTGGT GCTGAGCAGTTGCAGCAGGCCCTGGCACAACTGGACGAGGAAGACCCCTGCTTTGAGT TCCGGCAGCAGCAGCTCACTGTGCACCGTGTGCATGTCACTTTCCTGCCCCATGAACC GCCACCCCCCCGGCCTCACGATGTCACCCTTGTGGCCCAGCTGTCCATGGACCGGCTG CAGATGTTGGAAGCCCTGTGCAGGCACTGGCCTGGCCCCATGAGCCTGGCCTTGTACC TGACAGACGCAGAAGCTCAGCAGTTCCTGCATTTCGTCGAGGCCTCACCAGTGCTTGC TGCCCGGCAGGACGTGGCCTACCATGTGGTGTACCGTGAGGGGCCCCTATACCCCGTC AACCAGCT CGCAACGTGGCCTTGGCCCAGGCCCTCACGCCTTACGTCTTCCTCAGTG ACATTGACTTCCTGCCTGCCTAT CTCTCTACGACTACCTCAGGGCCTCCATTGAGCA GCTGGGGCTGGGCAGCCGGCGCAAGGCAGCACTGGTGGTGCCGGCAT TGAGACCCTG CGCTACCGCTTCAGCTTCCCCCATTCCAAGGTGGAGCTGTTGGCCTTGCTGGATGCGG GCACTCTCTACACCTTCAGGTACCACGAGTGGCCCCGAGGCCACGCACCCACAGACTA TGCCCGCTGGCGGGAGGCTCAGGCCCCGTACCGTGTGCAATGGGCGGCCAACTATGAA CCCTACGTGGTGGTGCCACGAGACTGTCCCCGCTATGATCCTCGCTTTGTGGGCTTCG GCTGGAACAAAGTGGCCCACATTGTGGAGCTGGATGCCCAGGAATATGAGCTCCTGGT GCTGCCCGAGGCCTTCACCATCCATCTGCCCCACGCTCCAAGCCTGGACATCTCCCGC TTCCGCTCCAGCCCCACCTATCGTGACTGCCTCCAGGCCCTCAAGGACGAATTCCACC AGGACTTGTCCCGCCACCATGGGGCTGCTGCCCTCAAATACCTCCCAGCCCTGCAGCA GCCCCAGAGCCCTGCCCGAGGCTGAGGCTGGGCCGGCGCTGCCCCTCATCTTAGCATT

GGGCAGACACCAGGGCAACCTGCCCTCCGCCATCCCTGCTATTTAAATTATTTAAGGT

CTCTGGGAAGGGCTGGGGCAGAGCATCTGTGGGGTGGGGTCTTCCCCTTGCTGCTATTJ

GTATGGCTGGGGACTGGTCTCTCTCTGCCCCAGCCAGTTTGGGGCTGGTTCCCCCATC

TTGAATTGTTTATCCCTTTTTCATAATTAAA

ORF Start: ATG at 260 ORF Stop: TGA at 2111

SEQ ID NO: 146 617 aa MW at 70921.7kD

NOV31g, M LLGP RLPLCPPKRK P H HLVTDAVAR I ETLFHT MVPAIDPXVSFYHADQ KPQVSWIPNiαreSG YGLMK VLPNA PAELARVIVLDTDVTFASDISEL A FAHF CG50595-01 SDTQAIGLVENQSD YLG] _l _αvπjRPWPALGRGFN GVILLRLDRLRQAGWEQM R T Protein ARREL S PATSLADQDIFNAVIKEHPGLVQR PCVW VQ SDHTLAERCYSEASD K Sequence VIHWNSPKK RVKMOlVEFFRlvIFYLTFLEYDG IiLRRELFVCPSQPPPGAEQLQQA A QLDEEDPCFEFRQQQL VHRVHVTFLPHEPPPPRPHDVT VAQLSMDR QMLEALCRH WPGPMS ALY TDAEAQQF HFVEASPVI___lQDVAYrrvVYREGPLYPVNQLRNVA_A QALTPYVF SDIDFLPAYS YDY RASIEQLGLGSRRKAALWPAFETLRYRFSFPHS KVE ]_ALLDAGTLYTFRYHE PRGHAPTDYARWREAQAPYRVQ AANYEPYVVVPRDC PRYDPRFVGFGWNKVAHIVE DAQEYE LVLPEAFTIH PHAPSLDISRFRSSPTYRD CLQALKDEFHQDLSRHHGAAA KY PALQQPQSPARG

SEQ ID NO: 147 1926 bp

NOV31h, AACCGCTCCGACTGCGGCCCGCAGCCGCCGCCGCCGCCCAAGTGCGAGCTCTTGCATG TGGCCATCGTGTGTGCGGGGCATAACTCCAGCCGAGACGTCATCACCCTGGTGAAGTC CG50595-03 CATGCTCTTCTACAGGAAAAATCCACTGCACCTCCACTTGGTGACTGACGCCGTGGCC DNA Sequence AGAAACATCCTGGAGACGCTCTTCCACACATGGATGGTGCCTGCTGTCCGTGTCAGCT TTTATCATACCGACCAGCTCAAGCCCCAGGTCTCCTGGATCCCCAACAAGCACTACTC CGGCCTCTATGGGCTAATGAAGCTGGTGCTGCCCAGTGCCTTGCCTGCTGAGCTGGCC CGCGTCATTGTCCTGGACACGGATGTCACCTTCGCCTCTGACATCTCGGAGCTCTGGG CCCTCTGTGCTCACTTTTCTGACACGCAGGCGATCGGTCTTGTGGAGAACCAGAGTGA CTGGTACCTGGGCAACCTCTGGAAGAACCACAGGCCCTGGCCTGCCTTGGGCCGGGGA TTTAACACAGGTGTGATCCTGCTGCGGCTGGACCGGCTCCGGCAGGCTGGCTGGGAGC AGATGTGGAGGCTGACAGCCAGGCGGGAGCTCCTTAGCCTGCCTGCCACCTCACTGGC TGACCAGGACATCTTCAACGCTGTGATCAAGGAGCACCCGGGGCTAGTGCAGCGTCTG CCTTGTGTCTGGAATGTGCAGCTGTCAGATCACACACTGGCCGAGCGCTGCTACTCTGi

AGGACGTGGCCTACCATGTGGTGTACCGTGAGGGGCCCCTATACCCCGTCAACCAGCT TCGCAACGTGGCCTTGGCCCAGGCCCTCACGCCTTACGTCTTCCTCAGTGACATTGAC TTCCTGCCTGCCTATTCTCTCTACGACTACCTCAGGGCCTCCATTGAGCAGCTGGGGC TGGGCAGCCGGCGCAAGGCAGCACTGGTGGTGCCGGCATTTGAGACCCTGCGCTACCG CTTCAGCTTCCCCCATTCCAAGGTGGAGCTGTTGGCCTTGCTGGATGCGGGCACTCTC TACACCTTCAGGTACCACGAGTGGCCCCGAGGCCACGCACCCACAGACTATGCCCGCT GGCGGGAGGCTCAGGCCCCGTACCGTGTGCAATGGGCGGCCAACTATGAACCCTACGT GGTGGTGCCACGAGACTGTCCCCGCTATGATCCTCGCTTTGTGGGCTTCGGCTGGAAC AAAGTGGCCCACATTGTGGAGCTGGATGCCCAGGAATATGAGCTCCTGGTGCTGCCCG AGGCCTTCACCATCCATCTGCCCCACGCTCCAAGCCTGGACATCTCCCGCTTCCGCTC CAGCCCCACCTATCGTGACTGCCTCCAGGCCCTCAAGGACGAATTCCACCAGGACTTG TCCCGCCACCATGGGGCTGCTGCCCTCAAATACCTCCCAGCCCTGCAGCAGCCCCAGA GCCCTGCCCGAGGCTGAGGCTGGGCCGGCGCTGCCCCTCATCTTAGCATTGGGCAGAC ACCAGGGCAACCTGCCCTCCGCCATCCCTGCTATTTAAATTATTTAAGGTCTCTGGGA

AGGGCTGGGGCAGAGCATCTGTGGGGTGGGGTCTTCCCCTTGCTGCTATTGTATGGCT GGGGACTGGTCTCTCTCTGCCCCAGCCAGTTTGGGGCTGGTTCCCCCATCTTGAATTG TTTATCCCTTTTTCATAATTAAAGTTTTAAAACATCA

ORF Start: ATG at 18 ORF Stop: TGA at 2103

SEQ ID NO: 150 695 aa MW at 79345.3kD

|NOV31i, M PRGRPRALGAAALLLLLLLLGFL FGGDLGREAAESRRPRRIvIPGGPAPGTTTAPTA ARSRRRPPKCELLHVAIVCAGHNSSRDVII VKSMLFYRKNPLHLHLVTDAVARNILE CG50595-05 T FHT 1WPAVRVSFYHADQLKPQVS IP KHΥSG YGLMKLVLPSALPAE ARVIVL Protein DTDVTFASDISE ALFAHFSDTQAIGLVΞNQSDV_LGNLWK_IHRPWPALGRGFNTGV Sequence IL RLDRLRQAGWEQM RLTARRELLSLPATSLADQDIFNAVIKEHPGLVQR PCVWN VQ SDHT AERCYSEASDLKVIHVrøSPKKLRVKNKHVEFFR FYLTFLEYDG LLRRE LFVCPSQPPPGAEQ QQALAQ DEEDPCFEFRQQQLTVHRVHVTFLPHEPPPPRPHDV T VAQLSMDRLQMLEALCRH PGPMSLALY TDAEAQQFLHFVEASPV AARQDVAYH WYREGP YPVNQLRNVALAQALTPYVFLSDIDFLPAYSLYDY RASIEQLG GSRRK AALWPAFETLRYRFSFPHSKVEL ALLDAGT YTFRYHE PRGHAPTDYAR REAQA PYRVQ AA YEPYVV tPRDCPRYDPRFVGFGWπSIKVAHIVE DAQEYELIiVIiPEAFTIH LPHAPSLDISRFRSSPTYRDCLQAL DEFHQDLSRHHGAAALKYLPALQQPQSPARG

SEQ ID NO: 151 2115 bp

NOV31J, ATAGCGGCCGCCACCATGCTGCCCCGAGGGCGCCCCCGGGCGCTGGGGGCCGCCGCGC TGTTGCTGCTGCTGCTGCTGCTCGGATTCCTCCTGTTCGACGGGAGGCTGCGGAGAGC CG50595-09 CGCCGCCCTCGACGGAGACCCGGGGGCCGGCCCCGGGGACCACAACCGCTCCGACTGC DNA Sequence GGCCCGCAGCCGCCGCCGCCGCCCAAGTGCGAGCTCTTGCATGTGGCCATCGTGTGTG CGGGGCATAACTCCAGCCGAGACGTCATCACCCTGGTGAAGTCCATGCTCTTCTACAG GAAAAATCCACTGCACCTCCACTTGGTGACTGACGCCGTGGCCAGAAACATCCTGGAG ACGCTCTTCCACACATGGATGGTGCCTGCTGTCCGTGTCAGCTTTTATCATGCCGACC AGCTCAAGCCCCAGGTCTCCTGGATCCCCAACAAGCACTACTCCGGCCTCTATGGGCT AATGAAGCTGGTGCTGCCCAGTGCCTTGCCTGCTGAGCTGGCCCGCGTCATTGTCCTG GACACGGATGTCACCTTCGCCTCTGACATCTCGGAGCTCTGGGCCCTCTTTGCTCACT TTTCTGACACGCAGGCGATCGGTCTTGTGGAGAACCAGAGTGACTGGTACCTGGGCAA CCTCTGGAAGAACCACAGGCCCTGGCCTGCCTTGGGCCGGGGATTTAACACAGGTGTG ATCCTGCTGCGGCTGGACCGGCTCCGGCAGGCTGGCTGGGAGCAGATGTGGAGGCTGA CAGCCAGGCGGGAGCTCCTTAGCCTGCCTGCCACCTCACTGGCTGACCAGGACATCTT CAACGCTGTGATCAAGGAGCACCCGGGGCTAGTGCAGCGTCTGCCTTGTGTCTGGAAT GTGCAGCTGTCAGATCACACACTGGCCGAGCGCTGCTACTCTGAGGCGTCTGACCTCA AGGTGATCCACTGGAACTCACCAAAGAAGCTTCGGGTGAAGAACAAGCATGTGGAATT CTTCCGCAATTTCTACCTGACCTTCCTGGAGTACGATGGGAACCTGCTGCGGAGAGAG CTCTTTGTGTGCCCCAGCCAGCCCCCACCTGGTGCTGAGCAGTTGCAGCAGGCCCTGG CACAACTGGACGAGGAAGACCCCTGCTTTGAGTTCCGGCAGCAGCAGCTCACTGTGCA CCGTGTGCATGTCACTTTCCTGCCCCATGAACCGCCACCCCCCCGGCCTCACGATGTC ACCCTTGTGGCCCAGCTGTCCATGGACCGGCTGCAGATGTTGGAAGCCCTGTGCAGGC ACTGGCCTGGCCCCATGAGCCTGGCCTTGTACCTGACAGACGCAGAAGCTCAGCAGTT CCTGCATTTCGTCGAGGCCTCACCAGTGCTTGCTGCCCGGCAGGACGTGGCCTACCAT GTGGTGTACCGTGAGGGGCCCCTATACCCCGTCAACCAGCTTCGCAACGTGGCCTTGG CCCAGGCCCTCACGCCTTACGTCTTCCTCAGTGACATTGACTTCCTGCCTGCCTATTC TCTCTACGACTACCTCAGGGCCTCCATTGAGCAGCTGGGGCTGGGCAGCCGGCGCAAG GCAGCACTGGTGGTGCCGGCATTTGAGACCCTGCGCTACCGCTTCAGCTTCCCCCATT CCAAGGTGGAGCTGTTGGCCTTGCTGGATGCGGGCACTCTCTACACCTTCAGGTACCA CGAGTGGCCCCGAGGCCACGCACCCACAGACTATGCCCGCTGGCGGGAGGCTCAGGCC CCGTACCGTGTGCAATGGGCGGCCAACTATGAACCCTACGTGGTGGTGCCACGAGACT GTCCCCGCTATGATCCTCGCTTTGTGGGCTTCGGCTGGAACAAAGTGGCCCACATTGT GGAGCTGGATGCCCAGGAATATGAGCTCCTGGTGCTGCCCGAGGCCTTCACCATCCAT CTGCCCCACGCTCCAAGCCTGGACATCTCCCGCTTCCGCTCCAGCCCCACCTATCGTG ACTGCCTCCAGGCCCTCAAGGACGAATTCCACCAGGACTTGTCCCGCCACCATGGGGC TGCTGCCCTCAAATACCTCCCAGCCCTGCAGCAGCCGCAGAGCCCTGCCCGAGGCCAC CATCACCACCATCACTGACTCGAGCGG

ORF Start: ATG at 16 ORF Stop: TGA at 2104

SEQ ID NO: 152 696 aa MW at 79522.4kD

NOV31J, MLPRGRPRALGAAALL LLL GF LFDGRLRRAAALDGDPGAGPGDH RSDCGPQPP

PPPKCELLHVAIVCAGH SSRDVITLVKSMLFYRK P HLHLVTDAVARNI ETLFHT CG50595-09 WWPA VSFYHADQ_KPQVSWIPNKHYSGLYG _ζ_V^ Protein FASDISEL A FAHFSDTQAIGLVENQSDWY GNL KJSIHRP PALGRGF TGVILLR Sequence DRLRQAG EQM R TARRELLSLPATSLADQDIFNAVIKEHPGLVQRLPCVWNVQLSD

HTLAERCYSEASDLKVIrlWNSPKXLRV-O^HvΕFFRNFYLTFLEYDGKL RRE FVCP

SQPPPGAEQLQQALAQ DEEDPCFEFRQQQ TVHRVHVTFLPHEPPPPRPHDVTLVAQ

LSI_)RLQMLEA CRHWPGPMSLALYLTDAEAQQFLHFVEASPVLAARQDVAYHVVYRE

GPLYPV QLRNVALAQA TPYVFLSDIDFLPAYSLYDY RASIEQLGLGSRRKAA W

PAFETLRYRFSFPHSKVE LAL DAGTLYTFRYHE PRGHAPTDYAR REAQAPYRVQ AATSTYEPYVVVPRDCPRYDPRFVGFG'WNKVAHIVELDAQEYELLVLPEAFTIHLPHAP

S DISRFRSSPTYRDCLQALKDEFHQD SRHHGAAALKYLPA QQPQSPARGHHHHHH

SEQ ID NO: 153 2094 bp

NOV31 , ATAGCGGCCGCCACCATGCTGCCCCGAGGGCGCCCCCGGGCGCTGGGGGCCGCCGCGC

TGTTGCTGCTGCTGCTGCTGCTCGGATTCCTCCTGTTCGACGGGAGGCTGCGGAGAGC CG50595-10 CGCCGCCCTCGACGGAGACCCGGGGGCCGGCCCCGGGGACCACAACCGCTCCGACTGC DNA Sequence GGCCCGCAGCCGCCGCCGCCGCCCAAGTGCGAGCTCTTGCATGTGGCCATCGTGTGTG CGGGGCATAACTCCAGCCGAGACGTCATCACCCTGGTGAAGTCCATGCTCTTCTACAG GAAAAATCCACTGCACCTCCACTTGGTGACTGACGCCGTGGCCAGAAACATCCTGGAG ACGCTCTTCCACACATGGATGGTGCCTGCTGTCCGTGTCAGCTTTTATCATGCCGACC AGCTCAAGCCCCAGGTCTCCTGGATCCCCAACAAGCACTACTCCGGCCTCTATGGGCT AATGAAGCTGGTGCTGCCCAGTGCCTTGCCTGCTGAGCTGGCCCGCGTCATTGTCCTG GACACGGATGTCACCTTCGCCTCTGACATCTCGGAGCTCTGGGCCCTCTTTGCTCACT TTTCTGACACGCAGGCGATCGGTCTTGTGGAGAACCAGAGTGACTGGTACCTGGGCAA CCTCTGGAAGAACCACAGGCCCTGGCCTGCCTTGGGCCGGGGATTTAACACAGGTGTG ATCCTGCTGCGGCTGGACCGGCTCCGGCAGGCTGGCTGGGAGCAGATGTGGAGGCTGA CAGCCAGGCGGGAGCTCCTTAGCCTGCCTGCCACCTCACTGGCTGACCAGGACATCTT CAACGCTGTGATCAAGGAGCACCCGGGGCTAGTGCAGCGTCTGCCTTGTGTCTGGAAT GTGCAGCTGTCAGATCACACACTGGCCGAGCGCTGCTACTCTGAGGCGTCTGACCTCA AGGTGATCCACTGGAACTCACCAAAGAAGCTTCGGGTGAAGAACAAGCATGTGGAATT CTTCCGCAATTTCTACCTGACCTTCCTGGAGTACGATGGGAACCTGCTGCGGAGAGAG CTCTTTGTGTGCCCCAGCCAGCCCCCACCTGGTGCTGAGCAGTTGCAGCAGGCCCTGG CACAACTGGACGAGGAAGACCCCTGCTTTGAGTTCCGGCAGCAGCAGCTCACTGTGCA CCGTGTGCATGTCACTTTCCTGCCCCATGAACCGCCACCCCCCCGGCCTCACGATGTC ACCCTTGTGGCCCAGCTGTCCATGGACCGGCTGCAGATGTTGGAAGCCCTGTGCAGGC ACTGGCCTGGCCCCATGAGCCTGGCCTTGTACCTGACAGACGCAGAAGCTCAGCAGTT CCTGCATTTCGTCGAGGCCTCACCAGTGCTTGCTGCCCGGCAGGACGTGGCCTACCAT GTGGTGTACCGTGAGGGGCCCCTATACCCCGTCAACCAGCTTCGCAACGTGGCCTTGG CCCAGGCCCTCACGCCTTACGTCTTCCTCAGTGACATTGACTTCCTGCCTGCCTATTC TCTCTACGACTACCTCAGGGCCTCCATTGAGCAGCTGGGGCTGGGCAGCCGGCGCAAG GCAGCACTGGTGGTGCCGGCATTTGAGACCCTGCGCTACCGCTTCAGCTTCCCCCATT CCAAGGTGGAGCTGTTGGCCTTGCTGGATGCGGGCACTCTCTACACCTTCAGGTACCA CGAGTGGCCCCGAGGCCACGCACCCACAGACTATGCCCGCTGGCGGGAGGCTCAGGCC CCGTACCGTGTGCAATGGGCGGCCAACTATGAACCCTACGTGGTGGTGCCACGAGACT GTCCCCGCTATGATCCTCGCTTTGTGGGCTTCGGCTGGAACAAAGTGGCCCACATTGT GGAGCTGGATGCCCAGGAATATGAGCTCCTGGTGCTGCCCGAGGCCTTCACCATCCAT CTGCCCCACGCTCCAAGCCTGGACATCTCCCGCTTCCGCTCCAGCCCCACCTATCGTG ACTGCCTCCAGGCCCTCAAGGACGAATTCCACCAGGACTTGTCCCGCCACCATGGGGC TGCTGCCCTCAAATACCTCCCAGCCCTGCAGCAGCCCCAGAGCCCTGCCCGAGGCCTC GAGGGC

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

in Table 3 lC.

Table 31C. Protein Sequence Properties NOV31a

SignalP analysis: Cleavage site between residues 37 and 38

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

N-region: length 8; pos.chg 3; neg.chg 0 H-region: length 19; peak value 11.71 PSG score: 7.31

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -₂.1): 0.8₂ possible cleavage site: between ₂7 and ₂8

»> Seems to have a cleavable signal peptide (1 to 27) ALOM: Klein et al's method for TM region allocation Init position for calculation: 28

Tentative number of TMS(s) for the threshold 0.5: 1 Number of TMS(s) for threshold 0.5: 0 PERIPHERAL Likelihood = 4.61 (at 478) ALOM score: -1.38 (number of TMSs: 0)

MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 13 Charge difference: -4.0 C( 0.0) - N( 4.0) N >= C: N-terminal side will be inside

MITDISC: discrimination of mitochondrial targeting seq R content: 3 Hyd Moment (75) : 7.34 Hyd Momen (95) : 7.37 G content: 3 D/E content: 1 S/T content: 0 Score: -3.37

Gavel: prediction, of cleavage sites for mitochondrial preseq R-2 motif at 18 PRA|LG

NUCDISC: discrimination of nuclear localization signals pat4 : none pat7: PKKLRVK (5) at 312 bipartite: none content of basic residues: 10.1% NLS Score: -0.04

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

ER Membrane Retention Signals :

XXRR-like motif in the N-terminus: LPRG

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

PTS2 : 2nd peroxisomal targeting signal: none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

A search of the NOV31a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 3 ID.

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

PFam analysis predicts that the NOV31a protein contains the domains shown in the Table 3 IF.

Example 32.

