CA2485089A1 - Therapeutic polypeptides, nucleic acids encoding same, and methods of use - Google Patents

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 molecule s 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

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THERAPEUTIC POLYPEPTIDES, NUCLEIC ACIDS ENCODING
SAME, AND METHODS OF USE
FIELD OF THE INVENTION
The present invention relates to both novel polypeptides, and the nucleic acids encoding them as well as polypeptides that are targets of small molecule drugs. Those polypeptides 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 OF THE INVENTION
Eukaryotic cells are characterized by biochemical and physiological processes which under normal conditions are exquisitely balanced to achieve the preservation and propagation of the cells. When such cells are components of multicellular organisms such as vertebrates, or more particularly organisms such as mammals, the regulation of the biochemical and physiological processes involves intricate signaling pathways. Frequently, such signaling pathways involve extracellular signaling proteins, cellular receptors that bind the signaling proteins, and signal transducing components located within the cells.
Signaling proteins may be classified as endocrine efFectors, paracrine effectors or autocrine effectors. Endocrine effectors are signaling molecules secreted by a given organ into the circulatory system, which are then transported to a distant target organ or tissue. The target cells include the receptors for the endocrine effector, and when the endocrine effector binds, a signaling cascade is induced. Paracrine effectors involve secreting cells and receptor cells in close proximity to each other, for example two different classes of cells in the same tissue or organ. One class of cells secretes the paracrine effector, which then reaches the second class of cells, for example by diffusion through the extracellular fluid. The second class of cells contains the receptors for the paracrine effector; binding of the effector results in induction of the signaling cascade that elicits the corresponding biochemical or physiological effect. Autocrine effectors are highly analogous to paracrine effectors, except that the same cell type that secretes the autocrine effector also contains the receptor. Thus the autocrine effector binds to receptors on the same cell, or on identical neighboring cells. The binding process then elicits the characteristic biochemical or physiological effect.
Signaling processes may elicit a variety of effects on cells and tissues including by way of nonlimiting example induction of cell or tissue proliferation, suppression of growth or proliferation, induction of differentiation or maturation of a cell or tissue, and suppression of differentiation or maturation of a cell or tissue.
Many pathological conditions involve dysregulation of expression of important effector proteins. In certain classes of pathologies the dysregulation is manifested as diminished or suppressed level of synthesis and secretion of protein effectors. In other classes of pathologies the dysregulation is manifested as increased or up-regulated level of synthesis and secretion of protein effectors. In a clinical setting a subject may be suspected of suffering from a condition brought on by altered or mis-regulated levels of a protein effector of interest. Therefore there is a need to assay for the level of the protein effector of interest in a biological sample from such a subject, and to compare the level with that characteristic of a nonpathological condition. There also is a need to provide the protein effector as a product of manufacture. Administration of the effector to a subject in need thereof is useful in treatment of the pathological condition. Accordingly, there is a need for a method of treatment of a pathological condition brought on by a diminished or suppressed levels of the protein effector of interest. Tn 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 taxget 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 fox 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 of the 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.

Antibodies are multichain proteins that bind specifically to a given antigen, and bind poorly, or not at all, to substances deemed not to be cognate antigens.
Antibodies are comprised of two short chains termed light chains and two long chains termed heavy chains.
These chains are constituted of immunoglobulin domains, of which generally there are two classes: one variable domain per chain, one constant domain in light chains, and three or more constant domains in heavy chains. The antigen-specific portion of the immunoglobulin molecules resides in the variable domains; the variable domains of one light chain and one heavy chain associate with each other to generate the antigen-binding moiety.
Antibodies that bind immunospecifically to a cognate or target antigen bind with high affinities.
Accordingly, they are useful in assaying specifically for the presence of the antigen in a sample. In addition, they have the potential of inactivating the activity of the antigen.
Therefore there is a need to assay for the level of a protein effector of interest in a biological sample from such a subject, and to compare this level with that characteristic of a nonpathological condition. In particular, there is a need for such an assay based on the use of an antibody that binds immunospecifically to the antigen. There further is a need to inhibit the activity of the protein effector in cases where a pathological condition arises from elevated or excessive levels of the effector based on the use of an antibody that binds immunospecifically to the effector. Thus, there is a need for the antibody as a product of manufacture. There further is a need for a method of treatment of a pathological condition brought on by an elevated or excessive level of the protein effector of interest based on administering the antibody to the subject.
SUMMARY OF THE INVENTION
The invention is based in part upon the discovery of isolated polypeptides including amino acid sequences selected from mature forms of the amino acid sequences selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between l and 174. The novel nucleic acids and polypeptides are referred to herein as NOV 1 a, NOV 1 b, NOV
1 c, NOV 1 d, NOV2a, NOV2b, NOV2c, NOV2d, NOV3a, NOV3b, etc. These nucleic acids and polypeptides, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as "NOVX" nucleic acid or polypeptide sequences.
The invention also is based in part upon variants of a mature form of the amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 174, wherein any amino acid in the mature form is changed to a different amino acid, provided that no more than 15°~0 of the amino acid residues in the sequence of the mature form are so changed. In another embodiment, the invention includes the amino acid sequences selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 174. In another embodiment, the invention also comprises variants of the amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between l and 174 wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed. The invention also involves fragments of any of the mature forms of the amino acid sequences selected from the group consisting of SEQ ID
N0:2n, wherein n is an integer between l and 174, or any other amino acid sequence selected from this group. The invention also comprises fragments from these groups in which up to 15% of the residues are changed.
In another embodiment, the invention encompasses polypeptides that are naturally occurring allelic vaxiants of the sequence selected from the group consisting of SEQ ID
N0:2n, wherein n is an integer between l and 174. These allelic vaxiants include amino acid sequences that are the translations of nucleic acid sequences differing by a single nucleotide from nucleic acid sequences selected from the group consisting of SEQ ID NOS:
2n-l, wherein n is an integer between l and 174. The variant polypeptide where any amino acid changed in the chosen sequence is changed to provide a conservative substitution.
In another embodiment, the invention comprises a pharmaceutical composition involving a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between l and 174 and a pharmaceutically acceptable carrier. In another embodiment, the invention involves a kit, including, in one or more containers, this pharmaceutical composition.
In another embodiment, the invention includes the use of a therapeutic in the manufacture of a medicament for treating a syndrome associated with a human disease, the disease being selected from a pathology associated with a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 174 wherein said therapeutic is the polypeptide selected from this group.
In another embodiment, the invention comprises a method for determining the presence or amount of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 174 in a sample, the method involving providing the sample; introducing the sample to an antibody that binds immunospecifically to the polypeptide; and determining the presence or amount of antibody bound to the polypeptide, thereby determining the presence or amount of polypeptide in the sample.
In another embodiment, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between l and 174 in a first mammalian subject, the method involving measuring the level of expression of~the polypeptide in a sample from the first mammalian subject;
and comparing the amount of the polypeptide in this sample to the amount of the polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, the disease, wherein an alteration in the expression level of the polypeptide in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.
In another embodiment, the invention involves a method of identifying an agent that binds to a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 174, the method including introducing the polypeptide to the agent; and determining whether the agent binds to the polypeptide. The agent could be a cellular receptor or a downstream effector.
In another embodiment, the invention involves a method for identifying a potential therapeutic agent for use in treatment of a pathology, wherein the pathology is related to aberrant expression or aberrant physiological interactions of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 174, the method including providing a cell expressing the polypeptide of the invention and having a property or function ascribable to the polypeptide;
contacting the cell with a composition comprising a candidate substance; and determining whether the substance alters the property or function ascribable to the polypeptide; whereby, if an alteration observed in the presence of the substance is not observed when the cell is contacted with a composition devoid of the substance, the substance is identified as a potential therapeutic agent.
In another embodiment, the invention involves a method for screening for a modulator of activity or of latency or predisposition to a pathology associated with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
N0:2n, wherein n is an integer between l and 174, the method including administering a test compound to a test animal at increased risk for a pathology associated with the polypeptide of the invention, wherein the test animal recombinantly expresses the polypeptide of the invention; measuring the activity of the polypeptide in the test animal after administering the test compound; and comparing the activity of the protein in the test animal with the activity of the polypeptide in a control animal not administered the polypeptide, wherein a change in the activity of the polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency of, or predisposition to, a pathology associated with the polypeptide of the invention. The recombinant test animal could express a test protein transgene or express the transgene under the control of a promoter at an increased level relative to a wild-type test animal The promoter may or may not b the native gene promoter of the transgene.
In another embodiment, the invention involves a method for modulating the activity of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID
NO:2n, wherein n is an integer between l and 174, the method including introducing a cell sample expressing the polypeptide with a compound that binds to the polypeptide in an amount sufficient to modulate the activity of the polypeptide.
In another embodiment, the invention involves a method of treating or preventing a pathology associated with a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 174, the method including administering the polypeptide to a subject in which such treatment or prevention is desired in an amount sufficient to treat or prevent the pathology in the subject. The subject could be human.
In another embodiment, the invention involves a method of treating a pathological state in a mammal, the method including administering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95% identical to a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 174 or a biologically active fragment thereof.
In another embodiment, the invention involves an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ
ID N0:2n, wherein n is an integer between l and 174; 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 174 wherein any amino acid in the mature form of the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence of the mature form are so changed; the amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between l and 174; a variant of the amino acid sequence selected from the group consisting of SEQ
ID N0:2n, wherein n is an integer between 1 and 174, in which any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15%
of the amino acid residues in the sequence are so changed; a nucleic acid fragment encoding at Ieast a portion of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between l and 174 or any variant of the polypeptide wherein any amino acid of the chosen sequence is changed to a different amino acid, provided that no more than 10% of the amino acid residues in the sequence are so changed; and the complement of any of the nucleic acid molecules.
In another embodiment, the invention comprises an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ
ID N0:2n, wherein n is an integer between 1 and 174, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally occurring allelic nucleic acid variant.
In another embodiment,'~the invention involves an isolated nucleic acid molecule including a nucleic acid sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ
ID N0:2n, wherein n is an integer between 1 and 174 that encodes a variant polypeptide, wherein the variant polypeptide has the polypeptide sequence of a naturally occurring polypeptide variant.
In another embodiment, the invention comprises an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ
ID N0:2n, wherein n is an integer between 1 and 174, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 2n-l, wherein n is an integer between 1 and 174.
In another embodiment, the invention includes an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ
ID NO:2n, wherein n is an integer between 1 and 174, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence selected from the group consisting of SEQ ID N0:2n-1, wherein n is an integer between 1 and 174; a nucleotide sequence wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID N0:2n-1, wherein n is an integer between 1 and 174 is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed; a nucleic acid fragment of the sequence selected from the group consisting of SEQ
ID N0:2n-1, wherein n is an integer between 1 and 174; and a nucleic acid fragment wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID N0:2n-l, wherein n is an integer between 1 and 174 is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed.
In another embodiment, the invention includes an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ
ID N0:2n, wherein n is an integer between 1 and 174, wherein the nucleic acid molecule hybridizes under stringent conditions to the nucleotide sequence selected from the group consisting of SEQ ID N0:2n-1, wherein n is an integer between 1 and 174, or a complement of the nucleotide sequence.
In another embodiment, the invention includes an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ
ID N0:2n, wherein n is an integer between 1 and 174, wherein the nucleic acid molecule has a nucleotide sequence in which any nucleotide specified in the coding sequence of the chosen nucleotide sequence is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides in the chosen coding sequence are so changed, an isolated second polynucleotide that is a complement of the first polynucleotide, or a fragment of any of them.
In another embodiment, the invention includes a vector involving the nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID N0:2n, wherein n is an integer between l and 174. This vector can have a promoter operably linked to the nucleic acid molecule. This vector can be located within a cell.
In another embodiment, the invention involves a method fox determining the presence or amount of a nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID N0:2n, wherein n is an integer between 1 and 174 in a sample, the method including providing the sample; introducing the sample to a probe that binds to the nucleic acid molecule; and determining the presence or amount of the probe bound to the nucleic acid molecule, thereby determining the presence or amount of the nucleic acid molecule in the sample. The presence or amount of the nucleic acid molecule is used as a marker for cell or tissue type. The cell type can be cancerous.
In another embodiment, the invention involves a method for determining the presence of or predisposition for a disease associated with altered levels of a nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ
ID N0:2n, wherein n is an integer between 1 and 174 in a first mammalian subject, the method including measuring the amount of the nucleic acid in a sample from the first mammalian subject; and comparing the amount of the nucleic acid in the sample of step (a) to the amount of the nucleic acid present in a control sample from a second mammalian subject known not to have or not be predisposed to, the disease; wherein an alteration in the level of the nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.
The invention further provides an antibody that binds immunospecifically to a NOVX
polypeptide. The NOVX antibody may be monoclonal, humanized, or a fully human antibody. Preferably, the antibody has a dissociation constant for the binding of the NOVX
polypeptide to the antibody Less than 1 ac 10-9 M. More preferably, the NOVX
antibody neutralizes the activity of the NOVX polypeptide.
In a further aspect, the invention provides for the use of a therapeutic in the manufacture of a medicament for treating a syndrome associated with a human disease, associated with a NOVX polypeptide. Preferably the therapeutic is a NOVX
antibody.
In yet a further aspect, the invention provides a method of txeating or preventing a NOVX-associated disorder, a method of treating a pathological state in a mammal, and a method of treating or preventing a pathology associated with a polypeptide by administering a NOVX antibody to a subject in an amount sufficient to treat or prevent the disorder.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a bar diagram showing the activation of 786-0 epithelial cell BrdU
incorporation by CG51051-06 protein.
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.

NOV3b CG110590-O1 ' 17 18 Neuralin precursor (Ventroptin) -Homo ... . ...... .. . . saPiens..... . _......

NOV3c 13382325 19 20 Neuralin precursor (Ventroptin) - Homo '~ . __.- ..... . . ... .~ . .. ~Sal?iensV.__._~_.~_-,__- .
... . ~ . . . ......, NOV3d 13382326 21 22 Neuralin precursor (Ventroptin) - Homo sapiens NOV4a ' CG114555-O1 ' 23 ' 24 Solute carrier family 2, facilitated glucose transporter, member 9 (Glucose transporter type 9) - Homo sapiens NOV4b '247847074 25 26 Solute carrier family 2, facilitated glucose transporter, member 9 (Glucose _ transporter type 9) - Homo Sapiens ~
~
~

NOV4c ;247847070 Solute carrier family 2, facilitated glucose transporter, member 9 (Glucose ~.... ~...~.. ._. . .. .... .. transporter type 9).' Homo Sapiens.
.. .. ~ ..... . ...... . . .. ...

NOV4d 247847055 29 30 Solute carrier family 2, facilitated glucose transporter, member 9 (Glucose _ transporter type 9) Homo sapiens p "
~ ~~~
~

NOV4e Solute carrier family 2, 247847059 facilitated ' 31 glucose transporter, member 9 (Glucose transporter type 9) - Homo sapiens ~

NOV4f 247847047 33 34 Solute carrier family 2, facilitated glucose transporter, member 9 (Glucose _ _ transporter type 9) - Homo Sapiens ~ T' ~

NOV4g CG114555-02 Solute carrier family 2, facilitated glucose transporter, member 9 (Glucose __.. . .. ...... .. .. ~. . ~'~sporter,type.9). Homo.
Sapiens .. ~..

NOV4h CG114555-03 ' 37 38 Solute carrier family 2, facilitated glucose transporter, member 9 (Glucose .. _ _ . . _ _-.H '. ~.~ .. ~.u ~.. transporter type f9) . .Homo _ _ _ . . _ __ . ~ _ _._ . _._. Sapiens-__. _._ _ . _ .
~ w ._ . _ _ NOV4i 'CG114555-04 39 40 Solute carrier family 2, facilitated glucose transporter, member ~ 9 (Glucose .

. . . __ . ~~... _ . . transporter type 9) Homo Sapiens . .. .
~
~

NOV4j 13379365 41 Solute carrier family 2, facilitated glucose transporter, member 9 (Glucose _ _ transporter type 9) - Homo sapiens ~~ A m~~
Y
E ~
~ ~

NOV4k Solute carrier family 2, facilitated glucose transporter, member 9 (Glucose transporter type 9) - Homo sapiens 9363 olute carrier f NOV41 1337 ~ 45 ' 46 S amily 2, facilitated glucose transporter, member 9 (Glucose _ _ _ transporter type 9) - Homo ~ ~ Sapiens ~ ~~ ~ ~~
~ J~ . ~ y y. ' ~N

13379362 Solute carrier famil NOV4m 2, facilitated glucose transporter, member 9 (Glucose transporter type 9) - Homo Sapiens _.. ....._. ~ _. .... __ ...... .. . .~... ... . . . . . .
.... _ NOV4n 13379620 49 ' S0 Solute carrier family 2, facilitated glucose transporter, member 9 (Glucose ~_ ~ ~ ~~~ ~ transporter type 9) - Homo Sapiens NOVSa CG181662-O1 S 1 52 Protein farnesyltransferase alpha subunit (EC 2.5.1.-) (CAAX farnesyltransferase alpha subunit) (RAS proteins " prenyltransferase alpha) (FTase-alpha) -Homo, Sapiens. . . .. .. .

NOVSb CG181662-02 53 54 'Protein farnesyltransferase alpha subunit (EC 2.5.1.-) (CAAX farnesyltransferase alpha subunit) (R.AS proteins prenyltransferase alpha) (FTase-alpha) -Homo Sapiens ~

NOVSc '307686795 55 56 Protein farnesyltransferase alpha subunit (EC 2.5.1.-) (CAAX farnesyltransferase alpha subunit) (RAS proteins prenyltransferase alpha) (FTase-alpha) -Homo Sapiens V

NOVSd CG181662-03 58 Protein farnesyltransferase alpha 57 subunit (EC 2.5.1.-) (CAAX farnesyltransferase alpha subunit) (RAS proteins prenyltransferase alpha) (FTase-alpha) -Homo sapiens NOVSe CG181662-04 59 60 Protein farnesyltransferase alpha subunit (EC 2.5.1.-) (CAAX farnesyltransferase alpha subunit) (R.AS proteins prenyltransferase alpha) (FTase-alpha) -_ _ _ . u... ._... _.. Homo.,sapiens .._ _ OVSf 13382357 61 62 Protein farnesyltransferase alpha subunit (EC 2.5.1.-) (CAAX farnesyltransferase alpha subunit) (RAS proteins prenyltransferase alpha) (FTase-alpha) -Homo Sapiens NOVSg 13377970 63 64 Protein farnesyltransferase alpha subunit (EC 2.5.1.-) (CAAX farnesyltransferase alpha subunit) (R.AS proteins prenyltransferase alpha) (FTase-alpha) -Homo sapiens NOVSh 13378241 65 66 Protein farnesyltransferase alpha subunit (EC 2.5.1.-) (CAAX farnesyltransferase alpha subunit) (RAS proteins ~prenyltransferase alpha) (FTase alpha) Homo Sapiens NOVSi 13377901 ' 67 68 Protein farnesyltransferase alpha subunit (EC 2.5.1.-) (CAAX farnesyltransferase alpha subunit) (RAS proteins prenyltransferase alpha) (FTase-alpha) -Homo Sapiens ~
~

NOVSj 13377900 69 Protein farnesyltransferase alpha subunit ~(EC 2 5.1.-) (CAAX farnesyltransferase alpha subunit) (RAS proteins 'prenyltransferase alpha) (FTase-alpha) -_. . _ . . . Homo Sapiens .... .

NOV6a CG182223-O1 ' 71 ' 72 Human neurotransmission-associated -V.~_ ~ .,...~protem NTRAN8~Homo Sapiens ~
~

NOV7a CG1 74 Adult male liver tumor cDNA, full-length enriched library, clone:C730027017 product:hypothetical protein, full insert sequence - Mus musculus NOVBa CG183860 Ol 75 '76 Human secreted protein HNTNC20 -Homo Sapiens _ t _ _... .. _~ __ _.w... ~ . .. . . .. . .._ . _ .
_ .~ ..._.. _ _ . .. ... .
_ NOV9a CG184416-O1 77 78 ~MMP-23 (MIFR/FEMALYS1N) (DJ283E3.2.1) (Matrix metalloproteinase MMP2I/22A (MIFR1)) (Matrix ~metalloproteinase 23B) - Homo sapiens ~

NOV 10a CG 18520 - 1 ; 0 ec ete tr ' 7 , $ Human s r dl ansmembrane protein, ._ .. . _._.. . . _. .._._ _ . .PR01377 - Homo sapiens, _ _.. _._ .. _ .~... _ . ._.. .. . ._.... _ ____ _ . _ .._._ .. _ .
._.
_..

NOV 10b ' CG185200-02 81 82 Human secreted/transmembrane protein, . _. '.. . ~... _ . pRO1,37,7 - H_omo Sapiens. _.._ .. ~

NOV l CG50513-O1 83 ~ 84 central Ia nervous system protein #236 -_ __. . ... _... _ ._ ... ... Homo sap~ens .._.. _ ... _. _.... . . ..... _ .. _._ . _....
._. . ... ~

NOV 1 273654175 8S ; 86 central nervous system protein lb #236 -Homo sapiens _ __ ......... ... .. .. _ NOV1 __ 88 central nervous system protein lc CG50513-02 87 #236 -Homo Sapiens _ _ ~. __ _.. _. .__ ~ . .. _.. . ..._ _ _. .._.
. _ _ _ __. ..~ . _ _... _ _ _. . ~.... _ ._ _ ~...._.

'NOVlld CG50513-03 89 90 central nervous system protein #236 -. Homo Sapiens _ .. . ._ _ . ._. ~.

NOVlla CG50513-04 91 92 central nervous system protein #236 -_ _.. . _~ __. . ~......Homo.. Sapiens _. _ . _... .
. . _ .. . .. _ NOVllf CGSOS13-OS '93 94 central nervous system protein #236 -Homo sapiens _.. _.

NOVl CG50513-06 95 96 central nervous system protein#236 lg -~_ ~_ ._ _._.. __.~~____~.._.~~__.._.~.Homo,sapiens40.~ _... ___.
N.__..___.____~..__ .__ _._..__.... ___ NOV 1 ! CG50513-07 ~ 98 central nervous system protein lh 97 #236 -_.. _... Homo Sapiens _ . .
. _ NOVl 13376798 '99 100 central nervous system protein li #236 -~.. . ... ... _ _ .....~ . _Homo Sapiens. _. . _ ..
._W .. _ _ .._..

NOVl 13376799 101 102 central nervous system protein lj #236 -' . . ......Homo Sapiens .,....... ... ..
.. . ... . ... ..
.

NOV 12a CG50949-03 103 104 Membrane-type mosaic serine protease -_.... _ . _ ___.._ _~, ...____~V~_~~Homo_sapiens.._.~_________~._ _~_..N.__.._.. _ _.~_~~ _._ _._. ___._..._..__ -_ ~

NOVl2b 197192399 105 106 Membrane-type mosaic serine protease -_ . ' _ . Homo Sapiens _ . _ _ NOV 12c ' 257499999 ' 107 108 Membrane-type mosaic serine protease -_ . . _ _ _ _ ... .. Homo Sapiens _ ... ....
_..._ . .
. .

NOV ltd 257450010 109 110 Membrane-type mosaic serine protease -Homo Sapiens __ ., a ..... ~ ~._ ~ _. ... ...

'NOV 252417780 111 ~ 112 Membrane-type mosaic serine l2e protease -.... ~ Homo Sapiens ..... . .

NOV 12f 252417791 113 114 Membrane-type mosaic serine protease -_ ~ ~Ho'mo Sapiens . -....'.__",~,~_~_ .. .
NO ' 252417821 _.___.
12g . .116 V Membrane-type 115 mosaic serine protease -_ Homo Sapiens ~ .

NOV 12h 252417840 117 118 Membrane-type mosaic serine protease -Homo Sapiens NOV 12i 257474313 ' 120 Membrane-type mosaic serine 119 protease -Homo Sapiens ~
~

NOV l2j 257474324 121 122 Membrane-type mosaic serine protease -: ~ Homo Sapiens . i ._~ .;:_ . . ..
NOV 12k . ....... ...
~ CG50949-06. . ......
123 . ..
~ . ....
....
....
...
124 Membrane-type mosaic serine protease -_ Homo sapiens w ~ ~ ~
' m NOV 121 268669017 125 126 ~ Membrane-type mosaic serine protease -.. ~;;"_."~ ~
.",",~;,~~ .. Homo Sapiens .
w NOVl2m CG50949-OS 127 128 Membrane-type mosaic serine protease -~ Homo Sapiens .
_..~,_,~,:_;~,,~_, .. .
_ ..
_ _ NOV 12n ' 317431859 Membrane-type mosaic serine protease -. . . ~ .. Homo Sapiens . ...... .. ....
. ... . . . ..
.

NOVl2o CG50949-O1 131 132 'Membrane-type mosaic serine '-- protease -~

~mo Sapiens y NOV l2p CG50949-02 133 134 Membrane-type mosaic serine protease-y" w, ~;,~"~..Homo Sapiens Y ..
.~,~,;~~

NOVl2q CG50949-04 X135 136 Membrane-type mosaic serine protease-. ..... ... ~ . ._ . ..Homo Sapiens ..
. .

OVl2r CG50949-07 137 138 Membrane-type mosaic serine protease -_.. ..... .. Homo Sapiens ... _ . _ .. . .

NOVl2s 13374729 139 140 Membrane-type mosaic serine protease -_ _ ' Homo Sapiens ~ _ H.4~ N~
~
~

NOVl2t 13374730 141 142 Membrane-type mosaic serine protease -.. . . .. _ ~ .. . .. Homo Sapiens .. . . . _ ... _ .
. .

NOVl2u 13374731 143 144 Membrane-type mosaic serine protease -. _..... _._.Homo sapiens ,. .. ~; .~,.,,~, ~ : _ ~ ... ....,~ .. ...' ~ ..
_. .

NOV 13a CGS 1018-O1 145 146 ' Matrilin-2 precursor - Homo . .. sapiens .

27405127 4 .
NO 1 3 147 1 8 M trilin-2 precursor - Homo . . ~ .... ...... ~ . _. Sapiens ~~, . . ~ . , . _.~ .... ~.. . .. _ .. .. .... _ .. . .~._. . . ....

. 274051251 149 150 Matrilin-2 precursor -NOVl3c ' Homo sapiens ~

3d 27405125 _ _ NOV 1 3 ~ 1 5 a~
. S 1 2 M trilin 2 precursor - Homo 1 ...... Sapiens ~ __.. .. ~ ... .... . . .
..._ .. ......
...

NOVl3e 306562753 153 154 .
.. . .. .... .
Matrilin-2 precursor- Homo sapiens 3 f ' S Ma NOV 1 CG51 1 155 1 6 tnlm 2 precursor - Homo sapiens 0 8 02 ~. . i I. .. _ ....... _...
. . _...
..._.

NOVl3g CG51018-03 157 158 ;Matrilin-2 precursor-Homo sapiens ~

NOVl3h 13374217 ~ 160 Matrilin-2 precursor- Homo~sapiens _......._..,.. 159~ . _.... .. ., .. .~ ...... .. _ .. _...
.~ . .. . . ... .....
~ . .
.. _ .

NOVl4a 'CGSI051-07 ' _ Netrin-Gld - Mus musculus ~ 161 162 _ "
~

NOV l4b CGS I 051-14163 164 Netrin-G l d - Mus musculus NOVl4c 254537195 ' 165 166 Netrin-Gld - Mus musculus ' NOV l4d 254537282 167 168 Netrin-G_ld - Mus musculus t t ~ .. , _. , ~
_ NOVl4e . 169 , Netrm Gld - Mus musculus ,~,.,r. 170 'NOV 304965116 171 172 ' Netrin-Gld - Mus musculus 14f NOV l4g 273711018 173 174 Netrin-Gld - Mus musculus i . ~~~~~ ~ .

NOV l4h 273711053 175 176 Netrm Gld - Mus musculus NOVl4i 274051275 177 178 ~ Netrm Gld ~Mus musculus NOV l4j CG51051-O 179 I80 Netrin-Gld - Mus musculus 1 1.. 1 .,.. ..~
~ t . . ' ~ y ~
...
..

NOVl4k, ~CG51051-0 181 ,~, Netrm Gld Mus , musculus , ~, .

NOV 141 CG51051-03 183_ 184 N_etr_in-G_ld -_M_us_m_us_culus ~ ~ ~Y
~

NOVl4m CG51051-04 ' 185 186 Netrin-Gld H - Mus musculus NOVl4n GG51051-05 187 188 Netrm Gld Mus musculus _ _.

NOV 140 1051-06 1_89 190 Ne_tr_m_-Gl d - M_us musculus ~CG . ..
5 ~
y NOVl4p _ 191 192 Mus rnusculus ~ ~ _ Netrm-Gld -1-08 ~

NOVl4q _ 193 194 Netrm Gld Mus musculus ~ CG51051-10 _..

NOV l4r CG51051-11 :195 196 Netnn-Gld - Mus musculus NOVl4s CG51051-12 I97 198 ~ Netrm Gld Mus musculus , Y

NOV 14t CG51051-13 '.199 200 Netrin-Gld - Mus musculus z, _. ~ .. ~ .. _ , NOVl4u CG51051-15 201 202 Netrm Gld Mus musc_ulus ... ~~ ....

NOVl4v CG51051-16 203 a204 r Netnn-GId- Mus musculus _. _... _, _..w..

NOVl4w 13380736 '205 206 Netrin-G1d - Mus musculus ~ ~... ~ . _ _ _~ .. W . . . .
,. ~

NOVl4x 13380734 207 X208 Netrin-Gld- Mus musculus _ _.....G . . ~ . ..

NOVl4y 13382329 '209 210 ~ Netrm-Gld - Mus musculus .. ., . A .. .,. .., ....,. ........ ... .. . ..... ......
. . ., ..... #. ,9....... . _. . .. .. ,. . .. . ..
_. "... ., .."... .... . ., ....." . .. .......,.,...
, . . . ...
...,.. .
. ....
.

NOV l ' CG522 "211 ' 212 Netrin-Gl d - Mus musculus Sa 61 ~ _ 't .~ , ..
NOV l _ 213 x....... Netrm G l d Mus musculus Sb ...A.,..,~~ , .. a . .
~" ..... .,. 214 ~~
,~ _ ... , 268667469 .~ _. . .
.. . ... ~-~
~

" ", , NOVlSc CG52261-02 ~ ,~,_ , 215 216 ~~~
,~
.
.
.
Netrm-Gld - Mus musculus , _.

NOVlSd 13382342 217 218 Netrin-Gld - Mus musculus . _ , ~
.
~

NOVlSe 1338 -.~ , NetrmGld Mus musculus 1 ;2 0 NOV l6a _ _ _ Epidermal growth factor receptor-related _ 221u~ 222 _ protein homolog - Mus musculus ~

- el ed NOV l6b 305262879 223 224 s Epidermal growth factor rec ptor r . protein homolog, . Mus. musculus.......
_ ..

NOVl6c 319073326 225 226 Epidermal growth factor receptor-related _.. ~,.._ _ _ .._ _ Protein homolog,- Mus musculus m .__~ _._ . .__ .
.. _ ___~
..__~_ NOV l6d CG52414-O1 227 '228 Epidermal growth factor receptor-related protein homolog Mus musculus Y

NOV 16e CG52414-03 229 230 Epidermal growth factor receptor-related _ . ~ protem.homolog . IVIus musculus .., .~, ... ..

NOV 16f 13379509 ' 231 232 Epidermal growth factor receptor-related ' protein homolog - Mus musculus _ . .. _ . ... _ _.. .... _ ._. .... . ...
...._. . .. .. ....... . _....
.

NOVl6g 13381817 233 234 Epidermal growth factor receptor-related _" ~ _~., , protein homolog__Mus musculus y A _ _ , NOVl6 h 235 236 Epidermal growth factor receptor-related protein homolog - Mus musculus NOVl6i 13381560 237 238 Epidermal growth factor receptor-related ~. .,... protein homolog - Mus musculus .

NOVl7a CG52643-02 :239 240 Human follistatin-related protein NOV l7b 259341359 ' 241 242 Human follistatin-related .__. ~ . _. ... ~.. ~ ... protein . . t.. . . .... . .. . . .._ NOV I ' 268824728 243 244 Human follistatin-related 7c . o .. . _. ..... protein _ .. . . . . .
. ... ._. _ .
_ . .
.. _.
.

NOV 17d 268825987 245 246 Human follistatin-related . .. protein . ..

NOV l ~ 247 '2 8 Human follistatin-related 7e 26 y protein NOV l7f 275698334 ~ 249 r 250 Human follistatin-related ~ .. r_.....~ protein _. ~ .. t... .
~

NOV 17g CG52643-04 251 252 Human follistatin-related ... _ protein . ...... ~

NOV 17h g 253 254 Human follistatin-related 30 protein NOV l7i 289087852 255 ' 256 Human follistatin-related ~ ~ ~ protein ~

NOVl7j 289081920 257 258 Human follistatin-related _ ... __........ rotein _.. . p .............
NOVl7k 289098038 259 260 Human fol_listatin-relat_ed_protein _ , ~~_..
. _ ~ _.
.. . .~ _.
, , NOVI71 ~31I0608I8 261 '262 -_ . _. . ... . _ _._.. . Human folhstatm related protein . ._... _. .