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

Table 35A. NOV35 Sequence Analysis

SEQ ID NO: 155 3313 bp

NOV32a, TATAGCATCCATTGTGAAAGTGGAAAAGTAAAGATAATTCATCATGCCTGCTGTGGCT^ CG59406-02 TCAGTTCCTAAAGAACTCTACCTCAGTTCTTCACTAAAAGACCTTAATAAGAAGACAG AAGTTAAACCAGAGAAAATAAGCACTAAGAGTTATGTGCACAGTGCCCTGAAGATCTT DNA Sequence TAAGACAGCAGAAGAATGCAGATTAGATCGTGATGAGGAAAGGGCCTATGTACTATAT ATGAAATACGTGACTGTTTATAATCTTATCAAAAAAAGACCTGATTTCAAGCAACAGC AGGAT ATTTCCAT CAATACTTGGACCTGGAAACATCAAAAAAGCTGTCGAAGAAGC TGAAAGACTCTCTGAAAGCCTTAAATTAAGATATGAAGAAGCTGAAGTCCGGAAAAAA CTTGAGGAAAAAGACAGGCAGGAGGAAGCACAGCGGCTACAACAAAAAAGGCAGGAAA CAGGAAGAGAGGATGGTGGCACATTGGCTAAAGGCTCTTTGGAGAATGTTTTGGAT C CAAAGACAAAACCCAAAAGAGCAATGGTGAAAAGAATGAAAAATGTGAGACCAAAGAG AAAGGAGCAATCACAGCAAAGGAACTATACACAATGATGACGGATAAAAACATCAGCT TGATTATAATGGATGCTCGAAGAATGCAGGATTATCAGGATTCCTGTATTTTACATTC TCTCAGTGTTCCTGAAGAAGCCATCAGTCCAGGAGTCACTGCTAGTTGGATTGAAGCA CACCTGCCAGATGATTCTAAAGACACATGGAGGAAGAGGGGGAATGTGGAGTATGTGG TACTTCTTGACTGGTTTAGTTCTGCCAAAGATTTACAGATTGGAACAACTCTCCGGAG TCTGAAAGATGCACTTTTCAAGTGGGAAAGTAAAACTGTCCTGCGCAATGAGCCTTTG GTTTTAGAGGGAGGCTATGAAAACTGGCTCCTTTGTTATCCCCAGTATACAACAAATG CTAAGGTCACTCCACCCCCACGACGCCAGAATGAAGAGGTGTCTATCTCATTGGATTT TACTTATCCCTCATTGGAAGAATCAATTCCTTCTAAACCTGCTGCCCAGACGCCACCT GCATCTATAGAAGTAGATGAAAATATAGAATTGATAAGTGGTCAAAATGAGAGAATGG GACCACTGAATATATCAACTCCAGTTGAACCAGTTGCTGCTTCTAAATCTGATGTTTC ACCCATAATTCAGCCAGTGCCTAGTATAAAGAATGTTCCACAGATTGATCGTACTAAA AAACCAGCAGTCAAATTGCCTGAAGAGCATAGAATAAAATCTGAAAGTACAAACCATG AGCAACAATCTCCTCAGAGTGGAAAAGTTATTCCTGATCGT CCACCAAGCCAGTAGT TTTTTCTCCAACTCTCATGTTAACAGATGAAGAAAAGGCTCGTATTCATGCAGAAACT GCTCTTCTAATGGAAAAAAACAAACAAGAAAAAGAACTTCGGGAAAGGCAGCAAGAGG AACAGAAAGAGAAACTGAGGAAGGAAGAACAAGAACAAAAAGCCAAAAAGAAACAAGA AGCTGAAGAAAATGAAATTACAGAGAAGCAACAAAAAGCAAAAGAAGAAATGGAGAAG AAAGAAAGTGAACAGGCCAAGAAAGAAGATAAAGAAACCTCAGCAAAGAGGGGCAAAG AAATAACAGGAGTAAAAAGACAAAGTAAAAGTGAACATGAAACTTCTGATGCCAAGAA ATCTGTAGAAGATAGGGGGAAAAGGTGTCCAACCCCAGAAATACAGAAAAAGTCAACA GGAGATGTGTCCCATACATCTGTGACAGGGGATTCAGGTTCAGGCAAGGCTCAACGAG AACCTTTGACAAGAGCACGAAGTGAAGAAATGGGGAGGATCGTACCAGGACTGCC TC AGGCTGGGCCAAGTTTCTTGACCCAATCACTGGAACCTTTCGTTATTATCATTCACCC ACCAACACTGTTCATATGTACCCACCGGAAATGGCTCCTTCATCTGCACCTCCTTCCA CCCCTCCAACTCATAAAGCCAAGCCACAGATTCCTGCTGAGCGGGATAGGGAACCTTC CAAACTGAAGCGCTCCTACTCCTCCCCAGATATAACCCAGGCTATTCAAGAGGAAGAG AAGAGGAAGCCAACAGTAACTCCAACAGTTAATCGGGAAAACAAGCCAACATGTTATC CTAAAGCTGAGATCTCAAGGCTTTCTGCTTCTCAGATTCGGAACCTCAATCCTGTTTT TGGAGGTTCTGGACCAGCTCTTACTGGACTTCGTAACTTAGGAAATACTTGTTATATG AACTCAATATTGCAGTGCCTATGTAACGCTCCACATTTGGCTGATTATTTCAACCGAA ACTGTTATCAGGATGATATTAACAGGTCAAATTTGTTGGGGCATAAAGGTGAAGTGGC AGAAGAATTTGGTATAATCATGAAAGCCCTGTGGACAGGACAGTATAGATATATCAGT CCAAAGGACTTTAAAATCACCATTGGGAAGATCAATGACCAGTTTGCAGGATACAGTC AGCAAGATTCACAAGAATTGCTTCTGTTCCTAATGGATGGTCTCCATGAAGATCTAAA TAAAGCTGATAATCGGAAGAGATATAAAGAAGAAAATAATGATCATCTCGATGACTTT AAAGCTGCAGAACATGCCTGGCAGAAACACAAGCAGCTCAATGAGTCTATTATTGTTG CACTTTTTCAGGGTCAATTCAAATCTACAGTACAGTGCCTCACATGTCACAAAAAGTC TAGGACATTTGAGGCCTTCATGTATT GTCTCTACCACTAGCATCCACAAGTAAATGT ACATTACAGGATTGCCTTAGATTATTTTCCAAAGAAGAAAAACTCACAGATAACAACA GATTTTACTGCAGTCATTGCAGAGCTCGACGGGATTCTCTAAAAAAGATAGAAATCTG GAAGTTACCACCTGTGCTTTTAGTGCATCTGAAACGTTTTTCCTACGATGGCAGGTGG AAACAAAAATTACAGACATCTGTGGACTTCCCGTTAGAAAATC TGACTTGTCACAGT ATGTTATTGGTCCAAAGAACAATTTGAAGAAATATAATTTGTTTTCTGTTTCAAATCA CTACGGTGGGCTGGATGGAGGCCACTACACAGCCTATTGTAAAAATGCAGCAAGACAA CGGTGGTTTAAGTTTGATGATCATGAAGTTTCTGATATCTCCGTTTCTTCTGTGAAAT CTTCAGCAGCTTATATCCTCTTTTATACTTCATTGGGACCACGAGTAACTGATGTAGC CACATAA

ORF Start: ATG at 44 ORF Stop: TAA at 3311

SEQ ID NO: 156 1089 aa MW at 124221.2kD

NOV32a, MPAVASVPKE YLSSSLKDLNKKTEVKPEKISTKSYVHSALKIFKTAEECRLDRDEER AY^YMKYVTVYIJLIKKRPDFKQQQDYFHSILGPGNIKKAVEEAERLSESLKLRYEEA CG59406-02 EVTlKKLEEKDRQEEAQRLQQKRQETGREDGGTLA GS E VLDSKDKTQKSNGEKlvJEK Protein CETKEKGAITAKELYTM TDKNIS IIMDARRMQDYQDSCI HS SVPEEAISPGVTA Sequence S IEAHLPDDSKDTWRKRG VEYVV D FSSAKDLQIGTT RS KDALFKWESKTVL RNEP VLEGGYE fLLCYPQYTTNAKVTPPPRRQNEEVSISLDFTYPSLEESIPSKPA AQTPPASIEVDENIELISGQNERMGPLNISTPVEPVAASKSDVSPIIQPVPSIKNVPQ IDRTKKPAVKLPEEHRIKSESTNHEQQSPQSGKVIPDRSTKPVVFSPT M TDEEKAR IHAETALLMEK-vπQEKELRERQQEEQKEK RKEEQEQIU___QEAEEIv_ITEKQQKAK EEMEKKESEQAKKEDKETSAKRGKEITGVKRQSKSΞHETSDAKKSVEDRGKRCPTPEI QK STGDVSHTSVTGDSGSG AQREPLTRARSEEMGRIVPGLPSGWAKF DPITGTFR YYHSP N VHMYPPEMAPSSAPPSTPPTHKAKPQIPAERDREPSKLKRSYSSPDITQA IQEEEKRKP V P VTv EKuPTCYPKAEISRLSASQIR NPVFGGSGPALTGLRN G N CYMNSILQC CNAPH ADYFKQ_JCYQDDI1_SN LGHKGEVAEEFGII KAL TGQ YRYISPKDFKITIGKINDQFAGYSQQDSQEL LF MDGLHED ADNRKRYKEEN D H DDFKAAEHAWQKHKQLNESIIVALFQGQFKSTVQCLTCHKKSRTFEAFMYLSLP A STSKCT QDCLR FSKEEKLTDN1TOFYCSHCRARRDSLKKIEIWKLPPVLLVHLKRFS YDGR KQKLQTSVOFP EK—D SQYVIGPKlvlNLKKYNLFSVS HYGGLDGGHYTAYCK NAARQRWFKFDDHEVSDISVSSVKSSAAYI FYTSLGPRVTDVAT

SEQ ID NO: 157 4359 bp

NOV32b, GTGAGCTGGGCTGGCTTCCGTCCTGGTAGCCAAGGCTAATTCTCCCTCGAGTTCTTGG

GAGATGGGCATTTGGCGAGAAGGCTGGCGTTAGTGAAGCGCGCCCGGCGTCACGGTGA CG59406-01 GTGCGGGTCTTGGGCCCTAGCACCTGTTCTCTGGGAAGTCGTCCGCTGTGAACGATGA DNA Sequence ACGCCTTTCCTTCCACCAGCTGCTGGTTACCCCGGAGACAAGCTCTGTCCGCGGAGAG

GAGTGGGACAACTCCTAAAGGAAAGAAGCACTTGTAAGGAAATATAGCATCCATTGTG

AAAGTGGAAAAGTAAAGATAATTCATCATGCCTGCTGTGGCTTCAGTTCCTAAAGAAC TCTACCTCAGTTCTTCACTAAAAGACCTTAATAAGAAGACAGAAGTTAAACCAGAGAA AATAAGCACTAAGAGTTATGTGCACAGTGCCCTGAAGATCTTTAAGACAGCAGAAGAA TGCAGATTAGATCGTGATGAGGAAAGGGCCTATGTACTATATATGAAATACGTGACTG TTTATAATCTTATCAAAAAAAGACCTGAT TCAAGCAACAGCAGGATTATTTCCATTC AATACTTGGACCTGGAAACATCAAAAAAGCTGTCGAAGAAGCTGAAAGACTCTCTGAA AGCCTTAAATTAAGATATGAAGAAGCTGAAGTCCGGAAAAAACT GAGGAAAAAGACA GGCAGGAGGAAGCACAGCGGCTACAACAAAAAAGGCAGGAAACAGGAAGAGAGGAGG TGGCACATTGGCTAAAGGCTCTTTGGAGAATGTTTTGGATTCCAAAGACAAAACCCAA AAGAGCAATGGTGAAAAGAATGAAAAATGTGAGACCAAAGAGAAAGGAGCAATCACAG CAAAGGAACTATACACAATGATGACGGATAAAAACATCAGCTTGATTATAATGGATGC TCGAAGAATGCAGGATTATCAGGATTCCTGTATTTTACATTCTCTCAGTGTTCCTGAA GAAGCCATCAGTCCAGGAGTCACTGCTAGTTGGATTGAAGCACACCTGCCAGATGATT CTAAAGACACATGGAAGAAGAGGGGGAATGTGGAGTATGTGGTACTTCTTGACTGGTT TAGTTCTGCCAAAGATTTACAGATTGGAACAACTCTCCGGAGTCTGAAAGATGCACTT TTCAAGTGGGAAAGTAAAACTGTCCTGCGCAATGAGCCTTTGGTTTTAGAGGGAGGCT ATGAAAACTGGCTCCTTTGTTATCCCCAGTATACAACAAATGCTAAGGTCACTCCACC CCCACGACGCCAGAATGAAGAGG GTCTATCTCATTGGATTTTACTTATCCCTCATTG GAAGAATCAATTCCTTCTAAACCTGCTGCCCAGACGCCACCTGCATCTATAGAAGTAG ATGAAAATATAGAATTGATAAGTGGTCAAAATGAGAGAATGGGACCACTGAATATATC AACTCCAGTTGAACCAGTTGCTGCTTCTAAATCTGATGTTTCACCCATAATTCAGCCA GTGCCTAGTATAAAGAATGTTCCACAGATTGATCGTACTAAAAAACCAGCAGTCAAAT TGCCTGAAGAGCATAGAATAAAATCTGAAAGTACAAACCATGAGCAACAATCTCCTCA GAGTGGAAAAGTTATTCCTGATCGTTCCACCAAGCCAGTAGTTTTTTCTCCAACTCTC ATGTTAACAGATGAAGAAAAGGCTCGTATTCATGCAGAAACTGCTCTTCTAATGGAAA AAAACAAACAAGAAAAAGAACTTCGGGAAAGGCAGCAAGAGGAACAGAAAGAGAAACT GAGGAAGGAAGAACAAGAACAAAAAGCCAAAAAGAAACAAGAAGCTGAAGAAAATGAA AT ACAGAGAAGCAACAAAAAGCAAAAGAAGAAATGGAGAAGAAAGAAAGTGAACAGG CCAAGAAAGAAGATAAAGAAACCTCAGCAAAGAGGGGCAAAGAAATAACAGGAGTAAA AAGACAAAGTAAAAGTGAACATGAAACTTCTGATGCCAAGAAATCTGTAGAAGATAGG GGGAAAAGGTGTCCAACCCCAGAAATACAGAAAAAGTCAACAGGAGATGTGCCCCATA CATCTGTGACAGGGGATTCAGGTTCAGGCAAGCCATTTAAGATTAAAGGACAACCAGA AAGTGGAATTCTAAGGACAGGAACTTTTAGAGAGGATACAGACGATACCGAAAGAAAT AAAGCTCAACGAGAACCTTTGACAAGAGCACGAAGTGAAGAAATGGGGAGGATCGTAC CAGGACTGCCTTCAGGCTGGGCCAAGTTTCTTGACCCAATCACTGGAACCT TCGTTA TTATCATTCACCCACCAACACTGTTCATATGTACCCACCGGAAATGGCTCCTTCATCT GCACCTCCTTCCACCCCTCCAACTCATAAAGCCAAGCCACAGATTCCTGCTGAGCGGG ATAGGGAACCTTCCAAACTGAAGCGCTCCTACTCCTCCCCAGATATAACCCAGGCTAT TCAAGAGGAAGAGAAGAGGAAGCCAACAGTAACTCCAACAGTTAATCGGGAAAACAAG CCAACATGTTATCCTAAAGCTGAGATCTCAAGGCTTTCTGCTTCTCAGATTCGGAACC TCAATCCTGTTTTTGGAGGTTCTGGACCAGCTCTTACTGGACTTCGTAACTTAGGAAA TACTTGTTATATGAACTCAATATTGCAGTGCCTATGTAACGCTCCACATTTGGCTGAT TATTTCAACCGAAACTGTTATCAGGATGATATTAACAGGTCAAATTTGTTGGGGCATA AAGGTGAAGTGGCAGAAGAATTTGGTATAATCATGAAAGCCCTGTGGACAGGACAGTA TAGATATATCAGTCCAAAGGACTTTAAAATCACCATTGGGAAGATCAATGACCAGT T GCAGGATACAGTCAGCAAGATTCACAAGAATTGCTTCTGTTCCTAATGGATGGTCTCC ATGAAGATCTAAATAAAGCTGATAATCGGAAGAGATATAAAGAAGAAAATAATGATCA TCTCGATGACTTTAAAGCTGCAGAACATGCCTGGCAGAAACACAAGCAGCTCAATGAG TCTATTATTGTTGCACTTTTTCAGGGTCAATTCAAATCTACAGTACAGTGCCTCACAT GTCACAAAAAGTCTAGGACATTTGAGGCCTTCATGTATTTGTCTCTACCACTAGCATC CACAAGTAAATGTACATTACAGGATTGCCTTAGATTATTTTCCAAAGAAGAAAAACTC ACAGATAACAACAGATTTTACTGCAGTCATTGCAGAGCTCGACGGGATTCTCTAAAAA AGATAGAAATCTGGAAGTTACCACCTGTGCTTTTAGTGCATCTGAAACGTTTTTCCTA CGATGGCAGGTGGAAACAAAAATTACAGACATCTGTGGACTTCCCGTTAGAAAATCTT GACTTGTCACAGTATGTTATTGGTCCAAAGAACAATTTGAAGAAATATAATTTGTTTT CTGTTTCAAATCACTACGGTGGGCTGGATGGAGGCCACTACACAGCCTATTGTAAAAA TGCAGCAAGACAACGGTGGTTTAAGTTTGATGATCATGAAGTTTCTGATATCTCCG T TCTTCTGTGAAATCTTCAGCAGCTTATATCCTCTTTTATACTTCATTGGGACCACGAG TAACTGATGTAGCCACATAAGGAGACATAGGTTATAAACTAGTTATCTTTTAAAAGGC

TCAGCAACACAACTCTTGAAATGCTTATCAGGATAATGGTAGCTATAGCTGGCCATTT

AGAGGAATTCTAGGACAGTGGGAGCTGTGTTACTAGCACTATATAATTCCGGTCAGTG

CTGACAAATAACATTTAACAAGTATTGCAGTAATCATCACTTACAGGTACCATTTATT CAAAACAACTTTTTTAGTCTGCTCCAAAGTTAAAATAATTAACTAGCTAAGCATTAT!

TATTCAACTGGTCTAAAAACTATTGTTATCTTTTTTTTTCCTTTTCACTGTTATGGCC TTTTCACATTTCTAAATCCCATCTTGATATACTATGAATACTCTAGAATGATGTAAAG

CAGATAGGAATGTATGTGTACATATTTATTGCATACTTGCACATCAAATCGATGTACA

TAGTTTAACACGTGGTCCTTTTGTGAAACCTAGAACTCAGAGGATTGCTTTTTTTCTT

TCAGCCTATTTTGAGTTAACTTCAGTCCTTTCTTAGGGAAATGACAGGGCAAAGCAAT

TTTTCTGTTGGCTTTGGGCTGTATTTGTGCACTAAATCTTTATTCTAAAAAAAAAAAT GGAAACTTTAATTTTTTTAAAACGGGAAT TCATTTACAGCTACATTAAAATCTTAAT GAGAAAAAT

ORF Start: ATG at 318 ORF Stop: TAA at 3672

SEQ ID NO: 158 1118 aa MW at 127521.9kD tøOV32b, MPAVASVPKELYLSSS KDLNKKTEVKPEKISTKSYVHSALKIFKTAEECR DRDEER

AYVLY_SYVTVΥNLIKKRPDFKQQQDYFHSILGPGNIKKAVEEAERLSESLKLRYEEA CG59406- 01 EVRKK EEKDRQEEAQRLQQKRQETGREI3GTLAKGS ENv_,DSKDKTQKSNGEK EK Protein CETKEKGAITAKELYTMMTDKNISLIIMDARRMQDYQDSCILHS SVPEEAISPGVTA Sequence SWIEAH PDDSKD KIv^GNVEYVvliLDWFSSAKDLQIGTTLRS KDA F ESKTVL

RNEP VLEGGYElW LCYPQYT NAKvTPPPRRQ EEVSISLDFTYPSLEESIPSKPA

AQTPPASIEVDENIELISGQNERMGPNISTPVEPVAASKSDVSPIIQPVPSIKNVPQ

IDRTKKPAVKLPEEHRIKSESTNHEQQSPQSGKVIPDRSTKPWFSPT MLTDEEKAR

IHAETALLMEK QEKE RERQQEEQKEKLRKEEQEQKAKKKQEAEENEITEKQQKAK

EEMEK ESEQAKKEDKETSAKRGKEITGVKRQSKSEHETSDAKKSVEDRGKRCPTPEI

QI_STGDVPHTSV GDSGSGKPFKIKGQPESGI RTGTFREDTDDTERNKAQREPLTR

ARSEEMGRIVPGLPSG AKFLDPITGTFRYYHSPTNTVHMYPPE APSSAPPSTPPTH

KAKPQIPAERDREPS LKRSYSSPDITQAIQEEEKRKPTV PTVi EN PTCYPKAEI

SRLSASQIRI_NPVFGGSGPALTGLR LGN CYlrøSILQCLCNAPH ADYF- NCYQD

DINRSN GHKGEVAEEFGIIMKALW GQYRYISPKDFKITIGKINDQFAGYSQQDSQ

EL_LF_MDGLHΞDL]π__3NRKRYKEEN^

QFKS VQC TCHKKSRTFEAFMYLS PLASTSKCT QDC RLFSKEEKLTDNNRFYCS

HCRARRDSLKKIEIWK PPVL VH KΛFSYDGR KQKLQTSVDFP E D SQYVIGP

-V_^i KKYNLFSVSNHYGGLDGGrOT Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 32B.

Table 32B. Comparison of NOV32a against NOV32b.

NOV32a Residues/ Identities/

Protein Sequence Match Residues Similarities for the Matched Region

NOV32b 1..1089 1087/1118 (97%) 1..1118 1088/1118 (97%)

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

Table 32C. Protein Sequence Properties NOV32a

SignalP No Known Signal Sequence Predicted analysis:

PSORT II PSG: a new signal peptide prediction method

N-region: length 10; pos.chg 1; neg.chg 1 analysis: H-region: length 7; peak value 0.36 PSG score: -4.04

GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -12.27 possible cleavage site: between 16 and 17

>» Seems to have no N-terminal signal peptide

Tentative number of TMS(s) for the threshold 0.5: 0 number of TMS(s) .. fixed PERIPHERAL Likelihood = 4.40 (at 892) ALOM score: 4.40 (number of TMSs: 0)

MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment (75) : 0.88 Hyd Moment (95) : 1.75 G content: 0 D/E content: 2 S/T content: 4 Score: -7.07

NUCDISC: discrimination of nuclear localization signals pat4: KKRP (4) at 74 pat4: KRKP (4) at 702 pat7: PSKLKRS (4) at 681 bipartite: none content of basic residues: 15.9% NLS Score: 0.37

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

ER Membrane Retention Signals: none

VAC: possible vacuolar targeting motif: none

RNA-binding motif: none

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

NMYR: N-myristoylation pattern : none

Tyrosines in the tail: none

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

Small, acid-soluble spore proteins, alpha/beta type, signature 1 (PS00304): *** found *** KGEVAEEFGI at 794

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

92 G 0.99

93 N 1.00

94 I 1.00

95 K 1.00

96 K 1.00

97 A 1.00

98 V 1.00

99 E 1.00

100 E 1.00

101 A 1.00

102 E 1.00

103 R 1.00

104 L 1.00

105 S 1.00

106 E 1.00

107 S 1.00

108 L 1.00

109 K 1.00

110 L 1.00

111 R 1.00

112 Y 1.00

113 E 1.00

114 E 1.00

115 A 1.00

116 E 1.00

117 V 1.00

118 R 1.00

119 K 1.00

120 K 1.00

121 L 1.00

122 E 1.00

123 E 1.00

124 K 1.00

516 K 1.00

517 Q 1.00

518 Q 1.00

519 K 1.00

520 A 1.00

521 K 1.00

522 E 1.00

523 E 1.00

524 M 1.00

525 E 1.00

526 K 1.00

527 K 1.00

528 E 1.00

529 S 1.00

530 E 1.00

531 Q 1.00

532 A 1.00

533 K 1.00

534 K 1.00

535 E 1.00

536 D 1.00

537 K 1.00

538 E 1.00

539 T 1.00

540 S 0.98

541 A 0.98

542 K 0.98

543 R 0.97

544 G 0.97

545 K 0.97 .,

546 E 0.97

547 I 0.80 total : 132 residues

Final Results (k = 9/23 ) :

65.2 % : nuclear

17 .4 % : cytoplasmic

8.7 % : cytoskeletal

8.7 % : peroxisomal

» prediction for CG59406-02 is nuc (k=23 )

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

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

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

Example B: Sequencing Methodology and Identification of NOVX Clones

1. GeneCalling

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

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

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

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

Physical clone: the cDNA fragment derived by the screening procedure, covering the entire open reading frame is, as a recombinant DNA, cloned into pACT2 plasmid (Clontech) used to make the cDNA library. The recombinant plasmid is inserted into the host and selected by the yeast hybrid diploid generated during the screening procedure by the mating of both CuraGen Corporation proprietary yeast strains N106' and YULH (U. S. Patents 6,057,101 and 6,083,693).

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

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

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

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

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

The quantitative expression of various clones was assessed using microtiter plates containing RNA samples from a variety of normal and pathology-derived cells, cell lines and tissues using real time quantitative PCR (RTQ PCR). RTQ PCR was performed on an Applied Biosystems ABI PRISM® 7700 or an ABI PRISM® 7900 HT Sequence Detection System. Various collections of samples are assembled on the plates, and referred to as Panel 1 (containing normal tissues and cancer cell lines), Panel 2 (containing samples derived from tissues from normal and cancer sources), Panel 3 (containing cancer cell lines), Panel 4 (containing cells and cell lines from normal tissues and cells related to inflammatory conditions), Panel 5D/5I (containing human tissues and cell lines with an emphasis on metabolic diseases), AI_comprehensive_panel (containing normal tissue and samples from autoinflammatory diseases), Panel CNSD.01 (containing samples from normal and diseased brains) and CNS_neurodegeneration_panel (containing samples from normal and Alzheimer's diseased brains). RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s:18s) and the absence of low molecular weight RNAs that would be indicative of degradation products. Samples are controlled against genomic DNA contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.

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

In other cases, non-normalized RNA samples were converted to single strand cDNA (sscDNA) using Superscript II (Invitrogen 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 IX TaqMan® Universal Master mix (Applied Biosystems; catalog No. 4324020), following the manufacturer's instructions.

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

PCR conditions: When working with RNA samples, normalized RNA from each tissue and each cell line was spotted in each well of either a 96 well or a 384- well PCR plate (Applied Biosystems). PCR cocktails included either a single gene specific probe and primers set, or two multiplexed probe and primers sets (a set specific for the target clone and another gene-specific set multiplexed with the target probe). PCR reactions were set up using TaqMan® One-Step RT-PCR Master Mix (Applied Biosystems, Catalog No.4313803) following manufacturer's instructions. Reverse transcription was performed at 48°C for 30 minutes followed by amplification PCR cycles as follows: 95°C 10 min, then 40 cycles of 95°C for 15 seconds, 60°C for 1 minute. Results were recorded as CT values (cycle at which a given sample crosses a threshold level of fluorescence) using a log scale, with the difference in RNA concentration between a given sample and the sample with the lowest CT value being represented as 2 to the power of delta CT. The percent relative expression is then obtained by taking the reciprocal of this RNA difference and multiplying by 100.

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

Panels 1, 1.1, 1.2, and 1.3D

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

In the results for Panels 1, 1.1, 1.2 and 1.3D, the following abbreviations are used: ca. = carcinoma,

* = established from metastasis, met = metastasis, s cell var = small cell variant, non-s = non-sm = non-small, squam = squamous, pi. eff = pi effusion = pleural effusion, glio = glioma, astro = astrocytoma, and neuro = neuroblastoma.

General_screening_panel_vl.4, vl.5, vl.6 and 1.7

The plates for Panels 1.4, 1.5, 1.6 and 1.7 include 2 control wells (genomic DNA control and chemistry control) and 88 to 94 wells containing cDNA from various samples. The samples in Panels 1.4, 1.5, 1.6 and 1.7 are broken into 2 classes: samples derived from cultured cell lines and samples derived from primary normal tissues. The cell lines are derived from cancers ofthe following types: lung cancer, breast cancer, melanoma, colon cancer, prostate cancer, CNS cancer, squamous cell carcinoma, ovarian cancer, liver cancer, renal cancer, gastric cancer and pancreatic cancer. Cell lines used in Panels 1.4, 1.5, 1.6 and 1.7 are widely available through the American

Type Culture Collection (ATCC), a repository for cultured cell lines, and were cultured using the conditions recommended by the ATCC. The normal tissues found on Panels

1.4, 1.5, 1.6 and 1.7 are comprised of pools of samples derived from all major organ systems from 2 to 5 different adult individuals or fetuses. These samples are derived from the following organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various regions of the brain, the spleen, bone marrow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach, small intestine, colon, bladder, trachea, breast, ovary, uterus, placenta, prostate, testis and adipose. Abbreviations are as described for Panels 1, 1.1, 1.2, and 1.3D.

Panels 2D, 2.2, 2.3 and 2.4

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

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

ARDAIS Panel v 1.0

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

The plates for ARDAIS prostate 1.0 generally include 2 control wells and 68 test samples composed of RNA isolated from human tissue procured by surgeons working in close cooperation with Ardais 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.

Panel 3D, 3.1 and 3.2

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

Panels 4D, 4R, and 4.1D

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

Astrocytes, lung fibroblasts, dermal fibroblasts, coronary artery smooth muscle cells, small airway epithelium, bronchial epithelium, microvascular dermal endothelial cells, microvascular lung endothelial cells, human pulmonary aortic endothelial cells, human umbilical vein endothelial cells were all purchased from Clonetics (Walkersville, MD) and grown in the media supplied for these cell types by Clonetics. These primary cell types were activated with various cytokines or combinations of cytokines for 6 and/or 12-14 hours, as indicated. The following cytokines were used; IL-1 beta at approximately l-5ng ml, TNF alpha at approximately 5-10ng/ml, IFN gamma at approximately 20-50ng/ml, IL-4 at approximately 5-10ng/ml, IL-9 at approximately 5- lOng/ml, IL-13 at approximately 5-10ng/ml. Endothelial cells were sometimes starved for various times by culture in the basal media from Clonetics with 0.1% serum.

Mononuclear cells were prepared from blood of employees at CuraGen Corporation, using Ficoll. LAK cells were prepared from these cells by culture in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco/Life Technologies, Rockville, MD), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5x10^" ⁵M (Gibco), and lOmM Hepes (Gibco) and Interleukin 2 for 4-6 days. Cells were then either activated with 10-20ng/ml PMA and 1 -2μg/ml ionomycin, IL-12 at 5-lOng/ml, IFN gamma at 20-50ng/ml and IL-18 at 5-lOng/ml for 6 hours. In some cases, mononuclear cells were cultured for 4-5 days in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xl0^"5M (Gibco), and lOmM Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed mitogen) at approximately 5μg/ml. Samples were taken at 24, 48 and 72 hours for RNA preparation. MLR (mixed lymphocyte reaction) samples were obtained by taking blood from two donors, isolating the mononuclear cells using Ficoll and mixing the isolated mononuclear cells 1:1 at a final concentration of approximately 2xl0⁶cells/ml in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol (5.5x10^' M) (Gibco), and lOmM Hepes (Gibco). The MLR was cultured and samples taken at various time points ranging from 1- 7 days for RNA preparation.

Monocytes were isolated from mononuclear cells using CD14 Miltenyi Beads, +ve VS selection columns and a Vario Magnet according to the manufacturer's instructions. Monocytes were differentiated into dendritic cells by culture in DMEM 5% fetal calf serum (FCS) (Hyclone, Logan, UT), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xl0^"5M (Gibco), and lOmM Hepes (Gibco), 50ng/ml GMCSF and 5ng/ml IL-4 for 5-7 days. Macrophages were prepared by culture of monocytes for 5-7 days in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xlO^"5M (Gibco), lOmM Hepes (Gibco) and 10% AB Human Serum or MCSF at approximately 50ng/ml. Monocytes, macrophages and dendritic cells were stimulated for 6 and 12-14 hours with lipopolysaccharide (LPS) at lOOng ml. Dendritic cells were also stimulated with anti-CD40 monoclonal antibody (Pharmingen) at lOμg/ml for 6 and 12-14 hours.

CD4 lymphocytes, CD8 lymphocytes and NK cells were also isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi beads, positive VS selection columns and a Vario Magnet according to the manufacturer's instructions. CD45RA and CD45RO CD4 lymphocytes were isolated by depleting mononuclear cells of CD8, CD56, CD14 and CD19 cells using CD8, CD56, CD14 and CD19 Miltenyi beads and positive selection. CD45RO beads were then used to isolate the CD45RO CD4 lymphocytes with the remaining cells being CD45RA CD4 lymphocytes. CD45RA CD4, CD45RO CD4 and CD8 lymphocytes were placed in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xl0^"5M (Gibco), and lOmM Hepes (Gibco) and plated at 10⁶cells/ml onto Falcon 6 well tissue culture plates that had been coated overnight with 0.5μg/ml anti-CD28 (Pharmingen) and 3ug ml anti-CD3 (OKT3, ATCC) in PBS. After 6 and 24 hours, the cells were harvested for RNA preparation. To prepare chronically activated CD8 lymphocytes, we activated the isolated CD8 lymphocytes for 4 days on anti-CD28 and anti-CD3 coated plates and then harvested the cells and expanded them in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xl0^"5M (Gibco), and lOmM Hepes (Gibco) and IL-2. The expanded CD8 cells were then activated again with plate bound anti-CD3 and anti- CD28 for 4 days and expanded as before. RNA was isolated 6 and 24 hours after the second activation and after 4 days ofthe second expansion culture. The isolated NK cells were cultured in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xl0^"5M (Gibco), and lOmM Hepes (Gibco) and IL -2 for 4-6 days before RNA was prepared.

To obtain B cells, tonsils were procured from NDRI. The tonsil was cut up with sterile dissecting scissors and then passed through a sieve. Tonsil cells were then spun down and resupended at 10⁶cells/ml in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5x10^" ⁵M (Gibco), and lOmM Hepes (Gibco). To activate the cells, we used PWM at 5μg/ml or anti-CD40 (Pharmingen) at approximately lOμg ml and IL-4 at 5-10ng/ml. Cells were harvested for RNA preparation at 24,48 and 72 hours.

To prepare the primary and secondary Thl/Th2 and Trl cells, six-well Falcon plates were coated overnight with lOμg/ml anti-CD28 (Pharmingen) and 2μg/ml OKT3 (ATCC), and then washed twice with PBS. Umbilical cord blood CD4 lymphocytes (Poietic Systems, German Town, MD) were cultured at 10⁵-10⁶cells/ml in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xlO^"5M (Gibco), lOmM Hepes (Gibco) and E -2 (4ng/ml). IL-12 (5ng ml) and anti-IL4 (lμg/ l) were used to direct to Thl, while IL-4 (5ng ml) and anti-IFN gamma (lμg/ml) were used to direct to Th2 and IL-10 at 5ng ml was used to direct to Trl. After 4-5 days, the activated Thl, Th2 and Trl lymphocytes were washed once in DMEM and expanded for 4-7 days in DMEM 5% FCS (Hyclone), lOOμM non essential amino acids (Gibco), ImM sodium pyruvate (Gibco), mercaptoethanol 5.5xl0^"5M (Gibco), lOmM Hepes (Gibco) and IL-2 (lng ml). Following this, the activated Thl, Th2 and Trl lymphocytes were re-stimulated for 5 days with anti-CD28/OKT3 and cytokines as described above, but with the addition of anti-CD95L (lμg/ml) to prevent apoptosis. After 4-5 days, the Thl, Th2 and Trl lymphocytes were washed and then expanded again with IL-2 for 4-7 days. Activated Thl and Th2 lymphocytes were maintained in this way for a maximum of three cycles. RNA was prepared from primary and secondary Thl, Th2 and Trl after 6 and 24 hours following the second and third activations with plate bound anti-CD3 and anti-CD28 mAbs and 4 days into the second and third expansion cultures in Interleukin 2.

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

For these cell lines and blood cells, RNA was prepared by lysing approximately 10⁷cells/ml using Trizol (Gibco BRL). Briefly, 1/10 volume of bromochloropropane (Molecular Research Corporation) was added to the RNA sample, vortexed and after 10 minutes at room temperature, the tubes were spun at 14,000 rpm in a Sorvall SS34 rotor. The aqueous phase was removed and placed in a 15ml Falcon Tube. An equal volume of isopropanol was added and left at -20°C overnight. The precipitated RNA was spun down at 9,000 rpm for 15 min in a Sorvall SS34 rotor and washed in 70% ethanol. The pellet was redissolved in 300μl of RNAse-free water and 35μl buffer (Promega) 5μl DTT, 7μl RNAsin and 8μl DNAse were added. The tube was incubated at 37°C for 30 minutes to remove contaminating genomic DNA, extracted once with phenol chloroform and re-precipitated with 1/10 volume of 3M sodium acetate and 2 volumes of 100% ethanol. The RNA was spun down and placed in RNAse free water. RNA was stored at - 80°C.

AI_comprehensive panel_vl.O

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

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

Surgical specimens of psoriatic tissues and adjacent matched tissues were provided as total RNA by Clinomics. Two male and two female patients were selected between the ages of 25 and 47. None of the patients were taking prescription drugs at the time samples were isolated.

Surgical specimens of diseased colon from patients with ulcerative colitis and Crohns disease and adjacent matched tissues were obtained from Clinomics. Bowel tissue from three female and three male Crohn's patients between the ages of 41-69 were used. Two patients were not on prescription medication while the others were taking dexamethasone, phenobarbital, or tylenol. Ulcerative colitis tissue was from three male and four female patients. Four of the patients were taking lebvid and two were on phenobarbital.

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

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

Al = Autoimmunity

Syn = Synovial

Normal = No apparent disease

Rep22 /Rep20 = individual patients

RA = Rheumatoid arthritis

Backus = From Backus Hospital

OA = Osteoarthritis

(SS) (B A) (MF) = Individual patients

Adj = Adjacent tissue

Match control = adjacent tissues

-M = Male

-F = Female

COPD = Chronic obstructive pulmonary disease

AI.05 chondrosarcoma

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

Panels 5D and 51

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

In the Gestational Diabetes study subjects are young (18 - 40 years), otherwise healthy women with and without gestational diabetes undergoing routine (elective)

Caesarean section. After delivery ofthe infant, when the surgical incisions were being repaired/closed, the obstetrician removed a small sample (<1 cc) of the exposed metabolic tissues during the closure of each surgical level. The biopsy material was rinsed in sterile saline, blotted and fast frozen within 5 minutes from the time of removal. The tissue was then flash frozen in liquid nitrogen and stored, individually, in sterile screw-top tubes and kept on dry ice for shipment to or to be picked up by

CuraGen. The metabolic tissues of interest include uterine wall (smooth muscle), visceral adipose, skeletal muscle (rectus) and subcutaneous adipose. Patient descriptions are as follows:

Patient 2: Diabetic Hispanic, overweight, not on insulin

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

Patient 10: Diabetic Hispanic, overweight, on insulin

Patient 11 : Nondiabetic African American and overweight

Patient 12: Diabetic Hispanic on insulin

Adiocyte differentiation was induced in donor progenitor cells obtained from

Osirus (a division of Clonetics/BioWhittaker) in triplicate, except for Donor 3U which had only two replicates. Scientists at Clonetics isolated, grew and differentiated human mesenchymal stem cells (HuMSCs) for CuraGen based on the published protocol found in Mark F. Pittenger, et al., Multilineage Potential of Adult Human Mesenchymal Stem

Cells Science Apr 2 1999: 143-147. Clonetics provided Trizol lysates or frozen pellets suitable for mRNA isolation and ds cDNA production. A general description of each donor is as follows:

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

Donor 2 and 3 AM: Adipose, AdiposeMidway Differentiated

Donor 2 and 3 AD: Adipose, Adipose Differentiated

Human cell lines were generally obtained from ATCC (American Type Culture

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

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

In the labels employed to identify tissues in the 5D and 51 panels, the following abbreviations are used:

GO Adipose = Greater Omentum Adipose

SK = Skeletal Muscle

UT = Uterus

PL = Placenta

AD = Adipose Differentiated

AM = Adipose Midway Differentiated

U = Undifferentiated Stem Cells

Human Metabolic RTQ-PCR Panel

The plates for the Human Metabolic RTQ-PCR Panel include two control wells (genomic DNA control and chemistry control) and 211 cDNAs isolated from human tissues and cell lines with an emphasis on metabolic diseases. This panel is useful for establishing the tissue and cellular expression profiles for genes believed to play a role in the etiology and pathogenesis of obesity and/or diabetes and to confirm differential expression of such genes derived from other methods. Metabolic tissues were obtained from patients enrolled in the CuraGen Gestational Diabetes study and from autopsy tissues from Type II diabetics and age, sex and race-matched control patients. One or more of the following were used to characterize the patients: body mass index [BMI = wt (kg) / ht (m²)], serum glucose, HgbAlc. Cell lines used in this panel are widely available through the American Type Culture Collection (ATCC), a repository for cultured cell lines. RNA from human Pancreatic Islets was also obtained.

In the Gestational Diabetes study, subjects are young (18-40 years), otherwise healthy women with and without gestational diabetes undergoing routine (elective) Caesarian section. After delivery of the infant, when the surgical incisions were being repaired/closed, the obstetrician removed a small sample (<lcc) of the exposed W

metabolic tissues during the closure of each surgical level. The biopsy material was rinsed in sterile saline, blotted, and then flash frozen in liquid nitrogen and stored, individually, in sterile screw-top tubes and kept on dry ice for shipment to or to be picked up by CuraGen. The metabolic tissues of interest include uterine wall (smooth muscle), visceral adipose, skeletal muscle (rectus), and subcutaneous adipose. Patient descriptions are as follows:

Patient 7 Non-diabetic Caucasian and obese

Patient 8 Non-diabetic Caucasian and obese

Patient 12 Diabetic Caucasian with unknown BMI and on insulin

Patient 13 Diabetic Caucasian, overweight, not on insulin

Patient 15 Diabetic Caucasian, obese, not on insulin

Patient 17 Diabetic Caucasian, normal weight, not on insulin

Patient 18 Diabetic Hispanic, obese, not on insulin

Patient 19 Non-diabetic Caucasian and normal weight

Patient 20 Diabetic Caucasian, overweight, and on insulin

Patient 21 Non-diabetic Caucasian and overweight

Patient 22 Diabetic Caucasian, normal weight, on insulin

Patient 23 Non-diabetic Caucasian and overweight

Patient 25 Diabetic Caucasian, normal weight, not on insulin

Patient 26 Diabetic Caucasian, obese, on insulin

Patient 27 Diabetic Caucasian, obese, on insulin

Total RNA was isolated from metabolic tissues of 12 Type II diabetic patients and 12 matched control patients included hypothalamus, liver, pancreas, small intestine, psoas muscle, diaphragm muscle, visceral adipose, and subcutaneous adipose. The diabetics and non-diabetics were matched for age, sex, ethnicity, and BMI where possible. The panel also contains pancreatic islets from a 22 year old male patient (with a BMI of 35) obtained from the Diabetes Research Institute at the University of Miami School of Medicine. Islet tissue was processed to total RNA at CuraGen. Cell lines used in this panel are widely available through the American Type Culture Collection (ATCC), a repository for cultured cell lines, and were cultured at an outside facility. The RNA was extracted at CuraGen according to CuraGen protocols. All samples were then processed at CuraGen to produce single stranded cDNA.

In the labels used to identify tissues in the Human Metabolic panel, the following abbreviations are used: PI ₌ placenta

Go = greater omentum

Sk = skeletal muscle

Ut = uterus

CC = Caucasian

HI = Hispanic

AA = African American

AS = Asian

Diab = Type II diabetic

Norm = Non-diabetic

Overwt = Overweight; med BMI

Obese = Hi BMI

Low BM = 20-25

Med BM = 26-30

Hi BMI Greater than 30

M = Male

# = Patient identifier

Vis. = Visceral

SubQ = Subcutaneous

Panel CNSD.01

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

Disease diagnoses are taken from patient records. The panel contains two brains from each ofthe following diagnoses: Alzheimer's disease, Parkinson's disease, Huntington's disease, Progressive 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 ofthe substantia nigra making this region more difficult to obtain. Normal control brains were examined for neuropathology and found to be free of any pathology consistent with neurodegeneration.

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

PSP = Progressive supranuclear palsy

Sub Nigra = Substantia nigra

Glob Palladus= Globus palladus

Temp Pole = Temporal pole

Cing Gyr = Cingulate gyrus 1

B A 4 = Brodman Area 4

Panel CNS_Neurodegeneration_V1.0

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

Disease diagnoses are taken from patient records. The panel contains six brains from Alzheimer's disease (AD) patients, and eight brains from "Normal controls" who showed no evidence of dementia prior to death. The eight normal control brains are divided into two categories: Controls with no dementia and no Alzheimer's like pathology (Controls) and controls with no dementia but evidence of severe Alzheimer's like pathology, (specifically senile plaque load rated as level 3 on a scale of 0-3; 0 = no evidence of plaques, 3 = severe AD senile plaque load). Within each of these brains, the following regions are represented: hippocampus, temporal cortex (Brodman Area 21), parietal cortex (Brodman area 7), and occipital cortex (Brodman area 17). These regions were chosen to encompass all levels of neurodegeneration in AD. The hippocampus is a region of early and severe neuronal loss in AD; the temporal cortex is known to show neurodegeneration in AD after the hippocampus; the parietal cortex shows moderate neuronal death in the late stages of the disease; the occipital cortex is spared in AD and therefore acts as a "control" region within AD patients. Not all brain regions are represented in all cases.

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

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

Control = Control brains; patient not demented, showing no neuropathology

Control (Path) = Control brains; pateint not demented but showing sever AD-like pathology

SupTemporal Ctx = Superior Temporal Cortex

Inf Temporal Ctx = Inferior Temporal Cortex

Panel CNS_Neurodegeneration_V2.0

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

Disease diagnoses are taken from patient records. The panel contains sixteen brains from Alzheimer's disease (AD) patients, and twenty-nine brains from "Normal controls" who showed no evidence of dementia prior to death. The twenty-nine normal control brains are divided into two categories: Fourteen controls with no dementia and no Alzheimer's like pathology (Controls) and fifteen controls with no dementia but evidence of severe Alzheimer's like pathology, (specifically senile plaque load rated as level 3 on a scale of 0-3; 0 = no evidence of plaques, 3 = severe AD senile plaque load). Tissue from the temporal cotex (Broddmann Area 21) was selected for all samples from the Harvard Brain Tissue Resource Center; from the two sample from the Human Brain and Spinal Fluid Resource Center (samples 1 and 2) tissue from the inferior and superior temporal cortex was used; each sample on the panel represents a pool of inferior and superior temporal cortex from an individual patient. The temporal cortex was chosen as W 03

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.

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

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

Control = Control brains; patient not demented, showing no neuropathology

AH3 = Control brains; pateint not demented but showing sever AD-like pathology

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

A. CG103404-01 and CG103404-02: Macrophage colony stimulating factor receptor.

Expression of gene CG103404-01 and CG103404-02 was assessed using the primer-probe set Ag5108, described in Table AA. Results ofthe RTQ-PCR runs are shown in Tables AB, AC, AD, AE and AF. Please note that CG103404-02 represents a full length physical clone.

Table AA. Probe Name Ag5108

Table AB. CNS_neurodegeneration_vl.0

Table AC. General_screening_panel_vl.5

Table AD. Oncology_cell_line_screening_panel_v3.1

Table AE. Panel 4. ID

CNS_neurodegeneration_vl.O Summary: Ag5108 This panel confirms the expression of this gene at high to moderate 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. Therefore, therapeutic modulation ofthe expression or function of this gene may decrease neuronal death and be of use in the treatment of this disease.

General_screening_panel_vl.5 Summary: AG5108 Two experiments with the same probe and primer produce results that are in excellent agreement. Highest expression of this gene is seen in the placenta (CTs=25-26), with prominent expression also detected in normal bladder.

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

This gene is also expressed at 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 neurological disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, stroke and epilepsy.

In addition, this gene is expressed at much higher levels in fetal lung tissue (CTs=27-29) when compared to expression in the adult counterpart (CTs=30-32). Thus, expression of this gene may be used to differentiate between the fetal and adult source of this tissue.

In addition, the relative overexpression of this gene in fetal lung suggests that the protein product may enhance lung growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation ofthe protein encoded by this gene could be useful in treatment of lung related diseases.

Oncology_celI_Iine_screening_panel_v3.1 Summary: Ag5108 Two experiments with the same probe and primer set produce results that are in excellent agreement. Highest expression is seen in tissue derived from the cerebellum, with prominent expression also seen in a bladder derived sample. This expression is in agreement with expression in previous panels. Please see those panels for discussion of utility of this gene in disease.