NOVl7m 311885703 263 264 Human follistatin-related ~ protein ~~~

OV 17n ~' CG52 643 01 265 266 Human follistatin related protein NOVl7o CG526_43-03 267 '268 Human follistatin-related~_protein ~ ~
~ ~ ~

- ' 269 270 Human follistatin-related . ..._._.....6 3 OS . .._.... ... protein _ W ...... . . _._....__... . .. _...... _..... _.
. .... .... .....

NOV 17q CG52643-06 271 272 Human follistatin-related ~t. _._ . _~. ~ .. . protein .. _.
~

,, ,~ ,~ 274 Human follistatin-related NOVl7r 13382322 273 .. ... protein _ ..... . __ .... ..... ~.... _.... . _ . ... _........
_ . _. .
...._ NOVl7s 13382324 275 276 Human follistatin-related 4 y A ~ protein ~

NOVl7t 13381678 277 278 Human follistatin-related ___ i _ ._..... . .. r .........protein ..... . ~.. .... . . . .. _..
_ ... .. . ~
. .

... . ......... ._ _ ... .
NOV 18a CG53270-O1 279 280 . .
Serine/threonine kinase FKSG81 (Testis-specific serine/threonine kinase 1 ) -__ _ Homo Sapiens _ ~ w~ ~ ~~
Y ~~ ~

NOV l 274089779 281 ' 282 Serine/threonine kinase FKSG81 8b : Testis-( a specific serine/threonine kinase 1) -_ Homo Sapiens ~ y ~

NOVIBc CG53270-02 283 284 Serine/threonine kinase FKSG81 (Testis-specific serine/threonine kinase 1 ) -_ Homo sapiens ~ ~~ ~mm y ~

NOVl8d 13382344 285 286 Serine/threonine kinase FKSG81 (Testis-specific serine/threonine ~ kinase 1) -. _ .... .. . Homo...sapiens . ... . ....
... ..... ._. .. . . ........ .
._ ..
....

l8e 13382345 V 288 Serine/threonine kinase FKSG81 NO '287 (Testis-specific serine/threonine kinase 1 ) -_. _ . .__ . . _.. _. _ ~ Homo Sapiens,. . . ._._ ..._ ____ ..._. __. _._, _ __ __. __ _ .. ._ __~.. .__..w..._ __.. __ _..~
_ NOV 18f 13376391 ' 289 290 ' Serine/threonine kinase FKSG81 (Testis-specific serine/threonine kinase 1) -_ _ ~ Homo Sapiens NOV l 13376390 291 292 ' Serinelthreonine kinase FKSGB
Bg 1 (Testis-specific serine/threonine kinase 1) -~ Homo sapiens ~..... . ...~..w.
. _..._ ..

NOVl8h ' 13376389 293 294 Serine/threonine kinase FKSG81 ~ ' (Testis-specific serine/threonine kinase 1 ) -Homo sapiens NOV I9a CG54254-04 295 296 Fibronectin Ieucine rich ' transmembrane protein Y ~~ ~ ~

NOVl9b 247846813 297 298 Fibronectin leucine rich transmembrane . ",~,.~~~~ ~~,_~ Protein _ .. . . . .. ~_ . _ . .. , . . . .:.

NOV 19c 247846825 299 300 -~~ ~
' Fibronectin leucine rich transmembrane _... __. ... _ .. . . . protem ... . . ... _ ~..
.. ... .... . ... . ... . _ _ ... _ .... . _.
._ . : . ~

NOV I9d 247846967 301 302 ' Fibronectin leucine rich ' transmembrane _. .. . _.......protein...

_ .
NOV 19e 283841186 303 304 Fibronectin leucine rich transmembrane _ . __.._ ._._ . _._._~~.___ __. _ . ____._.protein , _. _.. .._. ....
_. .._ _. __. __.._ __. . .
. __. . .
_ . _ NOV 19f CG54254-O 305 306 _ 1 ' .
Fibronectin leucine rich transmembrane protein ..

NOV l9g CG54254-02 307 308 Fibronectin leucine rich ' ' transmembrane _..._... _. I?rotem ,....._ .... . ...
............. . _.. ...
. ..

NOV 19h CG54254-03 309 310 Fibronectin leucine rich ~ transmembrane ... . protein NOV 19i CG54254-OS 3 l 312 Fibronectin leucine rich ' l transmembrane . _. _ _ _ : protein ~ .. _. _ Y Y ~
y NOVI9j CG54254-06 313 3I4 Fibronectin leucine rich ! transmembrane .... _ .. _ ~ , I?rotem _ _ _ .. _ NOV 19k CG54254-07 315 316 Fibronectin leucine rich ' transmembrane _ _. _._~ ,~_Y".~~. ....... .. Protein ,...... ._ .... ...
. _..
.

NOV 191 13375078 317 318 Fibronectin leucine rich ' ' ' transmembrane .. . protein _........ . .......
.. ...

NOV 19m 13376406 319 320 Fibronectin leucine rich : transmembrane . . __._ ._ _.. . . _ ...... Protein . x _ .___.. _.._ . . __.._..._...._ ._ _.... _.__m_ _ .. .. .. ... ._ ~ _ . ..

NOV 19n 13375079 321 322 Fibronectin leucine rich ! transmembrane _ ... . .. _ . .. .. ~ .._ protein ... _ _ _ ~ _. . _ ., NOV 190 13376405 323 324 Fibronectin leucine rich transmembrane _ . ......._ ._ . __ protem... .._ . ... _ ...
.. . __. .. ... .
.......
~

NOV20a CG96778-02 325 326 , Acyl-CoA dehydrogenase, medium-chain specific, mitochondrial precursor {EC

_ _ 1.3,99.3) (MCAD) - Homo sapiens ry y rv ~

NOV20b CG96778-O1 327 328 Acyl-CoA
dehydrogenase, medmm-chain specific, mitochondrial precursor (EC

1.3.99.3) (MCAD) - Homo sapiens ~

NOV20c 276657466 329 330 Acyl-CoA dehydrogenase, medium-chain specific, mitochondria) precursor (EC

_ _ _ _ _ _ ' 1.3.99.3) (MCAD) - Homo Sapiens ~ ~ T ~' ~ y ~

NOV20d 276657530 331 332 Acyl-CoA dehydrogenase, medium-chain specific, mitochondria) precursor ~ ~ (EC

.. _... _ ...._ . .... ~ .3.99 3) (MCAD) Homo Sapiens _ _. . ...

NOV20e 276657538 333 334 Acyl CoA dehydrogenase, medmm-chain 1s specific, mitochondria) precursor (EC

.3.99.3) (MCAD) - Homo Sapiens NOV20f276657616 335 336 Acyl-CoA dehydrogenase, medium-chain specific, mitochondria) precursor (EC

1.3.99.3) (MCAD) -- Homo Sapiens 'NOV20g ~CG96778-03 '337 338 Acyl-CoA dehydrogenase, medium-chain specific, mitochondria) precursor (EC

1.3.99.3) (MCAD) - Homo sapiens ~~ ~

NOV20h 13382351 339 340 Acyl-CoA dehydrogenase, medium-chain specific, mitochondria) precursor (EC

_ _ 1.3.99.3) (MCAD) - Homo sapiens_ ~~

NOV20i 13382352 342 Acyl-CoA dehydrogenase, medmm-chain ~341 specific, mitochondria) precursor (EC

1.3.99.3) (MCAD) -_Ho_mo sapie_ns _ . .
~

v ..
NO 20j 13382353 343 344 Acyl-CoA dehydrogenase, medmm-chain specific, mitochondria) precursor (EC

1.3.99.3) (MCAD) - Homo sap_iens_ w a ~ ~
~~
~~

NOV20k 13382354 3 S 4 4 c 3 6 A yl-CoA dehydrogenase, medmm-chain specific, mitochondria) precursor (EC

1.3.99.3) (MCAD) - Homo Sapiens _ . .
...... ..
-...... ...

34 34 c NOV201 h 7 8 A yl oA de ydrogenase, medium-chain specific, mitochondria) precursor (EC

1.3.99.3) (MCAD) - Homo Sapiens 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, atria) septa) defect (ASD), vascular calcification, fibrosis, atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septa) defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, metabolic disturbances associated with obesity, transplantation, osteoarthritis, rheumatoid arthritis, osteochondrodysplasia, adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer, diabetes, metabolic disorders, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, glomerulonephritis, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, psoriasis, skin disorders, graft versus host disease, AIDS, bronchial asthma, lupus, Crohn's disease; inflammatory bowel disease, ulcerative colitis, multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias, schizophrenia, depression, asthma, emphysema, allergies, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers, as well as conditions such as transplantation, neuroprotection, fertility, or regeneration (in vitro and in vivo).
NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various NOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
Additionally, NOVX
nucleic acids and polypeptides can also be used to identify proteins that axe members of the family to which the NOVX polypeptides belong.
Consistent with other known members of the family of proteins, identified in column of Table A, the NOVX polypeptides of the present invention show homology to, and contain domains that are characteristic of, other members of such protein families. Details of the sequence relatedness and domain analysis for each NOVX are presented in Example A.
The NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit diseases associated with the protein families listed in Table A.
The NOVX nucleic acids and polypeptides are also useful for detecting specific cell types. Details of the expression analysis for each NOVX are presented in Example C.
Accordingly, the NOVX nucleic acids, polypeptides, antibodies and related compounds according to the invention will have diagnostic and therapeutic applications in the detection of a variety of diseases with differential expression in normal vs. diseased tissues, e.g.
detection of a variety of cancers.
Additional utilities for NOVX nucleic acids and polypeptides according to the invention are disclosed herein.
NOVX clones NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various NOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
Additionally, NOVX
nucleic acids and polypeptides can also be used to identify proteins that are members of the family to which the NOVX polypeptides belong.
The NOVX genes and their corresponding encoded proteins are useful for preventing, treating or ameliorating medical conditions, e.g., by protein or gene therapy.
Pathological conditions can be diagnosed by determining the amount of the new protein in a sample or by determining the presence of mutations in the new genes. Specific uses are described for each of the NOVX genes, based on the tissues in which they are most highly expressed. Uses include developing products for the diagnosis or treatment of a variety of diseases and disorders.
The NOVX nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration i~ vitro and ih 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 174; (b) a variant of a mature form of the amino acid sequence selected from the group consisting of SEQ ID NO: Vin, wherein n is an integer between 1 and 174, 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 l and 174; (d) a variant of the amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between l and 174 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 174; (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 174 wherein any amino acid in the mature form of the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence of the mature form are so changed; (c) the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between l and 174; (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 174, in which any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed; (e) a nucleic acid fragment encoding at least a portion of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between I and 174 or any variant of said polypeptide wherein any amino acid of the chosen sequence is changed to a different amino acid, provided that no more than 10% of the amino acid residues in the sequence are so changed; and (f) the complement of any of said nucleic acid molecules.
In yet another specific embodiment, the invention includes an isolated nucleic acid molecule, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of (a) the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-1, wherein n is an integer between l and 174; (b) a nucleotide sequence wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-I, wherein n is an integer between 1 and 174 is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed; (c) a nucleic acid fragment of the sequence selected from the group consisting of SEQ ID NO: 2n-1, wherein n is an integer between l and 174; and (d) a nucleic acid fragment wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO:
2n-1, wherein n is an integer between 1 and 174 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. Altenlatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein. The product "mature" form arises, by way of nonlimiting example, as a result of one or more naturally occurring processing steps that may take place within the cell (e.g., host cell) in which the gene product arises. Examples of such processing steps leading to a "mature"
form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+1 to residue N remaining. Further as used herein, a "mature".
form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
The term "probe", as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), about 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences.
Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single-stranded or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
The term "isolated" nucleic acid molecule, as used herein, is a nucleic acid that is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i. e., sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NOVX nucleic acid molecules can contain less than about kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an "isolated"
nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium, or of chemical precursors or other chemicals.
A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID N0:2n-1, wherein n is an integer between 1 and 174, 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 N0:2n-1, wherein n is an integer between 1 and 174, as a hybridization probe, NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et al., (eds.), MOLECULAR
CLONING: A
LABORATORY MANUAL 2°a Ed., Cold Spring Harboi 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 amplif ed can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides comprise a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment of the invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEQ ID N0:2~-l, wherein n is an integer between 1 and 174, 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
N0:2n-1, wherein n is an integer between 1 and 174, 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 N0:2~z-l, wherein n is an integer between 1 and I74, is one that is sufficiently complementary to the nucleotide sequence of SEQ ID N0:2u-1, wherein n is an integer between 1 and 174, that it can hydrogen bond with few or no mismatches to the nucleotide sequence shown in SEQ ID N0:2~-l, wherein n is an integer between 1 and 174, 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 ionzc, 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 suff cient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, and is at most some portion less than a full length sequence.
Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice.
A full-length NOVX clone is identified as containing an ATG translation start codon and an in-frame stop codon. Any disclosed NOVX nucleotide sequence lacking an ATG
start codon therefore encodes a truncated C-terminal fragment of the respective NOVX
polypeptide, and requires that the corresponding full-length cDNA extend in the 5' direction of the disclosed sequence. Any disclosed NOVX nucleotide sequence lacking an in-frame stop codon similarly encodes a truncated N-terminal fragment of the respective NOVX
polypeptide, and requires that the corresponding full-length cDNA extend in the 3' direction of the disclosed sequence.
A "derivative" is a nucleic acid sequence or amino acid sequence formed from the native compounds either directly, by modification or partial substitution. An "analog" is a nucleic acid sequence or amino acid sequence that has a structure similar to, but not identical to, the native compound, e.g. they differs from it in respect to certain components or side chains. Analogs may be synthetic or derived from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. A
"homolog" is a nucleic acid sequence or amino acid sequence of a particular gene that is derived from different species.
Derivatives and analogs may be full length or other than full length.
Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95%
identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT
PROTOCOLS nr MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences include those sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for a NOVX
polypeptide of species other than humans, including, but not limited to:
vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms.
Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein.
A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ
ID N0:2n-l, wherein n is an integer between 1 and 174, as well as a polypeptide possessing NOVX
biological activity. Various biological activities of the NOVX proteins are described below.
A NOVX polypeptide is encoded by the open reading frame ("ORF") of a NOVX
nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon. An ORF that represents the coding sequence for a full protein begins with an ATG "start" codon and terminates with one of the three "stop" codons, namely, TAA, TAG, or TGA. For the purposes of this invention, an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both. For an ORF to be considered as a good candidate for coding for a bona fide cellular protein, a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
The nucleotide sequences determined from the cloning of the human NOVX genes allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX
homologues from other vertebrates. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEQ ID N0:2h-l, wherein n is an integer between l and 174; or an anti-sense strand nucleotide sequence of SEQ ID
N0:2n-1, wherein n is an integer between 1 and 174; or of a naturally occurring mutant of SEQ ID N0:2n-1, wherein n is an integer between 1 and 174.
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 2~

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
N0:2n-1, wherein n is an integer between 1 and 174, 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 vit~~o) and assessing the activity of the encoded portion of NOVX.
NOVX Single Nucleotide Polymorphisms Variant sequences are also included in this application. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP
originates as a cDNA. A SNP can arise in several ways. For example, a SNP may be due to a substitution of one nucleotide for another at the polymorphic site. Such a substitution can be either a transition or a transversion. A SNP can also arise from a deletion of a nucleotide or an insertion of a nucleotide, relative to a reference allele. In this case, the polymorphic site is a site at which one allele bears a gap with respect to a particular nucleotide in another allele.
SNPs occurring within genes may result in an alteration of the amino acid encoded by the gene at the position of the SNP. Intragenic SNPs may also be silent, when a codon including a SNP encodes the same amino acid as a result of the redundancy of the genetic code. SNPs occurring outside the region of a gene, or in an intron within a gene, do not result in changes in any amino acid sequence of a protein but may result in altered regulation of the expression pattern. Examples include alteration in temporal expression, physiological response regulation, cell type expression regulation, intensity of expression, and stability of transcribed message.
SeqCalling assemblies produced by the exon linking process were selected and extended using the following criteria. Genomic clones having regions with 98%
identity to all or part of the initial or extended sequence were identified by BLASTN
searches using the relevant sequence to query human genomic databases. The genomic clones that resulted were selected for further analysis because this identity indicates that these clones contain the genomic locus for these SeqCalling assemblies. These sequences were analyzed for putative coding regions as well as for similarity to the known DNA and protein sequences. Programs used for these analyses include Grail, Genscan, BLAST, 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 2s 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 axe reported individually but any combination of all or a select subset of variants are also included as contemplated NOVX embodiments of the invention.
NOVX Nucleic Acid and Polypeptide Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences of SEQ ID N0:2~r-1, wherein n is an integer between I and 174, due to degeneracy of the genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences of SEQ ID N0:2n-1, wherein n is an integer between I
and I74. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence of SEQ ID N0:2fZ, wherein n is an integer between 1 and 174.
In addition to the human NOVX nucleotide sequences of SEQ ID N0:2n-1, wherein n is an integer between 1 and 174, it will be appreciated by those skilled in the art that DNA
sequence polymorphisms that lead to changes in the amino acid sequences of the NOVX
polypeptides may exist within a population (e.g., the human population). Such genetic polymorphism in the NOVX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF) encoding a NOVX
protein, preferably a vertebrate NOVX protein. Such natural allelic variations can typically result in 1-5°lo variance in the nucleotide sequence of the NOVX genes. Any and all such nucleotide variations and resulting amino acid poIymorphisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the NOVX
polypeptides, are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from a human SEQ ID N0:2n-1, wherein n is an integer between 1 and 174, are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the NOVX
cDNAs of the invention can be isolated based on their homology to the human NOVX
nucleic acids disclosed herein using the hiunan cDIVAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ TD NO:2n-1, wherein n is an integer between 1 and 174. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or mare 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°lo 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 S
°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 formarnide.
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 SO°C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a sequence of SEQ ID N0:2n-l, wherein n is an integer between 1 and 174, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occarring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:2n-1, wherein n is an integer between 1 and 174, 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, SX Reinhardt's solution, 0.5% SDS
and 100 mglml denatured salmon sperm DNA at 55 °C, followed by one or more washes in 1X SSC, 0.1 % SDS at 37 °C. Other conditions of moderate stringency that may be used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT
PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Krieger, 1990; GENE TRANSFER AND
EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences of SEQ ID NO:2n-1, wherein n is an integer between 1 and 174, 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, SX 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%
(wtlvol) 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 lVatl 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 TD NO:2h-l, wherein n is an integer between l and 174, 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 N0:2~c, wherein n is an integer between 1 and 174. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences of the NOVX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity.
For example, amino acid residues that are conserved among the NOVX proteins of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
Another aspect of the invention pertains to nucleic acid molecules encoding NOVX
proteins that contain changes in amino acid residues that are not essential for activity. Such NOVX proteins differ in amino acid sequence from SEQ ID NO:2n-1, wherein n is an integer between 1 and 174, 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 174.
Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID
N0:2h, wherein n is an integer between 1 and 174; more preferably at least about 70%
homologous to SEQ ID N0:2n, wherein n is an integer between 1 and I 74; still more preferably at least about 80% homologous to SEQ ID N0:2~, wherein n is an integer between 1 and 174; even more preferably at least about 90% homologous to SEQ ID N0:2n, wherein n is an integer between 1 and 174; and most preferably at least about 95%
homologous to SEQ ID N0:2n, wherein n is an integer between 1 and 174.
An isolated nucleic acid molecule encoding a NOVX protein homologous to the protein of SEQ ID N0:2n, wherein n is an integer between 1 and 174, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID N0:2n-1, wherein n is an integer between 1 and 174, 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 N0:2n-1, wherein n is an integer between 1 and 174, by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino. acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonune, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Thus, a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX
biological activity to identify mutants that retain activity. Following mutagenesis of a nucleic acid of SEQ ID N0:2n-l, wherein n is an integer between 1 and 174, 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 axe 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, STNI~, 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 I9-25 nt long segment of the NOVX gene transcript, including the 5' untranslated (UT) region, the ORF, or the 3' UT region. See, e.g., PCT applications WO00/44895, W099/32619, WO01/75164, WO01/92513, WO 01/29058, WOOI/89304, W002/I6620, and W002/29858, each incorporated by reference herein in their entirety. Targeted genes can be a NOVX gene, or an upstream or downstream modulator of the NOVX gene.
Nonlimiting examples of upstream or downstream modulators of a NOVX gene include, e.g., a transcription factor that binds the NOVX gene promoter, a kinase or phosphatase that interacts with a NOVX polypeptide, and polypeptides involved in a NOVX
regulatory pathway.
According to the methods of the present invention, NOVX gene expression is silenced using short interfering RNA. A NOVX polynucleotide according to the invention includes a siRNA polynucleotide. Such a NOVX siRNA can be obtained using a NOVX
polynucleotide sequence, for example, by processing the NOVX ribopolynucleotide sequence in a cell-free system, such as but not limited to a Drosophila extract, or by transcription of recombinant double stranded NOVX RNA or by chemical synthesis of nucleotide sequences homologous to a NOVX sequence. See, e.g., Tuschl, Zamore, Lehmann, Bartel and Sharp (1999), Genes & Dev. 13: 3191-3197, incorporated herein by reference in its entirety. When synthesized, a typical 0.2 micromolar-scale RNA synthesis provides about 1 milligram of siRNA, which is sufficient for 1000 transfection experiments using a 24-well tissue culture plate format.

The most efficient silencing is generally observed with siRNA duplexes composed of a 21-nt sense strand and a 21-nt antisense strand, paired in a manner to have a 2-nt 3' overhang. The sequence of the 2-nt 3' overhang makes an additional small contribution to the specificity of siRNA target recognition. The contribution to specificity is localized to the unpaired nucleotide adjacent to the first paired bases. In one embodiment, the nucleotides in the 3' overhang are ribonucleotides. In an alternative embodiment, the nucleotides in the 3' overhang are deoxyribonucleotides. Using 2'-deoxyribonucleotides in the 3' overhangs is as efficient as using ribonucleotides, but deoxyribonucleotides are often cheaper to synthesize and are most likely more nuclease resistant.
A contemplated recombinant expression vector of the invention comprises a NOVX
DNA molecule cloned into an expression vector comprising operatively-linked regulatory sequences flanking the NOVX sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands. An RNA molecule that is antisense to NOVX mRNA is transcribed by a first promoter (e.g., a promoter sequence 3' of the cloned DNA) anal an RNA molecule that is the sense strand for the NOVX mRNA is transcribed by a second promoter (e.g., a promoter sequence 5' of the cloned DNA). The sense and antisense strands may hybridize in vivo to generate siRNA constructs for silencing of the NOVX gene. Alternatively, two constructs can be utilized to create the sense and anti-sense strands of a siRNA construct. Finally, cloned DNA can encode a construct having secondary structure, wherein a single transcript has both the sense and complementary antisense sequences from the target gene or genes. In an example of this embodiment, a hairpin RNAi product is homologous to alI or a portion of the target gene. In another example, a hairpin RNAi product is a siRNA. The regulatory sequences flanking the NOVX sequence may be identical or may be different, such that their expression rnay 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 nucleax RNA (snRNA) U6 or the human RNase P RNA H1. One example of a vector system is the GeneSuppressor~ RNA Interference kit (commercially available from Imgenex). The U6 and Hl promoters are members of the type III class of Pol III
promoters.
The +1 nucleotide of the U6-like promoters is always guanosine, whereas the +1 for H1 promoters is adenosine. The termination signal for these promoters is defined by five consecutive thymidines. The transcript is typically cleaved after the second uridine. Cleavage at this position generates a 3' UU overhang in the expressed siRNA, which is similar to the 3' overhangs of synthetic siRNAs. Any sequence less than 400 nucleotides in length can be transcribed by these promoter, therefore they are ideally suited for the expression of around 21-nucleotide siRNAs in, e.g., an approximately 50-nucleotide RNA stem-loop transcript.
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. ~n 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 to100 nt downstream of the start codon.
Alternatively, 5' or 3' UTRs and regions nearby the start codon can be used but are generally avoided, as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP or RISC
endonuclease complex. An initial BLAST homology search for the selected siRNA sequence is done against an available nucleotide sequence library to ensure that only one gene is targeted.
Specificity of target recognition by siRNA duplexes indicate that a single point mutation, located in the paired region of an siRNA duplex is sufficient to abolish target mRNA
degradation. See, Elbashir et al. 2001 EMBO J. 20(23):6877-88. Hence, consideration should be taken to accommodate SNPs, polymorphisms, allelic variants or species-specific variations when targeting a desired gene.
In one embodiment, a complete NOVX siRNA experiment includes the proper negative control. A negative control siRNA generally has the same nucleotide composition as the NOVX siRNA but lack significant sequence homology to the genome.
Typically, one would scramble the nucleotide sequence of the NOVX siRNA and do a homology search to make sure it lacks homology to any other gene.
Two independent NOVX siRNA duplexes can be used to knock-down a target NOVX
gene. This helps to control for specificity of the silencing effect. In addition, expression of two independent genes can be simultaneously knocked down by using equal concentrations of different NOVX siRNA duplexes, e.g., a NOVX siRNA and an siRNA for a regulator of a NOVX gene or polypeptide. Availability of siRNA-associating proteins is believed to be more limiting than target mRNA accessibility.
A targeted NOVX region is typically a sequence of two adenines (AA) and two thymidines (TT) divided by a spacer region of nineteen (N19) residues (e.g., AA(N19)TT).
A desirable spacer region has a G/C-content of approximately 30% to 70%, and more preferably of about 50%. If the sequence AA(N19)TT is not present in the target sequence, an alternative target region would be AA(N21). The sequence of the NOVX sense siRNA
corresponds to (N19)TT or N21, respectively. In the latter case, conversion of the 3' end of the sense siRNA to TT can be performed if such a sequence does not naturally occur in the NOVX polynucleotide. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3' overhangs.
Symmetric 3' overhangs may help to ensure that the siRNPs are formed with approximately equal ratios of sense and antisense target RNA-cleaving siRNPs. See, e.g., Elbashir, Lendeckel and Tuschl (2001). Genes & Dev. 15: 188-200, incorporated by reference herein in its entirely. The modification of the overhang of the sense sequence of the siRNA duplex is not expected to affect targeted mRNA recognition, as the antisense siRNA
strand guides target recognition.
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 S' (N19)TT, as it is believed that the sequence of the 3'-most nucleotide of the antisense siRNA does not contribute to specificity.
Unlike antisense or ribozyme technology, the secondary structure of the target mRNA does not appear to have a strong effect on silencing. See, Harborth, et al. (2001) J. Cell Science 114: 4557-4565, incorporated by reference in its entirety.
Transfection of NOVX siRNA duplexes can be achieved using standard nucleic acid transfection methods, for example, OLIGOFECTAMINE Reagent (commercially available from Invitrogen). An assay for NOVX gene silencing is generally performed approximately 2 days after transfection. No NOVX gene silencing has been observed in the absence of transfection reagent, allowing for a comparative analysis of the wild-type and silenced NOVX phenotypes. In a specific embodiment, for one well of a 24-well plate, approximately 0.84 p,g 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 pg single-stranded sense NOVX
siRNA
will have no effect on NOVX silencing, and 0.84 p,g antisense siRNA has a weak silencing effect when compared to 0.84 p,g of duplex siRNAs. Control experiments again allow for a comparative analysis of the wild-type and silenced NOVX phenotypes. To control for transfection efficiency, targeting of common proteins is typically performed, for example targeting of lamin A/C or transfection of a CMV-driven EGFP-expression plasmid (e.g.
commercially available from Clontech). In the above example, a determination of the fraction of lamin A/C knockdown in cells is determined the next day by such techniques as immunofluorescence, Western blot, Northern blot or other similar assays for protein expression or gene expression. Lamin A/C monoclonal antibodies may be obtained from Santa Cruz Biotechnology.
Depending on the abundance and the half life (or turnover) of the targeted NOVX
polynucleotide in a cell, a knock-down phenotype may become apparent after 1 to 3 days, or even later. In eases where no NOVX knock-down phenotype is observed, depletion of the NOVX polynucleotide may be observed by immunofluorescence ox Western blotting.
If the NOVX polynucleotide is still abundant after 3 days, cells need to be split and transferred to a fresh 24-well plate for re-transfection. If no knock-down of the targeted protein is observed, it may be desirable to analyze whether the target mRNA (NOVX or a NOVX
upstream or downstream gene) was effectively destroyed by the transfected siRNA duplex.
Two days after transfection, total RNA is prepared, reverse transcribed using a target-specific primer, and PCR-amplified with a primer pair covering at least one exon-exon junction in order to control for amplification of pre-mRNAs. RT/PCR of a non-targeted mRNA is also needed as control. Effective depletion of the mRNA yet undetectable reduction of target protein may indicate that a large reservoir of stable NOVX protein may exist in the cell.
Multiple transfection in sufficiently Long intervals may be necessary until the target protein is finally depleted to a point where a phenotype may become apparent. If multiple transfection steps are required, cells are split 2 to 3 days after transfection. The cells may be transfected immediately after splitting.
An inventive therapeutic method of the invention contemplates administering a NOVX siRNA construct as therapy to compensate for increased or aberrant NOVX
expression or activity. The NOVX ribopolynucleotide is obtained and processed into siRNA
fragments, or a NOVX siRNA is synthesized, as described above. The NOVX siRNA
is administered to cells or tissues using known nucleic acid transfection techniques, as described above. A NOVX siRNA specific for a NOVX gene will decrease or knockdown NOVX transcription products, which will lead to reduced NOVX polypeptide production, resulting in reduced NOVX polypeptide activity in the cells or tissues.
The present invention also encompasses a method of treating a disease or condition associated with the presence of a NOVX protein in an individual comprising administering to the individual an RNAi construct that targets the mRNA of the protein (the mRNA that encodes the protein) for degradation. A specif c RNAi construct includes a siRNA or a double stranded gene transcript that is processed into siRNAs. Upon treatment, the target protein is not produced or is not produced to the extent it would be in the absence of the treatment.
Where the NOVX gene function is not correlated with a known phenotype, a control sample of cells or tissues from healthy individuals provides a reference standard for determining NOVX expression levels. Expression levels are detected using the assays described, e.g., RT-PCR, Northern blotting, Western blotting, ELISA, and the like. A subject sample of cells or tissues is taken from a mammal, preferably a human subject, suffering from a disease state. The NOVX ribopolynucleotide is used to produce siRNA
constructs, that are specific for the NOVX gene product. These cells or tissues are treated by administering NOVX siRNA's to the cells or tissues by methods described for the transfection of nucleic acids into a cell or tissue, and a change in NOVX
polypeptide or polynucleotide expression is observed in the subject sample relative to the control sample, using the assays described. This NOVX gene knockdown approach provides a rapid method for determination of a NOVX minus (NOVX-) phenotype in the treated subject sample. The NOVX- phenotype observed in the treated subject sample thus serves as a marker for monitoring the course of a disease state during treatment.
In specific embodiments, a NOVX siRNA is used in therapy. Methods for the generation and use of a NOVX siRNA are known to those skilled in the art.
Example techniques are provided below.
Production of RNAs Sense RNA (ssRNA) and antisense RNA (asRNA) of NOVX are produced using known methods such as transcription in RNA expression vectors. In the initial experiments, the sense and antisense RNA are about 500 bases in length each. The produced ssRNA and asRNA (0.5 p,M) in 10 mM Tris-HCl (pH 7.5) with 20 mM NaCI 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 3aP-ATP. Reactions are stopped by the addition of 2 X
proteinase I~ 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, I88-200 (2001)), followed by Sep-Pak C18 cartridge (Waters, Milford, Mass., USA) purification (Tuschl, et al., Biochemistry, 32:11658-11668 (1993)).
These RNAs (20 ~,M) single strands are incubated in annealing buffer (100 mM
potassium acetate, 30 mM HEPES-I~OH 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 e~cient 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 ribozyrne gene targeting experiments.
The above method provides a way both for the deduction of NOVX siRNA sequence and the use of such siRNA for in vitro suppression. In vivo suppression may be performed using the same siRNA using well known in vivo transfection or gene therapy transfection techniques.
Antisense Nucleic Acids Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID N0:2n-1, wherein n is an integer between 1 and 174, 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 N0:2n, wherein n is an integer between 1 and 174, or antisense nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ID N0:2n-1, wherein n is an integer between 1 and 174, are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a NOVX protein.
The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding the NOVX protein. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding the NOVX protein disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of NOVX mRNA.
For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).

Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, S-bromouracil, 5-chlorouracil, S-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-carboxymethylaminomethyl-2-thiouridine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 5-methoxyuracil, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 2-thiouracil, 4-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a NOVX protein to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementaxity 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 a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual (3-units, the strands run parallel to each other. See, e.g., Gaultier, et al., 1987.
Nucl. Acids Res. 15:
6625-6641. The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (See, e.g., Inoue, et al. 1987. Nucl. Acids Res. 15:
6131-6148) or a chimeric RNA-DNA analogue (See, e.g., moue, 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 sugax 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 N0:2r~-1, wherein n is an integer between 1 and 174). For example, a derivative of a Tet~-ahyme~za L-19 IVS RNA
can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a NOVX-encoding mRNA. See, e.g., U.S.
Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al. NOVX mRNA
can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA
molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the NOVX nucleic acid (e.g., the NOVX promoter and/or enhancers) to form triple helical structures that prevent transcription of the NOVX gene in target cells. See, e.g., Helene, 1991. Anticancer Drug Des. 6: 569-84;
Helene, et al. 1992. Anh. N. Y Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.
In various embodiments, the NOVX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al., 1996.
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., S1 nucleases (See, Hyrup, et al., 1996.supra); or as probes or primers for DNA sequence and hybridization (See, Hyrup, et al., 1996, supra;
Perry-O'Keefe, et al., 1996. supra).
In another embodiment, PNAs of NOVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA
chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleotide bases, and orientation (see, Hyrup, et al., .1996.
supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al., 1996. supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA
chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g., Mag, et al., 1989. Nucl Acid Res 17:
5973-5988.
PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al., 1996. supra.
Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA
segment. See, e.g., Petersen, et al., 1975. Bioorg. Med. Chem. Lett. 5: 1119-11124.
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g:, for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al., 1989. Proc. Natl.
Acad. Sci. U.SA. 86:
6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT
Publication No.
W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO
89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al., 1988. BioTech~iques 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
N0:2n, wherein n is an integer between l and 174. 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 N0:2n, wherein n is an integer between l and 174, 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 ofcellular material~or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of NOVX proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the language "substantially free of cellular material" includes preparations of NOVX proteins having less than about 30%
(by dry weight) of non-NOVX proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-NOVX proteins, still more preferably less than about 10% of non-NOVX proteins, and most preferably less than about 5% of non-NOVX
proteins.
When the NOVX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the NOVX protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of NOVX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals"
includes preparations of NOVX proteins having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20% chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals.
Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the NOVX proteins (e.g., the amino acid sequence of SEQ ID N0:2n, wherein n is an integer between l and 174) that include fewer amino acids than the full-length NOVX
proteins, and exhibit at least one activity of a NOVX protein. Typically, biologically-active portions comprise a domain or motif with at least one activity of the NOVX protein. A
biologically-active portion of a NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.

Moreover, other biologically-active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native NOVX protein.
In an embodiment, the NOVX protein has an ammo acid sequence of SEQ ID N0:2n, wherein n is an integer between l and 174. In other embodiments, the NOVX
protein is substantially homologous to SEQ ID N0:2n, wherein n is an integer between 1 and I74, and retains the functional activity of the protein of SEQ ID N0:2n, wherein n is an integer between 1 and 174, 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 N0:2n, wherein n is an integer between l and 174, and retains the functional activity of the NOVX proteins of SEQ ID NO:2n, wherein n is an integer between l and 174.
Determining Homology Eetween Two or More Sequences To determine the percent homology'of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i. e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. JMoI Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP
extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence of SEQ ID NO:2n-1, wherein n is an integer between 1 and 174.
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
N0:2n, wherein n is an integer between 1 and 174, whereas a "non-NOVX
polypeptide"
refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism. Within a NOVX
fusion protein the NOVX polypeptide can correspond to all or a portion of a NOVX protein.
In one embodiment, a NOVX fusion protein comprises at least one biologically-active portion of a NOVX protein. In another embodiment, a NOVX fusion protein comprises at least two biologically-active portions of a NOVX protein. In yet another embodiment, a NOVX fusion protein comprises at least three biologically-active portions of a NOVX
protein. Within the fusion protein, the term "operatively-linked" is intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame with one another.
The non-NOVX polypeptide can be fused to the N-terminus or C-terminus of the NOVX
polypeptide.
In one embodiment, the fusion protein is a GST-NOVX fusion protein in which the NOVX sequences are fused to the C-terminus of the GST (glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant NOVX
polypeptides.

In another embodiment, the fusion protein is a NOVX protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of NOVX can be increased through use of a heterologous signal sequence.
In yet another embodiment, the fusion protein is a NOVX-immunoglobulin fusion protein in which the NOVX sequences are fused to sequences derived from a member of the immunoglobulin protein family. The NOVX-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a NOVX ligand and a NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction in vivo. The NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of a NOVX
cognate ligand. Inhibition of the NOVX ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the NOVX-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of NOVX with a NOVX ligand.
A NOVX chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT 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.
so 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.
Tetrahed~o~z 39: 3;
Itakura, et al., 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al., 1984.
Science 198: 1056;
Ike, et al., 1983. Nucl. Acids Res. 11: 477.
sl Polypeptide Libraries In addition, libraries of fragments of the NOVX protein coding sequences can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of a NOVX protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a NOVX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with Si nuclease, and ligating the resulting fragment library into an expression vector. By this method, expression libraries can be derived which encodes N-terminal and internal fragments of various sizes of the NOVX
proteins.
Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA
libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of NOVX proteins.
The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected.
Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify NOVX
variants: See, e.g., Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89:
7811-7815;
Delgrave, et al., 1993. Protein Engineering 6:327-331.
Anti-NOVX Antibodies Included in the invention are antibodies to NOVX proteins, or fragments of NOVX
proteins. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab> and F~$b~~2 fragments, and an Fab expression library. In general, antibody molecules obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain pxesent in the molecule.
Certain classes have subclasses as well, such as IgG~, 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 genexate 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
N0:2n, wherein n is an integer between 1 and 174, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length pxotein ox with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, ox 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 axe 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 axt, including, for example, the Kyle Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. LISA
78: 3824-3828;
Kyte and Doolittle 1982, J. Mol. Biol. 157: I05-142, each incorporated herein by reference in their entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. A NOVX polypeptide or a fragment thereof comprises at least one antigenic epitope. An anti-NOVX antibody of the present invention is said to specifically bind to antigen NOVX when the equilibrium binding constant (IUD) is <1 p,M, preferably 5100 nM, more preferably _< 10 nM, and most preferably S 100 pM to about 1 pM, as measured by assays including radioligand binding assays or similar assays known to skilled artisans.
A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies:
A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below.
Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.
Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar irnmunostimulatory agents.
Additional examples of adjuvants which can be employed include MPL-TDM
adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D.
Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
Monoclonal Antibodies The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.
MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell Iine 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 marine 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 imrnunoprecipitation 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 purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of marine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA
also can be modified, for example, by substituting the coding sequence for human heavy and Iight chain constant domains in place of the homologous marine sequences (U.S.
Patent No.
4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies axe chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')Z
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 of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).
s~

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. L1SS, IriC., 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 W094/02602). The endogenous genes encoding the heavy and light imrnunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as ss progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse~ as disclosed in PCT publications WO

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 cultuxe, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.
In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT
publication WO 99/53049.
Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab')2 fragment produced by pepsin digestion of an antibody molecule;
(ii) an Fab fragment generated by reducing the disulfide bridges of an F(~6~~2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F~ fragments.
Bispecifc 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 amity chromatography steps. Similar procedures are disclosed in WO
93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (I986).
According to another approach described in WO 96/2701 l, 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 chains) 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')a fragments.
These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB
derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')~
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 bispecif c antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. I~ostelny et al., J. Immunol.
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.

The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci.
USA
90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy fox 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, txispecific 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 (Fc~yR), such as FcyRI (CD64), Fc~yRII (CD32) and FcyRIII
(CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.
Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope 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 purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.
Patent No. 4,676,980.

Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residues) 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 imrnunoconjugates 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 2lzBi, i3ih i3iIn, 9oY, and is6Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled I-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.
See W094/ 11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
Immunoliposomes The antibodies disclosed herein can also be formulated as immunoliposomes.
Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985);
Hwang et al., Proc.
Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.
Liposomes with enhanced circulation time are disclosed in U.S. Patent No.
5,013,556.
Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al ., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. A
chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome.
See Gabizon et al., J. National Cancer Inst., 81(19): 1484 (1989).
Diagnostic Applications of Antibodies Directed Against the Proteins of the Invention In one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of an NOVX
protein is facilitated by generation of hybridomas that bind to the fragment of an NOVX
protein possessing such a domain. Thus, antibodies that are specific for a desired domain within an NOVX protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
Antibodies directed against a NOVX protein of the invention may be used in methods known within the art relating to the localization andlor 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 ox 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, (3-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 lash 1311, 3sS 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 specif c 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 taxget 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 fox 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 othex cases, promotes a physiological response. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.
Pharmaceutical Compositions of Antibodies Antibodies specifically binding a protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington : The Science And Practice Of Pharmacy 19th ed.
(Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa. : 1995; Drug Absorption Enhancement : Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.
If the antigenic protein is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. I~owever, Iiposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA
technology.
See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).
The formulation herein can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., f lms, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT ~ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
ELISA Assay An agent for detecting an analyte protein is an antibody capable of binding to an analyte protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F~ab)2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term "biological sample", therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. I~ 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;
"hnmunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-an analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

NOVX Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a NOVX protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector, wherein additional DNA
segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequences) 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 axe described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NOVX
proteins, mutant forms of NOVX proteins, fusion proteins, etc. ).
The recombinant expression vectors of the invention can be designed for expression of NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX
proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSTON TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated ih vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a Iigand in amity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL
(New England Biolabs, Beverly, Mass.) and pRITS (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 (Amrarm et al., (1988) Gene 69:301-315) and pET 11 d (Studier et al., GENE
EXPRESSION
TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
(1990) 60-89).
~o 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) I 19-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E.
coli (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2I 18). 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 al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30:
933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
Alternatively, NOVX can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:
2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987.
EMBO
J. 6: I87-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Gold 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 axe used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987.
Genes Dev. 1:
268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immuhol.
43:
235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J.

8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740;
Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. TISA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the marine hox promoters (Kessel and Grass, 1990. Science 249:
374-379) and the a-fetoprotein promoter-(Campes and Tilghman, 1989. Genes Dev.
3:
537-546).
The invention further provides a recombinant expression vector comprising a DNA
molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to NOVX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high eff ciency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub, et al., "Antisense RNA as a molecular tool for genetic analysis," Reviews-Treads i~c Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell"
and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, NOVX
protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
~2 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, DEAF-dextran-mediated transfection, Iipofection, or electroporation. Suitable methods for transforming or txansfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as 6418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding NOVX or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) NOVX protein. Accordingly, the invention further provides methods for producing NOVX protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NOVX protein has been introduced) in a suitable medium such that NOVX protein is produced. In another embodiment, the method further comprises isolating NOVX protein from the medium or the host cell.
Transgenic NOVX Animals The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which NOVX protein-coding sequences have been introduced.
Such host cells can then be used to create non-human transgenic animals in which exogenous NOVX sequences have been introduced into their genome or homologous recombinant animals in which endogenous NOVX sequences have been altered. Such animals are useful for studying the function and/or activity of NOVX protein and for identifying and/or evaluating modulators of NOVX protein activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NOVX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
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 N0:2rc-1, wherein n is an integer between 1 and 174, can be introduced as a transgene into the genome of a non-human animal.
Alternatively, a non-human homologue of the human NOVX gene, such as a mouse NOVX
gene, can be isolated based on hybridization to the human NOVX cDNA (described further supra) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A
tissue-specific regulatory sequences) 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 N0:2n-1, wherein n is an integer between 1 and 174), 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 N0:2n-1, wherein n is an integer between 1 and 174, can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NOVX gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NOVX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NOVX protein). In the homologous recombination vector, the altered portion of the NOVX gene is flanked at its 5'- and 3'-termini by additional nucleic acid of the NOVX gene to allow for homologous recombination to occur between the exogenous NOVX
gene carried by the vector and an endogenous NOVX gene in an embryonic stem cell. The additional flanking NOVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA
(both at the 5'- and 3'-termini) are included in the vector. See, e.g., Thomas, et al., 1987. Cell 51: 503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NOVX gene has homologously-recombined with the endogenous NOVX gene are selected.
See, e.g., Li, et al., 1992. Cell 69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND
EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp.
113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells 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. Carrr. Opin.
Biotechnol. 2:
~5 823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO
92/0968;
and WO 93104169.
In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, See, e.g., Lakso, et al., 1992. Proc. Natl. Acad.
Sci. USA 89:
6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al., 1991. Science 251:1351-1355.
If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required.
Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al., 1997. Nature 385: 810-813. In brief, a cell (e.g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The quiescent cell can.then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell (e.g., the somatic cell) is isolated.
Pharmaceutical Compositions ' The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies (also referred to herein as "active compounds") of the invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5%

human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components:
a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate;
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 ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a NOVX protein or anti-NOVX antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier.
They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch;
a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide;
a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal ~s sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal suspensions (including Iiposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No.
5,328,470) or by stereotactic injection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci.
USA 91:
3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Screening and Detection Methods The isolated nucleic acid molecules of the invention can be used to express NOVX
protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect NOVX mRNA (e.g., in a biological sample) or a genetic lesion in a NOVX gene, and to modulate NOVX activity, as described further, below. In addition, the NOVX proteins can be used to screen drugs or compounds that modulate the NOVX protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of NOVX protein or production of NOVX protein forms that have decreased or aberrant activity compared to NOVX wild-type protein (e.g.; diabetes (regulates insulin release);
obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias. In addition, the anti-NOVX antibodies of the invention can be used to detect and isolate NOVX proteins and modulate NOVX activity. In yet a further aspect, the invention can be used in methods to influence appetite, absorption of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.
The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.
Screening Assays The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i. e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOVX
protein activity.
The invention also includes compounds identified in the screening assays described herein.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a NOVX
protein or polypeptide or biologically-active portion thereof. The test compounds of the invention can be obtained using any of the numerous approaches in combinatorial libraxy 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 so 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 S 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 andlor biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. U.SA. 90: 6909;
Erb, ~et al., 1994.
Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med.
Chem. 37: 2678;
Cho, et al., 1993. Science 26I : 1303; Carrell, et al., 1994. Angew. Chem.
Int. Ed. Engl. 33:
2059; Carell, et al., 1994. Augew. Chem. lut. Ed. Engl. 33: 2061; and Gallop, et al., 1994. J.
Med. Chem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992.
Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: SSS-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al., 1992. P~oc. Natl. Acad. Sci.
USA 89:
1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sei. ZLS.A. 87: 6378-6382; Felici, 1991.
J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to a NOVX protein determined. The cell, for example, can of mammalian origin or a yeast cell.
Determining the ability of the test compound to bind to the NOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic Iabel such that binding of the test compound to the NOVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with lash 3sS~ I4C~ or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by s1 determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of NOVX
protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NOVX
protein, wherein determining the ability of the test compound to interact with a NOVX
protein comprises determining the ability of the test compound to preferentially bind to NOVX protein or a biologically-active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the NOVX
protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the NOVX protein to bind to or interact with a NOVX
target molecule. As used herein, a "target molecule" is a molecule with which a NOVX
protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a NOVX interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. A NOVX target molecule can be a non-NOVX molecule or a NOVX protein or polypeptide of the invention. In one embodiment, a NOVX target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound NOVX molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with NOVX.
Determining the ability of the NOVX protein to bind to or interact with a NOVX
target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the NOVX protein to bind to or interact with a NOVX taxget molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i. e.
intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a NOVX-responsive s2 regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay comprising contacting a NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to bind to the NOVX protein or biologically-active portion thereof. Binding of the test compound to the NOVX
protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX or biologically-active portion thereof as compared to the known compound.
In still another embodiment, an assay is a cell-free assay comprising contacting NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX can be accomplished, for example, by determining the ability of the NOVX protein to bind to a NOVX target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of NOVX
protein can be accomplished by determining the ability of the NOVX protein further modulate a NOVX
target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as described, supra.
In yet another embodiment, the cell-free assay comprises contacting the NOVX
protein or biologically-active portion thereof with a known compound which binds NOVX
protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the NOVX protein to preferentially bind to or modulate the activity of a NOVX target molecule.
The cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of NOVX protein. In the case of cell-free assays comprising the membrane-bound form of NOVX protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of NOVX protein is maintained in solution.
Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton~ X-100, Triton~ X-114, Thesit~, Isotridecypoly(ethylene glycol ether)", N-dodecyl--N,N-dimethyl-3-ammonia-1-propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either NOVX protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to NOVX
protein, or interaction of NOVX protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants.
Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-NOVX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or NOVX protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra. Alternatively, the complexes can be dissociated from the matrix, and the level of NOVX protein binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the NOVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated NOVX protein or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX protein or target molecules, but which do not interfere with binding of the NOVX protein to its target molecule, can be derivatized to the wells of the plate, and unbound target or NOVX protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the NOVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NOVX protein or target molecule.
In another embodiment, modulators of NOVX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of NOVX
mRNA or protein in the cell is determined. The level of expression of NOVX
mRNA or protein in the presence of the candidate compound is compared to the level of expression of NOVX mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of NOVX mRNA or protein expression based upon this comparison. For example, when expression of NOVX mRNA or protein is greater (i. e., statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of NOVX
mRNA or protein expression. Alternatively, when expression of NOVX mRNA or protein is ,less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of NOVX mRNA or protein expression.
The level of NOVX mRNA or protein expression in the cells can be determined by methods described herein for detecting NOVX mRNA or protein.
In yet another aspect of the invention, the NOVX proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317;
Zervos, et al., 1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Chem.
268:
12046-12054; Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993.
Oncogehe 8: 1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with NOVX ("NOVX-binding proteins" or "NOVX-by") 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 ss unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey"
proteins are able to interact, in vivo, forming a NOVX-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with NOVX.
The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
Detection Assays Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing);
and (iii) aid in forensic identification of a biological sample. Some ~of these applications are described in the subsections, below.
Chromosome Mapping Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the NOVX
sequences of SEQ ID N0:2~-1, wherein n is an integer between 1 and 174, or fragments or derivatives thereof, can be used to map the location of the NOVX genes, respectively, on a chromosome.
The mapping of the NOVX sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 by in length) from the NOVX sequences. Computer analysis of the NOVX, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes.
Only those hybrids containing the human gene corresponding to the NOVX
sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. See, e.g., D'Eustachio, et al., 1983. Scieyzce 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 ih 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 S00 or 600 bases.
However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see, Verma, et al., HUMAN
CHROMOSOMES:
A MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988).
Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
s~

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland; et al., 1987. Nature, 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NOVX gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
Tissue Typing The NOVX sequences of the invention can also be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences of the invention are useful as additional DNA
markers for RFLP ("restriction fragment length polymorphisms," described in U.S. Patent No.
5,272,057).
Furthermore, the sequences of the invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the NOVX sequences described herein can be used to prepare two PCR primers from the 5'- and 3'-termini of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identif cations, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the invention can be used to obtain such identification sequences from individuals and from tissue. The NOVX
sequences of the invention uniquely represent portions of the human genome.
Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater ss degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisrns (RFLPs).
Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If coding sequences, such as those of SEQ ID
NO:~n-1, wherein n is an integer between 1 and 174, are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
Predictive Medicine The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the invention relates to diagnostic assays for determining NOVX
protein and/or nucleic acid expression as well as NOVX activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant NOVX
expression or activity. The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, mutations in a NOVX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity.
Another aspect of the invention provides methods for determining NOVX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics").

Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.) Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX in clinical trials.
These and other agents are described in further detail in the following sections.
Diagnostic Assays An exemplary method for detecting the presence or absence of NOVX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting NOVX
protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is detected in the biological sample. An agent for detecting NOVX mRNA
or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX
mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length NOVX
nucleic acid, such as the nucleic acid of SEQ ID NO:2~-1, wherein n is an integer between 1 and 174, or a portion thereof, such as an oligonucleotide of at least 1 S, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.
An agent for detecting NOVX protein is an antibody capable of binding to NOVX
protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')Z) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-Labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample i~z vitro as well as in vivo.
For example, in vitro techniques for detection of NOVX mRNA include Northern hybridizations and in situ hybridizations. I~ vitro techniques for detection of NOVX protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques fox 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 farther 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 fox 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 Ieast one of an alteration affecting the integrity of a gene encoding a NOVX-protein, or the misexpression of the NOVX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from a NOVX gene; (ii) an addition of one or more nucleotides to a NOVX gene; (iii) a substitution of one or more nucleotides of a NOVX gene, (iv) a chromosomal rearrangement of a NOVX gene; (v) an alteration in the level of a messenger RNA transcript of a NOVX gene, (vi) aberrant modification of a NOVX gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild-type splicing pattern of a messenger RNA transcript of a NOVX gene, (viii) a non-wild-type level of a NOVX protein, (ix) allelic loss of a NOVX gene, and (x) inappropriate post-translational modification of a NOVX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in a NOVX
gene. A
preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et al., 1988. Science 241: 1077-1080; and Nakazawa, et al., 1994.
Proc. Natl.
Acad. Sci. USA 91: 360-364), the latter of which can be particularly useful for detecting point mutations in the NOVX-gene (see, Abravaya, et al., 1995. Nucl. Acids Res. 23:
675-682).
This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to a NOVX gene under conditions such that hybridization and amplification of the NOVX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al., 1990. Proc. Natl. Acaa'. Sci. USA 87: 1874-1878), transcriptional amplification system (see, I~woh, et al., 1989. Proc. Natl. Acad. Sci. USA 86:
1173-1177);
Q(3 Replicase (see, Lizardi, et al, 1988. BioTechuology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in a NOVX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA
indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Patent No. 5,493,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in NOVX can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays containing hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al., 1996. Human Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For example, genetic mutations in NOVX can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, et al., supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the NOVX gene and detect mutations by comparing the sequence of the sample NOVX with the corresponding wild-type (control) sequence.
Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc.
Natl. Acad. Sci.
USA 74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Naeve, et al., 1995. Biotechniques 19: 448), including sequencing by mass spectrometry (see, e.g., PCT
International Publication No. WO 94/16101; Cohen, et al., 1996. Adv.
Chromatography 36:
127-162; and Griffin, et al., 1993. Appl. Biochem. Biotechnol. 38: 147-159).
Other methods for detecting mutations in the NOVX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA
or RNA/DNA heteroduplexes. See, e.g., Myers, et al., 1985. Science 230: 1242. In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to 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 I nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, et al., 1988. Proc.

Natl. Acad. Sci. USA 85: 4397; Saleeba, et al., 1992. Methods Enzymol. 217:
286-295. In an embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA
mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in NOVX cDNAs obtained from samples of cells. For example, the mutt enzyme of E.
coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T
at G/T mismatches. See, e.g., Hsu, et al., 1994. Carcinogenesis 15: 1657-1662.
According to an exemplary embodiment, a probe based on a NOVX sequence, e.g., a wild-type NOVX
sequence, is hybridized to a cDNA or other DNA product from a test cell(s).
The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in NOVX genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids. See, e.g., Orita, et al., 1989. Proc. Natl. Acad. Sci. USA: 86:
2766; Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-79.
Single-stranded DNA fragments of sample and control NOVX nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA
(rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility.
See, e.g., Keen, et al., 1991. Trends Genet. 7: 5.
In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE). See, e.g., Myers, et al., 1985. Nature 313: 495.
When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 by 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. Chern. 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). Tn addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection. See, e.g., Gasparini, et al., 1992. Mol. Cell Probes 6: 1. It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification. See, e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189. In such cases, ligation will occur only if there is a perfect match at the 3'-terminus of the 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
'The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which 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.
Pharmacogenonnics Agents, or modulators that have a stimulatory or inhibitory effect on NOVX
activity (e.g., NOVX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders. The disorders include but are not limited to, e.g., those diseases, disorders and conditions listed above, and more particularly include those diseases, disorders, or conditions associated with homologs of a NOVX protein, such as those summarized in Table A.
In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype.
Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of NOVX protein, expression of NOVX
nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agents) 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;
Linden 1997. Clih.
Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT
2) and cytochrome pregnancy zone protein precursor enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizes (EM) and poor metabolizes (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
Thus, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agents) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polyrnorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a NOVX modulator, such as a modulator identified by one of the exemplary screening assays described herein.
Monitoring of Effects During Clinical Trials Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase NOVX gene expression, protein levels, or upregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting decreased NOVX gene expression, protein levels, or downregulated NOVX activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease NOVX gene expression, protein.levels, or downregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting increased NOVX gene expression, protein levels, or upregulated NOVX activity.
In such clinical trials, the expression or activity of NOVX and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be. used as a "read out" or markers of the immune responsiveness of a particular cell.
By way of example, and not of limitation, genes, including NOVX, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates NOVX activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of NOVX and other genes implicated in the disorder. The levels of gene expression (i. e. , a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of NOVX or other genes. In this manner, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a NOVX protein, mRNA, or genomic DNA in the preadministration sample;
(iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the NOVX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the NOVX
protein, mRNA, or genomic DNA in the pre-administration sample with the NOVX
protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of NOVX
to higher levels than detected, i.e., to increase the effectiveness of the agent.
Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of NOVX to lower levels than detected, i.e., to decrease the effectiveness of the agent.
Methods of Treatment The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant NOVX expression or activity. The disorders include but are not limited to, e.g., those diseases, disorders and conditions listed above, and more particularly include those diseases, disorders, or conditions associated with homologs of a NOVX protein, such as those summarized in Table A.
These methods of treatment will be discussed more fully, below.

Diseases and Disorders Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner.
Therapeutics that may be utilized include, but are not limited to: (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (iv) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" (i. e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to "knockout" endogenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989. Science 244: 1288-1292); or (v) modulators ( i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between an aforementioned peptide and its binding partner.
Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) Levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide andlor RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of an aforementioned peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like).
Prophylactic Methods In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NOVA expression or activity, by administering to the subject an agent that modulates NOVX expression or at least one NOVA
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 o~ symptoms characteristic of the NOVX aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending upon the type of NOVX aberrancy, for example, a NOVX agonist or NOVX antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. The prophylactic methods of the invention are further discussed in the following subsections.
Therapeutic Methods Another aspect of the invention pertains to methods of modulating NOVX
expression or activity for therapeutic purposes. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of NOVX protein activity associated with the cell. An agent that modulates NOVX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a NOVX protein, a peptide, a NOVX peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more NOVX protein activity.
Examples of such stimulatory agents include active NOVX protein and a nucleic acid molecule encoding NOVX that has been introduced into the cell. In another embodiment, the agent inhibits one or more NOVX protein activity. Examples of such inhibitory agents include antisense NOVX nucleic acid molecules and anti-NOVX antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, irz 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 identif ed 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 ox activity.
Stimulation of NOVX activity is desirable ih 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 Tn various embodiments of the invention, suitable in vitro or ih vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue.
Tn various specific embodiments, in vitro assays may be performed with representative cells of the types) involved in the patient's disorder, to determine if a given Therapeutic exerts the desired effect upon the cell type(s). Compounds for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model system known in the art may be used prior to administration to human subjects.
Prophylactic and Therapeutic Uses of the Compositions of the Invention The NOVX nucleic acids and proteins of the invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders. The disorders include but are not limited to, e.g., those diseases, disorders and conditions listed above, and more particularly include those diseases, disorders, or conditions associated with homologs of a NOVX protein, such as those summarized in Table A.
As an example, a cDNA encoding the NOVX protein of the invention may be useful in gene therapy, and the protein may be useful when administered to a subj ect in need thereof. By way of non-limiting example, the compositions of the invention will have efficacy for treatment of patients suffering from diseases, disorders, conditions and the like, including but not limited to those listed herein.
Both the novel nucleic acid encoding the NOVX protein, and the NOVX protein of the invention, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. A
further use could be as an anti-bacterial molecule (i.e., some peptides have been found to possess anti-bacterial properties). These materials are further useful in the generation of antibodies, which immunospecifically-bind to the novel substances of the invention for use in therapeutic or diagnostic methods.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES
Example A: Polynucleotide and Polypeptide Sequences, and Homology Data Example 1.
The NOV 1 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 1 A.
_ Table 1A. NOV1 Sequence Analysis OVIa, CG103910-02 SEQ ID NO: 1 _ ' 1224 bp~Y,y _ NA Sequence ORF Start: ATG at l ~ ~ORF Stop: TGA at 1041 TTTTGGGCTTGCCCCACCGCCCG
GCGGCGGGCCCGGCGGCCAGGGCTTCTCCTACCCCTACAAGGCCGTCTTCAGTACCC
'CTGGCCAGCCTGCAAGATAGCCATTTCCTCACCGACGCCGACATGGTCATGAGCTTC
GGAACATGACAAGGAATTCTTCCACCCACGCTACCACCATCGAGAGTTCCGGTTTGA
..TCCCAGAAGGGGAAGCTGTCACGGCAGCCGAATTCCGGATCTACAAGGACTACATCC
GACAATGAGACGTTCCGGATCAGCGTTTATCAGGTGCTCCAGGAGCACTTGGGCAGG
CACTGGGTGGTCAATCCGCGGCACAACCTGGGCCTGCAGCTCTCGGTGG
CATCAACCCCAAGTTGGCGGGCCTGATTGGGCGGCACGGGCCCCAGAAC
CAGAACCGCTCCAAGACGCCCAAGAACCAGGAAGCCCTGCGGATGGCCAACG
CATCTCCGTCCTCTACTTCGATGACAGCTCCAACGTCATCCTGAAGAAATACAGAAACATGGTGGTCC
GGGCCTGTGGCTGCCACTAGCTCCTCCGAGAATTCAGACCCTTTGGGGCCAAGTTTTTCTGGATCCTC
CATTGCTCGCCTTGGCCAGGAACCAGCAGACCAACTGCCTTTTGTGAGACCTTCCCCTCCCTATCCCC
la, CG103910-02 ~SEQ ID NO: 2 X347 as BMW at 39545.6kD
RSLRAAAPHSFVALWAPLFLLRSALADFSLDNEVHSSFIHRRLRSQERREMQREILSILGLPHRP
LQGKHNSAPMFMLDLYNAMAVEEGGGPGGQGFSYPYKAVFSTQGPPLASLQDSHFLTDADMVMSF
VEHDKEFFHPRYHHREFRFDLSKIPEGEAVTAAEFRIYKDYIRERFDNETFRISVYQVLQEHLGR
LFLLDSRTLWASEEGWLVFDITATSNHWVVNPRHNLGLQLSVETLDGQSINPKLAGLIGRHGPQN
FMVAFFKATEVHFRSIRSTGSKQRSQNRSKTPKNQEALRMANVAGPLHQPGNGAQALLCAHAAQC
lb, CG103910-03 SEQ ID NO. 3 .1226 by Sequence ORF Start: ATG at 1 ORF Stop: TGA at 976 CT
AGATGCAGCGCGAGATCCTCTCCATTTTGGGCTTGCCCCACCGCCCG
CACAACTCGGCACCCATGTTCATGCTGGACCTGTACAACGCCATGGC
CGGCGGCCAGGGCTTCTCCTACCCCTACAAGGCCGTCTTCAGTACCC
TGCAAGATAGCCATTTCCTCACCGACGCCGACATGGTCATGAGCTTC
TCTTTCCAAGATCCCAGAAGGGGAAGCTGTCACGGCAGCCGAATTCCGGATCTACAAGGACTACATCC
GGGAACGCTTCGACAATGAGACGTTCCGGATCAGCGTTTATCAGGTGCTCCAGGAGCACTTGGGCAGG
GAATCGGATCTCTTCCTGCTCGACAGCCGTACCCTCTGGGCCTCGGAGGAGGGCTGGCTGGTGTTTGA
CATCACAGCCACCAGCAACCACTGGGTGGTCAATCCGCGGCACAACCTGGGCCTGCAGCTCTCGGTGG
AGACGCTGGA'T'GGGCAGAGCATCAACCCCAAGTTGGCGGGCCTGATTGGGCGGCACGGGCCCCAGAAC
AAGCAGCCCT'Z'CATGGTGGCTTTCTTCAAGGCCACGGAGGTCCACTTCCGCAGCATCCGGTCCACGGG
GAGCAAACAGCGCAGCCAGAACCGCTCCAAGACGCCCAAGAACCAGGAAGCCCTGCGGATGGCCAACG