Panel 4.1D Summary: Ag5108 Highest expression is seen in resting monocytes (CT=26.6). High to moderate levels of expression are seen in a cluster of samples derived from monocytes, macrophages, dendritic cells and eosinophils. This transcript is expressed in macrophages and dendritic cells. Thus, the protein encoded by this transcript may be important in monocytic and dendritic cell differentiation and activation. Therefore, regulating the expression of this transcript or the function ofthe protein it encodes may alter the types and levels of monocytic cells regulated by cytokine and chemokine production and T cell activation. Therapeutics designed with the protein encoded by this transcript could therefore be important for the treatment of asthma, emphysema, inflammatory bowel disease, arthritis and psoriasis.

Panel 5 Islet Summary: Ag5108 Highest expression of this gene is seen in placenta (CT=27), in agreement with Panel 1.5. In addition, moderate levels of expression are seen in skeletal muscle and uterus. Expression in skeletal muscle is also consistent with the results for Panel 1.5. Please see that panel for discussion of role in metabolic disease.

B. CG108945-02: Cation-transporting ATPase 1.

Expression of gene CG108945-02 was assessed using the primer-probe set Ag6263, described in Table BA. Results ofthe RTQ-PCR runs are shown in Tables BB and BC.

Table BA. Probe Name Ag6263

Table BB. CNS_neurodegeneration_vl.O

Table BC. General_screening_panel_vl.5

CNS_neurodegeneration_vl.O Summary: Ag6263 This panel confirms the expression of this gene at low levels in the brain in an independent group of individuals. This gene is found to be slightly down-regulated in the temporal cortex of Alzheimer's disease patients. Therefore, up-regulation of this gene or its protein product, or treatment with specific agonists for this receptor may be of use in reversing the dementia/memory loss associated with this disease and neuronal death. General_screening_panel_vl.5 Summary: Ag6263 Highest expression of this gene is detected in fetal brain and all the adult brain region including amygdala, hippocampus, substantia nigra, thalamus, cerebellum, and cerebral cortex (CTs=30). Moderate expression of this gene is also seen in spinal cord. Therefore, therapeutic modulation of this gene product may be useful in the treatment of central nervous system disorders such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia and depression.

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

Among tissues with metabolic or endocrine function, this gene is expressed at low levels in pancreas, thyroid, skeletal muscle, and fetal liver. Therefore, therapeutic modulation of the activity of this gene may prove useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes.

Interestingly, this gene is expressed at much higher levels in fetal (CTs=34-34.8) when compared to adult lung and liver (CTs=38). This observation suggests that expression of this gene can be used to distinguish fetal from adult liver. In addition, the relative overexpression of this gene in fetal tissue suggests that the protein product may enhance liver growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation ofthe protein encoded by this gene could be useful in treatment of liver related diseases.

C. CGI 19035-03: Myotonin-protein kinase.

Expression of gene CGI 19035-03 was assessed using the primer-probe set Ag7786, described in Tables CA. Results ofthe RTQ-PCR runs are shown in Tables CB and C.

Table CA. Probe Name Ag7786

CNS_neurodegeneration_vl.O Summary: Ag7786 This panel confirms the expression of this gene at moderate levels in the brain in an independent group of individuals. This gene appears to be slightly down-regulated in the temporal cortex of Alzheimer's disease patients. Therefore, up-regulation of this gene or its protein product, or treatment with specific agonists for this receptor may be of use in reversing the dementia, memory loss, and neuronal death associated with this disease.

Panel 4.1D Summary: Ag7786 Highest expression is seen in untreated lung fibroblasts (CT=31). Moderate levels of expression are also seen in clusters of samples derived from lung and dermal fibroblasts, HPAEC, HUVEC and lung and dermal microvascular EC. 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.

D. CG124873-02: Voltage -dependent L-type calcium channel alpha- 1S subunit.

Expression of gene CG124873-02 was assessed using the primer-probe set Ag6185, described in Table DA. Results of the RTQ-PCR runs are shown in Table DB. Table DA. Probe Name Ag6185

Table DB. General_screening_panel_vl.5

General_screening_panel_vl.5 Summary: Ag6185 Highest expression of this gene is detected in skeletal muscle (CT=26.5). Moderate expression of this gene is also seen in fetal skeletal muscle. Interestingly, expression of this gene is higher in adult compared to fetal skeletal muscle (CT=29.1). Therefore, expression of this gene may be used to distinguish adult skeletal muscle from the fetal tissue and also other samples used in this panel. In addition, moderate to low expression of this gene is also seen in adipose. Therefore, therapeutic modulation of this gene or its protein product through the use of small molecule drug may be useful in the treatment of metabolically related diseases, such as obesity and diabetes.

Low expression of this gene is also seen in a melanoma Hs688(A).T cell line. Therefore, expression of this gene may be used as marker to detect the presence of melanoma and also therapeutic modulation of this gene or its protein product may be useful in the treatment of melanoma.

Low expression of this gene is also seen in fetal lung. Interestingly, this gene is expressed at much higher levels in fetal (CT=33.2) when compared to adult lung (CT=40). This observation suggests that expression of this gene can be used to distinguish fetal from adult lung. In addition, the relative overexpression of this gene in fetal tissue suggests that the protein product may enhance lung growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation of the protein encoded by this gene could be useful in treatment of lung related diseases.

E. CG138554-02: SIMILAR TO CHITINASE 3-LIKE 1.

Expression of gene CGI 38554-02 was assessed using the primer-probe set Ag6540, described in Table EA. Results of the RTQ-PCR runs are shown in Table EB. Table EA. Probe Name Ag6540

Table EB. General_screening_panel_vl.6

General_screening_panel_vl.6 Summary: Ag6540 Expression of this gene is restricted to a sample derived from a brain cancer cell line (CT=24.4). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel and as a marker to detect the presence of brain cancer. Furthermore, therapeutic modulation ofthe expression or function of this gene may be effective in the treatment of brain cancer. F. CG144744-02 and CG144744-03: Calcium channel alpha-lA subunit

Expression of gene CG144744-02 and CG144744-03 was assessed using the primer-probe sets Ag6183 and Ag6184, described in Tables FA and FB. Results of the RTQ-PCR runs are shown in Tables FC, FD, FE and FF. Please note that probe-primer set Ag6184 is specific for CG144744-02.

Table FA. Probe Name Ag6183

Table FB. Probe Name Ag6184

Table FC. CNS_neurodegeneration_yl.0

Table FD. General_screening_panel_vl.5

CNS_neurodegeneration_vl.O Summary: Ag6183/Ag6184 These profiles confirm the expression of this gene at moderate to low levels in the brain. Please see Panel 1.5 for discussion of this gene in the central nervous system.

General_screening_panel_vl.5 Summary: Ag6183/Ag6184 Two experiments with two different probe and primer sets produce results that are in very good agreement. Highest expression is seen in the cerebellum and a lung cancer cell line (CTs=27 -29). In addition, moderate levels of expression are seen in all regions of the CNS examined including the hippocampus, thalamus, substantia nigra, amygdala, cerebellum and cerebral cortex, as well as in a brain cancer cell line. Therefore, therapeutic modulation of the expression or function of this gene may be useful in the treatment of neurological disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, stroke and epilepsy.

Moderate levels of expression are also seen in two lung cancer cell lines. Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel and as a marker to detect the presence of lung cancer. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of lung cancer.

General_screening_panel_vl.6 Summary: Ag6183/Ag6184 Multiple runs with different probe and primer sets produce show a brain specific pattern of expression for this gene, with highest expression seen in a brain cancer cell line and the cerebellum (CTs=28-31). Prominent expression is also seen in two lung cancer cell lines. This profile is consistent with the results in Panel 1.5. Please see that panel for discussion of this gene.

Panel 4.1D Summary: Ag6183/Ag6184 Low expression is seen in a cluster of samples derived from dermal fibroblasts. Expression in TNFalpha treated dermal fibroblasts suggests that this gene product may be involved in skin disorders, including psoriasis.

G. CG151723-02 and CG151723-03: Activin receptor-like kinase 1.

Expression of gene CG151723-02 and CG151723-03 was assessed using the primer-probe sets Ag6283, and Ag7174, described in Tables GA, GB and GC. Results of the RTQ-PCR runs are shown in Tables GD, GE and GF. Please note that probe- primer sets Ag6283 is specific for CG151723-02 is specific for CG151723-03.

Table GA. Probe Name Ag6283

Table GC. Probe Name Ag7174

Table GD. General_screening_panel_vl.5

Tissue Name ReI. Eχp.(%) I Tissue Name | Rel. Eχp.( )

Table GE. General_screening_panel_vl.7

Table GF. Panel 4. ID

General_screening_panel_vl.5 Summary: Ag6283 Highest expression of this gene is seen mainly in fetal lung (CT=33.8). Interestingly, this gene is expressed at much higher levels in fetal when compared to adult lung (CT=40). This observation suggests that expression of this gene can be used to distinguish fetal from adult lung. In addition, the relative overexpression of this gene in fetal tissue suggests that the protein product may enhance lung growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation ofthe protein encoded by this gene could be useful in treatment of lung related diseases.

Low expression of this gene is also seen in placenta. Therefore, therapeutic modulation of this gene may be useful in the treatment of reproductive disorders including fertility.

This gene is expressed at low levels in two melanoma cell lines. Therefore, expression of this gene is may be used as marker to detect the presence of melanoma and therapeutic modulation of this gene or its protein product may be useful in the treatment of melanoma.

General_screening_panel_vl.7 Summary: Ag7174 Highest expression of this gene is seen in HUVECs, with high expression also detected in adult lung (CTs=22). This gene is widely expressed in this panel, with high to moderate expression seen mainly in normal tissues. Moderate to high expression of this gene is also seen in tissues with metabolic function, including pituitary, adipose, adrenal gland, pancreas, thyroid, and adult and fetal skeletal muscle, heart, and liver. This widespread expression among these tissues suggests that this gene product may play a role in normal neuroendocrine and metabolic function and that disregulated expression of this gene may contribute to neuroendocrine disorders or metabolic diseases, such as obesity and diabetes.

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

Panel 4.1D Summary: Ag6283 Highest expression of this gene is seen mainly in starved HUVEC cells (CT=33.8). Moderate to low expression of this gene is mainly seen in endothelial cells including HUVEC, HPAEC, lung and dermal microvascular endothelial cells and also in interferon gamma or IL-4 activated dermal fibroblast cells. Therefore, antibodies and small molecules that antagonize the function of this gene product may reduce or eliminate the symptoms in patients with autoimmune and inflammatory diseases that involve endothelial cells, such as lupus erythematosus, asthma, emphysema, Crohn's disease, ulcerative colitis, rheumatoid arthritis, osteoarthritis, and psoriasis.

H. CG159280-01: Glycerol -3 phosphate dehydrogenase 1 (soluble).

Expression of gene CGI 59280-01 was assessed using the primer-probe set Ag5536, described in Table HA. Results of the RTQ-PCR runs are shown in Tables HB and HC.

Table HA. Probe Name Ag5536

Table HB. General_screemng_panel_vl.5

Renal ca. UO-31 0.7 iPancreas Pool 0.1

Table HC. Panel 5 Islet

General_screening_panel_vl.5 Summary: Ag5536 Highest expression of this gene is seen in skeletal muscle (CT=28). In addition, this gene is expressed at moderate levels in adipose, liver, and regions of the CNS. This expression suggests that this gene product may be involved in metabolic disease and that modulation ofthe expression or function of this gene product may be useful in the treatment of obesity and/or diabetes.

Panel 5 Islet Summary: Ag5536 Highest expression of this gene is seen in skeletal muscle (CT=29.5), with moderate levels of expression also seen in adipose. This expression is consistent with the results seen in Panel 1.5. Please see panel 1.5 for further discussion of this gene.

I. CGI 65915-02: Serine/threonine-protein kinase receptor R2.

Expression of gene CG165915-02 was assessed using the primer-probe set Ag6218, described in Table IA. Results of the RTQ-PCR runs are shown in Tables IB, IC and ID.

Table IA. Probe Name Ag6218

Table IB. CNS_neurodegeneration_vl.O

Tissue Name Rel. EXD. % ) Aε6218. Tissue Name Rel. EXD. % ) Aε6218.

Table IC. General_screening_panel_vl.5

CNS_neurodegeneration_vl.0 Summary: Ag6218 This profile confirms the expression of this gene at moderate levels in the brain. Please see Panel 1.5 for discussion of this gene in the central nervous system.

General_screening_panel_vl.5 Summary: Ag6218 Highest expression of this gene is seen in an ovarian cancer cell line (CT=30). This gene is widely expressed in this panel, with moderate expression seen in brain, colon, gastiic, lung, breast, ovarian, and melanoma cancer cell lines. This expression profile suggests a role for this gene product in cell survival and proliferation. Modulation of this gene product may be useful in the treatment of cancer.

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

This gene is also expressed at low but 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.

Panel 4.1D Summary: Ag6218 Highest expression is seen in LPS treated monocytes (CT=32.3). Upon activation with pathogens such as LPS, monocytes contribute to the innate and specific immunity by migrating to the site of tissue injury and releasing inflammatory cytokines. This release contributes to the inflammation process. Therefore, modulation of the expression ofthe protein encoded by this transcript may prevent the recruitment of monocytes and the initiation ofthe inflammatory process, and reduce the symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, or rheumatoid arthritis.

J. CGI 9667-01: Proprotein convertase subtilisin/kexin type 5 precursor. Expression of gene CGI 69667-01 was assessed using the primer-probe set Ag6124, described in Table JA. Results of the RTQ-PCR runs are shown in Tables JB, JC and JD.

Table JA. Probe Name Ag6124

Table JC. General_screening_panel_vl.5

Table JD. Panel 4. ID

CNS_neurodegeneration_vl.O Summary: Ag6124 This profile confirms the expression of this gene at moderate levels in the brain. Therefore, therapeutic modulation ofthe expression or function of this gene may be useful in the treatment of neurologic disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, stroke and epilepsy.

General_screening_panel_vl.5 Summary: Ag6124 Highest expression of this gene is seen in a renal cancer cell line (CT=28). Prominent levels of expression are also seen in cell lines derived from colon cancer and ovarian cancer. Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel and as a marker of these cancers.

Among tissues with metabolic function, this gene is expressed at low but significant levels in adipose, pancreas, thyroid, 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.

In addition, this gene is expressed at much higher levels in fetal lung, and kidney tissue (CTs=30-31) when compared to expression in the adult counterparts (CTs=36). Thus, expression of this gene may be used to differentiate between the fetal and adult source of these tissues. Furthermore, the relative overexpression of this gene in fetal lung and kidney suggests that the protein product may enhance lung and 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 lung and kidney related diseases.

Panel 4.1D Summary: Ag6124 Highest expression is seen in the kidney (CT=32), with prominent expression also detected in liver cirrhosis and a cluster of samples derived from dendritic cells. Therefore, therapeutic utilization of the protein encoded by this transcript may be important in immune modulation, organ/bone marrow transplantation, and the treatment of diseases where antigen presentation, a function of mature dendritic cells, plays an important role such as asthma, rheumatoid arthritis, IBD, and psoriasis.

K. CG169754-01: protein tyrosine phosphatase, non-receptor type 18.

Expression of gene CGI 69754-01 was assessed using the primer-probe set Ag6211, described in Table KA. Results of the RTQ-PCR runs are shown in Table KB. Table KA. Probe Name Ag6211

Table KB. General_screening_panel_vl.5

01

General_screening_panel_vl.5 Summary: Ag6211 This gene shows a restricted expression profile in this panel, with highest expression detected in a lung cancer cell line (CT=29.5). Prominent expression is also detected in a breast cancer cell line. Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel and as a marker of these cancers. Furthermore, therapeutic modulation of the expression or function of this gene may be useful in the treatment of these cancers.

L. CG170764-01: D-GLUCURONYL C5 EPIMERASE.

Expression of gene CG170764-01 was assessed using the primer-probe set Ag6136, described in Table LA. Results ofthe RTQ-PCR runs are shown in Tables LB, LC and LD.

Table LA. Probe Name Ag6136

Table LB. CNS_neurodegeneration_vl.O

1

TU 03/03401

Table LD. Panel 4. ID

CNS_neurodegeneration_vl.0 Summary: Ag6136 This panel does not show differential expression of this gene in Alzheimer's disease. However, this profile confirms the expression of this gene at high to moderate levels in the brain. Please see Panel 1.5 for discussion of this gene in the central nervous system.

General_screening_panel_vl.5 Summary: Ag6136 Highest expression of this gene is seen in the cerebellum (CT=23.7). In addition, this gene is expressed at high to moderate levels in all regions ofthe CNS examined, including the hippocampus, thalamus, substantia nigra, amygdala, cerebellum and cerebral cortex. High expression in the cerebellum suggests that this gene may be a useful and specific target of drugs for the treatment of CNS disorders that have this brain region as the site of pathology, such as autism and the ataxias.

Prominent levels of expression are also seen in a lung cancer cell line (CT=24). This gene is widely expressed in this panel, with high to moderate expression seen in brain, colon, gastiic, lung, 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.

In addition, this gene is expressed at much higher levels in fetal liver tissue (CT=28) when compared to expression in the adult counterpart (CT=31). Thus, expression of this gene may be used to differentiate between the fetal and adult source of this tissue. Furthermore, the relative overexpression of this gene in fetal liver 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.

Panel 4.1D Summary: Ag6136 This gene is widely expressed in this panel, with highest expression in IFN-gamma treated HUVECs (CT=29). In addition, moderate levels of expression are seen in a cluster of treated and untreated samples derived from HUVECs, as well as in the KU-812 basophil cell line, lung and dermal microvascular epithelial cells, small airway and bronchial epithelium, HPAECS and lung and dermal fibroblasts. The expression of this gene in cells derived from or within the lung suggests that this gene may be involved in normal conditions as well as pathological and inflammatory lung disorders that include chronic obstructive pulmonary disease, asthma, allergy and emphysema.

M. CG170882-01: Cyclin G-associated kinase.

Expression of gene CG170882-01 was assessed using the primer-probe sets Agl761 and Ag6215, described in Tables MA and MB. Results ofthe RTQ-PCR runs are shown in Tables MC, MD, ME and MF.

Table MA. Probe Name Agl761

Table MB. Probe Name Ag6215

Table MC. CNS_neurodegeneration_vl.O

Table MD. General_screening_panel_vl.5

Table ME. Panel 1.3D

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

Tissue Name Tissue Name Aεl761. Run Ael761. Run

Table MF. Panel 4.1D

CNS_neurodegeneration_vl.O Summary: Ag6215 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.

General_screenmg_panel_vl.5 Summary: Ag6215 Highest expression of this gene is seen in a gastric cancer cell line (CT=31), with prominent expression seen in a colon cancer cell line as well. Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel, and as a marker of these cancers.

Panel 1.3D Summary: Agl761 Highest expression of this gene is seen in the hippocampus (CT=27.3). 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.

This gene is widely expressed in this panel, with moderate expression seen in brain, colon, gastric, lung, 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.

Panel 4.1D Summary: Agl761 Highest expression of this gene is seen in TNF- a/ILl-b treated HPAECs (CT=28.7). This gene appears to be more highly expressed in activated T cells, when compared to expression in resting T cells. In addition, widespread and moderate levels of expression are seen in most ofthe samples on this panel, including B-cells, endothelial cells, macrophages/monocytes, and members of the peripheral blood mononuclear cell family, as well as epithelial and fibroblast cell types from lung and skin, and normal tissues represented by colon, lung, thymus and kidney. This ubiquitous pattern of expression suggests that this gene product may be involved in homeostatic processes for these and other cell types and tissues. This pattern is in agreement with the expression profile in General_screening_panel_vl.3D 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 ofthe symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, and osteoarthritis.

Ag6215 shows lower levels of expression in this panel, with highest expression in chronically activated Th2 cells (CT=33.4). N. CG170882-02: Cyclin G-associated kinase.

Expression of gene CG170882-02 was assessed using the primer-probe sets Agl761 and Ag6216, described in Tables NA and NB. Results of the RTQ-PCR runs are shown in Tables NC, ND,NE and NF.

Table NA. Probe Name Agl761

Table NB. Probe Name Ag6216

Table NC. CNS_neurodegeneration_vl.O

Table ND. General_screening_panel_vl.5

Table NE. Panel 1.3D

CNS_neurodegeneration_vl.O Summary: Ag6216 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.

General_screening_panel_vl.5 Summary: Ag6216 This panel shows low widespread expression of CG170882-02, with highest expression in a gastiic cancer cell line (CT=33). Please see panel 1.3D for discussion of this gene.

Panel 1.3D Summary: Agl761 Highest expression of this gene is seen in the hippocampus (CT=27.3). This gene is also expressed at moderate levels in the CNS, including the hippocampus, thalamus, substantia nigra, amygdala, cerebellum and cerebral cortex. Therefore, therapeutic modulation ofthe expression or function of this gene may be useful in the treatment of neurologic disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, stroke and epilepsy.

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. Panel 4.1D Summary: Agl761 Highest expression of this gene is seen in TNF- a/ILl-b treated HPAECs (CT=28.7). This gene appears to be more highly expressed in activated T cells, when compared to expression in resting T cells. In addition, widespread and moderate levels of expression are seen in most ofthe samples on this panel, including B-cells, endothelial cells, macrophages/monocytes, and members of the peripheral blood mononuclear cell family, as well as epithelial and fibroblast cell types from lung and skin, and normal tissues represented by colon, lung, thymus and kidney. This ubiquitous pattern of expression suggests that this gene product may be involved in homeostatic processes for these and other cell types and tissues. This pattern is in agreement with the expression profile in General_screening_panel_vl.3D 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 ofthe symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, and osteoarthritis.

Ag6216 shows lower levels of expression in this panel, with highest expression in ionomycin treated Ramos B cells (CT=32.5).

O. CG170882-03: Cyclin G-associated kinase.

Expression of gene CG170882-03 was assessed using the primer-probe sets Agl761 and Ag6212, described in Tables OA and OB. Results of the RTQ-PCR runs are shown in Tables OC, OD and OE.

Table OA. Probe Name Agl761

Forward 5 ' -gtatgcattaaagggcagct-3 20 195 217

ITET-5 ' -acacggttctgaagatcttctaccagacg-

Probe 3 ' -TAMRA 29 262 218

Reverse 15 ' -cttgaagtccctgtggatga-3 ' 20 331 219

Table OC. General_screening_panel_vl.5

Table OP. Panel 1.3D

Panel 1.3D Summary: Agl761 Highest expression of this gene is seen in the hippocampus (CT=27.3). This gene is also expressed at moderate levels in the CNS, including the hippocampus, thalamus, substantia nigra, amygdala, cerebellum and cerebral cortex. Therefore, therapeutic modulation ofthe expression or function of this 03 03401

gene may be useful in the treatment of neurologic disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, stroke and epilepsy.

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.

Panel 4.1D Summary: Agl761 Highest expression of this gene is seen in TNF- a/ILl-b treated HPAECs (CT=28.7). This gene appears to be more highly expressed in activated T cells, when compared to expression in resting T cells. In addition, widespread and moderate levels of expression are seen in most of the samples on this panel, including B-cells, endothelial cells, macrophages/monocytes, and members of the peripheral blood mononuclear cell family, as well as epithelial and fibroblast cell types from lung and skin, and normal tissues represented by colon, lung, thymus and kidney. This ubiquitous pattern of expression suggests that this gene product may be involved in homeostatic processes for these and other cell types and tissues. This pattern is in agreement with the expression profile in General_screening_panel_vl.3D and also suggests a role for the gene product in cell survival and proliferation. Therefore, modulation ofthe gene product with a functional therapeutic may lead to the alteration of functions associated with these cell types and lead to improvement ofthe symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, and osteoarthritis.

P. CG171205-01: malic enzyme. Expression of gene CG171205-01 was assessed using the primer-probe set Ag6217, described in Table PA. Results ofthe RTQ-PCR runs are shown in Tables PB, PC and PD.

Table PA. Probe Name Ag6217

Table PB. CNS_neurodegeneration_vl.O

Table PC. General_screening_panel_vl.5

Table PP. Panel 4. ID

CNS_neurodegeneration_vl.O Summary: Ag6217 This panel does not show differential expression of this gene in Alzheimer's disease. However, this profile confirms the expression of this gene at moderate levels in the brain. Please see Panel 1.5 for discussion of this gene in the central nervous system.

General_screening_panel_vl.5 Summary: Ag6217 Highest expression of this gene is seen in the cerebellum (CT=30.8), with moderate levels of expression seen in all regions ofthe brain examined, 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.