..TCATCGCGCCTGAAGGCTACGCCGCCTACTACTGTGAGGGGGAGTGTGCCTTCCC
Vlb, CG103910-03 ~SEQ ID NO: 4 325 as ~MW at 37269.9kD
tein Sequence RSLRAAAPHSFVALWAPLFLLRSALADFSLDNEVHSSFIHRRLRSQERREMQREILSILGLPHRP
LQGKHNSAPMFMLDLYNAMAVEEGGGPGGQGFSYPYKAVFSTQGPPLASLQDSHFLTDADMVMSF
VEHDKEFFHPRYHHREFRFDLSKIPEGEAVTAAEFRIYKDYIRERFDNETFRISVYQVLQEHLGR
LFLLDSRTLWASEEGWLVFDITATSNHWVVNPRHNLGLQLSVETLDGQSINPKLAGLIGRHGPQN
FMVAFFKATEVHFRSIRSTGSKQRSQNRSKTPKNQEALRMANVAGLDHRA
lc, CG103910-Ol ~SEQ ID NO: 5 .1878 by Sequence ORF Start: ATG at 123 ORF~Stop: TAG at 1418 GAGATGCAGCGCGAGATCCTCTCCATTTTGGGCTTGCCCCACCGCCCGCGCCCGCACCTCCA
GCACAACTCGGCACCCATGTTCATGCTGGACCTGTACAACGCCATGGCGGTGGAGGAGGGCG
CCGGCGGCCAGGGCTTCTCCTACCCCTACAAGGCCGTCTTCAGTACCCAGGGCCCCCCTCTG
CTGCAAGATAGCCATTTCCTCACCGACGCCGACATGGTCATGAGCTTCGTCAACCTCGTGGA
CAAGGAATTCTTCCACCCACGCTACCACCATCGAGAGTTCCGGTTTGATCTTTCCAAGATCC
GGGAAGCTGTCACGGCAGCCGAATTCCGGATCTACAAGGACTACATCCGGGAACGCTTCGAC
ACGTTCCGGATCAGCGTTTATCAGGTGCTCCAGGAGCACTTGGGCAGGGAATCGGATCTCTT
CCCAAGAACCAGGAAGCCCTGCGGATGGCCAACGTGGCAGAGAACAGC
CTGTAAGAAGCACGAGCTGTATGTCAGCTTCCGAGACCTGGGCTGGCA
CAACCACGCCATCGTGCAGACGCTGGTCCACTTCATCAACCCGGAAACG
CGCCCACGCAGCTCAATGCCATCTCCGTCCTCTACTTCGATGACAGCTC
TACAGAAACATGGTGGTCCGGGCCTGTGGCTGCCACTAGCTCCTCCGAG
CAAGTTTTTCTGGATCCTCCATTGCTCGCCTTGGCCAGGAACCAGCAGA
TATGGCTTTTGA
'TGCAGGCAAAACCTAGCAGGAAAAAAAAACAACGCATAAAGAAAAATGGCCGGGCCAGGTCATTGGCT
TAAATGTCACAA
lc, CG103910-O1 SEQ ID NO: 6 X431 as BMW at 49312.4kD
RSLRAAAPHSFVALWAPLFLLRSALADFSLDNEVHSSFIHRRLRSQERREMQREILSILGLPHRP
LQGKHNSAPMFMLDLYNAMA.VEEGGGPGGQGFSYPYKAVFSTQGPPLASLQDSHFLTDADMVMSF
VEHDKEFFHPRYHHREFRFDLSKIPEGEAVTAAEFRIYKDYIRERFDNETFRISVYQVLQEHLGR
LFLLDSRTLWASEEGWLVFDITATSNHWVVNPRHNLGLQLSVETLDGQSINPKLAGLIGRHGPQN
FMVAFFKATEVHFRSIRSTGSKQRSQNRSKTPKNQEALRMANVAENSSSDQRQACKKHELYVSFR
WQDWIIAPEGYAAYYCEGECAFPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQLNAISVLY
FDDS SNVILKKy'E2NMVVRACGCH
Vld, CG103910-04 ~SEQ ID NO: 7 997 bp.. _ A Sequence Ogp' Start: ATG at l4~ORF Ston. end of CACCGGATCCACCATGCACGTGCGCTCACTGCGAGCTGCGGCGCCGCACAGCTTCGTGGCGCTCTGGG

CACCCCTGTTCCTGCTGCGCTCCGCCCTGGCCGACTTCAGCCTGGACAACGAGGTGCACTCGAGCTTC
ATCCACCGGCGCCTCCGCAGCCAGGAGCGGCGGGAGATGCAGCGCGAGATCCTCTCCATTTTGGGCTT
GCCCCACCGCCCGCGCCCGCACCTCCAGGGCAAGCACAACTCGGCACCCATGTTCATGCTGGACCTGT
ACAACGCCATGGCGGTGGAGGAGGGCGGCGGGCCCGGCGGCCAGGGCTTCTCCTACCCCTACAAGGCC
GTCTTCAGTACCCAGGGCCCCCCTCTGGCCAGCCTGCAAGATAGCCATTTCCTCACCGACGCCGACAT
GGTCATGAGCTTCGTCAACCTCGTGGAACATGACAAGGAATTCTTCCACCCACGCTACCACCATCGAG
AGTTCCGGTTTGATCTTTCCAAGATCCCAGAAGGGGAAGCTGTCACGGCAGCCGAATTCCGGATCTAC
GCACTTGGGCAGGGAATCGGATCTCTTCCTGCTCGACAGCCGTACCCTCTGGGCCTCGGAGGAGGGCT
GGCTGGTGTTTGACATCACAGCCACCAGCAACCACTGGGTGGTCAATCCGCGGCACAACCTGGGCCTG
CAGCTCTCGGTGGAGACGCTGGATGGGCAGAGCATCAACCCCAAGTTGGCGGGCCTGATTGGGCGGCA
CGGGCCCCAGAACAAGCAGCCCTTCATGGTGGCTTTCTTCAAGGCCACGGAGGTCCACTTCCGCAGCA
TCCGGTCCACGGGGAGCAAACAGCGCAGCCAGAACCGCTCCAAGACGCCCAAGAACCAGGAAGCCCTG
CGGATGGCCAACGTGGCAGGACTGGATCATCGCGCC
Vld, CG103910-04 ~SEQ ID NO: 8 325 as ~MW at :ein Sequence 37269.9kD
RSLRAAAPHSFVALWAPLFLLRSALADFSLDNEVHSSFIHRRLRSQERREMQREILSILGLPHRP
LQGKHNSAPMFMLDLYNAMAVEEGGGPGGQGFSYPYKAVFSTQGPPLASLQDSHFLTDADMVMSF
VEHDKEFFHPRYHHREFRFDLSKIPEGEAVTAAEFRIYKDYIRERFDNETFRISVYQVLQEHLGR
LFLLDSRTLWASEEGWLVFDITATSNHWVVNPRHNLGLQLSVETLDGQSINPKLAGLIGRHGPQN
FMVAFFKATEVHFRSIRSTGSKQRSQNRSKTPKNQEALRMANVAGLDHRA
le, 13382317 SNP CG103910-02 ~SEQ ID NO: 9 SNP a_ t position 1193 Sequence ~pRF 4Start ATG~at l '=ORF Stop. TGA at ~~

AGCTGCGGCGCCGCACAGCTTCGTGGCGCTCTGGGCACCCCTGTTCCTGC
TTCAGCCTGGACAACGAGGTGCACTCGAGCTTCATCCACCGGCGCCTCCG
TGCAGCGCGAGATCCTCTCCATTTTGGGCTTGCCCCACCGCCCGCGCCCG
CTCGGCACCCATGTTCATGCTGGACCTGTACAACGCCATGGCGGTGGAGG
CAGGGCTTCTCCTACCCCTACAAGGCCGTCTTCAGTACCCAGGGCCCCCC
GCCATTTCCTCACCGACGCCGACATGGTCATGAGCTTCGTCAACCTCGTG
CCACCCACGCTACCACCATCGAGAGTTCCGGTTTGATCTTTCCAAGATCC
GATCAGCGTTTATCAGGTGCTCCAGGAGCACTTGGGCAGGGAATCGGATCTCTTCCTG
ACCCTCTGGGCCTCGGAGGAGGGCTGGCTGGTGTTTGACATCACAGCCACCAGCAACC
ATCCGCGGCACAACCTGGGCCTGCAGCTCTCGGTGGAGACGCTGGATGGGCAGAGCAT
GGCGGGCCTGATTGGGCGGCACGGGCCCCAGAACAAGCAGCCCTTCATGGTGGCTTTC
~TTCAGACCCTTTGGGGCCAAGTTTTTCTGGATCCTCCATTGCTCGCCTTGGCCAGGAACCAGCAGACCAA
OVle, 13382317 SNP CG103910-02SEQ ID NO: 10325 aa~SNP: No change in APHSFVALWAPLFLLRSALADFSLDNEVHSSFIHRRLRSQERREMQREILSILGLPHRP
SAPMFMLDLYNAMAVEEGGGPGGQGFSYPYKAVFSTQGPPLASLQDSHFLTDADMVMSF
FFHPRYHHREFRFDLSKIPEGEAVTAAEFRIYKDYIRERFDNETFRISVYQVLQEHLGR
RTLWASEEGWLVFDITATSNHWVVNPRHNLGLQLSVETLDGQSINPKLAGLIGRHGPQN
KATEVHFRSIRSTGSKQRSQNRSKTPKNQEALRMANVAGPLHQPGNGAQALLCAHAAQC
A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table 1B.

Table 1B. Comparison of the NOVl protein sequences.
NOVla MHVRSLRAAAPHSFVALWAPLFLLRSALADFSLDNEVHSSFIHRRLRSQERREMQREILS
NOVlb MHVRSLRAAAPHSFVALWAPLFLLRSALADFSLDNEVHSSFIHRRLRSQERREMQREILS
NOVIc MHVRSLRAAAPHSFVALWAPLFLLRSALADFSLDNEVHSSFIHRRLRSQERREMQREILS
NOVld MHVRSLRAAAPHSFVALWAPLFLLRSALADFSLDNEVHSSFIHRRZ,RSQERREMQREILS
NOVla ILGLPHRPRPHLQGKHNSAPMFMLDLYNAMAVEEGGGPGGQGFSYPYKAVFSTQGPPLAS
NOVlb ILGLPHRPRPHLQGKHNSAPMFMLDLYNAMAVEEGGGPGGQGFSYPYKAVFSTQGPPLAS
NOVlc ILGLPHRPRPHLQGKHNSAPMFMLDLYNAMAVEEGGGPGGQGFSYPYKAVFSTQGPPLAS
NOVld ILGLPHRPRPHLQGKHNSAPMFMLDLYNAMAVEEGGGPGGQGFSYPYKAVFSTQGPPLAS
NOVla LQDSHFLTDADMVMSFVNLVEHDKEFFHPRYHHREFRFDLSKIPEGEAVTAAEFRIYKDY
NOVlb LQDSHFLTDADMVMSFVNLVEHDKEFFHPRYHHREFRFDLSKIPEGEAVTAAEFRIYKDY
NOVlc LQDSHFLTDADMVMSFVNLVEHDKEFFHPRYHHREFRFDLSKIPEGEAVTAAEFRTYKDY
NOVld LQDSHFLTDADMVMSFVNLVEHDKEFFHPRYHHREFRFDLSKIPEGEAVTAAEFRIYKDY
NOVla IRERFDNETFRISVYQVLQEHLGRESDLFLLDSRTLWASEEGWLVFDITATSNHWWNPR
NOVlb IRERFDNETFRISVYQVLQEHLGRESDLFLLDSRTLWASEEGWLVFDITATSNHWVVNPR
NOVlc IRERFDNETFRISVYQVLQEHLGRESDLFLLDSRTLWASEEGWLVFDITATSNHWWNPR
NOVld IRERFDNETFRISVYQVLQEHLGRESDLFLLDSRTLWASEEGWLVFDITATSNHWVVNPR
NOVla HNLGLQLSVETLDGQSINPKLAGLIGRHGPQNKQPFMVAFFKATEVHFRSIRSTGSKQRS
NOVlb HNLGLQLSVETLDGQSINPKLAGLIGRHGPQNKQPFMVAFFKATEVHFRSIRSTGSKQRS
NOVlc HNLGLQLSVETLDGQSINPKLAGLIGRHGPQNKQPFMVAFFKATEVHFRSIRSTGSKQRS
NOVld HNLGLQLSVETLDGQSINPKLAGLIGRHGPQNKQPFMVAFFKATEVHFRSIRSTGSKQRS
NOVla QNRSKTPKNQEALRMANVAG----PLHQPGN---------------------GAQALLCA
NOVlb QNRSKTPKNQEALRMANVAG----LDHRA-------------------------------NOVlc QNRSKTPKNQEALRMANVAENSSSDQRQACKKHELYVSFRDLGWQDWIIAPEGYAAYYCE
NOVld' QNRSKTPKNQEALRMANVAG---LDHRA--------------------------------NOVla HAAQCHLRPLLR-_______-_____________-____-_-___-_-____________ NOVlb ___-___-_____-__-_-__________________________-_-______-_____ NOVlc GECAFPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQLNAISVLYFDDSSNVILKKY
NOVld _________-_-_______________________-__-_-_________________-_ NOVla -----------NOVlb -----------NOVlc RNMVVRACGCH
NOVld ---- . -----NOVla (SEQ ID N0: 2) NOVlb (SEQ ID NO: 4) NOVlc (SEQ ID N0: 6) NOVId (SEQ ID NO: 8) Further analysis of the NOV 1 a protein yielded the following properties shown in Table 1 C.
Table 1C. Protein Sequence Properties NOVla SignalP analysis: Cleavage site between residues 30 and 31 PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length 7; pos.chg 2; neg.chg 0 H-region: length 17; peak value 9.51 PSG score; 5.11 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 0.94 possible cleavage site: between 29 and 30 »> Seems to have a cleavable signal peptide (1 to 29) ALOM: Klein et al's method for TM region allocation Init position for calculation: 30 Tentative number of TMS(s) for the threshold 0.5: 0 number of TMS(s) .. fixed PERIPHERAL Likelihood = 6.10 (at 124) ALOM score: 6.10 (number of TMSs: 0) ~MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 14 Charge difference: -5.5 C(-1.5) - N( 4.0) N >= C: N-terminal side will be inside ~MTTDISC: discrimination of mitochondrial targeting seq R content: 3 Hyd Moment(75): 6.00 Hyd Moment(95): 9.57 G content: 0 D/E content: 1 S/T content: 3 ..
Score: -0.96 Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 35 LRSIAL
NUCDISC: discrimination of nuclear localization signals pat4: none pat7: none bipartite: none content of basic residues: 17..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: HVRS
none (SKL: peroxisomal targeting signal in the C-terminus: none r f ~PTS2: 2nd peroxisomal targeting signal: none I
tVAC: possible vacuolar targeting motif: none i ;RNA-binding motif: none 'Actinin-type actin-binding motif:
type l: none type 2: none NMYR: N-myristoylation pattern : none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none 'Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: cytoplasmic Reliability: 70.6 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues Final Results (k = 9/23):
44.4 %: extracellular, including cell wall 22.2 %: Golgi 11.1 0: vacuolar 11.1 0: nuclear 11.1 %: endoplasmic reticulum » prediction for CG103910-02 is exc (k=9) A search of the NOV 1 a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 1D.
los Table 1D. Geneseq Results for NOVla NOVla Identities/ 3 Geneseq Protein/Organism/Length Residues/Similarities Expect [Patent for Identifier#, Date] Match the Matched Value Residues Region ABU56730 v Lung cancer-associated 1..319 319/319 (100%)0.0 polypeptide #323 - Unidentified, 431 1..319 319/319 (100%) aa.

[WO200286443-A2, 31-OCT-2002]

AAU97017 ' Human osteogenic protein1..319 319/319 (100%)0.0 (OP-1) -Horno Sapiens, 431 aa. 1..319 319/319 (100%) [US2002049159-Al, 25-APR-2002]

AAE25993 ' Hurnan osteogenic protein1..319 319/319 (100%)0.0 1 (hOP-1) - Homo Sapiens, 431 1..319 319/319 (100%) aa.

[LTS6407060-Bl, 18-JUN-2002]

ABB82416 ' Human osteogenic protein-11..319 319/319 (100%)0.0 (OP-1) - Homo Sapiens, 431 aa. 1..319 319/319 (I00%) [W0200270029-A2, 12-SEP-2002]

AAB37614 Human OP-1 - Homo Sapiens,1..319 319/319 (100%)0.0 aa. [W0200066620-A2, 09-NOV-1..319 319/319 (100%) 2000]

Tn a BLAST search of public sequence databases, the NOV 1 a protein was found to have homology to the proteins shown in the BLASTP data in Table lE.

Table lE. Public BLASTP
Results for NOVla . _. . . ... ~~., Protein NOVla Identities/

AccessionP~otein/Organism/Length Residues/ Similarities Expect for Number Match the Matched Value Residues Portion ___ ' Q9BTB3 Similar to bone morphogenetic1..319 319/319 (100%)' 0.0 protein 7 (Osteogenic 1..319 319/319 (100%) protein 1) -Homo sapiens (Human), 412 aa.

P18075 Bone morphogenetic protein1..319 319/319 (100%)0 0 precursor (BMP-7) (Osteogenic1..319 3191319 (100%) protein 1) (OP-1) - Homo Sapiens (Human), 431 aa.

P23359 Bone morphogenetic protein1..319 309/319 (96%)~' e-180 precursor (BMP-7) (Osteogenic1..318 313/319 (97%) protein 1) (OP-1) - Mus musculus (Mouse), 430 aa.

JQ1184 osteogenic protein 1 precursor1. 319 308/319 (96%)e-179 -mouse, 430 aa. 1..318 312/319 (97%) ~

Q9I8T6 Bone morphogenetic protein39..319 246/285 (86%)e-143 7 - - ~

Gallus gallus (Chicken), 2..286 264/285 (92%) 398 as (fragment).

PFam analysis predicts that the NOV 1 a protein contains the domains shown in the Table 1 F
Table 1F. Domain Analysis of NOVla Identities/
Pfam Domain NOVla Match Region Similarities Expect Value for the Matched Region TGFb~ropeptide 37..281 104/269 (39%) 3e-1OO y 223/269 (83%) Example 2.
The NOV2 clone was analyzed, and the nucleotide and encoded polypeptide llo sequences are shown in Table 2A.

CCATACTTTCCAGTAGCTGTAGGACAATCTTACTCCTATTACTGTGACCAAAATTTTGTGACTCCT
AGGAAGTTACTGGGATTACATTCACTGCACACAAGATGGGTGGTTGCCAACAGTCCCATGCCTCAG
CATGCTCAAAATCAGATATAGAAATTGAAAATGGATTCATTTCTGAATCTTCCTCTATTTATATTT
AATAAAGAAATACAATATAAATGTAAACCAGGATATGCAACAGCAGATGGAAATTCTTCAGGTTCA
TACATGTTTGCAAAATGGATGGTCAGCACAACCAATTTGCATTAAATTTTGTGATATGCCTGTTTT
AGAATTCCAGAGCCAAGAGTAATGGCATGCGGTTTAAGCTCCATGACACATTGGACTACGAATGCT
GATGGATATGAAATCAGTTATGGAAACACCACAGGTTCCATAGTGTGTGGTGAAGATGGGTGGTCC
TTTCCCAACATGTTATAATTCTTCAGAA.AAGTGTGGGCCTCCTCCACCTATTAGCAATGGTGATAC
CCTCCTTTCTACTAAAAGTGTATGTGCCACAGTCAAGAGTCGAGTACCAATGCCAGTCCTACTATG
CACTTCTGCAGATGATCATGTCCAAGTTTGAGCTCCAAACTATGCAAGTGGCAAGACTGAAGAAG
TTAGTATCCTCAAATCA.AAATAGTTTACAAGTATCTTCAAACTTGATTTCATAGAAAAGTGTTAG
CTAAGATGGGTTT

CG106298-02 ~SEO ID NO: 12 X271 as BMW at 30635.1kD
LLTNVILTLWVSCANGQVKPCDFPDIKHGGLFHENMRRPYFPVAVGQSYSYYCDQNFVTPSGSYWD
HCTQDGWLPTVPCLRTCSKSDIEIENGFISESSSIYILNKEIQYKCKPGYATADGNSSGSITCLQN
SAQPTCTKFCDMPVFENSRAKSNGMRFKLHDTLDYECYDGYEISYGNTTGSTVCGEDGWSHFPTCY
SEKCGPPPPISNGDTTSFLLKVYVPQSRVEYQCQSYYELQGSNYVTCSNGEWSEPPRCIRIHFCR
CG106298-O1 ~SEO ID NO: 13 X2033 O ~ -A..Se.. uence,.... .- _ .~ .~ ~ S..ATG at 78 .~ ~ ~ T~ at 1812 . .. .....
......
'AATAATGAAAGATTTCAAACCCCAAACAGTGCAACTGAAACTTTTGCATTACTATACTACTGAGA
.TCTAACATGTTGTTACTAATCAATGTCATTCTGACCTTGTGGGTTTCCTGTGCTAATGGACAAGA
'GAAACCTTGTGATTTTCCAGAAATTCAACATGGAGGTCTATATTATAAGAGTTTGCGTAGACTAT
AGTTACTGGGATTACATTCATTGCACACAAGATGGTTGGTCACCAACGGTCCCATGCCTCAGAACATG
CTCAAAATCAGATGTAGA.AATTGAAAATGGATTCATTTCTGAATCTTCCTCTATTTATATTTTAAATG
AAGAAACACAATATAATTGTAAACCAGGATATGCAACAGCAGATGGAAATTCTTCAGGATCAATTACA
TGTTTGCAAA.ATGGATGGTCAACACAACCAATTTGCATTAAATTTTGTGATATGCCTGTTTTTGAGAA
TTCCAGAGCCAAGAGTAATGGCATGTGGTTTAAGCTCCATGACACATTGGACTATGAATGCTATGATG
GATATGAAAGCAGTTATGGAAACACCACAGATTCCATAGTGTGTGGTGAAGATGGCTGGTCCCATTTG
CCAACATGCTATAATTCTTCAGAAAGCTGTGGGCCTCCTCCACCTATTAGCAATGGAGATACCACGTC
CTTCCCGCAAAAAGTGTATCTGCCATGGTCAAGAGTCGAGTACCAGTGCCAGTCCTACTATGAACTTC
AGGGTTCTAAATATGTAACATGTAGTAATGGAGACTGGTCAGAACCACCAAGATGCATATCAATGAAA
CCTTGTGAGTTTCCAGAA.ATTCAACATGGACATCTATATTATGAGAATACGCGTAGACCATACTTTCC
TACATTCACTGCACACAAGATGGGTGGTTGCCAACAGTCCCATGCCTCAGAACATGCTCAAAA
TATAGAAATTGAAAATGGATTCATTTCTGAA.TCTTCCTCTATTTATATTTTAAATAAAGAAAT
ATAAATGTAA.ACCAGGATATGCAACAGCAGATGGAAATTCTTCAGGTTCAATTACATGTTTGC
GGATGGTCAGCACAACCAA,TTTGCATTAAATTTTGTGATATGCCTGTTTTTGAGAATTCCAGA
GAGTAATGGCATGCGGTTTAAGCTCCATGACACATTGGACTACGAATGCTACGATGGATATGA
GTTATGGAAACACCACAGGTTCCATAGTGTGTGGTGAAGATGGGTGGTCCCATTTCCCAACAT
AATTCTTCAGAAAAGTGTGGGCCTCCTCCACCTATTAGCAATGGTGATACCACCTCCTTTCTA
AGTGTATGTGCCACAGTCAAGAGTCGAGTACCAATGCCAGTCCTACTATGAACTTCAGGGTTC
ATGTAACATGTAGTAATGGAGAGTGGTCGGAACCACCAAGATGCATACATCCATGTATAATAA
GAAAACATGAATAAAAATAACATACAGTTAAAAGGAAAAAGTGACATAAAATATTATGCAAAA
GGATACCATTGAATTTATGTGTAAATTGGGATATAATGCGAATACATCAGTTCTATCATTTCA
TGTGTAGGGAAGGCATAGTGGAATACCCCAGATGCGAATAAGGCAGCATTGTTACCCTAAATG
CCAACTTCCACTTCTCACTCTTATGGTCTCAAAGCTTGCAAAGATAGCTTCTGATAfiTGTTGT
TART
OV2b, CG106298-O1 ~SEQ ID NO: 14 X578 as BMW at 65309.OkD
m MLLLINVILTLWVSCANGQEVKPCDFPEIQHGGLYYKSLRRLYFPAAAGQSYSYYCDQNFVTPSGSYW
DYIHCTQDGWSPTVPCLRTCSKSDVEIENGFISESSSIYILNEETQYNCKPGYATADGNSSGSITCLQ
NGWSTQPICIKFCDMPVFENSRAKSNGMWFKLHD'T'LDYECYDGYESSYGNTTDSIVCGEDGWSHLPTC
YNSSESCGPPPPTSNGDTTSFPQKVYLPWSRVEYQCQSYYELQGSKYVTCSNGDWSEPPRCISMKPCE
FPEIQHGHLYYENTRRPYFPVATGQSYSYYCDQNFVTPSGSYWDYIHCTQDGWLPTVPCLRTCSKSDT
EIENGFISESSSTYTLNKEIQYKCKPGYATADGNSSGSITCLQNGWSAQPICIKFCDMPVFENSRAKS
NGMRFKLI-~7TLDYECYDGYEISYGNTTGSIVCGEDGWSHFPTCYNSSEKCGPPPPISNGDTTSFLLKV
YVPQSRVEYQCQSYYELQGSNYVTCSNGEWSEPPRCTHPCIITEENMNKNNIQLKGKSDIKYYAKTGD
TIEFMCKLGYNANTSVLSFQAVCREGIVEYPRCE
A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table 2B.
Table 2B. Comparison of the NOV2 protein sequences.
NOV2a MLLLINVILTLWVSCANGQ-VKPCDFPDIKHGGLFHENMRRPYFPVAV6QSYSYYCDQNF
NOV2b MLLLINVILTLWVSCANGQEVKPCDFPEIQHGGLYYKSLRRLYFPAAAGQSYSYYCDQNF
NOV2a VTPSGSYWDYIHCTQDGWLPTVPCLRTCSKSDIEIENGFISESSSIYILNKEIQYKCKPG
NOV2b VTPSGSYWDYIHCTQDGWSPTVPCLRTCSKSDVEIENGFISESSSIYILNEETQYNCKPG
NOV2a YATADGNSSGSITCLQNGWSAQPICIKFCDMPVFENSRAKSNGMRFKLHDTLDYECYDGY
NOV2b YATADGNSS6SITCLQNGWSTQPICIKFCDMPVFENSRAKSNGMWFKLHDTLDYECYDGY
NOV2a EISYGNTTGSIVCGEDGWSHFPTCYNSSEKCGPPPPISNGDTTSFLLKVYVPQSRVEYQC
NOV2b ESSYGNTTDSIVCGEDGWSHLPTCYNSSESCGPPPPISNGDTTSFPQKVYLPWSRVEYQC
NOV2a QSYYELQGSNYVTCSNGEWSEPPRCIRIHFCR----------------------------NOV2b QSYYELQGSKYVTCSNGDWSEPPRCISMKPCEFPEIQHGHLYYENTRRPYFPVATGQSYS
NOV2a ________________________-___________________________-_______ NOV2b YYCDQNFVTPSGSYWDYIHCTQDGWLPTVPCLRTCSKSDIEIENGFISESSSTYILNKET
NOV2a -_________________-_________________________________-_______ NOV2b QYKCKPGYATADGNSSGSTTCLQNGWSAQPICIKFCDMPVFENSRAKSNGMRFKLHDTLD
NOV2a -_-___________________________________-___________-_-_______ NOV2b YECYDGYEISYGNTTGSIVCGEDGWSHFPTCYNSSEKCGPPPPTSNGDTTSFLLKVYVPQ
NOV2a -_-_______________________________________-_________________ NOV2b SRVEYQCQSYYELQGSNYVTCSNGEWSEPPRCIHPCIITEENMNKNNTQLKGKSDIKYYA
NOV2a ______________________________________ NOV2b KTGDTIEFMCKLGYNANTSVLSFQAVCREGIVEYPRCE
NOV2a (SEQ ID NO: 12) NOV2b (SEQ ID NO: 14) Further analysis of the NOVZa protein yielded the following properties shown in Table 2C.
Table 2C. Protein Sequence Properties NOV2a SignalP analysis: ' Cleavage site between residues 19 and 20 PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length 0; pos.chg 0; neg.chg 0 H-region: length 20; peak value 9.20 PSG score: 4.80 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 1.38 possible cleavage site: between 18 and 19 »> Seems to have a cleavable signal peptide (1 to 18) ALOM: Klein et al's method for TM region allocation Init position for calculation: 19 Tentative number of TMS(s) for the threshold 0.5: 0 number of TMS(s) .. fixed PERIPHERAL Likelihood = 8.27 (at 137) ALOM score: 8.27 (number of TMSs: O) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 9 Charge difference: 0.0 C( 1.0) - N( 1.0) N >= C: N-terminal side will be inside MITDTSC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment(75); 3.43 Hyd Moment(95): 4.91 G content: 1 D/E content: 1 S/T content: 2 Score: -5.38 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: 7.4°s 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 jmemYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none ~Dileucine motif in the tail: none (checking 63 PROSITE DNA binding motifs: none 'checking 71 PROSITE ribosomal protein motifs: none ,checking 33 PROSITE prokaryotic DNA binding motifs: none ',NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: nuclear Reliability: 76.7 ,COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues Final Results (k = 9/23):
44.4 %: extracellular, including cell wall 33.3 %: nuclear 22.2 0: mitochondrial » prediction for CG106298-02 is exc (k=9) A search of the NOV2a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 2D.

Table 2D. Geneseq Results for NOV2a NOV2a Identities/

Geneseq Protein/Organism/Length Residues/ Similarities' Expect [Patent for Identifier#, Date] Match the MatchedValue Residues Region AAY09065 ' Human complement factor 12..265 241/263 e-152 H (91%) homolog protein - Homo Sapiens,251..513 250/263 578 (94%) aa. [W09918200-A1, 15-APR-1999]

ABU07436 Protein differentially regulated10..253 135/303 9e-68 in (44%) prostate cancer #39 - Homo 312..611 171/303 Sapiens, (55%) 1231 aa. [W0200281638-A2, ~CT-2002]

AAB43738 Human cancer associated 1..265 108/270 2e-56 protein (40%) sequence SEQ ID NO:l 183 13..275 154/270 - Homo (57%) Sapiens, 342 aa. [W0200055350-Al, 21-SEP-2000]

ABB80571 Human sbg614126complfH protein1..265 113/269 2e-56 (42%) #2 - Homo sapiens, 327 aa. 1..262 154/269 (57%) [W0200222802-A1, 21-MAR-2002]

ABB80570 Human sbg614126complfH protein23 .265 102/247 Se-51 ' (41%) #1 - Homo Sapiens, 364 aa. 60..299 141/247 (56%) [W0200222802-Al, 21-MAR-2002]

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

Table 2E. Public BLASTP Results for NOV2a ~

Protein NOV2a Identities/

Accession' Protein/Organism/Length Residues/SimilaritiesExpect for Number Match the Matched Value Residues Portion -Q92496 Complement factor H-related 1..265 2SS1266~ e-160 protein (9S%) ~

4 precursor (FHR-4) - Homo 1..266 262/266 (97%) Sapiens (Human), 331 aa.

Q0298S ! Complement factor H-related1..265 172/270 (63%)e-101 protein 3 precursor (FHR-3) (H factor-like1..265 198/270 (72%) protein 3) (DOWNI6) - Homo sapiens (Human), 330 aa.

A4S222 complement factor H-related 1..265 173/269 (64%)e-101 protein DOWNI6 precursor - human, 1..266 196/269 (72%) 331 aa.

Q8ROI8 Hypothetical SB.I kDa protein10..270 150/320 (46%)6e-81 - Mus musculus (Mouse), S09 aa. 130..447 181/320 (SS%) .,~.,4,, ' .~"

,"~w Q61407 ' Complement factor H-related10..270 l SO/320 6e-81 protein - (46%) ' Mus musculus'(Mouse), 4S2 73..390 181/320 (SS%) as (fragment).

PFam analysis predicts that the NOV2a protein contains the domains shown in the Table 2F.
Table 2F. Domain Analysis of NOV2a Identities/

Pfam DomainNOV2a Match RegionSimilarities Expect Value ~

for the Matched Region Sushi 23..83 17/69 (2S%) 1.2e-08 47/69 (68%) Sushi 87..144 -22/68_(32.%) 2.2e-12..

4S/68 (66%) Sushi 148..203 22/66 (33%) 3.9e-08 44/66 (67%) Sushi 210..264 23/65 (3S%) S.4e-1S

42/65 (65%) .