In addition, prominent levels of expression are detected in a gastric cancer cell line (CT=30). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel and as a marker of this cancer.

Panel 4.1D Summary: Ag6217 Highest expression is seen in TNF-a treated dermal fibroblasts, suggesting that this gene product could be used involved in the pathogenesis and/or treatment of psoriasis.

Low expression of this gene is also seen in in activated CD45RA CD4 lymphocyte and CD45RO CD4 lymphocyte (CTs=33-34.7), which represent activated naive and memory T cells respectively. In activated CD4 Thl or Th2 cells, resting CD4 cells (CTs>35), the expression of this gene is down regulated suggesting a role for this putative protein in differentiation or activation of naive and memory T cells. In addition, low expression of this gene is also seen in activated primary Trl cells, activated lung fibroblasts, activated and starved HUVEC cells and lung microvascular endothelial cells. Therefore, modulation of the expression and/or activity of this putative protein encoded by this gene might be beneficial for the control of autoimmune diseases and T cell mediated diseases such as rheumatoid arthritis, osteoarthritis, psoriasis, allergy, asthma, and inflammatory bowel disease like Crohns and ulcerative colitis.

Q. CG171793-01: L-LACTATE DEHYDROGENASE.

Expression of gene CG171793-01 was assessed using the primer-probe set Ag6646, described in Table QA. Results of the RTQ-PCR runs are.shown in Table QB. Table OA. Probe Name Ag6646

Table OB. General_screening_panel_vl.6

General_screening_panel_vl.6 Summary: Ag6646 Highest expression of this gene is seen in an ovarian cancer cell line (CT=27). This gene is widely expressed in this panel, with high to moderate expression seen in brain, colon, gastric, lung, breast, prostate, ovarian, and melanoma cancer cell lines. This expression profile suggests a role for this gene product in cell survival and proliferation. Modulation of this gene product may be useful in the treatment of cancer.

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

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.

R. CG172979-01: EPHA4, Ephrin type-A receptor 4. Expression of gene CGI 72979-01 was assessed using the primer-probe set Ag6219, described in Table RA. Results ofthe RTQ-PCR runs are shown in Tables RB, RC and RD.

Table RA. Probe Name Ag6219

Table RC. General_screening_panel_vl.5

401

Table RD. Panel 4.1D

303401

03/03401

CNS_neurodegeneration_vl.O Summary: Ag6219 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 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.

General_screening_panel_vl.5 Summary: Ag6129 Highest expression of this gene is seen in the thalamus (CT=28.4). Moderate levels of expression are seen throughout the CNS, including hippocampus, thalamus, substantia nigra, amygdala, cerebellum and cerebral cortex. Therefore, therapeutic modulation ofthe expression or function of this gene may be useful in the treatment of neurologic disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, stroke and epilepsy.

Prominent levels of expression are seen in cancer cell lines derived from brain, colon, lung, renal, breast, ovarian and melanoma cell lines. This expression suggests that this gene product may be involved in these cances and that expression of the gene or the protein product may be used as a diagnostic marker.

Among tissues with metabolic function, this gene is expressed at low but significant levels in adipose, pancreas, thyroid, 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. Panel 4.1D Summary: Ag6129 Highest expression of this gene is seen in TNF- a treated dermal fibroblast (CT=31.2) suggesting that this gene product may be useful in the diagnosis and/or treatment of inflammatory skin disorders.

S. CG172979-02: Ephrin type-A receptor 4.

Expression of gene CG172979-02 was assessed using the primer-probe set Ag6220, described in Table SA. Results ofthe RTQ-PCR runs are shown in Table SB. Table SA. Probe Name Ag6220

Table SB. General_screening_panel_vl.5

General_screening_panel_vl.5 Summary: Ag6220 Highest expression is seen in a breast cancer cell line (CT=32.8). Significant expression is also seen in cell lines derived from melanoma, lung, gastric, colon, and brain cancers. Therefore, expression of this gene may be used as diagnostic marker to detect the presence of these cancers and also, therapeutic modulation of this gene may be useful in the treatment of melanoma, lung, gastric, colon, and brain cancers.

T. CG173488-01: Discoidin domain receptor 2.

Expression of gene CG173488-01 was assessed using the primer-probe sets Ag4022 and Ag6213, described in Tables TA and TB. Results ofthe RTQ-PCR runs are shown in Tables TC, TD, TE, TF, TG and TH.

Table TA. Probe Name Ag4022

Table TB. Probe Name Ag6213

Table TD. CNS_neurodegeneration_vl.O

W 03

Table TE. General_screening_panel_vl.4

Table TG. Panel 4. ID

0303401

Table TH. general oncology screening panel_v_2.4

AI_comprehensive panel_vl.O Summary: Ag4022 This gene shows prominent expression in a cluster of bone, synovium, and cartilage OA samples. Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel and as a marker of OA. Furthermore, therapeutic modulation of the expression or function of this gene may be useful in the treatment of OA. CNS_neurodegeneration_vl.O Summary: Ag4022 This panel confirms the expression of this gene at high to moderate in the brain in an independent group of individuals. This gene appears to be slightly upregulated in the temporal cortex of Alzheimer's disease patients. Therefore, therapeutic modulation ofthe expression or function of this gene may decrease neuronal death and be of use in the treatment of this disease.

General_screening_panel_vl.4 Summary: Ag4022 Highest expression of this gene is seen in a brain cancer cell line (CT=22). This gene is widely expressed at high levels in this panel, with moderate expression seen in brain, colon, prostate, gastric, lung, breast, ovarian, and melanoma cancer cell lines. This expression profile suggests a role for this gene product in cell survival and proliferation. Modulation of this gene product may be useful in the treatment of cancer.

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

This gene is also expressed at high 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.

General_screening_panel_vl.6 Summary: Ag6213 Highest expression is seen in a brain cancer cell line (CT=30.37). Moderate levels of expression are seen in a cluster of brain cancer derived cell lines. Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel, and as a marker of brain cancer. Furthermore, modulation of the expression or function of this gene product may be useful in the treatment of brain cancer.

Panel 4.1D Summary: Ag4022 Highest expression of this gene is seen in untreated lung fibroblasts (CT=26). Prominent expression is also seen in HPAECS, and treated and untreated lung and dermal fibroblast samples. Thus, this gene product may be involved in inflammatory conditions ofthe lung and skin.

Ag6213 Highest expression is seen in resting dermal fibroblasts (CT=34). general oncology screening panel_v_2.4 Summary: Ag4022 This gene is widely expressed at high levels in this panel, with highest expression detected in a melanoma sample (CT=24). High levels of expression are seen in melanoma and prostate carcinoma samples, suggesting that this gene product may be involved in these cancers. Therefore, expression of this gene may be useful in the diagnosis of these cancers and modulation of the gene or protein product may be beneficial in their treatment.

U. CG173939-01: Novel Pyrin domain containing protein.

Expression of gene CG173939-01 was assessed using the primer-probe sets Ag2181 and Ag6226, described in Tables UA and UB. Results ofthe RTQ-PCR runs are shown in Table UC.

Table UA. Probe Name Ag2181

Table UB. Probe Name Ag6226

Start SEQ ID

Primers Sequences Length Position No

Forward ' -ctgtatgaagctaaaataaagaatctggta-3 ' 30 1699 241

JTET-5 ' -catttcccatcagggtcaccacataa-3 '

Probe TAMRA 26 1737 242

Reverse |5 ' -gtttaaggagatgatgactgtaatgat-3 27 1765 243

Table UC. Panel 4D

Panel 4D Summary: Ag2181 This transcript is most highly expressed in NCI- H292 cells stimulated by IL-4(CT=34.1). The gene is also expressed in a cluster of tieated and untreated NCI-H292 mucoepidermoid cell line samples and in small airway epithelium treated with IL-lbeta and TNFalpha. In comparison, expression in the normal lung is very low. The expression of the transcript in activated normal epithelium as well as a cell line that is often used as a model for airway epithelium (NCI-H292 cells) suggests that this transcript may be important in the proliferation or activation of airway epithelium. Therefore, therapuetics designed with the GPCR encoded by the transcript could be important in the treatment of diseases which include lung airway inflammation such as asthma and chronic obstructive pulmonary disorder.

V. CG174799-02: RTK TIEl splice variant.

Expression of gene CG174799-02 was assessed using the primer-probe set Ag6300, described in Table VA. Results of the RTQ-PCR runs are shown in Tables VB, VC and VD.

Table VA. Probe Name Ag6300

Table VB. CNS_neurodegeneration_vl.O

Table VC. General_screening_panel_vl.5

Table VD. Panel 4. ID

3401

CNS_neurodegeneration_vl.O Summary: Ag6300 Expression is restricted to a single sample from the hippocampus of an Alzheimer's patient (CT=30.8).

General_screening_panel_vl.5 Summary: Ag6300 Expression of this gene is restricted to a sample derived from a colon cancer cell line (CT=27.2). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel and as a marker to detect the presence of colon cancer. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of colon cancer.

Panel 4.1D Summary: Ag6300 This transcript is most highly expressed untreated lung microvascular endothelial cells (CT=33). Low but significant levels of expression are also seen in TNF-a/ILl-b treated HPAECs, and IL-1 beta, IL-11, and IFN-gamma treated HUVECs. 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.

W. CG175427-01: ADAMTS-12 precursor. Expression of gene CG175427-01 was assessed using the primer-probe set Ag6329, described in Table WA. Results ofthe RTQ-PCR runs are shown in Tables WB, WC, WD, WE and WF.

Table WA. Probe Name Ag6329

Table WB. AI_comprehensive panel_vl.O

Table WC. CNS_neurodegeneration_vl.O

Table WD. General_screening_panel_vl.7

Table WE. Panel 4. ID

Table WF. Panel 5 Islet

AI_comprehensive panel_vl.O Summary: Ag6329 Highest expression of this gene is seen in an OA bone sample (CT=27.7). This gene is widely expressed at moderate to low levels in this panel, with prominent expression seen in a cluster of samples derived from OA. This gene encodes a protein with homology to members of the ADAMTS family. ADAMTS proteins have been implicated in extracellular proteolysis and may play a critical role in the tissue degradation seen in arthritis and other inflammatory conditions. (Martel-Pelletier J. (2001) Best Pract Res Clin Rheumatol 15(5):805-29 ) Therefore, therapeutic modulation ofthe expression or function of this gene through the use of human monoclonal antibodies or small molecule drugs may be effective in the treatment of osteoarthritis and other autoimmune diseases.

CNS_neurodegeneration_vl.O Summary: Ag6329 This panel does not show differential expression of this gene in Alzheimer's disease. However, this profile confirms the expression of this gene at moderate levels in the brain. Please see Panel 1.7 for discussion of this gene in the central nervous system.

General_screening_panel_vl.7 Summary: Ag6329 Highest expression of this gene is seen in the thymus (CT = 27.6). This gene is widely expressed in this panel, with moderate expression seen in lung and renal cancer cell lines. Modulation of this gene product may be useful in the treatment of these cancers. Among tissues with metabolic function, this gene is expressed at moderate to low 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.

In addition, this gene is expressed at much higher levels in fetal liver tissue (CT=30) when compared to expression in the adult counterpart (CT=33). Thus, expression of this gene may be used to differentiate between the fetal and adult source of this tissue. Furthermore, the relative overexpression of this gene in fetal liver 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.

This gene is also expressed at moderate to low 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.

Panel 4.1D Summary: Ag6329 Two experiments with the same probe and primer show expression of this gene is restricted to the thymus and kidney on this panel (CTs=34). The protein encoded by this gene has homology to ADAMTS family of molecules suggesting that it may function as an enzyme. Based on its homology, it may contribute to the tissue destruction and remodeling processes associated with asthma, ulcerative colitis, emphysema and osteoarthritis. (Kuno K. J Biol Chem 1997 Jan 3;272(l):556-62;) Therefore, blocking the function of the protein encoded by this gene may reduce or inhibit tissue destruction in the kidney and thymus.

Panel 5 Islet Summary: Ag6329 Detectable levels of expression are seen only in skeletal muscle (CT=34.7).

X. CG175516-01: TGFbRII splice variant. Expression of gene CG175516-01 was assessed using the primer-probe sets Ag6323 and Ag6350, described in Tables XA and XB. Results ofthe RTQ-PCR runs are shown in Tables XC and XD.

Table XA. Probe Name Ag6323

Table XB. Probe Name Ag6350

Table XC. General_screening_panel_vl.5

Table XD. Panel 4. ID

General_screening_panel_vl.5 Summary: Ag6323/Ag6350 Two experiments with the same probe and primer set show highest expression of this gene in a gastric cancer cell line (CT=33.5). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel. In addition, the expression of this gene may be used as diagnostic marker to detect the presence of gastric cancer.

Panel 4.1D Summary: Ag6350 Detectable expression is seen only in untreated lung microvascular endothelial cells (CT=34). Expression in lung microvascular endothelial cells suggests that the protein encoded by this transcript may also be involved in lung disorders including asthma, allergies, chronic obstructive pulmonary disease, and emphysema. Therefore, therapeutic modulation ofthe protein encoded by this gene may lead to amelioration of symptoms associated with psoriasis, asthma, allergies, chronic obstructive pulmonary disease, and emphysema.

Y. CG175869-01: Canalicular multispecific organic anion transporter 1.

Expression of gene CG175869-01 was assessed using the primer-probe set Ag6358, described in Table YA. Results of the RTQ-PCR runs are shown in Table YB. Table YA. Probe Name Ag6358

Table YB. General_screening_panel_vl.6

General_screening_panel_vl.6 Summary: Ag6358 Highest expression of this gene is seen in a lung cancer cell line (CT=30.6). Moderate to low levels of experssion are also seen in cell lines from colon, renal, melanoma, brain, and liver cancers, as well as in fetal liver. Thus, expression of this gene might be useful as a marker of these cancers.

Z. CG175900-01: Phospholipid transporting ATPase Class II type 9A.

Expression of gene CG175900-01 was assessed using the primer-probe set Ag6538, described in Table ZA. Results of the RTQ-PCR runs are shown in Tables ZB, ZC and ZD.

Table ZA. Probe Name Ag6538

Reverse ' -cagaaaccaggctgtgatgtac-3 ' 22 4497 J261

Table ZB. CNS_neurodegeneration_vl.O

Table ZC. General_screening_panel_vl.6

Table ZD. Panel 5 Islet

401

CNS_neurodegeneration_vl.O Summary: Ag6538 This panel does not show differential expression of this gene in Alzheimer's disease. However, this profile confirms the expression of this gene at high levels in the brain. Please see Panel 1.6 for discussion of this gene in the central nervous system.

General_screening_paneι_vl.6 Summary: Ag6538 Highest expression of this gene is seen in the cerebellum (CT=26). This gene is also expressed at high levels throughout 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, as well as CNS disorders that have this brain region as the site of pathology, such as autism and the ataxias.

This gene is widely expressed in the cancer cell lines on this panel, with moderate expression seen in brain, colon, gastric, lung, breast, ovarian, and melanoma cancer cell lines. This expression profile suggests a role for this gene product in cell survival and proliferation. Modulation of this gene product may be useful in the treatment of cancer.

Among tissues with metabolic function, this gene is expressed at moderate to low levels in pituitary, adipose, adrenal gland, pancreas, thyroid, and adult and fetal 3 03401

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.

In addition, this gene is expressed at much higher levels in fetal liver tissue (CT=29.5) when compared to expression in the adult counterpart (CT=33.5). Thus, expression of this gene may be used to differentiate between the fetal and adult source of this tissue. Furthermore, the relative overexpression of this gene in fetal liver 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.

Panel 5 Islet Summary: Ag6538 Highest expression of this gene is seen in human pancreatic islet cells (Bayer patient 1) (CT=30). Moderate levels of expression are also seen in adipose derived samples, suggesting that this gene product may be involved in metabolic disease, including obesity and diabets.

A A. CG176069-01: hepatocyte nuclear factor 4 alpha2 chain.

Expression of gene CG176069-01 was assessed using the primer-probe sets Ag6374 and Ag7795, described in Tables AAA and AAB. Results ofthe RTQ-PCR runs are shown in Table AAC.

Table AAA. Probe Name Ag6374

Table AAB. Probe Name Ag7795

Table AAC. Panel 4. ID

LAK cells (CT=26). These cells are involved in tumor immunology and cell clearance of virally and bacterial infected cells as well as tumors. Therefore, modulation ofthe function of the protein encoded by this gene through the application of a small molecule drug or antibody may alter the functions of these cells and lead to improvement of symptoms associated with these conditions.

AB. CG50595-02, CG50595-04, CG50595-06 and CG50595-07: ACETYLGLUCOSAMINYLTRANSFERASE.

Expression of gene CG50595-02, CG50595-04, CG50595-06 and CG50595-07 was assessed using the primer-probe sets Agl933, Ag2683, Ag795, Ag872, Ag915, Ag58 and Ag058b, described in Tables ABA, ABB, ABC, ABD, ABE, ABF and ABG. Results ofthe RTQ-PCR runs are shown in Tables ABH, ABI, ABJ, ABK, ABL, ABM, ABN, ABO and ABP.

Table ABA. Probe Name Agl933

Table ABB. Probe Name Ag2683

Table ABC. Probe Name Ag795

Table ABD. Probe Name Ag872

Primers Sequences Length Start SEO ID

Table ABE. Probe Name Ag915

Table ABF. Probe Name Ag58

Table ABH. Ardais Panel v.1.0

Table ABI. Panel 1

01

TU 03/03401

Table ABK. Panel 1.3D

Table ABL. Panel 2.2

Table ABO. Panel 4D

Rel. Rel. Rel. Rel. Rel. ReL

Tissue Name Exp.(%) Exp.( ) Exp.(%) Tissue Name Exp.(%) Exp.(%) Exp.( ) A_1933. Ae2683. A_795. A_1933. A_2683. A_795.

Ardais Panel v.1.0 Summary: Ag795 Highest expression of this gene is detected in a lung cancer(369) sample (CT=29.5). Moderate to low expression of this gene is detected in number of lung cancer samples and two normal adjacent tissue samples. Expression of this gene is higher in cancer compared to normal lung. Therefore, expression of this gene may be used as diagnostic marker to detect the presence of this cancer and also, therapeutic modulation of this gene through the use of small molecule or antibodies may be useful in the treatment of lung cancer.

Panel 1 Summary: Ag58/Ag58b Two experiments with same probe-primer sets are in good agreement with highest expression of this gene detected in placenta (CTs=23-26.8). Therefore, therapeutic modulation of this gene may be useful in the treament of placenta related diseases including reproductive disorders. High to moderate expression of this gene is also seen in number of cancer cell lines derived from ovarian, breast, lung, liver, gastric, and colon cancer. Interestingly, this gene is upregulated in ovarian, breast, lung, liver and colon carcinoma cell lines comparing to their normal tissues. The normal tissue expression of this gene includes prostate, placenta, mammary gland, kidney, trachea, stomach, lymph node, pituitary gland, salivary gland, thyroid and pancreas. The specific high expression in ovarian, lung and colon cancer cell lines indicates a role in ovarian, lung and colon cancer progression. More importantly, the expression of this gene is higher in colon SW620 (the metastatic variant of SW480; CTs=29-34) than that in colon SW480 colon cancer cells (CTs=32-40). This may indicate its important role in colon tumor metastasis. This is further supported by the analysis of panel 2.2 that reveals how this gene is upregulated in several cancer tissues, specifically colon, lung and ovarian cancers, comparing to the normal adjacent tissues. Based upon its profile, the expression of this gene may be used as a marker to detect the presence of these cancers in a subject, specifically in a subject blood and more specifically for colon, lung and ovarian cancers. In addition, down- regulation ofthe activity of this gene, through the use of antibodies or small molecule drugs, specifically antisense oligonucleotide, might be of use in the treatment of colon, lung and ovarian cancers.

In addition, moderate expression of this gene is also seen in hypothalamus region of the brain. Therefore, therapeutic modulation of this gene may be useful in the treatment of neurological disorders.

Moderate to low expression of this gene is also seen in number of tissues with metabolic/endocrine functions including pancreas, thyroid, and fetal liver. Therefore, therapeutic modulation of the activity of this gene may prove useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes.

Panel 1.2 Summary: Ag795 Two experiments with same probe-primer sets are in good agreement with highest expression of this gene detected in placenta (CTs=25.5). The expression profile in this panel is in concordance with expression seen in panel 1. Please see panel 1 for further discussion of this gene. Panel 1.3D Summary: Ag2683 Highest expression of this gene is detected in placenta (CT=28.7). The expression profile in this panel is in concordance with expression seen in panel 1. Please see panel 1 for further discussion of this gene.

Panel 2.2 Summary: Ag795 Two experiments with same probe-primer sets are in good agreement. Highest expression of this gene is detected in normal kidney and prostate samples (CTs=30-32.5). Moderate to low expression of this gene is seen in number of cancer and normal samples derived from bladder, colon, gastric, liver, thyroid, kidney, lung and ovary. Expression of this gene is upregulated in gastric, colon, liver, breast, lung and ovarian cancers. Interestingly expression of this gene is downregulated in six kidney cancer samples. Please see panel 1 for further discussion of this gene.

Panel 2D Summary: Ag2683/Ag795 Two experiments with same probe-primer sets are in good agreement. Highest expression of this gene is detected in ovarian cancer (CTs=26-27). Expression profile of this gene in this panel is in agreement with that seen in panel 2.2. Please see panel 2.2 for further discussion of this gene.

Panel 3D Summary: Ag795 Highest expression of this gene is detected in a lung cancer NCI-UMC-11 cell line (CT=28). Moderate to low expression of this gene is detected in number of cell lines derived from lung, tongue, breast, bladder, pancreatic, gastric, colon, and brain cancers. Please see panel 1 for further discussion of this gene.

Panel 4D Summary: Agl933/Ag2683/Ag795 Three experiments with two different probe-primer sets are in good agreement. Highest expression of this gene is seen in thymus, resting and activated keratinoncytes (CTs=27-30). Moderate to low expression of this gene is also detected in mucoepidermoid cell line NCI-H292, resting and activated small airway epithelius, activated bronchial epithelium. Therefore, therapeutics designed with the protein encoded by the transcript may reduce or eliminate symptoms caused by inflammation in lung epithelia in chronic obstructive pulmonary disease, asthma, allergy, and emphysema.

In addition, moderate to low expression of this gene is also seen in activated B lymphocytes, resting and activated PBMC cells, Two Way MLR 3, activated LAK cells, resting IL -2 treated NK cells, memory and naive CD4 lymphocytes, lupus kidney and normal tissues represented by kidney, lung and colon. 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.

General oncology screening panel_v_2.4 Summary: Ag795 Two experiments with same probe-primer sets are in good agreement. Highest expression of this gene is detected in colon cancer (CTs=30-31.7). Moderate expression of this gene is also seen in number of samples derived from colon and lung cancers. Expression of this gene is higher in cancer compared to normal adjacent colon and lung tissues. Interestingly, expression of this gene is downregulated in kidney cancer. The expression profile in this panel correlates with that seen in panel 2.2, please see panel 2.2 and panel 1 for further discussion of this gene.

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

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

SeqCalling assemblies produced by the exon linking process were selected and extended using the following criteria. Genomic clones having regions with 98% identity to all or part ofthe initial or extended sequence were identified by BLASTN searches using the relevant sequence to query human genomic databases. The genomic clones that resulted were selected for further analysis because this identity indicates that these clones contain the genomic locus for these SeqCalling assemblies. These sequences were analyzed for putative coding regions as well as for similarity to the known DNA and protein sequences. Programs used for these analyses include Grail, Genscan, BLAST, HMMER, FASTA, Hybrid and other relevant programs.

Some additional genomic regions may have also been identified because selected SeqCalling assemblies map to those regions. Such SeqCalling sequences may have overlapped with regions defined by homology or exon prediction. They may also be included because the location of the fragment was in the vicinity of genomic regions identified by similarity or exon prediction that had been included in the original predicted sequence. The sequence so identified was manually assembled and then may have been extended using one or more additional sequences taken from CuraGen Corporation's human SeqCalling database. SeqCalling fragments suitable for inclusion were identified by the CuraTools™ program SeqExtend or by identifying SeqCalling fragments mapping to the appropriate regions of the genomic clones analyzed.