Example 3.
The NOV3 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 3A.
Table 3A. NOV3 V3a, CG110590-02 (SEQ ID NO: 1S1487 by A Sequence ORF Start: ATG at I~12 ' ORF Stot~: JTGA~at 1303 CAGTGCCCAGGTTAGTGAGCAGTGCCCGGCGCCCGCTTCCCTCACCTCCTTTTCCAGCC
CTTGAAGGTTCTGTCACCTTTTGCAGTGGTCCAAATGAGAAAAAAGTGGAAAATGGGAG
ACATCTTTTCGTTGTTGTTCTTTCTTTTGCTAGAAGGAGGCAAAACAGAGCAAGTAAAA
ACATATTGCATGTTTCAAGACAAGAAGTACAGAGTGGGTGAGAGATGGCATCCTTACCT
..TGGGTTGGTTTACTGCGTGAACTGCATCTGCTCAGAGAATGGGAATGTGCTTTGCAGCC
CCAGACTCCTTACCCCCAGTGAACAATAAGGTGACCAGCAAGTCTTGCGAGTACAATGGGACAACTTA
CCAACATGGAGAGCTGTTCGTAGCTGAAGGGCTCTTTCAGAATCGGCAACCCAATCAATGCACCCAGT
GCAGCTGTTCGGAGGGAAACGTGTATTGTGGTCTCAAGACTTGCCCCAAATTAACCTGTGCCTTCCCA
GTCTCTGTTCCAGATTCCTGCTGCCGGGTATGCAGAGGAGATGGAGAACTGTCATGGGAACATTCTGA
TGGTGATATCTTCCGGCAACCTGCCAACAGAGAAGCAAGACATTCTTACCACCGCTCTCACTATGATC
CTCCACCAAGCCGACAGGCTGGAGGTCTGTCCCGCTTTCCTGGGGCCAGAAGTCACCGGGGAGCTCTT
ATGGATTCCCAGCAAGCATCAGGAACCATTGTGCAAATTGTCATCAATAACAAACACAAGCATGGACA
AGTGTGTGTTTCCAATGGAAAGACCTATTCTCATGGCGAGTCCTGGCACCCAAACCTCCGGGCATTTG
CCTGCAAGTATCCTCAAAAAATAGACGGAAAATGCTGCAAGGTGTGTCCAGGT.AAAAA
CTTCCAGGCCAAAGCTTTGACAATAAAGGATACTTCTGCGGGGAAGAAACGATGCCTG
TGTATTCATGGAGGATGGGGAGACAACCAGAAAAATAGCACTGGAGACTGAGAGACCA
TCTCCAGCACTTCCATATTGAGAAGATCTCCAAGAGGATGTTTGAGGAGCTTCCTCAC
TGACCAGAACAACCCTGAGCCAGTGGAAGATCTTCACCGAAGGAGAAGCTCAGATCAG
TTGGATAGGGTAAAGCAAGAA
CG110590-02 1SEO ID NO: 16 X397 as BMW at 44841.9kD
MRKKWKMGGMKYIFSLLFFLLLEGGKTEQVKHSETYCMFQDKKYRVGERWHPYLEPYGLVYCVNCICS
ENGNVLCSRVRCPNVHCLSPVHIPHLCCPRCPDSLPPVNNKVTSKSCEYNGTTYQHGELFVAEGLFQN
RQPNQCTQCSCSEGNVYCGLKTCPKLTCAFPVSVPDSCCRVCRGDGELSWEHSDGDIFRQPANREARH
SYHRSHYDPPPSRQAGGLSRFPGARSHRGALMDSQQASGTIVQIVINNKHKHGQVCVSNGKTYSHGES
WHPNLRAFGIVECVLCTCNVTKQECKKTHCPNRYPCKYPQKIDGKCCKVCPGKKAKELPGQSFDNKGY
FCGEETMPVYESVFMEDGETTRKIALETERPPQAFSSTSILRRSPRGCLRSFLTSSW
V3b, CGI 10590-OI SEQ ID NO 17 1440 by A Sequence ~Ogp .Start ...ATG at.l.8~ORF Stop TAG at 1374 GAA.AAAAGTGGAAAATGGGAGGCATGAAATACATCTTTTCGTTGTTGTTCTTTCTTTTGCTAGAA
.GGCAAAACAGAGCAAGTAAAACATTCAGAGACATATTGCATGTTTCAAGACAAGAAGTACAGAGT
PTGAGAGATGGCATCCTTACCTGGAACCTTATGGGTTGGTTTACTGCGTGAACTGCATCTGCTCAG
AmrrrnAmrmrrmmTGCAGCCGAGTCAGAmGmrGAAATGTTCATTGCCTTTCTCCTGTGCATATT
ATCGGCAACCCAATCAATGCACCCAGTGCAGCTGTTCGGAGGGAAACGTGTATTGTGGTCTCAAGACT
TGCCCCAAATTAACCTGTGCCTTCCCAGTCTCTGTTCCAGATTCCTGCTGCCGGGTATGCAGAGGAGA
TGGAGAACTGTCATGGGAACATTCTGATGGTGATATCTTCCGGCAACCTGCCAACAGAGAAGCAAGAC
TGTCACCAAG
TAGACGGAAA

.TGAGTCTGTATTCATGGAGGATGGGGAGACAACC
TATTGAGAAGATCTCCAAGAGGATGTTTGAGGAGCTTCCTCACTTCAAGC
.TGCAGAACAGAGCTTGAAGATTTAGTCAAGGTTTTGTACCTGGAGAGATCTGA
TTGGATAGGGTAAAGCAAGAAAACTCAAGCTGCAGCT
OV3b, CGl 10590-O1 ~SEQ ID NO: 18 X452 as BMW at 51425.SkD
LLFFLLLEGGKTEQVKHSETYCMFQDKKYRVGERWHPYLEPYGLVYCVNCICSENGNVL
HCLSPVHIPHLCCPRCPEDSLPPVNNKVTSKSCEYNGTTYQHGELFVAEGLFQNRQPNQ
NVYCGLKTCPKLTCAFPVSVPDSCCRVCRGDGELSWEHSDGDIFRQPANREARHSYHRS
HGESWHPNL
IVECVLCTCNVTKQECKKIHCPNRYPCKYPQKIDGKCCKVCPGKKAKEELPGQSFDNKGYFCGE
VYESVFMEDGETTRKIALETERPPQVEVHVWTIRKGILQHFHIEKISKRMFEELPHFKLVTRTT
KIFTEGEAQISQMCSSRVCRTELEDLVKVLYLERSEKGHC
OV3c, 13382325 SNP SEQ ID NO: 19 1487 bp, SNP T/C at 6110590-02 ". position 454 NA Sequence ; ORF Start: ATG at 112 ORF Stop: 1303 CCAGGTT
TGAGAAAAAAGTGGAAAATGGGAGGCATGA
CATCTTTTCGTTGTTGTTCTTTCTTTTGCTAGAAGGAGGCAAAACAGAGCAAGTAAAACATTCAGAG
ATTGCATGTTTCAAGACAAGAAGTACAGAGTGGGTGAGAGATGGCATCCTTACCTGGAACCTTATGG
GTTTACTGCGTGAACTGCATCTGCTCAGAGAATGGGAATGTGCTTTGCAGCCGAGTCAGATGTCCAA
ATTCCTCATCTGTGCTGCCCTCGCTGCCCAGACTCCTTACCCCCA
CAGTGCAGCTGTTCGGAGGGAAACGTGT
GCCTTCCCAGTCTCTGTTCCAGATTCCTGCTGCCGG
TTCTGATGGTGATATCTTCCGGCAACCTGCCAACAG
CATTCTTACCACCGCTCTCACTATGATCCTCCACCAAGCCGACAGGCTGGAGGTCTGTCCC
GGCCAGAAGTCACCGGGGAGCTCTTATGGATTCCCAGCAAGCATCAGGAACCATTGTGCAA
ATAACAAACACAAGCATGGACAAGTGTGTGTTTCCAATGGAAAGACCTATTCTCATGGCGA
CCAAACCTCCGGGCATTTGGCATTGTGGAGTGTGTGCTATGTACTTGTAATGTCACCAAGC
TCGATACCCCTGCAAGTATCCTCAAAAAATAGACGGAAAATGC
TGCAAGGTGTGTCCAGGTAAAAAAGCAAAAGAACTTCCAGGCCAAAGCTTTGACAATAAAGGATACTTCTG
TGAGTCTGTATTCATGGAGGATGGGGAGACAACCAGAAAAATAGCAC
CACCTCAGGCATTCTCCAGCACTTCCATATTGAGAAGATCTCCAAGAGGATGTTTG
OV3c, 13382325 SNP SEQ ID NO: 20 397, as SNP: Cys to Arg at 115 KKWKMGGMKYIFSLLFFLLLEGGKTEQVKHSETYCMFQDKKYRVGERWHPYLEPYGLVYCVNCICSENG
LCSRVRCPNVHCLSPVHTPHLCCPRCPDSLPPVNNKVTSKS_REYNGTTYQHGELFVAEGLFQNRQPNQC
CSCSEGNVYCGLKTCPKLTCAFPVSVPDSCCRVCRGDGELSWEHSDGDIFRQPANREARHSYHRSHYDP
SRQAGGLSRFPGARSHRGALMDSQQASGTTVQIVINNKHKHGQVCVSNGKTYSHGESWHPNLRAFGIVE
LCTCNVTKQECKKIHCPNRYPCKYPQKIDGKCCKVCPGKKAKELPGQSFDNKGYFCGEETMPVYESVFM
GETTRKIALETERPPQAFSSTSILRRSPRGCLRSFLTSSW
OV3d, 13382326 SNP SEQ ID NO 21 1440 by SNP: A/G at NA Sequence ORF Start: ATG at ORF Stop: end of 112 seauence 11s TGA
ACATCTTTTCGTTGTTGTTCTTTCTTTTGCTAGAAGGAGGCAAAACAGAGCAAGTAAAACATTCAGAG
TATTGCATGTTTCAAGACAAGAAGTACAGAGTGGGTGAGAGATGGCATCCTTACCTGGAACCTTATGG
TGTGCTTTGCAGCCGAGTCAGATGTCCAA
TGTTCATTGCCTTTCTCCTGTGCATATTCCTCATCTGTGCTGCCCTCGCTGCCCAGACTCCTTACCCCCA
TGGAGAGCTGTTCGT
TCAATGCACCCAGTGCAGCTGTTCGGAGGGAAACGTGT
TGGGAACATTCTGATGGTGATATCTTCCGGCAACCTGCCAACAG
TCAGGAACCATTGTGCAA
TGGAAAGACCTATTCTCATGGCGA
TGTCACCAAGC
TACTTCTG
ATTCATGGAGGATGGGGAGACAACCAGAAAAATAGCAC
CACCTCAGGCATTCTCCAGCACTTCCATATTGAGAAGATCTCCAAGAGGATGTTTG
.TCTGAAAAGGGCCACTGTTAGGCAAGACAGACAGTATTGGATAGGGTAAAGCAAGAA
V3d, 13382326 SNP ~SEQ ID NO: 22 397 as SNP: No change in protein 110590-02 see~uence RKKWKMGGMKYIFSLLFFLLLEGGKTEQVKHSETYCMFQDKKYRVGERWHPYLEPYGLVYCVNCICSENG
VLCSRVRCPNVHCLSPVHIPHLCCPRCPDSLPPVNNKVTSKSCEYNGTTYQHGELFVAEGLFQNRQPNQC
QCSCSEGNVYCGLKTCPKLTCAFPVSVPDSCCRVCRGDGELSWEHSDGDIFRQPANREARHSYHRSHYDP
_PSRQAGGLSRFPGARSHRGALMDSQQASGTIVQIVINNKHKHGQVCVSNGKTYSHGESWHPNLRAFGIVE
VLCTCNVTKQECKKIHCPNRYPCKYPQKIDGKCCKVCPGKKAKELPGQSFDNKGYFCGEETMPVYESVFM
PRGCLRSFLTSSW
A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table 3B.

Table 3B. Comparison of the NOV3 protein sequences.
NOV3a MRKKWKMGGMKYIFSLLFFLLLEGGKTEQVKHSETYCMFQDKKYRVGERWHPYLEPYGLV
NOV3b ------MGGMKYIFSLLFFLLLEGGKTEQVKHSETYCMFQDKKYRVGERWHPYLEPYGLV
NOV3a YCVNCICSENGNVLCSRVRCPNVHCLSPVHIPHLCCPRCP-DSLPPVNNKVTSKSCEYNG
NOV3b YCVNCICSENGNVLCSRVRCPNVHCLSPVHIPHLCCPRCPEDSLPPVNNKVTSKSCEYNG
NOV3a TTYQHGELFVAEGLFQNRQPNQCTQCSCSEGNVYCGLKTCPKLTCAFPVSVPDSCCRVCR
NOV3b TTYQHGELFVAEGLFQNRQPNQCTQCSCSEGNVYCGLKTCPKLTCAFPVSVPDSCCRVCR
NOV3a GDGELSWEHSDGDIFRQPANREARHSYHRSHYDPPPSRQAGGLSRFPGARSHRGALMDSQ
NOV3b GDGELSWEHSDGDIFRQPANREARHSYHRSHYDPPPSRQAGGLSRFPGARSHRGALMDSQ
NOV3a QASGTIVQIVINNKHKHGQVCVSNGKTYSHGESWHPNLRAFGIVECVLCTCNVTKQECKK
NOV3b QASGTIVQIVINNKHKHGQVCVSNGKTYSHGESWHPNLRAFGIVECVLCTCNVTKQECKK
NOV3a IHCPNRYPCKYPQKIDGKCCKVCPGKKAK-ELPGQSFDNKGYFCGEETMPVYESVFMEDG
NOV3b IHCPNRYPCKYPQKIDGKCCKVCPGKKAKEELPGQSFDNKGYFCGEETMPVYESVFMEDG
NOV3a ETTRKIALETERPP--------------QAFSSTSILRRS----PRGCLRS-FLTSSW--NOV3b ETTRKIALETERPPQVEVHVWTTRKGILQHFHIEKISKRMFEELPHFKLVTRTTLSQWKI
NOV3a ______________________________________ NOV3b FTEGEAQISQMCSSRVCRTELEDLVKVLYLERSEKGHC
NOV3a (SEQ TD NO: 16) NOV3b (SEQ ID NO: 18) Further analysis of the NOV3a protein yielded the following properties shown in Table 3C.
Table 3C. Protein Sequence Properties NOV3a SignalP analysis: Cleavage site between residues 2S and 29 PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length 11; pos.chg 5; neg.chg 0 H-region: length 11; peak value 12.14 PSG score: 7.74 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -1.64 possible cleavage site: between 27 and ~8 »> 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: 0 number of TMS(s) .. fixed PERIPHERAL Likelihood = 1.80 (at 277) ALOM score: 1.80 (number of TMSs: 0) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 13 Charge difference: -6.5 C(-0.5) - N( 6.0) N >= C: N-terminal side will be inside MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment(75): 6.12 Hyd Moment(95): 9.66 G content: 2 D/E content: 1 S/T content: 1 Score: -4.32 Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 12 MRK~KW
NUCDISC: discrimination of nuclear localization signals pat4: none pat7: PGKKAKE (4) at 323 j bipartite: none content of basic residues: 13.1%
NLS Score: -0.13 [KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals:
XXRR-like motif in the N-terminus: RKKW
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 fActinin-type actin-binding motif:
type 1: none type 2: none ~NMYR: N-myristoylation pattern : none ~Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none 'Dileucine motif in the tail: none ',checking 63 PROSITE DNA binding motifs: none ',checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: nuclear Reliability: 94.1 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues Final Results (k = 9/23):
66.7 0: extracellular, including cell wall 11.1 s: mitochondrial ll.l o: vacuolar 11.1 0: nuclear » prediction for CG110590-02 is exc (k=9) A search of the NOV3a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 3D.
Table 3D. Geneseq Results for NOV3a NOV3a Identities/

Geneseq Protein/Organism/Length Residues/SimilaritiesExpect [Patent ~ for Identifier#, Date] Match the Matched Value Residues Region AAY53035 Human secreted protein clone1..373 373/374 (99%)0.0 dw665_4 protein sequence 1..374 3731374 (99%) SEQ ID

N0:76 - Homo Sapiens, 457 aa.

[WO9957132-A1, I1-NOV-1999]

AAY82777 Human chordin related protein1..373 373/374 (99%)0.0 (Clone ' dw665_4) - Homo Sapiens, 1..374 373/374 (99%) 457 aa.

[W0200009551-Al, 24-FEB-2000]

AAM39408 Human polypeptide SEQ ID 1..373 371/375 (98%)0.0 - Homo Sapiens, 458 aa. 1..375 372/375 (98%) [W0200153312-AI, 26-JUL-2001]

AAB65027 Gene #1 associated peptide 1..373 368/374 (98%)0.0 #2 -Homo Sapiens, 489 aa. 37..406 369/374 (98%) [W0200075375-Al, 14-DEC-2000]

AAB64993 Human secreted protein #1 1..373 368/374 (98%)0.0 - Homo Sapiens, 453 aa. [W0200075375-AI,1..370 369/374 (98%) 14-DEC-2000]

In a BLAST search of public sequence databases, the NOV3a protein was found to have homology to the proteins shown in the BLASTP data in Table 3E.
Table 3E. Public BLASTP Results for NOV3a Protein NOV3a Identities/

Accession Protein/Organism/Length Residues/ Similarities ' Expect for Number Match the Matched Value Residues Portion Q9BLJ40 Neuralin precursor (Ventroptin)7..373 367/367 (100%)0.0 -Homo Sapiens (Human), 1..367 367/367 (100%) 450 aa.

,. CAC43868Sequence 7 from Patent 7..373 367/369 (99%)0.0 W00142465 precursor - 1..369 367/369 (99%) Homo Sapiens (Human), 452 aa.

GAC43869 ' Sequence 11 from Patent7..373 362/368 (98%)0.0 W00142465 precursor - 1..364 363/368 (98%) Homo Sapiens (Human), 447 aa.

(fragment).

t Q920C1 Neuralin precursor (Ventroptin)7..373 334/368 (90%)0.0 -Mus musculus (Mouse), 1..364 351/368 (94%) 447 aa.

CAC43867 Sequence 4 from Patent 7..377 327/372 (87%)0.0 WO0142465 precursor - 1..368 346/372 (92%) Rattus norvegicus (Rat), 382 aa.

PFam analysis predicts that the NOV3a protein contains the domains shown in the Table 3F.
Table 3F. Domain Analysis of NOV3a Identities/

Pfam DomainNOV3a Match Region ~ Similarities Expect Value for the Matched Region Vwc 37..99 25/84 (30%) ~ ~l.Se-10 39/84 (46%) ~

Vwc 115..178 8e-09 26/90 (29%) ~

48/90 (53%) Vwc 260..322 27/84 (32%) l.Se-11 ~. . .. .. .... _.. . .. . .. .... ~ . 41/84... . . .
(49%) .....

Example 4.
The NOV4 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shoum in Table 4A.
Table 4A. NOV4 4a, CG114SSS-O1 ~SEQ, ID NO: 231710 by Sequence O~' Start:~ATG at 14 ORF Stop TAA at 1534 TAGGAATTCCAAGGAACTGGGCCTAGTTCCCCTCACAG
CG
'GGGACGCTTCATCATGGGCATAGATGGAGGCGTCGCCCTCAGTGTGCTCCCCATGT
.TCTCACCCAAGGAGATCCGTGGCTCTCTGGGGCAGGTGACTGCCATCTTTATCTGC
ACTGGGCAGCTTCTGGGCCTGCCCGAGCTGCTGGGAAAGGAGAGTACCTGGCCATA
'GATTGTGGTCCCTGCCGTTGTCCAGCTGCTGAGCCTTCCCTTTCTCCCGGACAGCC
CGTCCGCTGGCAGGTGGTCACCGTGATTGTCACCA
TTTGGTTCTATACCAACAGCATCTTTGGAAAAGCT
CTTCTCTGGTTTGGTCATTGAGCACCTGGGACGGAGACCCCTCCTCATTGGTGGCTTTGGGCTCATGG
GCCTCTTCTTTGGGACCCTCACCATCACGCTGACCCTGCAGGACCACGCCCCCTGGGTCCCCTACCTG
AGTATCGTGGGCATTCTGGCCATCATCGCCTCTTTCTGCAGTGGGCCAGGTGGCATCCCGTTCATCTT
GACTGGTGAGTTCTTCCAGCAATCTCAGCGGCCGGCTGCCTTCATCATTGCAGGCACCGTCAACTGGC
TCTCCAACTTTGCTGTTGGGCTCCTCTTCCCATTCATTCAGAAAAGTCTGGACACCTACTGTTTCCTA
GTCTTTGCTACAATTTGTATCACAGGTGCTATCTACCTGTATTTTGTGCTGCCTGAGACCAAAAACAG
AACCTATGCAGAAATCAGCCAGGCATTTTCCAAAAGGAACAAAGCATACCCACCAGAAGAGAAAATCG
ACTCAGCTGTCACTGATGGTAAGATAAATGGAAGGCCTTAACAAGTTTCCTCCTCCACGTTGGACAAT
TATGTCAAAAACAGGATTGTCTACATGGATGATCTCACTTTTCAGGAAACTTAAAATTTACCCATTAT
TGGGAAGCTTAAATGAATTGAAGCTATGCAAGTCTTTTATATTATTAAATATTTAAAAGTAAACCTGT
AA
V4a, CGI I4SSS-Ol SEQ ID NO: 24 507 as MW at SS327.3kD
MARKQNRNSKELGLVPLTDDTSHAGPPGPGRALLECDHLRSGVPGGRRRKYIKAFYNESWERRHGRPI
DPDTLTLLWSVTVSIFAIGGLVGTLIVKMIGKVLGRKHTLLANNGFAISAALLMACSLQAGAFEMLIV
GRFIMGIDGGVALSVLPMYLSEISPKEIRGSLGQVTAIFICIGVFTGQLLGLPELLGKESTWPYLFGV
IWPAVVQLLSLPFLPDSPRYLLLEKIiNEARAVKAFQTFLGKADVSQEVEEVLAESRVQRSIRLVSVL
ELLRAPYVRWQVVTVIVTMACYQLCGLNAIWFYTNSIFGKAGIPLAKIPYVTLSTGGIETLAAVFSGL
VIEHLGRRPLLIGGFGLMGLFFGTLTITLTLQDHAPWVPYLSIVGILAIIASFCSGPGGIPFILTGEF
FQQSQRPAAFIIAGTVNWLSNFAVGLLFPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAE
ISQAFSKRNKAYPPEEKIDSAVTDGKINGRP
V4b, 247847074 SEQ ID NO: 2S 1203 b A Sequence per' Start at l ORF Stop: end of AATGGGTTTGCAATTTCTGCTGCATTGCTGATGGCCTGCTCGCTCCAGGCAGGAGCCTTTGAAATGC
.TCCGTGGCTCTCTGGGGCAGGTGACTGCCATCTTTATCTGCATTGG
TGGAGTGATTGTGGTCCCTGCCGTTGTCCAGCTGCTGAGCCTTCCCTTTCTCCCGGACAGCCCACGC
ACCTGCTCTTGGAGAAGCACAACGAGGCAAGAGCTGTGAAAGCCTTCCAAACGTTCTTGGGTAA.AGC
GACGTTTCCCAAGAGGTAGAGGAGGTCCTGGCTGAGAGCCGCGTGCAGAGGAGCATCCGCCTGGTGT

TGTGGCCTCAATGCAATTTGGTTCTATACCAACAGCATCTTTGGAA.AAGCTGGGAT
GATCCCATACGTCACCTTGAGTACAGGGGGCATCGAGACTTTGGCTGCCGTCTTCT
TTGAGCACCTGGGACGGAGACCCCTCCTCATTGGTGGCTTTGGGCTCATGGGCCTC
AGGTGGCATCCCGTTCATCTTGACTG
TTGCAGGCACCGTCAACTGGCTCTCC
4b,~247847074 SEQ ID NO: 26 401 as MW at 43391.7kD
LYKKAGSAAAPFTGTRKHTLLANNGFAISAALLMACSLQAGAFEMLIVGRFIMGIDGGVALSVLPMYL
SEISPKEIRGSLGQVTAIFICIGVFTGQLLGLPELLGKESTWPYLFGVIWPAWQLLSLPFLPDSPR
YLLLEKHNEARAVKAFQTFLGKADVSQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQWWIVTMA
CYQLCGLNAIWFYTNSZFGKAGIPPAKIPYVTLSTGGIETLAAV'FSGLVIEHLGRRPLLIGGFGLMGL
FFGTLTITLTLQDHAPWVPYLSIVGILAIIASFCSGPGGIPFILTGEFFQQSQRPAAFIIAGTVNWLS
NFAVGLLFPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAEISQAFLEGKGGRA
V4c, 247847070 SEQ ID NO 27 1087 by A Sequence O~' Start at 1 ORF Stop end of CGCGGCCGCCCCCTTCACCGGTACCAGGAAGCACACTTTGCTGGCCAATAATGGGTTTGCAA
GCTGCATTGCTGATGGCCTGCTCGCTCCAGGCAGGAGCCTTTGAAATGCTCATCGTGGGACGT
.TCTGCATTGGCGTGTTCACTGGGC
CCGGACAGCCCACGCTACCTGCTCTTGGA
AGGTAGAGGAGG'T'CCTGGCTGAGAGCCGCGTGCAGAGGAGCATCCGCCTGGTGTCCGTGCTGGAGCTG
CTGAGAGCTCCCTACGTCCGCTGGCAGGTGGTCACCGTGATTGTCACCATGGCCTGCTACCAGCTCTG
TGGCCTCAATGCAATTTGGTTCTATACCAACAGCATCTTTGGAAAAGCTGGGATCCCTCCGGCAAAGA
TCCCATACGTCACCTTGAGTACAGGGGGCATCGAGACTTTGGCTGCCGTCTTCTCTGACCACGCCCCC
TGGGTCCCCTACCTGAGTATCGTGGGCATTCTGGCCATCATCGCCTCTTTCTGCAGTGGGCCAGGTGG
CATCCCGTTCATCTTGACTGGTGAGTTCTTCCAGCAATCTCAGCGGCCGGCTGCCTTCATCATTGCAG
GCACCGTCAACTGGCTCTCCAACTTTGCTGTTGGGCTCCTCTTCCCATTCATTCAGAAAAGTCTGGAC
ACCTACTGTTTCCTAGTCTTTGCTACAATTTGTATCACAGGTGCTATCTACCTGTATTTTGTGCTGCC
TGAGACCAAAAACAGAACCTATGCAGAAATCAGCCAGGCATTTCTCGAGGGCAAGGGTGGGCGCGCC
OV4c, 247847070 SEQ ID NO: 28 362 as MW at 39164.SkD
.-otein Sequence ~SAAAPFTGTRKHTLLANNGFAISAALLMACSLQAGAFEMLIVGRFIMGIDGGVALSVLPMYLSEISP
KEIRGSLGQVTAIFICIGVFTGQLLGLPELLGKESTWPYLFGVIWPAWQLLSLPFLPDSPRYLLLE
f~HIQEARAVKAFQTFLGKADISQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQWTVIVTMACYQLC
GLNAIWFYTNSIFGKAGIPPAKIPYVTLSTGGIETLAAVFSDHAPWVPYLSIVGILAIIASFCSGPGG
IPFILTGEFFQQSQRPAAFIIAGTVNWLSNFAVGLLFPFIQKSLDTYCFLVFATICITGAIYLYFVLP
ETKNRTYAEISQAFLEGKGGRA
OV4d, 247847055 SEQ ID NO:.29 .,. X1.189 by __ NA Sequence ~ORF Start at l ORF Ston: end of TGGCCTGCTCGCTCCAGGCAGGAGCCTTTGAAATGCTCATCGTGGGACGC
GGAGGCGTCGCCCTCAGTGTGCTCCCCATGTACCTCAGTGAGATCTCACC
TCTGGGGCAGGTGACTGCCATCTTTATCTGCATTGGCGTGTTCACTGGGC

TCGAGACfiTTGGCTGCCGTCTTCTCTGGTTTGGTCATT
TGGGCCTCTTCTTTGGGACCCT
TCATCGCCTCTTTCTGCAGTGGGCCAGGTGGCATCCCGTTCATCTTGACTGGTGAGTTCTTCCAG
TCTCAGCGGCCGGCTGCCTTCATCATTGCAGGCACCGTCAACTGGCTCTCCAACTTTGCTGTTGG
CCTCTTCCCATTCATTCAGAAAAGTCTGGACACCTACTGTTTCCTAGTCTTTGCTACAATTTGTA
.TTTCTCGAGGGCAAGGGTGGGCGCGCC
OV4d, 247847055 ~SEQ ID NO: 30 396 as MW at 42768.9kD
AAAPFTGTRKHTLLANNGFAISAALLMACSLQAGAFEMLIVGRFIMGIDGGVALSVLPMYLSEISP
IRGSLGQVTAIFICIGVFTGQLLGLPELLGKESTWPYLFGVIVVPAVVQLLSLPFLPDSPRYLLLE
NEARAVKAFQTFLGKADVSQEVEEVLAESHVQRSIRLVSVLELLRAPYVRWQVVTVIVTMACYQLC
NAIWFYTNSIFGKAGIPPAKIPYVTLSTGGIETLAAVFSGLVIEHLGRRPLLIGGFGLMGLFFGTL
TLTLQDHAPWVPYLSIVGILAIIASFCSGPGGIPFTLTGEFFQQSQRPAAFIIAGTVNWLSNFAVG
FPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAEISQAFLEGKGGR.A
4e, 247847059 SEQ ID NO: 31 1.189_bp Sequence Start: at l ORF Ston: end of CTTCACCGGTACCAGGAAGCACACTTTGCTGGCCAATAATGGGTTTGCAA
.TGGAGGCGTCGCCCTCAGTGTGCTCCCCATGTACCTCAGTGAGATCTCACC
CTTTATCTGCATTGGCGTGTTCACTGGGC
CCGGACAGCCCACGCTACCTGCTCTTGGA
CTTCCAAACGTTCTTGGGTAAAGCAGACATTTCCCAAG
ACGTCCGCTGGCAGGTGGTCACCGTGATTGTCACCATGGCCTGCTACCAGCTCTG
TCTTTGGAAAAGCTGGGATCCCTCCGGCAAAGA
ACGTCACCTTGAGTACAGGGGGCATCGAGACTTTGGCTGCCGTCTTCTCTGGTTTGGTCATT
TTGGTGGCTTTGGGCTCATGGGCCTCTTCTTTGGGACCCT
GCCCCCTGGGTCCCCTACCTGAGTATCGTGGGCATTCTGG
CTCTTTCTGCAGTGGGCCAGGTGGCATCCCGTTCATCTTGACTGGTGAGTTCTTCCAG
TCATTGCAGGCACCGTCAACTGGCTCTCCAACTTTGCTGTTGG
GCTCCTCTTCCCATTCATTCAGAAAAGTCTGGACACCTACTGTTTCCTAGTCTTTGCTACAATTTGTA
TCACAGGTGCTATCTACCTGTATTTTGTGCTGCCTGAGACCAAAAACAGAACCTATGCAGAAATCAGC
CAGGCATTTCTCGAGGGCAAGGGTGGGCGCGCC
V4e, 247847059 SEQ ID NO: 32 396 as MW at 42801.9kD
tein Sequence AAAPFTGTRKI3TLLANNGFAISAA.LLMACSLQAGAFEMLIVGRFIMGIDGGVALSVLPMYLSEISP
IRGSLGQVTAIFICIGVFTGQLLGLPELLGKESTWPYLFGVIVVPAVVQLLSLPFLPDSPRYLLLE
BTEARAVKAFQTFLGKADISQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQVVTVIVTMACYQLC
C~TAIWFYTNSIFGKAGIPPAKIPYVTLSTGGIETLAAVFSGLVIEHLGRRPLLIGGFGLMGLFFGTL
TLTLQDHAPWVPYLSIVGILAIIASFCSGPGGIPFILTGEFFQQSQRPAAFIIAGTVNWLSNFAVG
FPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAEISQAFLEGKGGRA
4f, 247847047 ~SEQ ID NO 33 1.189 bp_ _ _ Sequence ~ . pRF Start at 1 ~ORF Stop~end of TAATGGGTTTGCAA
TGGAGGCGTCGCCCTCAGTGTGCTCCCCATGTACCTCAGTGAGATCTCACC
ACCTGGCCATACCTGTTTGGAGTGATTGTG
CCTTTCTCCCGGACAGCCCACGCTACCTGCTCTTGGA

CTTCCAAACGTTCTTGGGTAAAGCAGACGTTTCCCAAG
CTGAGAGCTCCCTACGTCCGCTGGCAGGTGGTCACCGTGATTGTCACCATGGCCTGCTACCAGCTCTG
TACCAACAGCATCTTTGGAA.AAGCTGGGATCCCTCTGGCAAAGA
TACGTCACCTTGAGTACAGGGGGCATCGAGACTTTGGCTGCCGTCTTCTCTGGTTTGGTCATT
TTGGTGGCTTTGGGCTCATGGGCCTCTTCTTTGGGACCCT
CACCATCACGCTGACCCTGCAGGACCACGCCCCCTGGGTCCCCTACCTGAGTATCGTGGGCATTCTGG
CCATCATCGCCTCTTTCTGCAGTGGGCCAGGTGGCATCCCGTTCATCTTGACTGGTGAGTTCTTCCAG
CAATCTCAGCGGCCGGCTGCCTTCATCATTGCAGGCACCGTCAACTGGCTCTCCAACTTTGCTGTTGG
TTTGTA
.TCTACCTGTATTTTGTGCTGCCTGAGACCAAAAACAGAACCTATGCAGAAATCAGC
CAGGCATTTCTCGAGGGCAAGGGTGGGCGCGCC
OV4f, 247847047 ~SEQ ID NO: 34 X396 as BMW at 42803.9kD
SAAAPFTGTRKHTLLANNGFAISAALLMACSLQAGAFEMLTVGRFIMGTDGGVALSVLPMYLSEISP
EIRGSLGQVTAIFICIGVFTGQLLGLPELLGKESTWPYLFGVIWPAWQLLSLPFLPDSPRYLLLE
C~TEARAVKAFQTFLGKAi3VSQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQVVTVIVTMACYQLC
LNAIWFYTNSIFGKAGIPLAKIPYWLSTGGIETLAAVFSGLVIEHLGRRPLLIGGFGLMGLFFGTL
ITLTLQDHAPWVPYLSIVGILAIIASFCSGPGGIPFILTGEFFQQSQRPAAFIIAGTVNWLSNFAVG
LFPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAETSQAFLEGKGGRA
OV4g, CG114555-02 ~SEQ ID NO 35 ~1.682...bp.
NA Sequence ORF Start. ATG at 14 ORF Ston: TAA at 1634 ACATCAAGG
CTTTTACAATGAGTCATGGGAAAGAAGGCATGGACGTCCAATAGACCCAGACACTCTGACTCTGCTC
.TATTCGCCATCGGTGGACTTGTGGGGACATTAATTGTGAAGATGATTGG
TGTACCTCAGTGAGATCTCACCCAAGGAGATCCGTGGCTC
CG
ACCTGGCCATACCTGTTTGGAGTGATTGTGGTCCCTGCCGTTGTCCAG
CCTGGACAGCCCACGCTACCTGCTCTTGGAGAAGCACAACGAGGCAAG
GCTCCCTACGTC
CAATGCAATTTG
ACGTCACCTTGA
TTGAGCACCTGGGACGGAGA
.TTGGTGGCTTTGGGCTCATGGGCCTCTTCTTTGGGGCCCTCACCATCACGCTGACCCT
TTCTGGCCATCATCGCCTCTTTCT
CGGCT
CTACTGTTTCCTAGTCTTTGCTACAATTTGTATCACAGGTGCTATCTACC
TTAACAAGTTTCCTCCTCCACGTTGGACAATTATGTCAA.AA.ACAGGATTG
OV4g, CG114555-02 SEQ ID NO: 36 X540 as BMW at 58796.3kD
VPLTDDTSHARPPGPGRALLECVHLRSGVPGGRRRKDWSCSLLVASLAGAFGSP
FLYGYNLSVVNAPTPYIKAFYNESWERRHGRPIDPDTLTLLWSVTVSIFAIGGLVGTLIVKMIGKVLG
RKHTLLANNGFAISAALLMACSLQAGAFEMLIVGRFIMGIDGGVALSVLPMYLSEISPKEIRGSLGQV
TAIFICIGVFTGQLLGLPELLGKESTWPYLFGVIWPAWQLLSLPFLLDSPRYLLLEKHNEARAVKA
FQTFLGKADVSQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQWWIVTMACYQLCGLNAIWFYTN
SIFGKAGIPLAKIPWTLSTGGIETLAAVFSGLVIEHLGRRPLLIGGFGLMGLFFGALTITLTLQDHA