The regions defined by the procedures described above were then manually integrated and corrected for apparent inconsistencies that may have arisen, for example, from miscalled bases in the original fragments or from discrepancies between predicted exon junctions, EST locations and regions of sequence similarity, to derive the final sequence disclosed herein. When necessary, the process to identify and analyze SeqCalling assemblies and genomic clones was reiterated to derive the full length sequence (Alderborn et al., Determination of Single Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing. Genome Research. 10 (8) 1249-1265, 2000). Variants are reported individually but any combination of all or a select subset of variants are also included as contemplated NOVX embodiments of the invention.

NOVlb SNP Data (CG103404-01)

Seventeen polymorphic variants of NOVb have been identified and are shown in Table SNP1.

NOV2b SNP Data (CG108945-02)

Six polymorphic variants of NOV2b has been identified and is shown in Table SNP2. Table SNP2. Variant of NOV2b.

NOV4b SNP Data (CG124873-02)

One polymorphic variant of NOV4b has been identified and is shown in Table SNP3.

Table SNP3. Variant of NOV4b.

NOV5b SNP Data (CG138554-02)

Nine polymorphic variants of NOV5b have been identified and are shown in Table SNP4.

Table SNP4. Variants of NOV5b.

NOV6b SNP Data (CG144744-02)

One polymorphic variant of NOV6b have been identified and are shown in Table SNP5.

Table SNP5. Variants of NOV6b.

NOV7b SNP Data (CG151723-02)

Nine polymorphic variants of NOV7b have been identified and are shown in Table SNP6.

Table SNP6. Variants of NOV7b.

NOV8b SNP Data (CG159280-01)

Eight polymorphic variants of NOVδb have been identified and are shown in Table SNP7.

Table SNP7. Variants of NOV8b

NOVlOa SNP Data (CG169667-01)

One polymorphic variant of NOVlOa have been identified and are shown in Table SNP8. Table SNP8. Variants of NOVlOa

NOV14a SNP Data (CG170882-01)

Twelve polymorphic variants of NOV14a have been identified and are shown in Table SNP9. Table SNP9. Variants of NOV14a

NOV15a SNP Data (CG171205-01)

Three polymorphic variants of NOV15a have been identified and are shown in Table SNP10. Table SNP10. Variants of NOV15a

NOV16a SNP Data (CG171793-01)

One polymorphic variant of NOV16a have been identified and are shown in Table SNP11. Table SNP11. Variants of NOV16a

Nucleotides Amino Acids

NOV18a SNP Data (CG173488-01)

Three polymorphic variants of NOVl 8a have been identified and are shown in Table SNP12. Table SNP12. Variants of NOV18a

NOV20a SNP Data (CG174189-01)

Six polymorphic variants of NOV20a have been identified and are shown in Table SNP13. Table SNP13. Variants of NOV20a

NOV21a SNP Data (CG174799-01)

Three polymorphic variants of NOV21a have been identified and are shown in Table SNP14. Table SNP14. Variants of NOV21a

NOV22b SNP Data (CG175105-01)

One polymorphic variant of NOV22b have been identified and are shown in Table SNP15. Table SNP15. Variants of NOV22b

NOV25a SNP Data (CG175516-01)

Five polymorphic variants of NOV25a have been identified and are shown in Table SNP16. Table SNP16. Variants of NOV25a

NOV27a SNP Data (CG175869-01) One polymorphic variant of NOV27a have been identified and are shown in Table SNP17. Table SNP17. Variants of NOV27a

NOV28a SNP Data (CG175900-01)

Two polymorphic variants of NOV28a have been identified and are shown in Table SNPlδ. Table SNP18. Variants of NOV28a

NOV31b SNP Data (CG50595-02)

Seven polymorphic variants of NO V3 lb have been identified and are shown in Table SNP19. Table SNP19. Variants of NOV31b

NOV32c SNP Data (CG59325-01)

Nine polymorphic variants of NOV32c have been identified and are shown in Table SNP20. Table SNP20. Variants of NOV32c

Example E: Method of Use

Example El: Method of Use for NOVlb, CG103404-01.

Method of Identifying the Differentially Expressed Gene and Gene Product The GeneCalling ™ method is described in Example B.

SPECIES #1 A gene fragment of ak004947, clone 1300008N20 of the Mus musculus adult male liver cDNA RIKEN full-length enriched library, identical to the mouse Macrophage colony stimulating factor 1 receptor was initially found to be upregulated by 2.1 fold in the epidydimal adipose tissue of mouse euglycemic sd7 mice relative to euglycemic sdl mice using CuraGen's GeneCalling ™ method of differential gene expression. A differentially expressed mouse gene fragment migrating, at approximately 156 nucleotides in length (Table E1B. - solid vertical line) was definitively identified as a component ofthe mouse Macrophage colony stimulating factor 1 receptor cDNA (in the graphs, the abscissa is measured in lengths of nucleotides and the ordinate is measured as signal response). The method of comparative PCR was used for conformation of the gene assessment. The electropheric peaks corresponding to the gene fragment ofthe mouse Macrophage colony stimulating factor 1 receptor are ablated when a gene-specific primer (see below) competes with primers in the linker- adaptors during the PCR amplification. The peaks at 156 nt in length are ablated (dotted or dashed trace) in the sample from both the euglycemic sd7 mice and euglycemic sdl mice.

Table E1A. Competitive PCR Primer sequence for mouse Macrophage colony stimulating factor 1 receptor:

(fragment from 3080 to 3235 in bold, band size: 156)

SEQ ID NO:289:

2599 TGTGGTCCTA CGGCATCCTC CTCTGGGAGA TCTTCTCGCT TGGTCTGAAC CCCTACCCCG 2659 GCATCCTAGT GAACAACAAG TTCTACAAAC TGGTGAAGGA TGGATACCAA ATGGCCCAGC 2719 CTGTATTTGC ACCGAAGAAC ATATACAGCA TCATGCAGTC CTGCTGGGAC CTGGAGCCTA 2779 CCAGAAGACC CACCTTCCAA CAGATCTGCT TCCTCCTCCA GGAGCAGGCC CGACTGGAGA 2839 GGAGAGACCA GGACTATGCT AACCTGCCAA GCAGCGGTGG CAGCAGCGGC AGTGACAGTG 2899 GTGGTGGCAG CAGCGGTGGC AGCAGCAGTG AGCCAGAAGA GGAGAGCTCC AGTGAACACC 2959 TGGCCTGCTG TGAGCCAGGG GACATCGCCC AGCCCCTGCT GCAGCCTAAC AACTACCAGT 3019 TCTGCTGAAG TGGGAGGGAG AGCCGAGTCC TGCCGCTCTC TACGTCCCAG CTTGGCCTCC 3079 TCCATGGCAC GGGCGACATG GGGAGAACAT ATGGACTTCG CCCTCAGCT GGCCCΔGCTC 3139 TGACACT CA GAACATGAGG GGTCTGGGGA GGTCAGAGGC CCCGTTTGTT CCCAGAGCCT 3199 GGGCCATCAC TGCCAGTGGG GTTCTCACAG TGCTAGCCTC TATATTACT ATGCCAACTG 3259 GTGCACCCCT AGTTTTCTTT CTCCATCCTA TTCCCATTTT AAAAAACCCG TCC

(gene length is 3311, only region from 2599 to 3311 shown)

Table E1B

Table E1B represents confirmatory results of differentially expressed gene fragment activity of mouse macrophage colony stimulating factor 1 receptor.

Human Macrophage colony stimulating factor 1 receptor gene and protein sequences are shown above as NOV lb, CG103404-01 and variants NOVla, NOVlc, NOVld, NOVle, NOVlf, NOVlg

The following is an alignment of the protein sequences of NOVlb (CG103404- 01), rat (SEQ ID 290) and mouse (SEQ ID 291) versions of the Macrophage colony stimulating factor 1 receptor.

Table EIC. Clustal W, Protein Domains, Cellular Location and Locus

KFMS RAT iEAV P C-ITD.RAi-KB-SVS, CG103404-01 >§__ ,cxiτ__ gEEEEΪs' *

KFMS RAT ■ YTFK FmRVKASEAaO-P'-MAOMKAQ HHUTFELTLRYFP.E'yg.-V.TWMP.yj^^SDV-LF-C 418 CG103404-01 .SfrFilLaia iK sΕlrjjM 420 _04947_MCSF_l_R_ o_β TOWiii»:_ιmm. ■araenrein-wa 418

KFMS RAT 479 l_______dt____*_>^^ 538 KFMS RAT 5980 CG103404-01 Q 6008 si004947_MCSF_l_R_ino_(! E 598

KFMS RAT OR 31 CG103404-01 601 iia WT

H3s?_! _04947_MCSF_l_R_mousβ 599

KFMS RAT I ITEYCCYGDL HF RRKAEAMLGPSLSPGQDSEOE'SSYKMI HLEKKY.V RRDSGFSSQG V 718 CG103404-01 ITEYCCYGDLLHFL RKAEAMCGPSLSPGQ ijEGEEBYKHI HLEKK^'YVR'F.DSGFSSQG V 720 _04947 MCSF 1 R mouse VITEYCCYO_LLWF_RRKAEAM-.GPS SPGOD3EGDΞ3Yi:NrHLEKKYyRRDSGF.SSCιGV 718

KFMS RAT 779 M3H*iB*ιtoι;ιt>a:«mιι_H»a;<ιιιo«aι»Ma>_Mwif aw 838 CG103404-01 _04947 MCSF l_R_mouse 7 ⁷⁸79¹ r3 CT$aaWt_7wT»CTιι^τ;_fffl«JMιWiMafffi:irt_a_fl«TOιιtmιaι»Mi««_^at«»»^ 8⁸3⁴⁰8

KFMS RAT CG10340401 <&0O4947_MCSF_l_R_mo_e

KFMS RAT 899 MMΛK'l *IMMHAΛA;hW''iAy>lUHΛΛΛ ma&AΛ&ΛAΛ&SAΛiM 958 CG103404-01 AMJisFlMJiWoEPiira_RI»S.Ti„<J*l- . -^~HREKM- -t 953 „04947_MCSF_l_R_mouse 'TFOOT CFL'LOEOA'RLERRBODYANLPSSOGSSGSDSGGGS S 958

KFMS RAT CEPGDI AQPL QPHNYi FS 978 CG103404-01 CES3D'IΑQP__QPNNΫQ C 972 ai004947 MCSF 1 R mouse EPGDI^'AOFLLOPWNYOFC 977

Variants of Human Macrophage Colony Stimulating Factor 1-Receptor from Direct Cloning and/or Public Databases

In addition to the human version ofthe Macrophage colony stimulating factor 1 receptor identified as being differentially expressed in the experimental study, other variants have been identified by direct sequencing of cDNAs derived from many different human tissues and from sequences in public databases. Several amino acid- changing cSNPs were identified. These are found above in Table SNPl. The preferred variant of all those identified, to be used for screening purposes, is NOVlb (CG103404- 01).

Pathways relevant to the etiology and pathogenesis of obesity and/or diabetes.

Table E1D.

Cell proliferation r

Table E1D shows pathways relevant to the etiology and pathogenesis of obesity and/or diabetes. It suggests how alterations in expression ofthe human Macrophage colony stimulating factor 1 receptor and associated gene products function in the etiology and pathogenesis of obesity and/or diabetes. The scheme incorporates the unique findings of these discovery studies in conjunction with what has been reported in the literature. It shows how sigaling through the MCSF receptor influences three distinct pathways which have been shown to be involved in insulin resistance. In addition, it has been shown by other literature to be involved in adipose hyperplasia (Levine et al., 1998). The outcome of inhibiting the action of the human Macrophage . colony stimulating factor 1 receptor would be decrease in lipid accumulation, a major problem in obesity.

Rationale for use as a diagnostic and/or target for small molecule drugs and antibody therapeutics.

MSCF-l-R was found upregulated in both visceral fat pads in obese hyperinsulinemic and hyperglycemic mice. It is is stimulated by TNF-alpha and high- cholesterol diet. MSCF-l-R 1 has been identified as a factor actively promoting adipose hyperplasia, while it is know that its ligand CSF-1 is downregulated during adipose differentiation. Moreover, MCSF-l-R enhances proliferation and differentiation of monocytes and foamy macrophages, contributing to atherosclerosis. Therefore, inhibiting the MCSF pathway by targeting the MSCF receptor will improve various symptoms ofthe metabolic syndrome X phenotype, including obesity, hyperglycemia and atherosclerosis leading to heart disease.The protein similarity information, expression pattern, cellular localization, and map location for the protein and nucleic acid disclosed herein suggest that this protein may have important structural and/or physiological functions characteristic ofthe macrophage colony stimulating factor 1 receptor family. Therefore, the nucleic acids and proteins ofthe invention can be used 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. These also include 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), (v) an agent promoting tissue regeneration in vitro and in vivo, and (vi) a biological defense weapon.

The nucleic acids and proteins of the invention have applications in the diagnosis and/or treatment of various diseases and disorders. For example, the compositions of the present invention will have efficacy for the treatment of patients suffering from: obesity and/or diabetes.

See: Clinton SK, Underwood R, Hayes L, Sherman ML, Kufe DW, Libby P. 1992. Macrophage colony-stimulating factor gene expression in vascular cells and in experimental and human atherosclerosis. Am J Pathol 1992 Feb;140(2):301-16. PMID: 1739124; Dey A, She H, Kim L, Boruch A, Guris DL, Carlberg K, Sebti SM, Woodley DT, Imamoto A, Li W. 2000. Colony-stimulating factor- 1 receptor utilizes multiple signaling pathways to induce cyclin D2 expression. Mol Biol Cell 2000 Nov;ll(ll):3835-48. PMID: 11071910; Levine JA, Jensen MD, Eberhardt NL, O'Brien T. 1998. Adipocyte macrophage colony-stimulating factor is a mediator of adipose tissue growth. : J Clin Invest 1998 Apr 15;101(8): 1557-64. PMTD: 9541484; Umezawa A, Tachibana K, Harigaya K, Kusakari S, Kato S, Watanabe Y, Takano T. 1991 Colony- stimulating factor 1 expression is down-regulated during the adipocyte differentiation of H-l/A marrow stromal cells and induced by cachectin/tumor necrosis factor. Mol Cell Biol Feb;ll(2):920-7. PMID: 1990292

Example E2: Method of Use for NOV8b, CG159820-01.

Glycerol 3-Phosphate Dehydrogenase 1 (GPD1) is a cytoplasmic enzyme involved in the glycerol 3-phosphate shuttle. It accounts for rapid re-oxidation of cytoplasmic NADH and links glycolysis with lipid production. Mice which lack GPD1, show an increase in energy expenditure in skeletal muscle during exercise (MacDonald MJ, Marshall LK. (2001) Survey of normal appearing mouse strain which lacks malic enzyme and Nad+-linked glycerol phosphate dehydrogenase: normal pancreatic beta cell function, but abnormal metabolite pattern in skeletal muscle. Mol Cell Biochem. 220, 117-25; PMID: 11451371). GPDl has been implicated in adipocyte differentiation (Shahparaki A, Grander L, Sorisky A. (2002) Comparison of human abdominal subcutaneous versus omental preadipocyte differentiation in primary culture. Metabolism, 51(9): 1211-5; PMID: 12200769). GPDl is also known to induce by glucocorticoids, and the excess glucocorticoids are associated with obesity and diabetes (Nicols NR, Dokas L, Ting SM, Kumar S, de Vellis J, Shors TJ, Uenishi N, Thompson RF, Finch CE. (1996) Hippocampal responses to corticosterone and stress, one of which is the 35,000 M(r) protein, glycerol phosphate dehydrogenase. J. Neuroendocrinol. 8, 867-76; PMID: 8933364).

BP24.02

The predominant cause for obesity in clinical populations is excess caloric intake. This so-called diet-induced obesity (DIO) is mimicked in animal models by feeding high fat diets of greater than 40% fat content. The gene expression changes contributing to the development and progression of diet-induced obesity were observed. The factors that lead to the ability of certain individuals to resist the effects of a high fat diet and thereby prevent obesity were looked for. The sample groups had body weights +1 S.D., + 4 S.D. and + 7 S.D. ofthe chow-fed controls (below). In addition, the biochemical profile ofthe + 7 S.D. mice revealed a further stratification of these animals into mice that retained a normal glycemic profile in spite of obesity and mice that demonstrated hyperglycemia. Tissues examined included hypothalamus, brainstem, liver, retroperitoneal white adipose tissue (WAT), epididymal WAT, brown adipose tissue (BAT), gastrocnemius muscle (fast twitch skeletal muscle) and soleus muscle (slow twitch skeletal muscle). The differential gene expression profiles for these tissues should reveal genes and pathways that can be used as therapeutic targets for obesity.

MB.04

A large number of mouse strains have been identified that differ in body mass and composition. The AKR and NZB strains are obese, the SWR, C57L and C57BL 6 strains are of average weight whereas the SM/J and Cast/Ei strains are lean. Understanding the gene expression differences in the major metabolic tissues from these strains will elucidate the pathophysiologic basis for obesity. These specific strains of rat were chosen for differential gene expression analysis because quantitative trait loci (QTL) for body weight and related traits had been reported in published genetic studies. Tissues included whole brain, skeletal muscle, visceral adipose, and liver.

Species #1 Mouse Strains C57BL 6J

Species #2 Mouse Strains C57BL/6J, Cast Ei

MB.08

Human Mesenchymal Stem Cell Differentiation Study (MB.08)

Bone marrow-derived human mesenchymal stem cells have the capacity to differentiate into muscle, adipose, cartilage and bone. Culture conditions have been established that permit the differentiation in vitro along the pathway to adipose, cartilage and bone. Understanding the gene expression changes that accompany these distinct differentiation processes would be of considerable biologic value. Regulation of adipocyte differentiation would have importance in the treatment of obesity, diabetes and hypertension. Human mesenchymal stem cells from 3 donors were obtained and differentiated in vitro according to published methods. RNA from samples of the undifferentiated, mid-way differentiated and fully differentiated cells was isolated for analysis of differential gene expression.

Results of MB.04

A fragment of the mouse Glycerol 3-Phosphate Dehydrogenase 1 gene was initially found to be upregulated by approximately 2.0 fold in the skeletal muscle and adipose tissue of control strains of mice when compared to genetically thin strains using CuraGen's GeneCalling™ method of differential gene expression. These differentially expressed mouse gene fragments, migrating at approximately 85.3, 84.1, 120.9, 91.4, 91.6 nucleotides in length, were definitively identified as components of the mouse TR3 nuclear receptor cDNA. The method of TraPping was used for confirmation of the gene assessment. Table E1B depicting the fragment lengths, dysregulation, TraPping score and actual sequence data is displayed below. The numerator in the score represents the number of nucleotides matched in the gene fragment. The actual nucleotide sequence is displayed in the column labeled "Fragment TraP Data". The denominator in the score represents the total number of TraP nucleotides available for this fragment with the actual nucleotide sequence presented in the column labeled "Predicted Trap Nucleotide Sequence". A score of 3/3 or 4/4 is treated with high confidence that the band belongs to that gene.

Table E2A: The results ofthe trapping data show that the bands found to be dysregulated in skeletal muscle from C57 mice compared to Cast/Ei strains in Discovery Study MB .04 study are from the mouse Glycerol 3-Phosphate Dehydrogenase 1 gene.

Table E2B: The results of the trapping data show that the bands found to be dysregulated in adipose of NZB compared to C57 mice in Discovery Study MB .04 study are from the mouse Glycerol 3-Phosphate Dehydrogenase 1 gene.

Results of MB.08

A gene fragment ofthe human Glycerol 3-Phosphate Dehydrogenase 1 was found to be up-regulated by 2.4 fold in mesenchemal stem cells differentiated to adipocytes compared to undifferentiated cells using CuraGen's GeneCalling ™ method of differential gene expression. Additionally, the same fragment of human Glycerol 3- Phosphate Dehydrogenase 1 was also up-regulated by 1.8 fold in mesenchemal stem cells fully differentiated to adipocytes compared to mid-way differentiated cells. A differentially expressed human gene fragment migrating, at approximately 335,0 nucleotides in length (Table E1B set A. - solid vertical line) was definitively identified as a component of the human Glycerol 3-Phosphate Dehydrogenase 1 cDNA (in the graphs, the abscissa is measured in lengths of nucleotides and the ordinate is measured as signal response). The method of competitive PCR was used for conformation of the gene assessment. The electropherographic peak corresponding to the gene fragment of the human Glycerol 3-Phosphate Dehydrogenase 1 is ablated when a gene-specific primer (Fig. E1B set A) competes with primers in the linker-adaptors during the PCR amplification in fully differentiated mesenchemal cells (Table E1B set B).

SPECIES #1 A gene fragment of the mouse Glycerol 3-Phosphate Dehydrogenase 1 was initially found to be up-regulated by 2.1 fold in the slow twitch muscle in hyperglycemic animal relative to normal glycemic animal with similar weight using CuraGen's GeneCalling ™ method of differential gene expression. Additionally, the fragment mouse Glycerol 3-Phosphate Dehydrogenase 1 was also up-regulated in comparison between fast twitch and slow twitch muscle in hyperglycemic animals and animals on high fat diet. A differentially expressed mouse gene fragment migrating, at approximately 120.9 nucleotides in length (Table El set A. - solid vertical line) was definitively identified as a component of the mouse Glycerol 3-Phosphate Dehydrogenase 1 cDNA (in the graphs, the abscissa is measured in lengths of nucleotides and the ordinate is measured as signal response). The method of comparative PCR was used for conformation of the gene assessment. The electropherographic peaks corresponding to the gene fragment of the mouse Glycerol 3- Phosphate Dehydrogenase lare ablated when a gene-specific primer (see below) competes with primers in the linker-adaptors during the PCR amplification. The peaks at 120.9 nt in length are ablated (dotted or dashed trace) in the sample from both the hyperglycemic and euglycemic animals.

SPECIES #2 A gene fragment of the mouse Glycerol 3-Phosphate Dehydrogenase 1 was also found to be up-regulated by 2 fold in the muscle of C57 relative to Cast/Ei mouse using CuraGen's GeneCalling ™ method of differential gene expression. A differentially expressed mouse gene fragment was definitively identified as a component of the mouse Glycerol 3 -Phosphate Dehydrogenase 1 cDNA by computer analysis.

Competitive PCR Primer for the mouse Glycerol 3-Phosphate Dehydrogenase 1.

Table E2C. Sequence #1, fragment from 915 to 1034 in bold, band size: 120; primers used in competitive PCR are underlined. (SEQ ID NO:292)

434 GCGCCTTGGC ATTCCCATGA GCGTGCTGAT GGGGGCCAAC ATTGCCAGCG AGGTGGCTGA 494 GGAGAAGTTC TGTGAGACGA CCATCGGCTG CAAGGACCCG GCCCAGGGAC AGCTCCTGAA 554 GGACCTGATG CAGACACCCA ACTTTCGCAT CACTGTGGTA CAAGAGGTGG ACACAGTGGA 614 GATCTGTGGG GCCTTGAAGA ATATAGTGGC CG TGGGGCT GGCTTCTGTG ATGGGCTTGG 674 CTTCGGTGAC AACACCAAGG CGGCGGTGAT CCGGTTGGGG CTCATGGAGA TGATCGCCTT 734 CGCCAAGCTC TTCTGCAGTG GCACTGTGTC CTCGGCCACC TTCCTGGAGA GCTGCGGGGT 794 CGCAGACCTC ATCACGACCT GCTATGGGGG GCGGAACCGC AAGGTGGCAG AGGCCTTTGC 854 TCGAACTGGA AAGTCCATTG AGCAGCTGGA GAAGGAGATG CTAAATGGGC AGAAGCTACA 914 GGGGCCCCAG ACAGCCCGGG AGCTGCACAG CAT CTCCAA CACAAGGGCC GTGGACAA 974 G TCCCCTTG T CACTGCGG TGTACAAAGT GTGCTATGAG GGCCAGCCAG TGGOCGAATT 1034 CATCCGCTGT CTGCAGAACC ACCCGGAACA CATGTGAATG GGGCCAGGGC CCAGGACAGG 1094 CAGCCCCTTT GCCCCAACGG AGACAAGCGG AAGGAAGCAC GTGACACCCG TCATCAGGAC 1154 TGTCTCCAGG ACTCCCCATC TAGCAGAGTC CTCTCTGAAG GACCCTGAGG ACAGGAGGCT 1214 GCGGGGCTCA GCTACACACC TAGAGATGCT CGCCACAGAA TCCACACTTC CTTGCTGTCC 1274 TCTGGGAAGT GTAGAACTAA GCCCCAGTGG TGCCTGCTCC AGGGGTGGGG TTGGGGGAAG 1334 GGAAGGCGCC AAGTCAAGGG CTGCCGGTTG CTGCCTCACA CACACTGGGA ACCGGTCCCA 1394 AGTCCCATTA AGTGACTGAA GAAGCACTTC AGCCACAGGA AAGATGGGGC AAAGGCTGCC 1454 AAGGGAAGGG TCTGGATGCC TGAGCTGACA CAGACCCCAG AGACCCTTTG GCCGACCTCT 1514 G

Gene length is 2785, but only the region from 434 to 1514 is shown here.

Human Glycerol 3-Phosphate Dehydrogenase 1 , CG159280 DNA and amino acid sequences are shown above, N0V8a, 8b, 8c, 8d, 8e, 8f , 8g .

Table E2D Competitive PCR primer for the human Glycerol 3-Phosphate Dehydrogenase 1: The sequence of the 335.0 nucleotide long gene fragment (from 1740 to 2075 band size: 336) and the gene-specific primers used for competitive PCR are indicated on the cDNA sequence ofthe human Glycerol 3-Phosphate Dehydrogenase 1 and are shown below in bold. The gene-specific primers at the 5' and 3' ends ofthe fragment are underlined. Gene Seςtuβncθ (fragment from 1740 to 2075 in bold, band size: 336) SEQ ID 293)

1259 TGTTAACAAA CACTGAGATC CATCGAAGCT GTGTGGGTCC TGGCAGAGGT CAGCAAAGGG

1319 GTCCTTGGGG CCTGCATCAG CTCAGAAGCC CAGACCCCTC CCTGACAATC CTTGCCTCAT

1379 CGCTCCTGTG GCTAAGATAC TTCTTCAGCC CTTTAACAGG ATTACTGGTG TATGTGAGAC

1439 AGCCACTAGC AGCCCCTGCC CTACCGGCCT CTCCCCCAAC CCTTCTCCAC ATACATCACC

1499 CCCGCCACCA AGCAGGCAGC ACTGGGGCTT GATTCCACCC CTCCCAGAGG ATATGGAGAA

1559 AGCCCCACAT CTGGAGAGGG ACAGGGCAAC TGCATTTCTG GCAAATGTGG TGGCATGAGG

1619 CTGCCAGTGG CTTGACAGTT CAGGATCTCC AGGTGCGTAG CTGGGCCCCA TAGCCTCCTG

1679 TCCTCCAGTG AAAAGATGAG AAGACTCTGC TCCCTGGGAG TCCTGGAGAT CCTGGTGACT

1739 AGGTACCCCA GGCTTCTGCT GGTCTCCACT GGGGTAAAAA AGCGGCCTGG CCTGGGCCCT

1799 GGCCCCACTC ACATATGTTC TGGATGATTC TGCAGGCAGT GGATGAATTC ACCCACTGGC

1859 TGGCCCTCGT AGCACACCTT GTACACAGCC ATGAACAAGG GAAACTTGTC TACCAGGCCC

1919 TTGTGCTGGA GGATGCTGTA TAGCTCCCGG GCTGTCTCGG GCCCCTGCAG TTTCTGCCCA

1979 TTCAGCAACT CTTTCTCCAG CTGCTCAATG GACTTTCCTG TACGCGCAAA GGCCTCAGCC

2039 ACTTTCCGGT TCCGCCCTCC ATAGCAGGTA GTGATCAGGT CAGCAACACC ACAGCTCTCC

2099 AGGAAGGTGG CGGAGGACAC AGAGCCACTG CAGAAGAGCT TGGCGAAGGC TATCATCTCC

2159 ATGAGCCCCA GCCGGATCAC TGCCGCCTTG GTGTTGTCAC CGAAGCCAAG CCCGTCACAG

2219 AAGCCAGCCC CAACAGCCAC TACATTCTTT AAGGCTCCAC AGATCTCTAC TGTGTCCACC

2279 TCTTGCACCA CTGTGATACG GAAGTTTGGT GTCTGCATCA GCTCTTTCAG GAGTTGTCCC

2339 TGGGCCGGGT CCTTGCAGCC AATGGTTGTC TCACAGAACT TCTCATCAGC CACCTCGCTG

2399 GCAATGTTGG CCCCCATCAG CACACTCATG GGGATGCCGA GGCGCTCCCC AATCACTTCC

2459 GAGATGAGCT TCAGCCCATT GGGGCCCTCG TCTACCCCCT TAATAAGAGA TATGCCAGTG

2519 GCGTTTGCCT TCAGATGGCC CTTGAGCTGG TCACAAG

(gene length is 2877, only region from 1259 to 2555 shown)

Table E2E: Differentially expressed gene fragment from Discovery Study MB .08 from the human Glycerol 3-Phosphate Dehydrogenase 1.

Table E2F Differentially expressed gene fragment for Sequence #1 in Discovery Study BP24.02 from mouse Glycerol 3-Phosphate Dehydrogenase 1.

Variants of the human Glycerol 3-Phosphate Dehydrogenase 1-like Protein are shown above as NOV8a,b,c,d,e,f,g. Splice-form variants are found above in table SNP7. The preferred variant of all those identified, to be used for screening purposes, is CG159280-01.

Biochemistry and Cell Line Expression

Glycerol 3-Phosphate Dehydrogenase 1 catalyzes the bi-directional reaction:

sn-glycerol 3-phosphate + nad(+) = glycerone phosphate + nadh.

It is one of the major enzyme in glycerol 3-phosphate shuttle, which control NAD+/NADH ratio in cytoplasm, e.g. red/ox state in the cell. The following illustrations summarizes the biochemistry surrounding the human Glycerol 3-Phosphate Dehydrogenase 1. Additional cell lines expressing the Glycerol 3-Phosphate Dehydrogenase can be obtained from the RTQ-PCR results shown above. These and other Glycerol 3-Phosphate Dehydrogenase expressing cell lines could be used for screening purposes.

Table E2G

Glycerol Phosphate Shuttle

GPDl links glycolysis to lipid synthesis in skeletal muscle and adipose. Glycerol 3-Phosphate Dehydrogenase 1, GPDl, CG159280-01 is up-regulated in obese and diabetic adipose and skeletal muscle suggesting that the role of GPDl in the development of obesity/diabetes phenotype. GPDl is induced during adipocyte differentiation proposing that inhibition of GPDl may interfere with adipose expansion and finally obesity. The inventors have found that GPDl is expressed more in glycolytic than in oxidative skeletal muscle fibers. Inhibition of GPDl would impair triglyceride production and favor oxidative muscle phenotype thus improving muscle metabolic profile by promoting glucose uptake and utilization of fat as an energy source.

• GPDl is up-regulated in skeletal muscle and adipose in hyperglycemic and obese states. It is expressed more in glycolytic than in oxidative muscle.

• Obese/diabetic skeletal muscle has a reduced oxidative enzyme activity, increased glycolytic activity and increased lipid content.

• GPDl is induced by glucocorticoids (Nicols et al.,J.Neuroendocrin.(1996) 8, 867) and during adipocyte differentiation (Gangon et al., J.Cell Physiol. (2001) 189,14; Seki et al., J.Cell Physiol. (2001) 189,72).

• Inhibition of GPDl may inhibit glycolysis, triglyceride production, and may favor utilization of fat for energy production and oxidative muscle phenotype.

Therefore, an antagonist of GPDl is beneficial for treatment of obesity/diabetes. See: Fink SC, Brosemer RW (1975) Glycerol-3-phosphate dehydrogenase from the honey bee. Methods Enzymol. 41, 240-5. PMID: 236444; MacDonald MJ, Marshall

LK. (2001) Survey of normal appearing mouse strain which lacks malic enzyme and

Nad+-linked glycerol phosphate dehydrogenase: normal pancreatic beta cell function, but abnormal metabolite pattern in skeletal muscle. Mol Cell Biochem. 220, 117-25.

PMTD: 11451371; Nicols NR, Dokas L, Ting SM, Kumar S, de Vellis J, Shors TJ,

Uenishi N, Thompson RF, Finch CE. (1996) Hippocampal responses to corticosterone and stress, one of which is the 35,000 M(r) protein, glycerol phosphate dehydrogenase. J

Neuroendocrinol. 8, 867-76. PMID: 8933364; Niesel DW, Pan YC, Bewley GC,

Armstrong FB, Li SS (1982) Structural analysis of adult and larval isozymes of sn- glycerol-3-phosphate dehydrogenase of Drosophila melanogaster. J Biol Chem. 257, 979-83. PMID: 6798037; Seki T, Miyasu T, Noguchi T, Hamasaki A, Sasaki R, Ozawa Y, Okukita K, Declerck PJ, Ariga T. (2001) Reciprocal regulation of tissue-type and urokinase-type plasminogen activators in the differentiation of murine preadipocyte line 3T3-L1 and the hormonal regulation of fibrinolytic factors in the mature adipocytes. J. Cell Physiol. 189, 72-8. PMID: 11573206

Example E3: Method of Use for NOV28a, CG175900-01

ATPases (also known as P-type) are a family of membrane proteins that use the free energy of ATP hydrolysis to drive uphill transport of ions across membranes. A subfamily includes NOV28a (CGI 75900-01), a potential aminophospholipid- transporting ATPase. Aminophospholipid translocase is an inward-directed pump specific for phosphatidylserine and phosphatidylethanolamine, although phosphatidylinositol may also be a substrate (Bevers EM, Comfurius P, Dekkers DW, Zwaal RF, 1999, Lipid translocation across the plasma membrane of mammalian cells. Biochim Biophys Acta. 1439(3):317-30. Review. PMID: 10446420). It's role is believed to be in the maintenance or regulation of membrane lipid asymmetry. This family of translocases may also be involved in body fat distribution and lipid or adipocyte metabolism. A novel P-type ATPase has been identified as a candidate gene for increased body fat in mice (Dhar M, Webb LS, Smith L, Hauser L, Johnson D, West DB., 2000, A novel ATPase on mouse chromosome 7 is a candidate gene for increased body fat. Physiol Genomics 4(1):93-100, PMID: 11074018; Dhar M, Hauser L, Johnson D, 2002, An aminophospholipid translocase associated with body fat and type 2 diabetes phenotypes. Obes Res. 2002 Jul;10(7):695-702. PMTD: 12105293). The precise metabolic role of this gene in adiposity and body weight regulation is not yet known.

The present mvention discloses novel associations of proteins and polypeptides and the nucleic acids that encode them with various diseases or pathologies. The proteins and related proteins that are similar to them, are encoded by a cDNA and/or by genomic DNA. The proteins, polypeptides and their cognate nucleic acids were identified by the inventors in certain cases. In particular, the phospholipid transporting ATPase Class II type 9A protein encoded by NOV28a (CG175900-01) and any variants, thereof, are suitable as diagnostic markers, targets for an antibody therapeutic and targets for small molecule drugs. CuraGen RTQ-PCR data documents that this gene is well-expressed in human adipose. In addition, CuraGen GeneCalling studies have also uncovered a role for this gene in whole-body adiposity in the mouse. The mouse homolog of NOV28a (CG175900-01) is upregulated 7.7-fold in AKR obese versus normal body weight C57L/J adipose. The mouse homolog is also dysregulated in the diet-induced obesity study. The response to a high fat diet in mice is accompanied by both up- and downregulations of multiple lipid-metabolizing genes. In the case of the mouse homolog of NOV28a (CG175900-01), there is a downregulation of this gene in adipose tissue. It is downregulated 2-fold in the comparison of ngsd7 versus sdl epididymal fat pad, and downregulated 1.6-fold in the comparison of ngsd7 versus sdl retroperitoneal fat pad. The multiple dysregulations of this gene in animal models of obesity suggest that it may be a therapeutic target. A preferred method ofthe invention is the use of the phospholipid transporting ATPase Class II type 9A for identifying an antagonist that would be beneficial in the treatment of obesity. As such the current invention embodies the use of recombinantly expressed and/or endogenously expressed protein in various screens to identify such therapeutic antibodies and/or therapeutic small molecules.

Mouse Obesity Study (MB.04)

A fragment ofthe mouse phospholipid transporting ATPase Class II type 9 A gene was initially found to be upregulated by 7.7 fold in the AKR obese versus normal body weight C57L/J adipose using CuraGen's GeneCalling ™ method of differential gene expression. A differentially expressed mouse gene fragment migrating, at approximately 277.7 nucleotides in length (see Table E3A) was definitively identified as a component ofthe mouse phospholipid transporting ATPase Class II type 9A cDNA (in the graphs, the abscissa is measured in lengths of nucleotides and the ordinate is measured as signal response). The method of competitive PCR was used for confirmation ofthe gene assessment. The electropherographic peaks corresponding to the gene fragment ofthe mouse phospholipid transporting ATPase Class II type 9A are ablated when a gene-specific primer (see Table E3A) competes with primers in the linker-adaptors during the PCR amplification. The peaks at 277.7 nt in length are ablated (dotted or dashed trace) in the sample from both the obese and the normal body weight C57L/J adipose (see Table E3B).

Mouse Dietary - Induced Obesity Study (BP24.02)

A fragment of the mouse phospholipid transporting ATPase Class II type 9A gene was initially found to be downregulated 2-fold in the comparison of ngsd7 versus sdl epididymal fat pad, and downregulated 1.6-fold in the comparison of ngsd7 versus sdl retroperitoneal fat pad using CuraGen's GeneCalling ™ method of differential gene expression. A differentially expressed mouse gene fragment migrating, at approximately 145 and 229 nucleotides in length (see Table E3D, respectively) was definitively identified as a component of the mouse phospholipid transporting ATPase Class II type 9A cDNA (in the graphs, the abscissa is measured in lengths of nucleotides and the ordinate is measured as signal response). The method of competitive PCR was used for confirmation of the gene assessment. The electropherographic peaks corresponding to the gene fragment of the mouse phospholipid transporting ATPase Class II type 9A are ablated when a gene-specific primer (see Table E3D) competes with primers in the linker-adaptors during the PCR amplification. The peaks at 145 and 229 nt in length are ablated (dotted or dashed trace) in the sample from the ngsd7 and sdl epididymal fat pad and sdl retroperitoneal fat pad (see Table E3D).

Competitive PCR primer for the mouse phospholipid transporting ATPase Class II type 9A: The sequence of the 277.7 nucleoti de-long gene fragment (from 1289 to 1567 band size: 279) and the gene-specific primers used for competitive PCR are indicated on the cDNA sequence ofthe mouse phospholipid transporting ATPase Class II type 9A and are shown below in bold. The gene-specific primers at the 5' and 3' ends ofthe fragment are underlined. Table E3A Gene Sequence (fragment from 1289 to 1567 in bold, band size: 279) SEQ ID 294:

808 GACTGGAAGC TTCGGCTCCC GGTGGCCTGC ACACAGAGGC TTCCCACGGC TGCTGACCTC 868 CTGCAGATTC GGTCCTATGT GTACGCTGAA AAACCCAACA TCGACATTCA CAACT CCTG 928 GGGACTTTCA CCAGGGAAAA CAGTGACCCT CCGATCAGTG AGAGTCTGAG CATTGAGAAC 988 ACGCTGTGGG CCGGCACCGT CATAGCATCA GGCACTGTTG TAGGCGTTGT TCTCTACACT 1048 GGCAGAAAAC TGCGGAGTGT CATGAATACT TCCGACCCCA GAAGTAAGAT TGGCCTGTTC 1108 GACCTGGAGG TGAACTGCCT CACCAAAATC CTGTT GGTG CGCTGGTGGT GGTGTCCCTG 1168 GTCATGGTGG CCCTGCAGCA CTTTGCCGGC CGCTGGTACC TGCAGATCAT CCGCTTCCTG 1228 CTCCTGTTTT CCAACATCAT TCCTATCAGC TTGCGTGTGA ACTTGGACAT GGGCAAGATC 1288 GTGTACAGCT GGGTGATCCG CAGGGATTCC AAAATCCCCG GGACCGTGGT TCGTTCCAGC 1348 ACAATTCCTG AGCAGCTGGG CAGGATTTCG TACTTGCTCA CAGACAAGAC AGGAACCCTG 1408 ACCCAGAATG AGATGGTGTT CAAGCGGCTG CACCTGGGTA CGGTGGCCTA CGGCCTGGAC 1468 TCCATGGACG AAGTGCAGAG TCACATCTTC AGCATTTACA CCCAGCAATC CCAGGATCCA 1528 CCTGCTCAGA AGGGCCCCAC GGTCACCACC AAGGTCCGGA GGACCATGAG CAGCCGTGTC 1588 CACGAGGCTG TGAAGGCCAT TGCACTCTGC CACAACGTGA CACCCGTGTA CGAGTCCAAT 1648 GGTGTGACGG ACCAGGCTGA GGCTGAGAAG CAGTTTGAGG ACTCCTGCCG AGTGTACCAG 1708 GCATCCAGCC CGGATGAGGT GGCTCTGGTC CAGTGGACAG AAAGTGTGGG ACTGACGCTG 1768 GTGGGTCGAG ACCAGTCCTC CATGCAGCTG AGGACCCCTG GTGACCAGGT CCTGAATCTC 1828 ACCATCCTTC AGGTCTTCCC GTTCACCTAT GAGAGCAAGC GGATGGGCAT CATCGTGCGG 1888 GATGAGTCCA CGGGGGAAAT CACGTTCTAC ATGAAGGGAG CAGACGTCGT CATGGCTGGC 1948 ATTGTCCAGT ACAACGACTG GCTGGAGGAG GAGTGTGGCA ACATGGCCCG GGAGGGACTA 2008 CGTGTGCTGG TGGTAGCCAA GAAGTCCCTC ACAGAGGAGC

(gene length is 3684, only region from 808 to 2047 shown)

Table E3B. Differentially expressed gene fragment from Discovery Study MB .04 from the mouse phospholipid transporting ATPase Class II type 9A

Competitive PCR primer for the mouse phospholipid transporting ATPase Class π type 9A in epididymal fat pad: The sequence of the 145 nucleotide-long gene fragment (from 2707 to 2851 band size: 145) and the gene-specific primers used for competitive PCR are indicated on the cDNA sequence ofthe mouse phospholipid transporting ATPase Class II type 9A and are shown below in bold. The gene-specific primers at the 5' and 3' ends of the fragment are underlined.

Table E3C Gene Sequence (fragment from 2707 to 2851 in bold, band size: 145) (SEQ ID 295)

2226 TGGCATCAAG GTTTGGATGC TAACAGGGGA CAAGCTGGAG ACAGCCACGT GCACAGCCAA 2286 GAACGCACAT CTGGTGACCA GAAACCAAGA TATCCATGTT TTCCGACTGG TGACCAACCG 2346 CGGGGAGGCC CACCTGGAGC TGAATGCCTT CCGTAGGAAG CATGACTGTG CCCTGGTCAT 2406 CTCTGGAGAC TCCCTGGAGG TTTGCCTCAA ATACTATGAG TACGAGTTCA TGGAACTGGC 2466 CTGCCAGTGC CCGGCTGTGG TGTGCTGCCG CTGTGCCCCA ACCCAGAAGG CCCAGATTGT 2526 TCGGCTGCTC CAGGAACGCA CCGGGAAGCT CACCTGTGCA GTAGGGGACG GAGGCAATGA 2586 CGTCAGCATG ATCCAGGAAT CCGACTGCGG CGTGGGCGTG GAGGGCAAGG AAGGGAAGCA 2646 GGCCTCGCTG GCAGCGGACT TCTCCATCAC CCAGTTCAAG CATCTCGGCC GCTTGCTCAT 2706 GGTGCACGGT CGGAACAGCT ACAAGCGCTC GGCGGCCCTC AGTCAGTTTG TGATCCACAG 2766 GAGCCTCTGC ATCAGCACCA TGCAGGCTGT CTTCTCGTCT GTGTTCTACT TTGCATCCGT 2826 TCCTCTCTAC CAAGGCTTCC TGATCATTGG GTATTCTACC ATCTACACGA TGTTTCCCGT 2886 GTTCTCCCTG GTTTTGGACA AAGACGTGAA GTCGGAAGTC GCCATGTTGT ATCCTGAGCT 2946 CTACAAGGAC CTGCTTAAGG GGCGGCCACT GTCCTACAAG ACGTTCTTAA TTTGGGTGTT 3006 AATCAGCATC TATCAAGGGA GCACCATCAT GTACGGGGCG CTGCTGCTGT TCGAGTCGGA 3066 GTTTGTACAC ATCGTGGCAA TCTCCTTCAC ATCCCTCATC CTCACTGAGC TACTGATGGT 3126 GGCGCTCACC ATCCAGACGT GGCACTGGCT CATGACAGTG GCCGAGCTAC TCAGCCTGGC 3186 CTGCTACATT GCCTCCCTGG TGTTCCTCCA TGAGTTCATC GATGTCTACT TCATTGCCAC 3246 CCTGTCATTC CTCTGGAAGG TGTCCGTCAT CACCTTGGTC AGCTGTCTCC CCCTCTATGT 3306 CCTCAAGTAC CTGCGGAGAC GGTTCT

(gene length is 4048, only region from 2226 to 3331 shown) Table E3D. This differentially expressed gene fragment in Discovery Study BP24.02 is from the mouse phospholipid transporting ATPase Class II type 9A.

Competitive PCR primer for the mouse phospholipid transporting ATPase Class II type 9A in retroperitoneal fat pad: The sequence of the 229 nucleotide-long gene fragment (from 2846 to 3074 band size: 229) and the gene-specific primers used for competitive PCR are indicated on the cDNA sequence ofthe mouse phospholipid transporting ATPase Class II type 9A and are shown below in bold. The gene-specific primers at the 5' and 3' ends of the fragment are underlined.

Table E3E. Gene Sequence (fragment from 2846 to 3074 in bold, band size: 229) (SEQ ID 296)

2365 CTGAATGCCT TCCGTAGGAA GCATGACTGT GCCCTGGTCA TCTCTGGAGA CTCCCTGGAG 2425 GTTTGCCTCA AATACTATGA GTACGAGTTC ATGGAACTGG CCTGCCAGTG CCCGGCTGTG 2485 GTGTGCTGCC GCTGTGCCCC AACCCAGAAG GCCCAGATTG TTCGGCTGCT CCAGGAACGC 2545 ACCGGGAAGC TCACCTGTGC AGTAGGGGAC GGAGGCAATG ACGTCAGCAT GATCCAGGAA 2605 TCCGACTGCG GCGTGGGCGT GGAGGGCAAG GAAGGGAAGC AGGCCTCGCT GGCAGCGGAC 2665 TTCTCCATCA CCCAGTTCAA GCATCTCGGC CGCTTGCTCA TGGTGCACGG TCGGAACAGC 2725 TACAAGCGCT CGGCGGCCCT CAGTCAGTTT GTGATCCACA GGAGCCTCTG CATCAGCACC 2785 ATGCAGGCTG TCTTCTCGTC TGTGTTCTAC TTTGCATCCG TTCCTCTCTA CCAAGGCTTC 2845 CTGATCATTG GGTATTCTAC CATCTACACG ATGTTTCCCG TGTTCTCCCT GGTTTTGGAC 2905 AAAGACGTGA AGTCGGAAGT CGCCATGTTG TATCCTGAGC TCTACAAGGA CCTGCTTAAG 2965 GGGCGGCCAC TGTCCTACAA GACGTTCTTA ATTTGGGTGT TAATCAGCAT CTATCAAGGG 3025 AGCACCATCA TGTACGGGGC GCTGCTGCTG TTCGAGTCGG AGTTTGTACA CATCGTGGCA 3085 ATCTCCTTCA CATCCCTCAT CCTCACTGAG CTACTGATGG TGGCGCTCAC CATCCAGACG 3145 TGGCACTGGC TCATGACAGT GGCCGAGCTA CTCAGCCTGG CCTGCTACAT TGCCTCCCTG 3205 GTGTTCCTCC ATGAGTTCAT CGATGTCTAC TTCATTGCCA CCCTGTCATT CCTCTGGAAG 3265 GTGTCCGTCA TCACCTTGGT CAGCTGTCTC CCCCTCTATG TCCTCAAGTA CCTGCGGAGA 3325 CGGTTCTCCC CACCCAGCTA CTCGAAGCTC ACTTCCTAAA GCTGCAGGGC TGCCTCGGGC 3385 AGGGCCTCCG GCCTCCGGCG CTCTCCCAGG AGGAGGTCAA GTTCCACACG CACGAGCCGC 3445 CTCAGCTGGA CGGTGCAGTC ATGGCTGGCA CATGAGGCTT CGCTGAGGCG ACACTGGGCA 3505 CCTAATGGGG ATGGAACATT GGTGGAACCG GAGGGAGGGG CCTGAGAGCT

(gene length is 4048, only region from 2365 to 3554 shown)

Table E3F. This differentially expressed gene fragment in Discovery Study BP24.02 is from the mouse phospholipid transporting ATPase Class II type 9A.