PWVPYLSIVGILAIIASFCSGPGGIPFILTGEFFQQSQRPAAFIIAGTVNWLSNFAVGLLFPFIQKSL
DTYCFLVFATICITGAIYLYFVLPETKNRTYAEISQAFSKRNKAYPPEEKIDSAVTDGKINGRP
NOV4h, CG114555-03 SEQ ID NO: 37 ' 1757 by ,DNA Sequence ORF Start ATG at 14 tORF Stop TAA at 170_9 _ __ TCACTGAGACCCATGGCAAGGAAGCAAAATAGGAATTCCAAGGAACTGGGCCTAGTTCCCCTCACAG
iATGACACCAGCCACGCCGGGCCTCCAGGGCCAGGGAGGGCACTGCTGGAGTGTGACCACCTGAGGAGT
GGGGTGCCAGGTGGAAGGAGAAGAAAGCAGCCTCTACGGAGCACCTCCTCTGCAGCAGGCTCCTCAAC
AACATATGTGGCCAGTGCTGCTATTAAGATCCCATTTCACAGGTGGGCAAGCTTAGCCCCAGAAAAGT
CAAGTCACTTGCTCAGACTCCTACAGCTGAGGGGACTGGCCCTGGAGGTAAAGCTGATATCACTTGGC
TCAAAGCCCCAAAGCTCTATCTCGTGGCTGGTGGCACTAGAGGAGACAAACGAGATTGGCAGAGACTG
GTCCTGCTCGCTCCTCGTGGCCTCCCTCGCGGGCGCCTTCGGCTCCTCCTTCCTCTACGGCTACAACC
TGTCGGTGGTGAATGCCCCCACCCCGCACACTTTGCTGGCCAATAATGGGTTTGCAATTTCTGCTGCA
TTGCTGATGGCCTGCTCGCTCCAGGCAGGAGCCTTTGAAATGCTCATCGTGGGACGCTTCATCATGGG
CATAGATGGAGGCGTCGCCCTCAGTGTGCTCCCCATGTACCTCAGTGAGATCTCACCCAAGGAGATCC
GTGGCTCTCTGGGGCAGG'TGACTGCCATCTTTATCTGCATTGGCGTGTTCACTGGGCAGCTTCTGGGC
CTGCCCGAGCTGCTGGGAAAGGAGAGTACCTGGCCATACCTGTTTGGAGTGATTGTGGTCCCTGCCGT
TGTCCAGCTGCTGAGCCTTCCCTTTCTCCTGGACAGCCCACGCTACCTGCTCTTGGAGAAGCACAACG
AGGCAAGAGCTGTGAAAGCCTTCCAAACGTTCTTGGGTAAAGCAGACGTTTCCCAAGAGGTAGAGGAG
GTCCTGGCTGAGAGCCGCGTGCAGAGGAGCATCCGCCTGGTGTCCGTGCTGGAGCTGCTGAGAGCTCC
CTACGTCCGCTGGCAGGTGGTCACCGTGATTGTCACCATGGCCTGCTACCAGCTCTGTGGCCTCAATG
CAATTTGGTTCTATACCAACAGCATCTTTGGAAAAGCTGGGATCCCTCTGGCAAAGATCCCATACGTC
ACCTTGAGTACAGGGGGCATCGAGACTTTGGCTGCCGTCTTCTCTGGTTTGGTCATTGAGCACCTGGG
ACGGAGACCCCTCCTCATTGGTGGCTTTGGGCTCATGGGCCTCTTCTTTGGGGCCCTCACCATCACGC
TGACCCTGCAGGACCACGCCCCCTGGGTCCCCTACCTGAGTATCGTGGGCATTCTGGCCATCATCGCC
TCTTTCTGCAGTGGGCCAGGTGGCATCCCGTTCATCTTGACTGGTGAGTTCTTCCAGCAATCTCAGCG
GCCGGCTGCCTTCATCATTGCAGGCACCGTCAACTGGCTCTCCAACTTTGCTGTTGGGCTCCTCTTCC
CATTCATTCAGAAAAGTCTGGACACCTACTGTTTCCTAGTCTTTGCTACAATTTGTATCACAGGTGCT
ATCTACCTGTATTTTGTGCTGCCTGAGACCAAAAACAGAACCTATGCAGAAATCAGCCAGGCATTTTC
CAAAAGGAACAAAGCATACCCACCAGAAGAGAAAATCGACTCAGCTGTCACTGATGGTAAGATAAATG
GAAGGCCTTAACAAGTTTCCTCCTCCACGTTGGACAATTATGTCAAAAACAGGATTG
NOV4h, CG114555~-03 ~ SEQ ID NO: 3~ 565 as MW at 61112.6kD , ~V~~~
Protein Sequence _ _ _ _ _.. .. ___ _d__ _~ _.._ _ ~_ . . . . _ __~_ _ - __~ ~ ._ _ -_ . . ~.
_. _ _ _ _ _._ _. _ ~ _ . ..
MARKQNRNSKELGLVPLTDDTSHAGPPGPGRALLECDHLRSGVPGGRRRKQPLRSTSSAAGSSTTYVA
SAAIKIPFHRWASLAPEKSSHLLRLLQLRGLALEVKLISLGSKPQSSISWLVALEETNEIGRDWSCSL
LVASLAGAFGSSFLYGYNLSVVNAPTPHTLLANNGFAISAALLMACSLQAGAFEMLIVGRFIMGIDGG
VALSVLPMYLSEISPKEIRGSLGQVTAIFICTGVFTGQLLGLPELLGKESTWPYLFGVIVVPAVVQLL
SLPFLLDSPRYLLLEKHNEARAVKAFQTFLGKADVSQEVEEVLAESRVQRSIRLVSVLELLRAPYVRW
QVVTVIVTMACYQLCGLNAIWFYTNSIFGKAGIPLAKIPYVTLSTGGIETLAAVFSGLVIEHLGRRPL
LIGGFGLMGLFFGALTITLTLQDHAPWVPYLSIVGILAIIASFCSGPGGIPFILTGEFFQQSQRPAAF
IIAGTVNWLSNFAVGLLFPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAEISQAFSKRNK
AYPPEEKIDSAVTDGKINGRP
NOV4i, CG114555-04SEQ ID NO 39 1502 by DNA Sequence ORF Start: ATG at 14 ORF Stop: TAA at 1454 CTGGGCCTAGTTC C A
GTCACTGAGACCCATGGCAAGGAAACAAAATAGGAATTCCAAGGAA C CTC CAG
ATGACACCAGCCACGCCAGGCCTCCAGGGCCAGGGAGGGCACTGCTGGAGTGTGTCCACCTGAGGAGT
GGGGTGCCAGGTGGAAGGAGAAGAAAGGACTGGTCCTGCTCGCTCCTCGTGGCCTCCCTCGCGGGCGC
CTTCGGCTCCCCCTTCCTCTACGGCTACAACCTGTCGGTGGTGAATGCCCCCACCCCGTACATCAAGG
CCTTTTACAATGAGTCATGGGAAAGAAGGCATGGACGTCCAATAGACCCAGACACTCTGACTCTGCTC
TGGTCTGTGACTGTGTCCATATTCGCCATCGGTGGACTTGTGGGGACATTAATTGTGAAGATGATTGG
AAAGGTTCTTGGGAGGAAGCACACTTTGCTGGCCAATAATGGGTTTGCAATTTCTGCTGCATTGCTGA
TGGCCTGCTCGCTCCAGGCAGGAGCCTTTGAGATGCTCATCGTGGGACGCTTCATCATGGGCATAGAT
GGAGGCGTCGCCCTCAGTGTGCTCCCCATGTACCTCAGTGAGATCTCACCCAAGGAGATCCGTGGCTC
TCTGGGGCAGGTGACTGCCATCTTTATCTGCATTGGCGTGTTCACTGGGCAGCTTCTGGGCCTGCCCG
AGCTGCTGGGAAAGGAGAGTACCTGGCCATACCTGTTTGGAGTGATTGTGGTCCCTGCCGTTGTCCAG
CTGCTGAGCCTTCCCTTTCTCCTGGACAGCCCACGCTACCTGCTCTTGGAGAAGCACAACGAGGCAAG

CTTCCAAACGTTCTTGGGTAAAGCAGACGTTTCCCAAGAGGTAGAGGAGGTCCTGG
TCCGCCTGGTGTCCGTGCTGGAGCTGCTGAGAGCTCCCTACGTC
CTGCTACCAGCTCTGTGGCCTCAATGCAATTTG
ATACCAACAGCATCTTTGGAAAAGCTGGGATCCCTCTGGCAAAGATCCCATACGTCACCTTGA
TCTTGACTGGTGAGTTC
TTCAGAAAAGTCTGGACACCTACTGTTTCCTAGTCTTTGCTACAA
TTGTATCACAGGTGCTATCTACCTGTATTTTGTGCTGCCTGAGACCAAA.AACAGAACCTATGCAGAA
TCAGCCAGGCATTTTCCAAAAGGAACAAAGCATACCCACCAGAAGAGAA.AATCGACTCAGCTGTCAC
TGGAAGGCCTTAACAAGTTTCCTCCTCCACGTTGGACAATTATGTCAAAAACA
V4i, CG114555-04 SEQ ID NO: 40 480 as jMW at 52522.9kD
PGPGRALLECVHLRSGVPGGRRRKDWSCSLLVASLAGAFGSP
LYGYNLSVVNAPTPYIKAFYNESWERRHGRPIDPDTLTLLWSVWSIFAIGGLVGTLIVKMIGKVLG
KHTLLANNGFAISAALLMACSLQAGAFEMLIVGRFIMGIDGGVALSVLPMYLSEISPKEIRGSLGQV
AIFICIGVFTGQLLGLPELLGKESTWPYLFGVIWPAWQLLSLPFLLDSPRYLLLEKHNEARAVKA
QTFLGKADVSQEVEEVLAESRVQRSIRLVSVLELLRAPYVR.WQWTVIVTMACYQLCGLNAIWFYTN
IFGKAGIPLAKIPYVTLSTGGIETLAAVFSGIPFILTGEFFQQSQRPAAFIIAGTVNWLSNFAVGLL
PFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAEISQAFSKRNKAYPPEEKIDSAVTDGKI
V4j, 13379365 SNP in SEQ _ID NO: 41 _ SNP: G/A at position 86 114555-O1 O~RF~Start: ATG at l4 ORF Stop: TAA~at 1535 TTCCAAGGAACTGGGCCTAGTTCCCCTCACAGATGA
CCAGGGCCAGGGAGGGCACTGCTGGAGTGTGACCACCTGAGGAGTGGGGTGCC
T
.TTGGAAAGGTTCTTGGGAGGAAGCACACTTTGCTGGCCAATAATGGGTTTGCAAT
CTTTGAAATGCTCATCGTGGGACGCTTCAT
TGGAGGCGTCGCCCTCAGTGTGCTCCCCATGTACCTCAGTGAGATCTCACCCAAGGAGAT
TCTTTATCTGCATTGGCGTGTTCACTGGGCAGCTTCTGGGCCT
CA
CCTTTCTCCCGGACAGCCCACGCTACCTGCTCTTGGAGAAGCACAACGAGGCAAGAGC
AGAGGAGGTCCTGGCTGAGAG
CCGCGTGCAGAGGAGCATCCGCCTGGTGTCCGTGCTGGAGCTGCTGAGAGCTCCCTACGTCCGCTGGCAGGT
CATCTTTGGAAAAGCTGGGATCCCTCTGGCAAAGATCCCATACGTCACCTTGAGTACAGGGGGCATCGAGAC
TTTGGCTGCCGTCTTCTCTGGTTTGGTCATTGAGCACCTGGGACGGAGACCCCTCCTCATTGGTGGCTTTGG
CTCTTCTTTGGGACCCTCACCATCACGCTGACCCTGCAGGACCACGCCCCCTGGGTCCCCTA
CCATTCATTCAGAAAAGTCTGGACACCTACTGTTTCCTA
TCAGCCAGGCATTTTCCAAAAGGAACAAAGCATACCCACCAGAAGAGAAAATCGACTCAGCT
GTAAGATAAATGGAAGGCCTTAACAAGTTTCCTCCTCCACGTTGGACAATTATGTCAAAAAC
ACATGGATGATCTCACTTTTCAGGAAACTTAAAATTTACCCATTATTGGGAAGCTTAAATGA
..TTAAATATTTAAAAGTAAACCTGTACTAATCTAA
V4j, 13379365 SNP in ~SEQ ID NO: 42 ~~507aa ~~SNP: Gly to 114555-O1 ~ j Arg at position MARKQNRNSKELGLVPLTDDTSHARPPGPGRALLECDHLRSGVPGGRRRKYIKAFYNESWERRHGRPIDPDT
LTLLWSVTVSIFAIGGLVGTLIVKMIGKVLGRKHTLLANNGFAISAALLMACSLQAGAFEMLIVGRFIMGTD
GGVALSVLPMYLSEISPKEIRGSLGQWAIFICIGVFTGQLLGLPELLGKESTWPYLFGVIWPAWQLLSL
PFLPDSPRYLLLEKHNEARAVKAFQTFLGKADVSQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQWTVI

TNSIFGKAGIPLAKIPYVTLSTGGIETLAAVFSGLVIEHLGRRPLLIGGFGLMGL
'VPYLSIVGILAIIASFCSGPGGIPFILTGEFFQQSQRPAAFIIAGTVNWLSNFAV
~VFATICITGAIYLYFVLPETKNRTY'AEISQAFSKRNKAYPPEEKIDSAVTDGKIN
OV4k, 13379364 SNP in ~SEQ-ID-N0:43,.. . ~,-y ~~SNP:"G/A at position 97 6114555-O1 , ORF Start: ATG at 4 ORF Stop: TAAyat 1535 NA Sequence GGAAACAAAATAGGAATTCCAAGGAACTGGGCCTAGTTCCCCTCACAGATGA
CAGGA_CCAGGGAGGGCACTGCTGGAGTGTGACCACCTGAGGAGTGGGGTGCC
ACATCAAGGCCTTTTACAATGAGTCATGGGAAAGAAGGCATGGACGTCCAAT
TTAATTGTGAAGATGATTGGAAAGGTTCTTGGGAGGAAGCACACTTTGCTGGCCAATAATGGGTTTGCAAT
TCTGCTGCATTGCTGATGGCCTGCTCGCTCCAGGCAGGAGCCTTTGAAATGCTCATCGTGGGACGCTTCAT
ATGGGCATAGATGGAGGCGTCGCCCTCAGTGTGCTCCCCATGTACCTCAGTGAGATCTCACCCAAGGAGAT
CGTGGCTCTCTGGGGCAGGTGACTGCCATCTTTATCTGCATTGGCGTGTTCACTGGGCAGCTTCTGGGCCT
CCCGAGCTGCTGGGAAAGGAGAGTACCTGGCCATACCTGTTTGGAGTGATTGTGGTCCCTGCCGTTGTCCA
TGTGAAAGCCTTCCAAACGTTCTTGGGTAAAGCAGACGTTTCCCAAGAGGTAGAGGAGGTCCTGGCTGAGAG
CCGCGTGCAGAGGAGCATCCGCCTGGTGTCCGTGCTGGAGCTGCTGAGAGCTCCCTACGTCCGCTGGCAGGT
.TCCCATACGTCACCTTGAGTACAGGGGGCATCGAGAC
CATCACGCTGACCCTGCAGGACCACGCCCCCTGGGTCCCCTA
CGCCTCTTTCTGCAGTGGGCCAGGTGGCATCCCGTTCATCTT
ACAATTTGTATCACAGGTGCTATCTACCTGTATTTTGTGCTGCCTGAGACCAAAAACAGAACCTATGCAGA
ATCAGCCAGGCATTTTCCAAAAGGAACAAAGCATACCCACCAGAAGAGAAAATCGACTCAGCTGTCACTGA
GGTAAGATAAATGGAAGGCCTTAACAAGTTTCCTCCTCCACGTTGGACAATTATGTCAAAAACAGGATTGT
TACATGGATGATCTCACTTTTCAGGAAACTTAAA.ATTTACCCATTATTGGGAAGCTTAAATGAATTGAAGC
ATGCAAGTCTTTTATATTATTAAATATTTAAAAGTAAACCTGTACTAATCTAA
V4k, 13379364 SNP ~SEQ ID NO: 507 as SNP : Gly to Gly at position 28 MARKQNRNSKELGLVPLTDDTSHAGPP_GPGRALLECDHLRSGVPGGRRRKYIKAFYNESWERRHGRPIDPDT
LTLLWSVWSIFAIGGLVGTLIVKMIGKVLGRKHTLLANNGFAISAALLMACSLQAGAFEMLIVGRFIMGID
GGVALSVLPMYLSEISPKEIRGSLGQVTATFICIGVFTGQLLGLPELLGKESTWPYLFGVIWPAWQLLSL
PFLPDSPRYLLLEKHNEARAVKAFQTFLGKADVSQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQWWI
VTMACYQLCGLNAIWFYTNSIFGKAGIPLAKIPWTLSTGGIETLAAVFSGLVIEHLGRRPLLIGGFGLMGL
FFGTLTITLTLQDHAPWVPYLSIVGILAITASFCSGPGGIPFILTGEFFQQSQRPAAFIIAGTVNWLSNFAV
GLLFPFIQKSLDTYCFLVFATICTTGATYLYFVLPETKNRTYAEISQAFSKRNKAYPPEEKIDSAVTDGKIN
OV41, 13379363 SNP CG114555-O1 ~SEQ TD N0:45 SNP A/G.at,position 289 NA Sequence ORF~Start: ATG at ORF Stop: TAA~at 1535 position 14 TAGGAATTCCAAGGAACTGGGCCTAGTTCCCCTCACAGATGA
TGGGAAAGAAGGCATGGACGTCCAAT
TTCGCCATCGGTGGACTTGTGGGGAC
ATTGCTGATGGCCTGCTCGCTCCAGGCAGGAGCCTTTGAAATGCTCATCGTGGGACGCTTCAT
AGATGGAGGCGTCGCCCTCAGTGTGCTCCCCATGTACCTCAGTGAGATCTCACCCAAGGAGAT
TCTGGGGCAGGTGACTGCCATCTTTATCTGCATTGGCGTGTTCACTGGGCAGCTTCTGGGCCT
GCTGGGAAAGGAGAGTACCTGGCCATACCTGTTTGGAGTGATTGTGGTCCCTGCCGTTGTCCA

GCTGCTGAGCCTTCCCTTTCTCCCGGACAGCCCACGCTACCTGCTCTTGGAGAAGCACAACGAGGCAAGAGC
TGTGAAAGCCTTCCAAACGTTCTTGGGTAAAGCAGACGTTTCCCAAGAGGTAGAGGAGGTCCTGGCTGAGAG
'CCGCGTGCAGAGGAGCATCCGCCTGGTGTCCGTGCTGGAGCTGCTGAGAGCTCCCTACGTCCGCTGGCAGGT
GGTCACCGTGATTGTCACCATGGCCTGCTACCAGCTCTGTGGCCTCAATGCAATTTGGTTCTATACCAACAG
CATCTTTGGAAAAGCTGGGATCCCTCTGGCAAAGATCCCATACGTCACCTTGAGTACAGGGGGCATCGAGAC
TTTGGCTGCCGTCTTCTCTGGTTTGGTCATTGAGCACCTGGGACGGAGACCCCTCCTCATTGGTGGCTTTGG
GCTCATGGGCCTCTTCTTTGGGACCCTCACCATCACGCTGACCCTGCAGGACCACGCCCCCTGGGTCCCCTA
CCTGAGTATCGTGGGCATTCTGGCCATCATCGCCTCTTTCTGCAGTGGGCCAGGTGGCATCCCGTTCATCTT
GACTGGTGAGTTCTTCCAGCAATCTCAGCGGCCGGCTGCCTTCATCATTGCAGGCACCGTCAACTGGCTCTC
CAACTTTGCTGTTGGGCTCCTCTTCCCATTCATTCAGAAAAGTCTGGACACCTACTGTTTCCTAGTCTTTGC
TACAATTTGTATCACAGGTGCTATCTACCTGTATTTTGTGCTGCCTGAGACCAAAAACAGAACCTATGCAGA
AATCAGCCAGGCATTTTCCAAAAGGAACAAAGCATACCCACCAGAAGAGAAAATCGACTCAGCTGTCACTGA
TGGTAAGATAAATGGAAGGCCTTAACAAGTTTCCTCCTCCACGTTGGACAATTATGTCAAAAACAGGATTGT
CTACATGGATGATCTCACTTTTCAGGAAACTTAAAATTTACCCATTATTGGGAAGCTTAAATGAATTGAAGC
TATGCAAGTCTTTTATATTATTAAATATTTAAAAGTAAACCTGTACTAATCTAA
NOV41, 13379363 SNP SEQ ID NO: S07 SNP: no change in the protein CG114SS5-O1 46 aa. sequence Protein. Sequence .... .. ~ . .. _... .. .... ~ ... ' .. ~ _ _ ._.
MAR.KQNRNSKELGLVPLTDDTSHAGPPGPGRALLECDHLRSGVPGGRRRKYIKAFYNESWERRHGRPIDPDT
LTLLWSVTVSIFAIGGLVG_LIVKMIGKVLGRKHTLLANNGFAISAALLMACSLQAGAFEMLIVGRFIMGID
GGVALSVLPMYLSEISPKEIRGSLGQVTAIFICIGVFTGQLLGLPELLGKESTWPYLFGVIWPAWQLLSL
PFLPDSPRYLLLEKHNEARAVKAFQTFLGKADVSQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQWTVI
VTMACYQLCGLNAIWFYTNSIFGKAGIPLAKIPWTLSTGGIETLAAVFSGLVIEHLGRRPLLIGGFGLMGL
FFGTLTITLTLQDHAPWVPYLSIVGILAIIASFCSGPGGIPFILTGEFFQQSQRPAAFIIAGTVNWLSNFAV
GLLFPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAEISQAFSKRNKAYPPEEKIDSAVTDGKIN
GRP
NOV4m, 13379362 SNP CG114SSS-O1 SEQ ID N0: 47 ~ SNP: C/T at.position 672 3......_.... w. ..,...... . .... , -....... . .....,.; .. .. . ... ......... -"... .....,..... .,. ... . .." ....... . ..
DNA Sequence ORF Start: ATG at ORF Stop: TAA at 1535 _.. position 14 GTCACTGAGACCCATGGCAA.GGAAACAAAATAGGAATTCCAAGGAACTGGGCCTAGTTCCCCTCACAGATGA
CACCAGCCACGCCGGGCCTCCAGGGCCAGGGAGGGCACTGCTGGAGTGTGACCACCTGAGGAGTGGGGTGCC
AGGTGGAAGGAGAAGAAAGTACATCAAGGCCTTTTACAATGAGTCATGGGAAAGAAGGCATGGACGTCCAAT
AGACCCAGACACTCTGACTCTGCTCTGGTCTGTGACTGTGTCCATATTCGCCATCGGTGGACTTGTGGGGAC
ATTAATTGTGAAGATGATTGGAAAGGTTCTTGGGAGGAAGCACACTTTGCTGGCCAATAATGGGTTTGCAAT
TTCTGCTGCATTGCTGATGGCCTGCTCGCTCCAGGCAGGAGCCTTTGAAATGCTCATCGTGGGACGCTTCAT
CATGGGCATAGATGGAGGCGTCGCCCTCAGTGTGCTCCCCATGTACCTCAGTGAGATCTCACCCAAGGAGAT
CCGTGGCTCTCTGGGGCAGGTGACTGCCATCTTTATCTGCATTGGCGTGTTCACTGGGCAGCTTCTGGGCCT
GCCCGAGCTGCTGGGAAAGGAGAGTACCTGGCCATACCTGTTTGGAGTGATTGTGGTCCCTGCCGTTGTCCA
GCTGCTGAGCCTTCCCTTTCTCC_TGGACAGCCCACGCTACCTGCTCTTGGAGAAGCACAACGAGGCAAGAGC
TGTGAAAGCCTTCCAAACGTTCTTGGGTAAAGCAGACGTTTCCCAAGAGGTAGAGGAGGTCCTGGCTGAGAG
CCGCGTGCAGAGGAGCATCCGCCTGGTGTCCGTGCTGGAGCTGCTGAGAGCTCCCTACGTCCGCTGGCAGGT
GGTCACCGTGATTGTCACCATGGCCTGCTACCAGCTCTGTGGCCTCAATGCAATTTGGTTCTATACCAACAG
CATCTTTGGAAAAGCTGGGATCCCTCTGGCAAAGATCCCATACGTCACCTTGAGTACAGGGGGCATCGAGAC
TTTGGCTGCCGTCTTCTCTGGTTTGGTCATTGAGCACCTGGGACGGAGACCCCTCCTCATTGGTGGCTTTGG
GCTCATGGGCCTCTTCTTTGGGACCCTCACCATCACGCTGACCCTGCAGGACCACGCCCCCTGGGTCCCCTA
CCTGAGTATCGTGGGCATTCTGGCCATCATCGCCTCTTTCTGCAGTGGGCCAGGTGGCATCCCGTTCATCTT
GACTGGTGAGTTCTTCCAGCAATCTCAGCGGCCGGCTGCCTTCATCATTGCAGGCACCGTCAACTGGCTCTC
CAACTTTGCTGTTGGGCTCCTCTTCCCATTCATTCAGAAAAGTCTGGACACCTACTGTTTCCTAGTCTTTGC
TACAATTTGTATCACAGGTGCTATCTACCTGTATTTTGTGCTGCCTGAGACCAAAAACAGAACCTATGCAGA
AATCAGCCAGGCATTTTCCAAAAGGAACAAAGCATACCCACCAGAAGAGAAA.ATCGACTCAGCTGTCACTGA
TGGTAAGATAAATGGAAGGCCTTAACAAGTTTCCTCCTCCACGTTGGACAATTATGTCAAAAACAGGATTGT
CTACATGGATGATCTCACTTTTCAGGAAACTTAAAATTTACCCATTATTGGGAAGCTTAAATGAATTGAAGC
TATGCAAGTCTTTTATATTATTAAATATTTAAAAGTAAACCTGTACTAATCTAA
NOV4m, 13379362 SEQ ID NO: 48 S07 as SNP: Pro to Leu at position 220 SNP CG114SSS-O1 . , .

~VPLTDDTSHAGPPGPGRALLECDHLRSGVPGGRRRKYIKAFYNESWERRHGRPIDPD
TLTLLWSVTVSIFAIGGLVGTLIVKMIGKVLGRKHTLLANNGFAISAALLMACSLQAGAFEMLIVGRFIMG
IDGGVALSVLPMXLSEISPKEIRGSLGQVTAIFICIGVFTGQLLGLPELLGKESTWPYLFGVIVVPAVVQL
LSLPFL_LDSPRYLLLEKHNEARAVKAFQTFLGKADVSQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQV
VTVIVTMACYQLCGLNAIWFYTNSIFGKAGIPLAKIPYVTLSTGGIETLAAVFSGLVIEHLGRRPLLIGGF
GLMGLFFGTLTITLTLQDHAPWVPYLSIVGILAIIASFCSGPGGIPFILTGEFFQQSQRPAAFIIAGTVNW
LSNFAVGLLFPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAEISQAFSKRNKAYPPEEKIDSA
V4n, 13379620 SNP ~SEQ ID NO 49 SNP: T/C at position 963 114555-Ol ORF Start ATG at ORF Stop: TAA at 1535 A Sequence Dosition 14 TGGCAAGGAAACAAAATAGGAATTCCAAGGAACTGGGCCTAGTTCCCCTCACAGATG
TCAAGGCCTTTTACAATGAGTCATGGGAAAGAAGGCATGGACGTCC
TTCGCCATCGGTGGACTTGTGG
TTGCTGATGGCCTGCTCGCTCCAGGCAGGAGCCTTTGAAATGCTCATCGTGGGACG
ACCTCAGTGAGATCTCACCCA
.TACCTGTTTGGAGTGATTGTGGTCCCTGC
AGAGGAGGTC
GCTCATGGGCCTCTTCTTTGGGACCCTCACCATCACGCTGACCCTGCAGGACCACG
ACCTGAGTATCGTGGGCATTCTGGCCATCATCGCCTCTTTCTGCAGTGGGCCAGGT
.TCTCAGCGGCCGGCTGCCTTCATCATTGCAGG
ATTCATTCAGAAAAGTCTGGACACCT
ACCTGTATTTTGTGCTGCCTGAGACC
CAGGCATTTTCCAAAAGGAACAAAGCATACCCACCAGAAGAGAA
GATAAATGGAAGGCCTTAACAAGTTTCCTCCTCCACGTTGGACA
TGGATGATCTCACTTTTCAGGAAACTTAAAATTTACCCATTATT
~GGGAAGCTTAAATGAATTGAAGCTATGCAAGTCTTTTATATTATTAAATATTTAAAAGTAAACCTGTACTA
~OV4n, 13379620 SNP CG114555- SEQ ID NO: 50507 as SNP: Leu to Pro at 1 t~osition 317 l2RHGRPIDPDTLTLLWSVTVSIFAIGGLVGTLIVKMIGKVLGRKHTLLANNGFAISAALLM
ACSLQAGAFEMLIVGRFIMGIDGGVALSVLPMYLSEISPKEIRGSLGQVTAIFICIGVFTG
QLLGLPELLGKESTWPYLFGVIVVPAVVQLLSLPFLPDSPRYLLLEKHNEARAVKAFQTFL
GKADVSQEVEEVLAESRVQRSIRLVSVLELLRAPYV'RWQVVTVIVTMACYQLCGLNAIWFY
TNSIFGKAGIP_PAKIPYVTLSTGGIETLAAVFSGLVIEHLGRRPLLIGGFGLMGLFFGTLT
ITLTLQDHAPWVPYLSIVGILAITASFCSGPGGIPFILTGEFFQQSQRPAAFIIAGTVNWL
SNFAVGLLFPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAEISQAFSKRNKAY
PPEEKIDSAVTDGKINGRP
A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table 4B.