Example E4: Method of Treating Cancer associated with NOV31 b, CG50595-02,

Five oligonucleotides were designed and synthesized as mixed-backbone oligonucleotides containing modified phosphorothioate segments at 5' and 3' ends and 2'-O-methyl RNA oligoribonucleotide segments located in the middle. The purity ofthe oligonucleotides was confirmed by Masspectrophometry. The oligonucleotide sequences for CG50595-02 are:

AS1: 5'-CAGCAGGAGCAACATGGGAC-3' (complementary to the sequence surrounding ATG starting codon) SEQ ID NO:297; AS2: 5'-CAAAGAGGAAGCCTGAGAGG-3' (complementary to the sequence 3' next to AS2) SEQ ID NO:298;

AS3: 5'-CCAAGTGGAGGTGCAGTGGA-3' (complementary to the sequence 3' next to AS3) SEQ ID NO: 299;

AS4: 5'-GAGGTGGCAGGCAGGCTAAG-3' (complementary to the sequence in the middle of SLPI ORF) SEQ ID NO: 300;

AS5: 5'-CTACAACTGCTC AGCACCAG-3' (complementary to the sequence flanking the 3' stop codon) SEQ ID NO:301;

10,000 cells were seeded in each well of a 96 well plate in complete medium 24 hr before transfection. Cells were cultured to reach 50% confluency on the day of transfection. Oligonucleotides were diluted with Optimen to 50, 100, 200 and 400 nM, and mixed with Oligofectamine (Invitrogen) according to manufacture's instructions. Cells were washed with serum-free medium. Cells were transfected with CG50595-02 antisense oligonucleotides mixtures 1) AS1, AS2 and AS3 or 2) AS1, AS2, AS3, AS4 and AS5 or irrelevant oligonucleotide, lipisome controls at 50, 100, 200 or 400nM. 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 in a proliferation assay performed 24 and 48 hr after transfection. Briefly, 20 DI of combined MTS/PMS solution were 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.

As shown in Table E4A, transfection of NOV31b (CG50595-02) antisense oligonucleotides inhibited SW620 cell growth more than 50%, whereas transfection of scramble antisense control had little effect on cell growth. The same growth inhibitory effects were also observed in LX-1 cancer cells (Table E4A). Whereas we observed almost no effect with NOV31b (CG50595-02) antisense oligo transfection in SW480 cells (in which the expression of NOV31b (CG50595-02) is much lower) (Table E4B) and NCI-H460 cells (in which there is no expression of CG50595-02) (Table E4B). Knockdown of NOV31b (CG50595-02) gene by antisense oligo at the transcriptional level was verified by RTQ-PCR (Table E4C). Comparing to scramble control, up to 80% of NOV31b (CG50595-02) gene expression was wiped out at the mRNA level by gene-specific antisense oligo.

Results show that NOV31b (CG50595-02) expression is involved in cancer cell proliferation. The inhibition of the activity of this gene through the use of antibodies or small molecule drugs, specifically antisense oligonucleotides, will be of use in the treatment of ovarian, thyroid, lung liver, kidney and melanoma cancers.

TABLE E4A Antisense Knockdown Of CG50595-02 Inhibits SW620 (A) And LX-1 (B) Cell Growth.

Antisense Knockdown of CG50595-02 Inhibits SW620 Colon Carcinoma Cell Growth

AS1,2,3 AS1 ,2,3,4,5 scramble lipisome untreated control control control

Antisense Knockdown of CG50595-02 Inhibits LX-1 Lung Carcinoma Cell Growth

AS1,2,3 AS1 ,2,3,4,5 scramble lipisome untreated control control control

Table E4B. Antisense knockdown of CG50595-02 has little effect on SW480 (A) or NCI-H460 (B) cell growth.

Antisense Knockdown of CG50595-02 Has Little Effect on SW480 Colon Carcinoma Cell Growth

AS1,2,3 AS1 ,2,3,4,5 scramble vehicle untreated control control control

Antisense Knockdown of CG50595-02 Has No Effect on Null Cell Type NCI-H460 Growth

AS1,2,3 AS1, 2,3,4,5 scramble vehicle untreated control control control

Table E4C. RTQ PCR analysis indicates antisense oligonuclotides effectively knockdown of CG50595-02 expression at mRNA level in SW620 (A) and LX-1 (B) cancer cell lines.

Example E5: NOV30a, b, c, CG50303.

The invention relates to an isolated nucleic acid molecule encoding a Novel GPCR-like protein NOV30c, CG50303-03 having a nucleotide polymorphism SNPl 3373946 where the T allele is indicative of an increased risk of an electrocardiographic ST segment, and therefore an increased risk for myocardial infarction and resultant complications in the cardiovascular and other organ systems. The invention also relates to a method for identifying individuals who are carriers of the genetic risk-altering factor or have an altered risk of the specified disease processes or related processes.. The method includes obtaining a biological sample from an individual and testing the individual for the nucleotide polymorphism, wherein the disease risk may increase with the dose of the T allele.

The gene for the Novel GPCR-like protein NOV30c (CG50303-03) is a gene encoding a protein.

Myocardial infarction is a common genetically complex trait in which the disease prevalence and progression are the product of environment and gene interaction. Electrocardiographic findings of an increased risk of an ST-segment indicates that the artery to an area of the heart is blocked, and that the full thickness of the heart muscle is damaged. Coronary arteries may gradually become partly obstructed by plaques in the chronic process of atherosclerosis. This condition produces ischemia when, even though the myocardial blood supply is sufficient at a resting workload, it becomes insufficient when the workload is increased by either emotional or physical stress. Partially obstructed atherosclerotic coronary arteries may suddenly become completely obstructed. Ischemia develops immediately unless the resting metabolic demands of the affected myocardial cells can be satisfied by any collateral blood flow. If the obstruction is relieved before the glycogen reserve of the affected cells is severely depleted, the cells promptly resume their contraction. However, if the acute, complete obstruction continues until the myocardial cells' glycogen is severely depleted, they become stunned. Even after blood flow is restored, these cells are unable to resume contraction until they have repleted their glycogen reserves. If the complete obstruction further persists until the myocardial cells' glycogen is entirely depleted, the cells are unable to sustain themselves, are irreversibly damaged, and become necrotic. This clinical process is termed a heart attack or myocardial infarction (MI).

The ECG changes caused by a potentially reversible decrease in coronary blood flow are typically termed "injury" when the level ofthe ST-segment baseline is deviated from the level of the TP and PR segment baseline. Shifting of the ST segment baseline occurs when insufficient perfusion causes the myocardial-cell membranes to become abnormally permeable to the flow of ions. The resulting difference in electrical potential between injured and uninjured myocardium causes a constant flow of injury current. In most cases patients go on to develop a full-blown heart attack, medically referred to as a Q-wave myocardial infarction. ST-elevations are good indicators for aggressive treatments (thrombolytic drugs or angioplasty) to reopen blood vessels. In a some cases, however, the patient's status drops to a non-Q-wave myocardial infarction, a less serious condition. Non-elevated ST segments indicate a normal heart beat.

The invention relates to the identification of a polymorphism in the gene encoding A Novel GPCR-like protein NOV30c (CG50303-03) in humans, particularly of Caucasian ethnicity, and a method for identifying individuals who are carriers of the genetic risk-altering factor for an elevated electrocardiographic ST segment, and therefore an increased risk for myocardial infarction and resultant cardiovascular complications, by detecting the presence of the polymorphism.

The invention has enabled a DNA-based diagnostic test for an an increased risk of an electrocardiographic ST segment, and therefore an increased risk for myocardial infarction and resultant cardiovascular complications, to be developed, and, therefore, a technique for identifying individuals for more frequent monitoring and earlier or more aggressive intervention.

The use of NOV 30c (CG50303-03), SNP13373946 or a haplotype containing SNPl 3373946 as a target for pharmaceutical intervention in elevated electrocardiographic ST segment and myocardial infarction, and as an aid in the design, testing, or evaluation of such pharmaceutical compounds.

On the genetic basis of complex traits: D. S. Falconer and T. F. C. Mackay, Introduction to quantitative genetics, 4^th edition, Prentice Hall, New York, 1996. On statistical methods: G. W. Snedecor and W. G. Cochran, Statistical Methods, 8^th edition, Iowa State University Press, Ames, Iowa, 1989.

On computational methods: W. H. Press, S. A. Teukolsky, W. T. Vetterling, and B. P. Flannery, Numerical Recipes in C, the Art of Scientific Computing, 2^nd edition, Cambridge University Press, New York, 1997.

On electrocardiographic ST segment elevation and its effects: G.Wagner, Marriott's Practical Electrocardiography 10^th ed., Lippincott Williams & Wilkins, Philadelphia, 2001.

On the genetics of myocardial infarction: M.S. Williams and P.F. Bray. Genetics of arterial prothrombotic risk states. Experimental Biology & Medicine, 226(5):409-19, 2001. B.R. Winkelmann, W. Marz, B.O. Boehm, et al, LURIC Study Group (LUdwigshafen Risk and Cardiovascular Health). Rationale and design of the LURIC study—a resource for functional genomics, pharmacogenomics and long-term prognosis of cardiovascular disease. Pharmacogenomics. 2(1 Suppl l):Sl-73, 2001.

On the population sample used for genotypic analysis: Hedstrand H. A study of middle-aged men with particular reference to risk factors for carrdiovascular disease. Upsala J Med Sci Suppl. 1975; 19.

The invention relates to the identification of a polymorphism in the gene encoding A Novel GPCR-like protein NOV30c (CG50303-03) in humans, particularly of Caucasian ethnicity, and a method for identifying individuals who have a genetic risk-altering factor an increased risk of an electrocardiographic ST segment, and therefore an increased risk for myocardial infarction and resultant cardiovascular complications, by detecting the presence ofthe polymorphism.

The invention corresponds to identification of SNP13373946 and to fragments of the nucleic acid molecules. The molecules must be long enough to serve in genotyping assays, for example as PCR primers. The nucleic acid molecules need not be identical to the sequence of SEQ ID NO: 131, excluding the polymorphism. Instead, the molecules need to have sufficient identity to the remainder of SEQ ID NO: 131 that the nucleic acid molecule may be used to differentiate between the presence or absence of SNP13373946. The invention also relates to a method for identifying individuals, particularly of Caucasian ethnicity, who have a genetic risk-altering factor an increased risk of an electrocardiographic ST segment, and therefore an increased risk for myocardial infarction and resultant cardiovascular complications, caused by presence of the T allele variant in their genome. The method includes obtaining a biological sample from an individual and testing the sample for T allele, wherein the allele dose correlates with increased disease risk.

Please see above Example B for a description of SeqCallingTM Technology.

Variant sequences are 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 ofthe amino acid encoded by the gene at the position of the SNP. Intragenic SNPs may also be silent, however, in the case that 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 ofthe expression pattern for example, alteration in temporal expression, physiological response regulation, cell type expression regulation, intensity of expression, stability of transcribed message.

Method of novel SNP Identification: SNPs are identified by analyzing SeqCalling sequence assemblies using CuraGen's proprietary SNPTool algorithm. SNPTool identifies variation in assemblies with the following criteria: SNPs are not analyzed within 10 base pairs on both ends of an alignment; Window size (number of bases in a view) is 10; The allowed number of mismatches in a window is 2; Minimum SNP base quality (PHRED score) is 23; Minimum number of changes to score an SNP is 2/assembly position. SNPTool analyzes the assembly and displays SNP positions, associated individual variant sequences in the assembly, the depth of the assembly at that given position, the putative assembly allele frequency, and the SNP sequence variation. Sequence traces are then selected and brought into view for manual validation. The consensus assembly sequence is imported into CuraTools along with variant sequence changes to identify potential amino acid changes resulting from the SNP sequence variation. Comprehensive SNP data analysis is then exported into the SNPCalling database.

Method of novel SNP Confirmation: SNPs are confirmed employing one of several validated methods, for instance Pyrosequencing (Pyrosequencing, Sweden), MassArray (Sequenom, San Diego, USA), or BeadArray (Illumina, San Diego, USA).

Results

The DNA and amino acid sequences for A Novel GPCR-like protein NOV30c (CG50303-03), and a variant polymorphic form SNPl 3373946, have been identified in humans. The reference DNA sequence for A Novel GPCR-like protein NOV30c (CG50303-03), which encodes PROTEIN- 1, is provided as SEQ ID NO: 131 above.

The polymorphism SNP13373946 is a change in the DNA sequence, a variant of SEQID 131. Nucleotide 126 is changed from C to T. This polymorphism lies within the amino acid coding sequence and alters the amino acid sequence.

The polymorphism SNPl 3373946 codes for a change at amino acid position 36 in SEQ ID 132 from a Proline to a Serine.

Genotype results for SNP13373946. homozygous major allele C/C 620 heterozygous C/T 25 homozygous minor allele T/T 0 Population, clinical measurements, and genotypes

The population providing evidence for the association between the genetic variants and the disease was comprised of middle-aged Caucasian males who took part in a longitudinal survey to identify risk factors for cardiovascular disease and to select high-risk individuals for preventive treatment. Between 1970 and 1973 all 50-year-old men living in the municipality of Uppsala were invited to participate in a health survey on risk factors for Coronary Heart Disease. Genotyping was performed on 825 subjects who were available for followup at age 70.

Clinical measurements were made, for traits in categories including but not limited to:supine systolic and diastolic blood pressure, fasting blood glucose, blood glucose tolerance test (oral and intravenous challenge), glucose uptake (hyperinsulinemic clamp), body mass index, fasting serum lipids, smoking, body height, physical activity, serum beta carotene, alpha tocopherol, selenium, serum fatty acids in cholesterol esters, serum insulin, serum creatinine, complete 2D & Doppler echocardiographic studies, and 12-lead electocardiographic studies, urinary albumin, medication history, and socioeconomic status.

For traits with quantitative values, each trait was standardized to approximate a univariate standard normal distribution. For most traits, this involved calculating the trait mean and standard deviation, then subtracting the mean for each trait score and dividing by the standard deviation to yield a trait with zero mean and unit variance. For some traits, the distribution appeared log-normal, and a log transform was applied prior to the standardization.

Genotypes were measured for each marker for at least 70% of the individuals with a discrepancy rate of 4% or less. Genotyping discrepancies do not increase the false-positive rate of a test, although they do increase the false-negative rate.

Statistical analysis for each marker/trait combination

Data collection

An individual was defined as informative if both the trait value and genotype were available. The markers we tested were all bi-allelic. The frequency of the minor allele, termed T, is denoted p, and the frequency of the major allele, termed allele B, is denoted q and equals 1-p.

Hardy- Weinberg tests

Hardy- Weinberg equilibrium (HWE) relates genotype frequencies to allele frequencies under general assumptions of an equilibrium population. Violations of HWE may indicate selection against the minor allele and population stratification.

Selection against the minor allele occurs when the minor allele detracts from evolutionary fitness and may result in having fewer homozygotes than would be expected by chance.

Population stratification arises when the population being studies is actually a mix of sub-populations with different frequencies of allele T. Stratification results in having more homozygotes than would be expected by chance. Stratification may increase the false-positive and false-negative rates for between-family tests but does not affect within-family tests (see below). Thus, if stratification is indicated, it is preferable to perform only within-family tests.

To perform Hardy- Weinberg tests, the counts of individuals with AA, AB, and BB genotypes in this population were termed N(AA), N(AB), and N(BB), respectively, and the allele frequency p was calculated as p = [N(AA) + 0.5 N(BB)]/N.

Next, the counts of individuals expected for each genotype under the null hypothesis of HWE were calculated as n(AA) = p²N n(AB) = 2pqN n(BB) = q²N Finally, two test statistics were calculated:

HW1 = [N(AA)-n(AA)]²/n(AA) + [N(AB)-n(AB)]²/n(AB) + [N(BB)-n(BB)]²/n(BB) HW2 = {[N(AA)+N(BB)]-[n(AA)+n(BB)]}²/{n(AA) +n(BB)} + [N(AB)- n(AB)]²/n(AB) Under the null hypothesis, both HW1 and HW2 follow χ² distributions with 1 degree of freedom. The critical values of χ² for p-values of 0.05 and 0.01 are 3.84 and 6.63 respectively. Values of χ larger than these indicate a 5% chance or a 1% chance of the HW assumptions being satisfied.

The HW1 test is the standard test, but it is not accurate when the smallest category, typically N(AA), has fewer than 5 individuals. The HW2 test is more robust but can be less sensitive for rare alleles.

If there is significant deviation from HWE, the sign of [N(AA)+N(BB)]- [n(AA)+n(BB)] indicates the reason: positive values indicate stratification and negative values indicate selection against the minor allele.

Association tests

Association tests were based on a genetic model for the marker as a quantitative trait locus (QTL),

X_fi = Y_f + Y_fi + m(G_fi) where Xg is the phenotypic value of individual i in family f, Y_f represents the contribution to Xβ from shared genetic and environmental effects excluding effects from the QTL , Y_f, represents the non-shared contributions excluding the QTL, and m(G_fi) represents the mean effect from the QTL and depends only on the genotype G_fl5 with m(AA) = a - c m(AB) = d - c m(BB) = - a- c, where the constant c is defined as (ρ-q)a + 2pqd.

Instead of testing for the significance of both a and d, we focused on just the additive contribution from the allele to the phenotype by testing the significance ofthe regression coefficient b in the model

Xi = Yi+ a + bpi where Xj is the phenotypic value for sample i, Yj represents the contributions to the phenotype excluding the QTL for sample i, and pi is the allele frequency for sample i.

Since pi takes a discrete number of values, the tests were performed by calculating the mean and standard error of Xi for each value of pi, then performing a regression test of the binned values to obtain b and its sampling standard deviation s. Under the null hypothesis of no association, b/s follows a standard normal distribution. The p-value for a significant association was calculated from a two-sided test of b/s.

A total of 6 tests of this nature were performed:

Unrelated Xj, and pi are from unrelated individuals.

Mean Each DZ pair yields a single sample, with Xj and pi equal to the mean phenotypic value and allele frequency of pair i.

Difference Each DZ pair yields a single sample, with Xi and pi equal to the difference in phenotypic value and allele frequency between the first and second sib. This test is robust to stratification.

Non-parametric difference Each DZ pair yields a single sample, with pi equal to the difference in allele frequency between the first and second sib, and Xi equal to 1, 0, or-1 if the phenotypic value ofthe first sib is greater than, equal to, or less than that ofthe second sib. This test is like a transmission disequilibrium test (TDT). Like the difference test, it is robust to stratification; it is also robust to non-normality and outliers, but is less sensitive to small effects than the difference test.

Total The total test combines the estimates of b from the unrelated, mean, and difference tests, which are statistically independent. A minimum variance estimator of b is built by weighting each of the three tests by the inverse of their sampling variance, and the variance of the combined estimator is the inverse of the sum of the inverse variances of the independent estimates. This test is more sensitive than either ofthe three independent tests in the absence of stratification, but is not as robust as the difference or non-parametric difference test in the presence of stratification. Stratification The test statistic for the stratification test is the square ofthe difference of the estimates of b from the mean and difference tests, normalized by the sum of the variances of the two estimators, follows a χ distribution with 1 degree of freedom. Large values of the test statistic indicate population stratification and that only the difference test and non-parametric difference test may be robust.

For the mean, difference, and total tests, the term b is related to the parameters of the genetic model as b = 2[a-(p-q)d].

The effect size was reported as the quantity a assuming additive inheritance (d = 0), then taking the ratio of a to the standard deviation of the trait value.

We also applied a multiple testing correction by requiring a p-value of less than approximately 10 for a significant test. The roughly 100 phenotypes tests correspond to approximately 20 independent tests because many of the phenotypes are correlated; this threshold corresponds to an approximate false-positive rate of 2% per marker tested.

Software

We wrote a computer program GOUDA to perform the statistical analysis. A copy ofthe program is provided, and the example contains program output.

Example output for analysis of SNP13373946

1337946 with Z732 (ST segment elevation)

Contingency table:

Genotype: C/C C/T no ST elevation: 618 23 ST elevation: 2 2

Chi-square using correction for continuity:

645(1190 - 322.5)^Λ2 / 641 x 4 x 620 x 25 = 12.2

P-value from two-sided chi-square, corrected for continuity: 0.00048

P-value from Fisher exact test, two-sided: 0.0017 Defined terms

Defined terms

OTHER EMBODIMENTS

Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims, which follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope ofthe invention as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge 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

What is claimed is:

1. An isolated polypeptide comprising the mature form of an amino acid sequenced selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 79.

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 79.

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 79.

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 79.

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 ofthe 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 79 or a biologically active fragment thereof.

20. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79.

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

22. A nucleic acid molecule, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 79.

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 79.

24. An isolated nucleic acid molecule comprising a nucleic acid selected from the group consisting of 2n-l, wherein n is an integer between 1 and 79.

25. The nucleic acid molecule of claim 20, wherein said nucleic acid molecule hybridizes under stringent conditions to the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-l, wherein n is an integer between 1 and 79, 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, wherem the antibody is a humanized antibody.

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

(a) providing said sample;

(b) introducing said sample to a probe that binds to said nucleic acid molecule; and (c) determining the presence or amount of said probe bound to said nucleic acid molecule, thereby determining the presence or amount ofthe nucleic acid molecule in said sample.

33. The method of claim 32 wherein presence or amount 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 ofthe nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.

36. A method of producing the polypeptide of claim 1 , the method comprising culturing a cell under conditions that lead to expression ofthe polypeptide, wherein said cell comprises a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2n-l, wherein n is an integer between 1 and 79.

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

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

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

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

41. A method of producing the polypeptide of claim 2, the method comprising culturing a cell under conditions that lead to expression ofthe polypeptide, wherein said cell comprises a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2n-l, wherem n is an integer between 1 and 79.

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

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

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

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

46. A method for identifying individuals who are carriers of the genetic risk-altering factor for an increased risk of an electrocardiographic ST segment, and therefore have an increased risk for myocardial infarction and resultant cardiovascular complications, comprising (i) obtaining a biological sample from an individual (ii) testing the sample for the present of the polymorphism SNP13373946 (iii) where the copy number of T allele is indicative of disease, disease risk, and carrier status.

47. The method of claim 46 where the test is conducted by various genotyping methods including but not limited to: oligonucleotide ligation, direct sequencing, mass spec, real time kinetic polymerase chain reaction (PCR), hybridization, pyrosequencing, fragment polymorphisms, hybridization, and fluorescence depolarization.

48. The method of claim 48 where the nucleic acid is genomic DNA or cDNA or mRNA.

49. The method of claim 46 where the biological sample is any tissue containing genomic DNA, including but not limited to: blood, hair, cheek scraping, saliva, biopsy, and semen.

50. The method of claim 46 in which other nucleotide markers that are correlated with SNPl 3373946, are used to indicate disease risk.

51. The method of claim 46 in which a multiple locus test corresponding to a haplotype containing the T allele of SNP13373946 is used to indicate disease risk.

52. A diagnostic kit for determining whether an individual is a carrier of the genetic risk-altering factor for an increased risk of an electrocardiographic ST segment, and therefore has an increased risk for myocardial infarction and resultant cardiovascular complications, comprising reagents that will determine the variant of NOV30c or its associated linkage disequilibrium group or haplotype in the individual's genome.