Table 4B. Comparison of the NOV4 protein sequences.
NOV4a MARKQNRNSKELGLVPLTDDTSHAGPPGPGRALLECDHLRSGVPGGRRRK----------NOV4b -_______-_____-________________-____________________________ NOV4c -_______________-___________________________________________ NOV4d ____________________________________________________________ NOV4e ______________________________________-_____________________ NOV4f ________________-_-__-___-__________________________________ NOV4g MARKQNRNSKELGLVPLTDDTSHARPPGPGRALLECVHLRSGVPGGRRRKDW--------NOV4h MARKQNRNSKELGLVPLTDDTSHAGPPGPGRALLECDHLRSGVPGGRRRKQPLRSTSSAA
NOV4i MARKQNRNSKELGLVPLTDDTSHARPPGPGRALLECVHLRSGVPGGRRRKDW--------NOV4a -_______-_______________________________________yI~FYNESWER
NOV4b __-___________________________-_____________________________ NOV4c -___________________________________________________________ NOV4d ___________-________________________________________________ NOV4e ______-______________________-___________-__________-_______ NOV4f -_-_________________________________________________________ NOV4g --------SCSLLV-- --ASLAGAFGSPFLYGYNLS----VVNAPTPYIKAFYNESWER
NOV4h GSSTTYVASAAIKIPFHRWASLAPEKSSHLLRLLQLRGLALEVKLISLGSKPQSSISWLV
N0V4i --------SCSLLV-----ASLAGAFGSPFLYGYNLS----WNAPTPYIKAFYNESWER
NOV4a RHGRPIDPDTLTLLWSVTVSIFAIGGLVGTLIVKMTGKVLGRKHTLLANNGFAISAALLM
NOV4b --------------------------LYKKAGSAAAPFTGTRKHTLLANNGFAISAALLM
NOV4c -------------------------------GSAAAPFTGTRKHTLLANNGFAISAALLM
NOV4d -------------------------------GSAAAPFTGTRKHTLLANNGFAISAALLM
NOV4e -------------------------------GSAAAPFTGTRKHTLLANNGFAISAALLM
NOV4f -------------------------------GSAAAPFTGTRKHTLLANNGFAISAALLM
NOV4g RHGRPIDPDTLTLLWSVTVSIFAIGGLVGTLIVKMIGKVLGRKHTLLANNGFAISAALLM
NOV4h ALEETNEIGRDWSCSLLVASLAGAFGSSFLYGYNLSWNAPTPHTLLANNGFAISAALLM
NOV4i RHGRPIDPDTLTLLWSVTVSIFAIGGLVGTLIVKMIGKVLGRKHTLLANNGFAISAALLM
NOV4a ACSLQAGAFEMLIVGRFIMGIDGGVALSVLPMYLSEISPKEIRGSLGQVTAIFICIGVFT
NOV4b ACSLQAGAFEMLIVGRFIMGIDGGVALSVLPMYLSEISPKEIRGSLGQVTAIFICIGVFT
NOV4c ACSLQAGAFEMLIVGRFIMGIDGGVALSVLPMYLSEISPKEIRGSLGQVTAIFICIGVFT
NOV4d ACSLQAGAFEMLIVGRFIMGIDGGVALSVLPMYLSEISPKEIRGSLGQVTAIFICIGVFT
NOV4e ACSLQAGAFEMLIVGRFIMGIDGGVALSVLPMYLSEISPKEIRGSLGQVTAIFICIGVFT
NOV4f ACSLQAGAFEMLIVGRFIMGIDGGVALSVLPMYLSEISPKEIRGSLGQVTAIFICIGVFT
NOV4g ACSLQAGAFEMLIVGRFIMGIDGGVALSVLPMYLSEISPKEIRGSLGQVTAIFICIGVFT
NOV4h ACSLQAGAFEMLIVGRFIMGIDGGVALSVLPMYLSEISPKEIRGSLGQVTAIFICIGVFT
NOV4i ACSLQAGAFEMLIVGRFIMGIDGGVALSVLPMYLSEISPKEIRGSLGQVTAIFICIGVFT
NOV4a GQLLGLPELLGKESTWPYLFGVIWPAWQLLSLPFLPDSPRYLLLEKHNEARAVKAFQT
NOV4b GQLLGLPELLGKESTWPYLFGVIVVPAVVQLLSLPFLPDSPRYLLLEKHNEARAVKAFQT
N0V4c GQLLGLPELLGKESTWPYLFGVIWPAVVQLLSLPFLPDSPRYLLLEKHNEARAVKAFQT
N0V4d GQLLGLPELLGKESTWPYLFGVIVVPAWQLLSLPFLPDSPRYLLLEKHNEARAVKAFQT
NOV4e GQLLGLPELLGKESTWPYLFGVIWPAVVQLLSLPFLPDSPRYLLLEKHNEARAVKAFQT
NOV4f GQLLGLPELLGKESTWPYLFGVIWPAVVQLLSLPFLPDSPRYLLLEKHNEARAVKAFQT
NOV4g GQLLGLPELLGKESTWPYLFGVIVVPAVVQLLSLPFLLDSPRYLLLEKHNEARAVKAFQT
NOV4h GQLLGLPELLGKESTWPYLFGVIVVPAWQLLSLPFLLDSPRYLLLEKHNEARAVKAFQT
NOV4i GQLLGLPELLGKESTWPYLFGVIVVPAVVQLLSLPFLLDSPRYLLLEKHNEARAVKAFQT
NOV4a FLGKADVSQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQVVTVIVTMACYQLCGLNAI
NOV4b FLGKADVSQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQWTVIVTMACYQLCGLNAI
NOV4c FLGKADISQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQVVTVIVTMACYQLCGLNAI
NOV4d FLGKADVSQEVEEVLAESHVQRSIRLVSVLELLRAPYVRWQWTVIVTMACYQLCGLNAI
NOV4e FLGKADISQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQVVTVIVTMACYQLCGLNAI

',NOV4f FLGKADVSQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQVVTVIVTMACYQLCGLNAI
NOV4g FLGKADVSQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQVVTVIVTMACYQLCGLNAI
NOV4h FLGKADVSQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQVVTVIVTMACYQLCGLNAI
NOV4i FLGKADVSQEVEEVLAESRVQRSIRLVSVLELLRAPYVRWQVVTVIVTMACYQLCGLNAI
NOV4a WFYTNSIFGKAGIPLAKIPYVTLSTGGIETLAAVFSGLVIEHLGRRPLLIGGFGLMGLFF
NOV4b WFYTNSIFGKAGIPPAKIPYVTLSTGGIETLAAVFSGLVIEHLGRRPLLIGGFGLMGLFF
NOV4c WFYTNSIFGKAGIPPAKIPYVTLSTGGIETLAAVFS------------------------NOV4d WFYTNSIFGKAGIPPAKIPYVTLSTGGIETLAAVFSGLVIEHLGRRPLLIGGFGLMGLFF
NOV4e WFYTNSIFGKAGIPPAKIPYVTLSTGGIETLAAVFSGLVIEHLGRRPLLIGGFGLMGLFF
NOV4f WFYTNSIFGKAGIPLAKIPYVTLSTGGIETLAAVFSGLVTEHLGRRPLLIGGFGLMGLFF
NOV4g WFYTNSIFGKAGIPLAKIPYVTLSTGGIETLAAVFSGLVIEHLGRRPLLIGGFGLMGLFF
NOV4h WFYTNSIFGKAGIPLAKIPYVTLSTGGIETLAAVFSGLVTEHLGRRPLLTGGFGLMGLFF
fNOV4i WFYTNSIFGKAGIPLAKIPYVTZSTGGIETLAAVfS------------------------~NOV4a GTLTITLTLQDHAPWVPYLSIVGILAIIASFCSGPGGIPFILTGEFFQQSQRPAAFITAG
~NOV4b GTLTITLTLQDHAPWVPYLSIVGILAIIASFCSGPGGIPFILTGEFFQQSQRPAAFIIAG
!NOV4c ----------DHAPWVPYLSIVGILAIIASFCSGPGGTPFILTGEFFQQSQRPAAFIIAG
~NOV4d GTLTITLTLQDHAPWVPYLSIVGILAIIASFCSGPGGIPFILTGEFFQQSQRPAAFIIAG
~NOV4e GTLTITLTLQDHAPWVPYLSIVGILAIIASFCSGPGGIPFILTGEFFQQSQRPAAFIIAG
f ~NOV4f GTLTITLTLQDHAPWVPYLSIVGILAIIASFCSGPGGIPFILTGEFFQQSQRPAAFIIAG
~NOV4g GALTITLTLQDHAPWVPYLSIVGILAIIASFCSGPGGIPFILTGEFFQQSQRPAAFIIAG
NOV4h GALTITLTLQDHAPWVPYLSIVGILAIIASFCSGPGGIPFILTGEFFQQSQRPAAFIIAG
~NOV4i ___________ _________________________GIPFILTGEFFQQSQRPAAFIIAG
NOV4a TVNWLSNFAVGLLFPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAEISQAFS
'NOV4b TVNWLSNFAVGLLFPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAEISQAFL
'NOV4c TVNWLSNFAVGLLFPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAEISQAFL
',NOV4d TVNWLSNFAVGLLFPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAEISQAFL
'NOV4e TVNWLSNFAVGLLFPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAEISQAFL
'NOV4f TVNWLSNFAVGLLFPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAEISQAFL
',NOV4g TVNWLSNFAVGLLFPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAEISQAFS
'NOV4h TVNWLSNFAVGLLFPFIQKSLDTYCFLVFATICITGAIYLYFVLPETKNRTYAETSQAFS
'NOV4i TVNWLSNFAVGLLFPFIQKSLDTYCFLVFATTCITGATYLYFVLPETKNRTYAEISQAFS
'NOV4aKRNKAYPPEEKIDSAVTDGKINGRP

'NOV4bE-----------------GKG-GRA

'NOV4cE-----------------GKG-GRA

'NOV4dE-----------------GKG-GRA

NOV4e E-____________-__-GKG-GRA

NOV4f E-----------------GKG-GRA

NOV4g KRNKAYPPEEKIDSAVTDGKINGRP

NOV4h KRNKAYPPEEKIDSAVTDGKINGRP

NOV4i KRNKAYPPEEKTDSAVTDGKINGRP

NOV4a(SEQ ID NO:24) NOV4b(SEQ ID NO:26) NOV4c(SEQ ID NO:28) NOV4d(SEQ ID NO:30) NOV4e(SEQ ID NO:32) NOV4f(SEQ ID NO:34) NOV4g(SEQ ID NO:33) NOV4h(SEQ ID NO:38) NOV4i(SEQ ID NO:40) Further analysis of the NOV4a protein yielded the following properties shown in Table 4C.
Table 4C. Protein Sequence Properties NOV4a SignalP analysis: ~ No Known Signal Sequence Predicted PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length 11; pos.chg 4; neg.chg Z
H-region: length 7; peak value 1.99 PSG score: -2.41 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -4.97 possible cleavage site: between 24 and 25 »> Seems to have no N-terminal signal peptide ALOM: Klein s method TM
et al' for region allocation Tnit position 1 for calculation:

Tentative number the threshold0.5; 9 of TMS(s) for INTEGRAL Likelihood-6.48Transmembrane79 -= 95 INTEGRAL Likelihood-1.75Transmembrane120 -= 136 INTEGRAL Likelihood0.47 Transmembrane140 -= 156 INTEGRAL Likelihood-3.40Transmembrane171 -= 187 INTEGRAL Likelihood-5.73Transmembrane200 -= 216 INTEGRAL Likelihood-0.32Transmembrane283 -= 299 INTEGRAL Likelihood-2.23Transmembrane351 -= 367 INTEGRAL Likelihood-5.89Transmembrane378 -= 394 INTEGRAL Likelihood-5.26Transmembrane449 -= 465 PERIPHERAL Likelihood1.01 (at 419) =

ALOM score: -6.48 (number TMSs: 9) of MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 86 Charge difference: 5.0 C( 4.5) - N(-0.5) C > N: C-terminal side will be inside »> membrane topology: type 3b MITDISC: discrimination of mitochondrial targeting seq R content: 2 Hyd Moment(75): 10.97 Hyd Moment(95): 13.25 G content: 1 D/E content: 2 S/T content: 2 Score: -3.59 Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 17 NRN~SK
NUCDISC: discrimination of nuclear localization signals pat4: RRRK (5) at 47 pat7: PGGRRRK (5) at 44 bipartite: none content of basic residues: 9.1%
NLS Score: 0.27 ~KDEL: ER retention motif in the C-terminus: none HER Membrane Retention Signals:
XXRR-like motif in the N-terminus: ARKQ
inone SILL: 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 l: none type 2: none NMYR: N-myristoylation pattern : none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none ~Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none ~NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: cytoplasmic Reliability: 94.1 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues ;Final Results (k = 9/23):
66.7 %: endoplasmic reticulum j 11.1 ~: vacuolar 11.1 %: mitochondria) 11.1 0: Golgi i » prediction for CG114555-O1 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.

Table 4D. Geneseq Results for NOV4a NOV4a Identities/

Geneseq ProteinlOrganism/L,ength Residues/Similarities' Expect [Patent ' for Identifier#, Date] Match the Matched Value Residues Region AAM79422 Human protein SEQ ID NO 1..507 506/540 (93%)0.0 Homo sapiens, SS8 aa. 19..558 5061540 (93%) [WO2001S7190-A2, 09-AUG-2001]

ABB11910 ' Human GLUTS homologue, 1..507 506/540 (93%)0.0 SEQ ID

N0:2280 - Homo sapiens, 19..558 506/540 (93%) SS8 aa. ' [W0200IS7I88-A2, 09-AUG-2001]

AAM41316 ; Human polypeptide SEQ 1..507 505/540 (93%)~ 0.0 ID NO

6247 - Homo sapiens, 558 19..558 505/540 (93%) aa.

[W0200153312-A1, 26-JUL-2001]

AAE16788 Human transporter and ion 1..504 500/537 (93%)0.0 channel-2S (TRICH-2S) protein - 1..537 501/537 (93%) Homo , Sapiens, 537 aa.. [W0200192304-A2, 06-DEC-2001 ]

AAE14611 ' Human glucose transporter1..500 498/533 (93%)0.0 protein GLUTX - Homo sapiens, 563 1..533 498/533 (93%) aa.

[US6346374-B1, 12-FEB-2002]

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

Table 4E. Public BLASTP
Results for NOV4a NOV4a Identities/

Protein Residues/Similarities ' Expect AccessionProtein/Organism/Length for Match the Matched Value Number ' Residues Portion ' Q9NRM0 Solute carrier family 2, 1..507 506/540 (93%)0.0 facilitated glucose transporter, member1..540 506/540 (93%) (Glucose transporter type 9) - Homa sapiens (Human), 540 aa.
_ ~..~..,..~......"~..-".,~.,.W. .
....~....

~ ...-,~.,.,.
Q8V~V30 Similar to solute carrier 51..507 ..~." 0.0 family 2 457/457 (100%) (Facilitated glucose transporter),SS..S1I 457!457 (I00%);

member 9 - Homa Sapiens (Human), 511 aa.

____ - , ~,._ _ P22732 Solute carrier family 2, 52..494 202/446 (4S%)e-112 facilitated ' glucose transporter, member46..491 291/446 (64%) S ~

(Glucose transporter type S, small intestine) (Fructose transporter) -Homo Sapiens (Human), 501 aa.

602864 fructose transporter - human,' 52..494201!446 (45%)e-111 481 aa. ~

26..471 290/446 (64%) Q8R1N7 Similar to solute carrier 50..493 201/447 (44%)e-111 family 2 (Facilitated glucose transporter),43..489 290/447 (63%) member 5 - Mus musculus (Mouse), SO1 aa.

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

Example 5.
The NOVS clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table SA.
Table SA. NOVS Sequence Sa, CG181662-O1 ~SEQ,ID NO: S1 _1492 by Sequence ~ORF Start: ATG at 4 ~ORF Stop TAA at 940 TGGCGGCCACCGAGGGGGTCGGGGAGGCTGCGCAAGGGGGCGAGCCCGGGCAGCCGGCGCAACC
CCCCAGCCGCACCCACCGCCGCCCCAGCAGCAGCACAAGGAAGAGATGGCGGCCGAGGCTGGGG
CGTGGCGTCCCCCATGGACGACGGGTTTGTGAGCCTGGACTCGCCCTCCTATGTCCTATACAGG
TCCGGAGAGTTCTTTTGAAGTCACTTCAGAAGGATCTACATGAGGAAATGAACTACATCACTGC
ATTGAGGAGCAGCCCAAAAACTATCAAGTTTGGCATCATAGGCGAGTATTAGTGGAATGGCTAA
TCCATCTCAGGAGCTTGAATTTATTGCTGATATTCTTAATCAGGATGCAAAGAATTATCATGCC
AGCATCGACAATGGGTTATTCAGGAATTTAAACTTTGGGATAATGAGCTGCAGTATGTGGACCA
CACATAATGAAAGTGCATGGAACTATTTGAAAGGGATTTTGCAGGATCGTGGTCTTTCCAAATATCC
AATCTGTTAAATCAATTACTTGATTTACAACCAAGTCATAGTTCCCCCTACCTAATTGCCTTTCTTG
GGATATCTATGAAGACATGCTAGAAAATCAGTGTGACAATAAGGAAGACATTCTTAATAAAGCATTA
AGTTATGTGAAATCCTAGCTAAAGAAAAGGACACTATAAGAAAGGAATATTGGAGATACATTGGAAG
TCCCTTCAAAGCAAACACAGCACAGAAAATGACTCACCAACAAATGTACAGCAATAACACCATCCAG
AGAACTTGATGGAATGCTTTTATTTTTTATTAAGGGACCCTGCAGGAGTTTCACACGAGAGTGGTCC
AATGTGATTCTTCT
GGAGGAAGAAAAAGTCCCATAAAGGAACTTTTGTAGTCTTATCAACATATAATCTAATCCCTTAGCAT
CAGCTCCTCCCTCAGTGGTACATGCGTCAAGATTTGTAGCAGTAATAACTGCAGGTCACTTGTATGTA
ATGGATGTGAGGTAGCCGAAGTTTGGTTCAGTAAGCAGGGAATACAGTCGTTCCATCAGAGCTGGTCT
TT
VSa, CGI8I662-O1 ~SEQ ID NO: S2 X312 as BMW at 36492.6kD
'GEAAQGGEPGQPAQPPPQPHPPPPQQQHKEEMAAEAGEAVASPMDDGFVSLDSPSYVLYRH
.SLQKDLHEEMNYITAIIEEQPKNYQVWHHRRVLVEWLRDPSQELEFIADILNQDAKNYHAW
QEFKLWDNELQYVDQLLKEDVRNNSVWNQRYFVISNTTGYNDRAVLEREVQYTLEMIKLVP
YLKGILQDRGLSKYPNLLNQLLDLQPSHSSPYLIAFLVDIYEDMLENQCDNKEDILNKALE
.EKDTIRKEYWRYIGRSLQSKHSTENDSPTNVQQ
VSb, CG181662-02 SEQ..ID NO 53.. .. .... ~1487..bp .. ..... ...
A Sequence ORF Start: ATG at 17 ~ORF Stop: TAA at 9S3 .TGGCG
T'rCCGGAGAGTTCTTTTGAAGTCACTTCAGAAGGATCTACATGAGGAAATGA
AATTGAGGAGCAGCCCAAA.AACTATCAAGTTTGGCATCATAGGCGAGTATTA
TTATCATGCCTGGCAGCATCGACAATGGGTTATTCAGGAATTTAAACTTTGGGATAATGAGCTGC
ATGTGGACCAACTTCTGAAAGAGGATGTGAGAAATAACTCTGTCTGGAACCAAAGATACTTCGTT
ATCCTAATCTGTTAAATCAATTACTTGATTTACAACCAAGTCATAGTTCCCCCTACCTA
AGCATTAGAGTTATGTGAAATCCTAGCTAAAGAAAAGGACACTATAAGAAAGGAATATTGGA
TTGGAAGATCCCTTCAAAGCAAACACAGCACAGAAAATGACTCACCAACAAATGTACAGCAA

ACTGATGCTCCTTGGGTGCTGCTGCTACTCAGACTAGCTCTAAGTAATGTGATTCTTCTAAAGCAAAG
TCATTGGATGGGAGGAGGAAGAAAA.AGTCCCATAAAGGAACTTTTGTAGTCTTATCAACATATAATCT
AATCCCTTAGCATCAGCTCCTCCCTCAGTGGTACATGCGTCAAGATTTGTAGCAGTAATAACTGCAGG
TCACTTGTATGTAATGGATGTGAGGTAGCCGAAGTTTGGTTCAGTAAGCAGGGAATACAGTCGTTCCA
TTGTAATAAAATTATAGCTGTATCTAAAAAC CCAAAAAAAT
Sb, CG181662-02 ~~SEQ ID NO: 54 X312 as BMW at 36492.6kD
uence PGQPAQPPPQPHPPPPQQQHKEEMAAEAGEAVASPMDDGFVSLDSPSYVLYRH
EMNYITAIIEEQPKNYQVWHHRRVLVEWLRDPSQELEFIADILNQDAKNYHAW
ELQYVDQLLKEDVRNNSVWNQRYFVISNTTGYNDRAVLEREVQYTLEMIKLVP
RGLSKYPNLLNQLLDLQPSHSSPYLIAFLVDIYEDMLENQCDNKEDILNKALE
YWRYIGRSLQSKHSTENDSPTNVQQ
OVSc, 307686795 SEQ ID NO: 55148_7 by NA Sequence ~ Start: at 2 ~ORF Ston: TAA at 953 'CGGCGCAACCCCCGCCCCAGCCGCACCCACCGCCGCCCCAGCAGCAGCACAAGGAAGAGATGGCG
'GAGGCTGGGGAAGCCGTGGCGTCCCCCATGGACGACGGGTTTGTGAGCCTGGACTCGCCCTCCTA
'CCTATACAGGCATTTCCGGAGAGTTCTTTTGAAGTCACTTCAGAAGGATCTACATGAGGAAATGA
'ACATCACTGCAATAATTGAGGAGCAGCCCAAAAACTATCAAGTTTGGCATCATAGGCGAGTATTA
'~GAATGGCTAAGAGATCCATCTCAGGAGCTTGAATTTATTGCTGATATTCTTAATCAGGATGCAAA
.TTATCATGCCTGGCAGCATCGACAATGGGTTATTCAGGAATTTAAACTTTGGGATAATGAGCTGC
''ATGTGGACCAACTTCTGAAAGAGGATGTGAGAAATAACTCTGTCTGGAACCAAAGATACTTCGTT
'TCTAACACCACTGGCTACAATGATCGTGCTGTATTGGAGAGAGAAGTCCAATACACTCTGGAAAT
'TAAACTAGTACCACATAATGAAAGTGCATGGAACTATTTGAAAGGGATTTTGCAGGATCGTGGTC
'CCAAATATCCTAATCTGTTAAATCAATTACTTGATTTACAACCAAGTCATAGTTCCCCCTACCTA
'GCCTTTCTTGTGGATATCTATGAAGACATGCTAGAAAATCAGTGTGACAATAAGGAAGACATTCT
.TAAAGCATTAGAGTTATGTGAAATCCTAGCTAAAGAAAAGGACACTATAAGAAAGGAATATTGGA
'ACATTGG.AAGATCCCTTCAAAGCAAACACAGCACAGAAAATGACTCACCAACAAATGTACAGCAA
.CACCATCCAGAAGAACTTGATGGAATGCTTTTATTTTTTATTAAGGGACCCTGCAGGAGTTTCAC
ACTGATGCTCCTTGGGTGCTGCTGCTACTCAGACTAGCTCTAAGTAATGTGATTCTTCTAAAGCAAAG
TCATTGGATGGGAGGAGGAAGAAAAAGTCCCATAAAGGAACTTTTGTAGTCTTATCAACATATAATCT
AATCCCTTAGCATCAGCTCCTCCCTCAGTGGTACATGCGTCAAGATTTGTAGCAGTAATAACTGCAGG
~TCAGAGCTGGTCTGCACACTCACATTATCTTGCTATCACTGTAACCAACTAATGCCAAAAGAACGGTT
TAGCTGTA
OVSc, 307686795 SEQ ID NO: 56 317 as MW at 37049.2kD
rotein Seauence DEMAATEGVGEAAQGGEPGQPAQPPPQPHPPPPQQQHKEEMAAEAGEAVASPMDDGFVSLDSPSY
'RHFRRVLLKSLQKDLHEEMNYITAIIEEQPKNXQVWHHRRVLVEWLRDPSQELEFIADILNQDAK
AWQHRQWVIQEFKLWDNELQYVDQLLKEDVRNNSVWNQRYFVISNTTGYNDRAVLEREVQYTLEM
~VPHNESAWNYLKGILQDRGLSKYPNLLNQLLDLQPSHSSPYLIAFLVDIYEDMLENQCDNKEDIL
.LELCEILAKEKDTIRKEYWRYIGRSLQSKHSTENDSPTNVQQ
OVSd, CG181662-03 ~SEQ ID NO. 57 (1344 by NA Sequence ORF Start: ATG at 17 ' ORF Stop: TAA at 1154 TGGCGGCCACCGAGGGGGTCGGGGAGGCTGCGCAAGGGGGCGAGCCCGGGC
.TCATTTATAGTGACAAATTTAGAGATGTTTATGATTACTTCCGAGCTGTCCTGCAG
AGTGAACGAGCTTTTAAGCTAACCCGGGATGCTATTGAGTTAAATGCAGCCAATTA
.TTTCCGGAGAGTTCTTTTGAAGTCACTTCAGAAGGATCTACATGAGGAAATGAACT

ACATCACTGCAATAATTGAGGAGCAGCCCAAAAACTATCAAGTTTGGCATCATAGGCGAGTATTAGTG
GAATGGCTAAGAGATCCATCTCAGGAGCTTGAATTTATTGCTGATATTCTTAATCAGGATGCAAAGAA
TTATCATGCCTGGCAGCATCGACAATGGGTTATTCAGGAATTTAAACTTTGGGATAATGAGCTGCAGT
ATGTGGACCAACTTCTGAAAGAGGATGTGAGAAATAACTCTGTCTGGAACCAAAGATACTTCGTTATT
TCTAACACCACTGGCTACAATGATCGTGCTGTATTGGAGAGAGAAGTCCAATACACTCTGGAAATGAT
TAAACTAGTACCACATAATGAAAGTGCATGGAACTATTTGAAAGGGATTTTGCAGGATCGTGGTCTTT
CCAAATATCCTAATCTGTTAAATCAATTACTTGATTTACAACCAAGTCATAGTTCCCCCTACCTAATT
GCCTTTCTTGTGGATATCTATGAAGACATGCTAGAAAATCAGTGTGACAATAAGGAAGACATTCTTAA
TAAAGCATTAGAGTTATGTGAAATCCTAGCTAAAGAAAAGGACACTATAAGAAAGGAATATTGGAGAT
AcUAGTTCCTTCCCTTTTGTGGTGTAAAAGTGCATCACACAGGTATTGCTTTTTACAGACTGATGCTCC
TTGGTGCTGCTGCATCTATCTCAGACTAGCTCTAGTATGTGATCTCTAAGCA
Sd,~CG181662-03 ~SEQ ID NO: 58 379 as MW at 44408.2kD
TEGVGEAAQGGEPGQPAQPPPQPHPPPPQQQHKEEMAAEAGEAVASPMDDGFVSLDSPSYVLYRD
WADIDPVPQNDGPNPWQIIYSDKFRDVYDYFRAVLQRDERSERAFKLTRDAIELNAANYTVWHF
LLKSLQKDLHEEMNYITAIIEEQPKNYQVWHHRRVLVEWLRDPSQELEFIADILNQDAKNYHAWQ
WVIQEFKLWDNELQYVDQLLKEDVRNNSVWNQRYFVISNTTGYNDRAVLEREVQYTLEMIKLVPH
AWNYLKGILQDRGLSKYPNLLNQLLDLQPSHSSPYLIAFLVDTYEDMLENQCDNKEDILNKALEL
LAKEKDTIRKEYWRYIGRSLQSKHSTENDSPTNVQQ
VSe, CG181662-04 ~SEQ ID NO: 59 11156 by A Sequence 0~' Start: ATG at l l ORF Stop: end of GCCGTGGCGTCCCCCATGGACGACGGGTTTGTGAGCCTGGACTCGCCCTCCTATGTCCT
CAGAGCAGAATGGGCTGATATAGATCCGGTGCCGCAGAATGATGGCCCCAATCCCGTGG
TTTATAGTGACAAATTTAGAGATGTTTATGATTACTTCCGAGCTGTCCTGCAGCGTGAT
GAACGAGCTTTTAAGCTAACCCGGGATGCTATTGAGTTAAATGCAGCCAATTATACAGT
CCGGAGAGTTCTTTTGAAGTCACTTCAGAAGGATCTACATGAGGAAATGAACTACATCA
CATCTCAGGAGCTTGAATTTATTGCTGATATTCTTAATCAGGATGCAAAGAATTATCA
CATCGACAATGGGTTATTCAGGAATTTAAACTTTGGGATAATGAGCTGCAGTATGTGG
GAAAGAGGATGTGAGAAATAACTCTGTCTGGAACCAAAGATACTTCGTTATTTCTAAC
ACAATGATCGTGCTGTATTGGAGAGAGAAGTCCAATACACTCTGGAAATGATTAAACT
AATGAAAGTGCATGGAACTATTTGAAAGGGATTTTGCAGGATCGTGGTCTTTCCAAAT
GTTAAATCAATTACTTGATTTACAACCAAGTCATAGTTCCCCCTACCTAATTGCCTTT
TCTATGAAGACATGCTAGAAAATCAGTGTGACAATAAGGAAGACATTCTTAATAAAGC
TGTGAAATCCTAGCTAAAGAAAAGGACACTATAAGAAAGGAATATTGGAGATACATTG
OVSe, CG181662-04 ~SEQ ID NO: 60 X379 as BMW at 44408.2kD
uence ATEGVGEAAQGGEPGQPAQPPPQPHPPPPQQQHKEEMAAEAGEAVASPMDDGFVSLDSPSYVLYRD
EWADIDPVPQNDGPNPWQIIYSDKFRDVYDYFRAVLQRDERSERAFKLTRDAIELNAANYTVWHF
VLLKSLQKDLHEEMNYITAIIEEQPKNYQVWHHRRVLVEWLRDPSQELEFIADILNQDAFCNYHAWQ
QWVIQEFKLWDNELQYVDQLLKEDVRNNSVWNQRYFVISNTTGYNDRAVLEREVQYTLEMIKLVPH
SAWNYLKGILQDRGLSKYPNLLNQLLDLQPSHSSPYLIAFLVDIYEDMLENQCDNKEDILNKALEL
ILAKEKDTIRKEYWRYIGRSLQSKHSTENDSPTNVQQ
)VSf, 13382357 SNP CG181662-O1 SEQ ID NO: 1492 bp, SNP at position JA Sequence 61 310 C/T
RF Start: ORF Stop: TAA at 940 TGat4 CCAGCAGCAGCACAAGGAAGAGATGGCGGCCGAGGCTGGGGAA

.TGGACGACGGGTTTGTGAGCCTGGACTCGCCCTCCTATGTCCTATACAGGCAT
TGAAGTCACTTCAGAAGGATCTACATGAGGAAATGAACTACATCACTGCAATA
TTCTTAATCAGGATGCAAAGAATTATCATGCCTGGCAG
GATGTGAGAAATAACTCTGTCTGGAACCAAAGATACTTCGTTATTTCTAACACCACTGGCTAC
CGTGCTGTATTGGAGAGAGAAGTCCAATACACTCTGGAAATGATTAAACTAGTACCACATAAT
GCATGGAACTATTTGAAAGGGATTTTGCAGGATCGTGGTCTTTCCAAATATCCTAATCTGTTA
TTACTTGATTTACAACCAAGTCATAGTTCCCCCTACCTAATTGCCTTTCTTGTGGATATCTAT
ATGCTAGAAAATCAGTGTGACAATAAGGAAGACATTCTTAATAAAGCATTAGAGTTATGTGAA
GCTAAAGAAAAGGACACTATAAGAAAGGAATATTGGAGATACATTGGAAGATCCCTTCAAAGC
~CAGACTAGCTCTAAGTAATGTGATTCTTCTAAAGCAAAGTCATTGGATGGGAGGAGGAAGAAAAArTrr_ ~TATCACTGTAACCAACTAATGCCAAAAGAACGGTTTTGTAATAAAATTATAGCTGTATCT~~~AAAAAAA
'OVSf, 13382357 SNP CG181662- SEQ ID NO: 3I2 as SNP: His to Tyr at 1 62 Dosition 103 MAATEGVGEAAQGGEPGQPAQPPPQPHPPPPQQQHKEEMAAEAGEAVASPMDDGFVSLDSPSYVLYRHF
RRVLLKSLQKDLHEEMNYITAIIEEQPKNYQVW_YHRRVLVEWLRDPSQELEFIADILNQDAI~NYHAWQH
RQWVIQEFKLWDNELQYVDQLLKEDVRNNSVWNQRYFVISNTTGYNDRAVLEREVQYTLEMIKLVPHNE
SAWNYLKGILQDRGLSKYPNLLNQLLDLQPSHSSPYLIAFLVDIYEDMLENQCDNKEDILNKALELCEI
Sg, 13377970 SNP CG181662-Ol SEQ ID 1492 bp, SNP at position Sequence NO: 63 457 G/C
RF Start: ORF Stop: TAA at 940 TG at 4 CGGGCAGCCGGCGCAACCC
P.CGGGTTTGTGAGCCTGGACTCGCCCTCCTATGTCCTATACAGGCAT
CACTTCAGAAGGATCTACATGAGGAAATGAACTACATCACTGCAATA
ATCAAGTTTGGCATCATAGGCGAGTATTAGTGGAATGGCTAAGAGAT
ACAATGGGTTATTCAGGAATTTAAACTTTGGGATAAT_CAGCTGCAGTATGTGGACCAACTTCTG
GGATGTGAGAAATAACTCTGTCTGGAACCAAAGATACTTCGTTATTTCTAACACCACTGGCTAC
TCGTGCTGTATTGGAGAGAGAAGTCCAATACACTCTGGAAATGATTAAACTAGTACCACATAAT
TGCATGGAACTATTTGAAAGGGATTTTGCAGGATCGTGGTCTTTCCAAATATCCTAATCTGTTA
ATTACTTGATTTACAACCAAGTCATAGTTCCCCCTACCTAATTGCCTTTCTTGTGGATATCTAT
CATGGTAGAAAATCAGTGTGACAATAAGGAAGACATTCTTAATAAAGCATTAGAGTTATGTGAA
AGCTAAAGAAAAGGACACTATAAGAAAGGAATATTGGAGATACATTGGAAGATCCCTTCAAAGC
AGCTCTAAGT
AAT
AAT
~NOVS~,~ 13377970 SNP ~SEQ ID NO: 64 X312 SNP: Glu to Gln at 181662-O1 ~ ~aa. position 152 GEAAQGGEPGQPAQPPPQPHPPPPQQQHKEEMAAEAGEAVASPMDDGFVSLDSPSYVLYRHF
LQKDLHEEMNYITAIIEEQPKNYQVWHHRRVLVEWLRDPSQELEFIADILNQDAIQJYHAWQH
FKLWDN_QLQYVDQLLKEDVRNNSVWNQRYFVISNTTGYNDRAVLEREVQYTLEMIKLVPHNE
GILQDRGLSKYPNLLNQLLDLQPSHSSPYLIAFLVDIYEDMLENQCDNKEDILNKALELCEI
IRKEYWRYIGRSLQSKHSTENDSPTNVQQ
VSh, 13378241 SNP CG181662-OI ~SEQ ID 1492 bp, SNP at position A Sequence . NO: 65 729 C/A
RF Start: ?ORF Stop: TAA at 940 TG at 4 CGCACCCACCGCCGCCCCAGCAGCAGCACAAGGAAGAGATGGCGGCCGAGGCTGGGGAA
CCCCCATGGACGACGGGTTTGTGAGCCTGGACTCGCCCTCCTATGTCCTATACAGGCAT
GAGCAGCCCAAAAACTATCAAGTTTGGCATCATAGGCGAGTATTAGTGGAATGGCTAAGAGAT
CAGGAGCTTGAATTTATTGCTGATATTCTTAATCAGGATGCAAAGAATTATCATGCCTGGCAG
CAATGGGTTATTCAGGAATTTAAACTTTGGGATAATGAGCTGCAGTATGTGGACCAACTTCTG
GATGTGAGAAATAACTCTGTCTGGAACCAAAGATACTTCGTTATTTCTAACACCACTGGCTAC
CGTGCTGTATTGGAGAGAGAAGTCCAATACACTCTGGAAATGATTAAACTAGTACCACATAAT
GCATGGAACTATTTGAAAGGGATTTTGCAGGATCGTGGTCTTTCCAAATATCCTAATCTGTTA
TTACTTGATTTACAACCAAGTCATAGTTCCCC_ATACCTAATTGCCTTTCTTGTGGATATCTAT
ATGCTAGAAAATCAGTGTGACAATAAGGAAGACATTCTTAATAAAGCATTAGAGTTATGTGAA
GCTAAAGAAAAGGACACTATAAGAAAGGAATATTGGAGATACATTGGAAGATCCCTTCAAAGC
AGCACAGAAAATGACTCACCAACAAATGTACAGCAATAACACCATCCAGAAGAACTTGATGGA
~GTGTAAAAGTGCATCACACAGGTATTGCTTTTTAACAAGAACTGATGCTCCTTGGGTGCTGCTGCTACT
CATAAAGGAACTTTTGTAGTCTTATCAACATATAATCTAATCCCTTAGCATCAGCTCCTCCCTCAGTGG
TACATGCGTCAAGATTTGTAGCAGTAATAACTGCAGGTCACTTGTATGTAATGGATGTGAGGTAGCCGA
~TATCACTGTAACCAACTAATGCCAAAAGAACGGTTTTGTAATAAAATTATAGCTGTATCTF~~AAAAAAA
Sh, 13378241 SNP CG181662-Ol ~SEQ ID 312 SNP: no change in the in Sequence NO: 66 as protein sequence EGVGEAAQGGEPGQPAQPPPQPHPPPPQQQHKEEMAAEAGEAVASPMDDGFVSLDSPSYVLYRHF
LKSLQKDLHEEMNYITAIIEEQPKNYQVWHHRRVLVEWLRDPSQELEFIADILNQDAKNYHAWQH
IQEFKLWDNELQYVDQLLKEDVRNNSVWNQRYFVISNTTGYNDRAVLEREVQYTLEMIKLVPHNE
YLKGILQDRGLSKYPNLLNQLLDLQPSHSS_PYLIAFLVDIYEDMLENQCDNKEDILNKALELCEI
KDTIRKEYWRYIGRSLQSKHSTENDSPTNVQQ
>i, 13377901 SNP CG181662-O1 ~SEQ ID 1492 bp, SNP at position Sequence NO: 67 1330 G/T
ORF ORF Stop: TAA at 940 Start:
ATG at TGGCGGCCACCGAGGGGGTCGGGGAGGCTGCGCAAGGGGGCGAGCCCGGGCAGCCGGCGCAACCC
CCCAGCCGCACCCACCGCCGCCCCAGCAGCAGCACAAGGAAGAGATGGCGGCCGAGGCTGGGGAA
TGGCGTCCCCCATGGACGACGGGTTTGTGAGCCTGGACTCGCCCTCCTATGTCCTATACAGGCAT
GGAGAGTTCTTTTGAAGTCACTTCAGAAGGATCTACATGAGGAAATGAACTACATCACTGCAATA
AGGAGCAGCCCAAAA.ACTATCAAGTTTGGCATCATAGGCGAGTATTAGTGGAATGGCTAAGAGAT
AATGAGCTGCAGTATGTGGACCAACTTCTG
TACTTCGTTATTTCTAACACCACTGGCTAC

AATGATCGTGCTGTATTGGAGAGAGAAGTCCAATACACTCTGGAAATGATTAAACTAGTACCACATAAT
GAAAGTGCATGGAACTATTTGAAAGGGATTTTGCAGGATCGTGGTCTTTCCAAATATCCTAATCTGTTA
AATCAATTACTTGATTTACAACCAAGTCATAGTTCCCCCTACCTAATTGCCTTTCTTGTGGATATCTAT
GAAGACATGCTAGAAAATCAGTGTGACAATAAGGAAGACATTCTTAATAAAGCATTAGAGTTATGTGAA
ATCCTAGCTAAAGAAAAGGACACTATAAGAAAGGAATATTGGAGATACATTGGAAGATCCCTTCAAAGC
AAACACAGCACAGAA.AATGACTCACCAACAAATGTACAGCAATAACACCATCCAGAAGAACTTGATGGA
ATGCTTTTATTTTTTATTAAGGGACCCTGCAGGAGTTTCACACGAGAGTGGTCCTTCCCTTTGCCTGTG
GTGTAAAAGTGCATCACACAGGTATTGCTTTTTAACAAGAACTGATGCTCCTTGGGTGCTGCTGCTACT
CAGACTAGCTCTAAGTAATGTGATTCTTCTAAAGCAAAGTCATTGGATGGGAGGAGGAAGAAAA.AGTCC
CATAAAGGAACTTTTGTAGTCTTATCAACATATAATCTAATCCCTTAGCATCAGCTCCTCCCTCAGTGG
TACATGCGTCAAGATTTGTAGCAGTAATAACTGCAGGTCACTTGTATGTAATGGATGTGAGGTAGCCGA
AGTTTGGTTCAGTAAGCATGGAATACAGTCGTTCCATCAGAGCTGGTCTGCACACTCACATTATCTTGC
TATCACTGTAACCAACTAATGCCAAAAGAACGGTTTTGTAATAAAATTATAGCTGTATCTAAAAAAAAA
CAAA
NOVSi, 13377901 SNP CG181662-01 ~ SEQ ID NO: 68 ~ 312 as ' SN Not in coding Protein Sequence . ____~.____._. .~.......___...' ~.__._~..__._W.__i____-~_.__..~~region __ ___ MAATEGVGEAAQGGEPGQPAQPPPQPHPPPPQQQHKEEMAAEAGEAVASPMDDGFVSLDSPSYVLYRHF
RRVLLKSLQKDLHEEN1~7~YITAIIEEQPKNYQVWHHRRVLVEWLRDPSQELEFIADILNQDAKNYHAWQH

SAWNYLKGILQDRGLSKYPNLLNQLLDLQPSHSSPYLIAFLVDIYEDMLENQCDNKEDILNKALELCEI
LAKEKDTIRKEYWRYIGRSLQSKHSTENDSPTNVQQ
-' .
NOVSj, 13377900 SNP~CG181662-Ol j SEQ ID i 1492 bp, SNP at position DNA Sequence 'NO: 69 ~ 1385 A/C
_.__....... ...... ......._. ....
........._................_........._........
ORF Start. ! ORF Stop: TAA at 940 _ _ _ __ _ _ _ _ . _ ~ A_T_G a_t 4 ~~~_ _ _ _ _ __ __ _GAGATGGCGGCCACCGAGGGGGTCGGGGAGGCTGCGCAAGGGGGCGAGCCCGGGCAGCCGGCGCAACCC
CCGCCCCAGCCGCACCCACCGCCGCCCCAGCAGCAGCACAAGGAAGAGATGGCGGCCGAGGCTGGGGAA
'GCCGTGGCGTCCCCCATGGACGACGGGTTTGTGAGCCTGGACTCGCCCTCCTATGTCCTATACAGGCAT
TTCCGGAGAGTTCTTTTGAAGTCACTTCAGAAGGATCTACATGAGGAAATGAACTACATCACTGCAATA
'ATTGAGGAGCAGCCCAAAAACTATCAAGTTTGGCATCATAGGCGAGTATTAGTGGAATGGCTAAGAGAT
CCATCTCAGGAGCTTGAATTTATTGCTGATATTCTTAATCAGGATGCAAAGAATTATCATGCCTGGCAG
~CATCGACAATGGGTTATTCAGGAATTTAAACTTTGGGATAATGAGCTGCAGTATGTGGACCAACTTCTG
AAAGAGGATGTGAGAAATAACTCTGTCTGGAACCAAAGATACTTCGTTATTTCTAACACCACTGGCTAC
AATGATCGTGCTGTATTGGAGAGAGAAGTCCAATACACTCTGGAAATGATTAAACTAGTACCACATAAT
GAAAGTGCATGGAACTATTTGAAAGGGATTTTGCAGGATCGTGGTCTTTCCAAATATCCTAATCTGTTA
AATCAATTACTTGATTTACAACCAAGTCATAGTTCCCCCTACCTAATTGCCTTTCTTGTGGATATCTAT
',GAAGACATGCTAGAAAATCAGTGTGACAATAAGGAAGACATTCTTAATAAAGCATTAGAGTTATGTGAA
'ATCCTAGCTAAAGAAAAGGACACTATAAGAAAGGAATATTGGAGATACATTGGAAGATCCCTTCAAAGC
'AAACACAGCACAGAAAATGACTCACCAACAAATGTACAGCAATAACACCATCCAGAAGAACTTGATGGA
ATGCTTTTATTTTTTATTAAGGGACCCTGCAGGAGTTTCACACGAGAGTGGTCCTTCCCTTTGCCTGTG
GTGTAAAAGTGCATCACACAGGTATTGCTTTTTAACAAGAACTGATGCTCCTTGGGTGCTGCTGCTACT
ICAGACTAGCTCTAAGTAATGTGATTCTTCTAAAGCAAAGTCATTGGATGGGAGGAGGAAGAAAAAGTCC
CATAAAGGAACTTTTGTAGTCTTATCAACATATAATCTAATCCCTTAGCATCAGCTCCTCCCTCAGTGG
TACATGCGTC.AAGATTTGTAGCAGTAATAACTGCAGGTCACTTGTATGTAATGGATGTGAGGTAGC_CGA
~AGTTTGGTTCAGTAAGCAGGGAATACAGTCGTTCCATCAGAGCTGGTCTGCACACTCACATTATCTTGC
TATCCCTGTAACCAACTAATGCCAAAAGAACGGTTTTGTAATAAAATTATAGCTGTATCTAAAAAAAAA
NOVS~, 13377900 SNP CGI8I662-O1 SEQ ID NO 70 312 as SNP: Not m coding Protein Sequence ~ region _ .. ... ____ ...._ _. ...
MAATEGVGEAAQGGEPGQPAQPPPQPHPPPPQQQHKEEMAAEAGEAVASPMDDGF'VSLDSPSYVLYRHF
RRVLLKSLQKDLHEEMNYITAIIEEQPKNYQVWHHRRVLVEWLRDPSQELEFIADILNQDAFCNYHAWQH
RQWVIQEFKLWDNELQYVDQLLKEDVRNNSVWNQRYFV'ISNTTGYNDRAVLEREVQYTLEMIKLVPHNE
SAWNYLKGILQDRGLSKYPNLLNQLLDLQPSHSSPYLTAFLVDIYEDMLENQCDNKEDILNKALELCEI
LAKEKDTIRKEYWRYIGRSLQSKHSTENDSPTNVQQ

A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table SB.
Table SB. Comparison of the NOVS protein sequences.
NOVSa -----MAATEGVGEAAQGGEPGQPAQPPPQPHPPPPQQQHKEEMAAEAGEAVASPMDDGF
NOV5b -----MAATEGVGEAAQGGEPGQPAQPPPQPHPPPPQQQHKEEMAAEAGEAVASPMDDGF
NOVSc GRVDEMAATEGVGEAAQGGEPGQPAQPPPQPHPPPPQQQHKEEMAAEAGEAVASPMDDGF
NOVSd -----MAATEGVGEAAQGGEPGQPAQPPPQPHPPPPQQQHKEEMAAEAGEAVASPMDDGF
NOVSe -----MAATEGVGEAAQGGEPGQPAQPPPQPHPPPPQQQHKEEMAAEAGEAVASPMDDGF
NOVSa VSLDSPSYVLYR-_______________________________________________ NOVSb VSLDSPSYVLYR-_________-__-__________________________________ NOVSC VSLDSPSYVLYR-________________-______________________________ NOVSd VSLDSPSYVLYRDRAEWADIDPVPQNDGPNPVVQIIYSDKFRDVYDYFRAVLQRDERSER
NOVSe VSLDSPSYVLYRDRAEWADIDPVPQNDGPNPWQIIYSDKFRDVYDYFRAVLQRDERSER
NOVSa -------------------HFRRVLLKSLQKDLHEEMNYITAIIEEQPKNYQVWHHRRVL
NOVSb -------------------HFRRVLLKSLQKDLHEEMNYITAITEEQPKNYQVWHHRRVL
NOVSc -------------------HFRRVLLKSLQKDLHEEMNYITAIIEEQPKNYQVWHHRRVL
NOVSd AFKLTRDATELNAANYTVWHFRRVLLKSLQKDLHEEMNYTTAITEEQPKNYQVWHHRRVL
NOVSe AFKLTRDATELNAANYTVWHFRRVLLKSLQKDLHEEMNYITAITEEQPKNYQVWHHRRVL
NOVSa VEWLRDPSQELEFIADILNQDAKNYHAWQHRQWVIQEFKLWDNELQYVDQLLKEDVRNNS
NOVSb VEWLRDPSQELEFIADILNQDAKNYHAWQHRQWVIQEFKLWDNELQYVDQLLKEDVRNNS
NOVSc VEWLRDPSQELEFIADILNQDAKNYHAWQHRQWVIQEFKLWDNELQYVDQLLKEDVRNNS
NOVSd VEWLRDPSQELEFIADILNQDAKNYHAWQHRQWVIQEFKLWDNELQYVDQLLKEDVRNNS
NOVSe VEWLRDPSQELEFIADILNQDAKNYHAWQHRQWVTQEFKLWDNELQYVDQLLKEDVRNNS
NOVSa VWNQRYFVISNTTGYNDRAVLEREVQYTLEMIKLVPHNESAWNYLKGTLQDRGLSKYPNL
NOVSb VWNQRYFVTSNTTGYNDRAVLEREVQYTLEMIKLVPHNESAWNYLKGTLQDRGLSKYPNL
NOVSc VWNQRYFVISNTTGYNDRAVLEREVQYTLEMIKLVPHNESAWNYLKGTLQDRGLSKYPNL
NOVSd VWNQRYFVISNTTGYNDRAVLEREVQYTLEMIKLVPHNESAWNYLKGTLQDRGLSKYPNL
NOVSe VWNQRYFVTSNTTGYNDRAVLEREVQYTLEMIKLVPHNESAWNYLKGTLQDRGLSKYPNL
NOVSa LNQLLDLQPSHSSPYLIAFLVDIYEDMLENQCDNKEDTLNKALELCETLAKEKDTIRKEY
NOVSb LNQLLDLQPSHSSPYLIAFLVDIYEDMLENQCDNKEDTLNKALELCEILAKEKDTIRKEY
NOVSc LNQLLDLQPSHSSPYLIAFLVDIYEDMLENQCDNKEDTLNKALELCEILAKEKDTIRKEY
NOVSd LNQLLDLQPSHSSPYLIAFLVDIYEDMLENQCDNKEDILNKALELCEILAKEKDTIRKEY
NOVSe LNQLLDLQPSHSSPYLIAFLVDIYEDMLENQCDNKEDTLNKALELCETLAKEKDTTRKEY
NOVSa WRYIGRSLQSKHSTENDSPTNVQQ
NOVSb WRYIGRSLQSKHSTENDSPTNVQQ
NOVSc WRYIGRSLQSKHSTENDSPTNVQQ
NOVSd WRYIGRSLQSKHSTENDSPTNVQQ
NOVSe WRYIGRSLQSKHSTENDSPTNVQQ
NOVSa (SEQ TD NO: 52) NOVSb (SEQ ID NO: 54) NOVSc (SEQ TD NO: 56) NOVSd (SEQ TD NO: 58) NOVSe (SEQ TD NO: 60j Further analysis of the NOVSa protein yielded the following properties shown in Table SC.

Table SC. Protein Sequence Properties NOVSa SignaIP analysis: ~ No Known Signal Sequence Predicted PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length 9; pos.chg 0; neg.chg 2 H-region: length 5; peak value 0.00 PSG score: -4.40 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -11.19 possible cleavage site: between 13 and 14 »> Seems to have no N-terminal signal peptide ALOM: Klein et al's method for TM region allocation Init position for calculation: 1 Tentative number of TMS(s) for the threshold 0.5: 0 number of TMS(s) .. fixed PERIPHERAL Likelihood = 6.42 (at 240) ALOM score: 6.42 (number of TMSs: 0) MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment(75): 6.27 Hyd Moment(95): 4.56 G content: 2 D/E content: 2 S/T content: 1 Score: -7.86 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: 9.9%
NLS Score: -0.47 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: none SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif:
type 1: none type 2.: none NMYR: N-myristoylation pattern : none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none 'Dileucine motif in the tail: none 'checking 63 PROSITE DNA binding motifs: none ',checking 71 PROSTTE ribosomal protein motifs: none ',checking 33 PROSTTE prokaryotic DNA binding motifs: none 'NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: nuclear Reliability: 55.5 COTL: Lupas's algorithm to detect coiled-coil regions j 249 D 0.58 250 I 0.58 251 Y 0.82 [ 252 E 0.82 i 253 D 0.93 254 M 0.93 255 L 0.97 256 E 0.97 257 N 0.97 258 Q 0.97 259 C 0.97 260 D 0. 97 262 N 0.97 262 K 0.97 263 E 0.97 264 D 0.97 265 I 0.97 266 L 0.97 267 N 0.97 268 K 0.97 269 A 0.97 270 L 0.97 271 E 0.97 272 L 0.97 273 C 0.97 274 E 0.97 275 I 0.97 276 L 0.97 277 A 0.97 278 K 0.97 279 E 0.97 280 K 0.97 281 D 0.97 282 T 0.97 283 I 0.86 284 R 0.70 285 K 0.70 286 E 0.70 287 Y 0.70 total: 39 residues Final Results (k = 9/23):
78.3 0: nuclear 8.7 %: mitochondria) 8.7 %: cytoplasmic 4.3 s: peroxisomal » prediction for CG181662-01 is nuc (k=23) A search of the NOVSa protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table SD.
Table SD. Geneseq Results for NOVSa NOVSa Identities/

Geneseq Protein/Organism/Lerigth Residues/Similarities Expect [Patent for Identifier#, Date] Match the Matched Value ResiduesRegion AAB58384 Lung cancer associated polypeptide1..312 278/380 (73%)e-152 sequence SEQ ID 722 - Homo 16..394 289/380 (75%) Sapiens, 394 aa. [W0200055180-A2, 21-SEP-2000]

ABB08436 ' Protein sequence 2 relative1..312 278/380 (73%)e-152 to the farnesyltransferase ofthe 1..379 289/380 (75%) invention -Unidentified, 379 aa. [KR98075770-A, 16-NOV-1998]

AAU77150 Human geranylgeranyltransferase1..312 278/380 (73%)e-152 ', type I related protein 1..379 289/380 (75%) #2 -Unidentified, 380 aa. [KR98075771-A, 16 NOV-1998]

AAW04431 Human farnesyl transferase 1..312 278/380 (73%)e-152 enzyme alpha subunit - Homo Sapiens,1..379 289/380 (75%) aa. [W09634113-A2, 31-OCT-1996]

AAR77841 Human farnesyl protein transferase1..312 278/380 (73%)e-152 alpha subunit - Homo Sapiens,1..379 289/380 (75%) aa. [LJS5420245-A, 30-MAY-1995]

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

Table SE. Public BLASTP Results fox NOVSa P NOVSa Identities/
t i ro Protein/Organism/Length Residues/SimilaritiesExpect e for n t-lccession Number Match the Matched Value Residues Portion P49354 Protein farnesyltransferase 1..312 278/380 (73%)e-152 alpha subunit (EC 2.5.1.-) (CAAX 1..379 289/380 (75%) farnesyltransferase alpha subunit) (RAS

proteins prenyltransferase alpha) (FTase-alpha) - Homo sapiens (Human), 379 aa.

P29702 Protein farnesyltransferase 56..312 242/257 (94%)e-143 alpha subunit (EC 2.5.1.-) (CAAX 85..340 251/257 (97%) farnesyltransferase alpha subunit) (RAS

proteins prenyltransferase alpha) (FTase-alpha) - Bos taurus (Bovine), 340 as (fragment).

Q04631 Protein farnesyltransferase 1..310 258/378 (68%)e-139 alpha subunit (EC 2.5.1.-) (CAAX 1..377 277/378 (73%) farnesyltransferase alpha subunit) (RAS

proteins prenyltransferase alpha) (FTase-alpha) - Rattus norvegicus (Rat), 377 aa.

Q61239 Protein farnesyltransferase 1..310 256/378 (67%)e-139 alpha 4 subunit (EC 2.5.1.-) (CAA~~ 1..377 277/378 (72%) farnesyltransferase alpha subunit) (RAS

proteins prenyltransferase alpha) (FTase-alpha) - Mus musculus (Mouse), 377 aa.

Q92IF7 Similar to farnesyltransferase,1..310 255/378 (67%)e-138 CAAX

box, alpha - Mus musculus 1..377 277/378 (72%) (Mouse), 377 aa.

PFam analysis predicts that the NOVSa~protein contains the domains shown in the Table SF.
iso Table 5F. Domain Analysis of NOVSa Identities/
Pfam Domain NOVSa Match Re i 'e g on Similariti s Expect Value for the Matched Region PPTA 83..113 12/31 (39%) 3.3e-11 28131 (90%) PPTA ~~~~117..147 12/31 (39%) 4e-12 29131 (94%) PPTA 151..181 9/31 (29%) 2.8e-09 29/31 (94%) PPTA 191..221 a 15/31 (48%) 1.7e-09 28/31 (90%) Example 6.
The NOV6 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 6A.
Table 6A. NOV6 NOV6a, CG182223-O1 ~SEQ ID..N0.,714683 bp.
DNA Sequence O~' Std: ATG at 7 ORF Stop: TAA at 4588 TTTTTATATGTTCGGGTTGATGG
TCCTTCCGATGTCATCGTCTCTA
TGACAAGGACGATCCCCGGTCCCACAGGATGCTTCTGCCCAGCGGATC
TGCACGGGCGCAGGAGTAAACCTGATGAAGGAAGCTACGTTTGTGTTG
GCAGTGAGTCGAAATGCGTCTCTGGAAGTGGCATTGTTACGAGATGAC
TGTTGTAGTGGCAGCTGGAGAGCCTGCAATCCTGGAGTGCCAGCCTCC
CCATCTACTGGAAAAAAGACAAAGTTCGAATTGATGACAAGGAAGAAA
.TTAACCAGGTGGTACTGGAGGAAGAAGCTGTAGAATTTCGTTGTCAAG
TGAAGGCACCTATATGTG
TTTCCCTGTGAAACTAAA
CAGTAGATGCTCAGTGTCACCAACTGGAGACCTCACAATCACCAACATTCAACGTT
ACTACATCTGCCAGGCTTTAACTGTGGCAGGAAGCATTTTAGCAAAAGCTCAACTG
.CAGCG'1'TACTGAAATGTAAAGCCACTGGTGATCCTCTTCCTGTAATTAGCTGGT
ACTTTTCCGGGTAGAGATCCAAGAGCAACAATTCAAGAGCAAGGCACACTGCAG
GATTTCTGATACTGGCACTTATACTTGTGTGGCTACAAGTTCAAGTGGAGAGAC
CAGCCAGGTACCCCTGGAACCCTTCCAGCAAGTGCATATATCATTGAGGCTTTCAGCCAATCAGTGAG
CAACAGCTGGCAGACCGTGGCAAACCATGTAAAGACCACCCTCTATACTGTAAGAGGACTGCGGCCCA

TCAACCCCCAAGGTCTCAGTGACCCAAGTCCCATGTCA
AGGAGATGTCCTTGTCCGTCTTCATAATCCAGTTGTGCTGACTCCCACCACGGTTCAGGTCACAT
CAGTTTATCCAAGGCTACCGAGTGATGTATCGTCAGACTTCAGGTCTG
CAGGCGACATCTTCGTGGCAGAATTTAGATGCCAAAGTCCCGACTGAACGAAGTGCTGTCTTAGTCAA
CCTGAAAAAGGGGGTGACTTATGAAATTAAAGTACGGCCATATTTTAATGAGTTCCAAGGAATGGATA
GTGAATCTAAAACGGTTCGTACTACTGAAGAAGCCCCAAGTGCCCCACCACAGTCTGTCACTGTACTG
CTCCAGATCACCAGAA
TTATCCAAGAATACAAGATCTGGTGTCTAGGAAATGAAACGCGATTCCATATCAACAAAACTG
TACCGGGTAGAGGTT
AGTACCAGTGCAGGGGTTGGAGTAAAGAGTGAGCCACAGCCAATAATAATAGGGAGACGCAA
TCACTGATGTGGTGAAGCAACCAG
TTGGTGGTGCCTGCTGGGTAATTCTGATGGGTTTTAGCATATGGTTGTATTGG
TAGTTACGTTTCAAAGAGG
GGTGATCCCAGCTATCCAT
TAATAGCAACAGTGGCCCAAATGAGATT
TGCT
TGGAGCCATTTATAGTAGCATTGACTTCACTACCAAAACCAGTTACAACAGTTCCAGCCAAA
AGGCTACCCCATATGCCACGACACAGATCTTGCATTCCAACAGCATACATGAATTGGCTGTC
TCAGAACAAAGGTAACAATTTACTTTACATTCCTGACTACCGATTGGCTGAGGGATTGT
TGCCACACAACCAGTCTCAGGATTTCAGCACCACCAGCTCTCACAACAGCTCAGAAAGG
ATGCAGTGGAACAACAAGAAAATGGGTATGACAGTGATAGCTGGTGCCCACCATTG
TGATGATAGGGTCCCAAC
TGCTGCAGGCTCACCTGGATGAGTTGACAAGAGCCTAT
ATTCAAAGCAATAATCAACCTCCACAGCCTCCAGTTCC
TTCTGATTTGGAAACGGATGTTGCAGATGATGATGCCG
.GGCCCCTGAGAGCACTGGACCAGACTCCTGGATCCAGC
.TGGACAATCTAGACAGCTCTGTGACAGGTAACGGAAGACCTCGACCTACCAGCCCATTTTCTACTGA
.TGGACCAACAACCAGCATTGCCTCATCGAAGGGAAGGAATGACAGATGATCTTCCACCACCACCA
TCAGGGTTTAAGGCAGCAAATAGGCCCGAGCCAGCAGGCTGGTAACGTGGAAAA
CTCAGCAGAGAGAAAAGGAAGCTCTCTAGAGAGACAACATGCATCCAGCTTAGAAGACACAAAGAGCT
CATTGGATTGTCCAGCTAGAACCTCCCTAGAGTGGCAGCGACAAACCCAGGAATGGATAAGCTCCACA
GAACGACAAGAAGATATACGGAAAGCCCCACACAAACAAGGTTTTTCAGAGGAGGCCTTGGTGCCCTA
TAGCAAGCCCAGTTTCCCATCTCCAGGTGGCCACAGCTCATCAGGAACAGCTTCTTCTAAGGGATCCA
ATGGGCTCCAACAGTCAAGGACAGTTTACAGGTGAATTATGTAAGTGCTTAGGTCATTTAAA
.TAACATTGCCACATTAAACAAATTTCAGATTAAT

6a, CG182223-O1 ~SEQ ID NO: 72 1527 as ~MW at 167842.2kD
YVRVDGSRLRQEDFPPRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDG
LLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRDDFR
SIRGGKLMISNTRKSDAGMYTCV
TDEGTYMCIAENRVGKMEASATLTVRAPPQFVVRPRDQIVAQGRTVTFPCETKGN
PTGDLTITNIQRSDAGYYICQALTVAGSILAKAQLEV
VLTDRPPPIILQGPANQTLAVDGTALLKCKATGDPLPVISWLKEGFTFPGRDPRATIQEQGTLQIK
RISDTGTYTCVATSSSGETSWSAVLDVTESGATISKNYDLSDLPGPPSKPQVTDVTKNSVTLSWQP
PGTLPASAYIIEAFSQSVSNSWQTVANHVKTTLYTVRGLRPNTIYLFMVRAINPQGLSDPSPMSDP
TQDISPPAQGVDHRQVQKELGDVLVRLHNPVVLTPTTVQVTWTVDRQPQFIQGYRVMYRQTSGLQA
SWONLDAKVPTERSAVLVNLKKGVTYEIKVRPYFNEFQGMDSESKTVRTTEEAPSAPPQSVTVLTV
GSYNSTSISVSWDPPPPDHQNGIIQEYKIWCLGNETRFHINKTVDAAIRSVIIGGLFPGIQYRVEVAA
STSAGVGVKSEPQPIIIGRRNEVVITENNNSTTEQITDVVKQPAFIAGIGGACWVILMGFSIWLYWRR

GRGDVLPPVPGQGDKTATMLSDGAIYSSIDFTTKTSYNSSSQITQATPYATTQILHSNSIHELAVDL
DPQWKSSIQQKTDLMGFGYSLPDQNKGNNLLYIPDYRLAEGLSNRMPHNQSQDFSTTSSHNSSERSG
LSGGKGGKKKKNKNSSKPQKNNGSTWANVPLPPPPVQPLPGTELEHYAVEQQENGYDSDSWCPPLPV
TYLHQGLEDELEEDDDRVPTPPVRGVASSPAISFGQQSTATLTPSPREEMQPMLQAHLDELTRAYQF
IAKQTWHIQSNNQPPQPPVPPLGYVSGALISDLETDVADDDADDEEEALETPRPLRALDQTPGSSMD
LDSSVTGNGRPRPTSPFSTDSNTSAALSQSQRPRPTKKHKGGRMDQQPALPHRREGMTDDLPPPPDP
PGQGLRQQIGPSQQAGNVENSAERKGSSLERQHASSLEDTKSSLDCPARTSLEWQRQTQEWISSTER
EDIRKAPHKQGFSEEALVPYSKPSFPSPGGHSSSGTASSKGSTGPRKTEVLRAGHQRNASDLLDIGY
GSNSQGQFTGELCKCLGHLKGYRDSERILG
Further analysis of the NOV6a protein yielded the following properties shown in Table 6B.
Table 6B. Protein Sequence Properties NOV6a SignalP analysis: Cleavage site between residues 22 and 23 PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length 0; pos.chg 0; neg.chg 0 H-region: length 17; peak value 9.00 PSG score: 4.60 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -3.73 possible cleavage site: between 15 and l6 »> Seems to have no N-terminal signal peptide ALOM: Klein et al's method for TM region allocation Init position for calculation: 1 Tentative number of TMS(s) for the threshold 0.5: 2 INTEGRAL Likelihood = -2.81 Transmembrane 1 - 17 INTEGRAL Likelihood = -3.98 Transmembrane 860 - 876 PER2PHERAL Likelihood = 1.01 (at 792) ALOM score: -3.98 (number of TMSs: 2) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 8 Charge difference: 0.0 C( 1.0) - N( 1.0) N >= C: N-terminal side will be inside »> membrane topology: type 3a MITDTSC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment(75): 4.50 Hyd Moment(95): 2.47 G content: 1 D/E content: 1 S/T content: 2 Score: -4.61 Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 28 VRV~DG
NUCDISC: discrimination of nuclear localization signals pat4: RRKK (5) at 883 ' pat4: RKKR (5) at 884 pat4: KKRK (5) at 885 pat4: KKKK (5) at 1097 pat4: KKHK (3) at 1330 pat7: PTVRWKK (3) at 254 pat7: PTKKHKG (4) at 1328 I bipartite: none content of basic residues: 10.30 NLS Score : 1.. 57 i E
E
~KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: none SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif:
type 1: none type 2: none NMYR: N-myristoylation pattern : none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none ~Tyrosines in the tail: none Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: nuclear Reliability: 94.1 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues Final Results (k = 9/23):
39.1 0: mitochondrial 34.8 s: nuclear 17.4 0: endoplasmic reticulum 4.3 ~: cytoplasmic 4.3 0: peroxisomal » prediction for CG182223-01 is mit (k=23) A search of the NOV 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 6C.
Table 6C. Geneseq Results for NOV6a NOV6a Tdentities/
Geneseq Protein/Organism/Length Residues/ Similarities for Expeet Identifier ° ~ [Patent #, Date] Match the Matched Value Residues Region AA019185 Human neurotransmission- 1..1509 1501/1520 (98%) 0.0 associated protein NTR.ANB - 1..1515 150111520 (98%) Homo Sapiens, 1515 aa.
[W0200266646-A2, 29-AUG-2002]

_.

AA019179 Human neurotransmission- 1..1400 1356/1413 (95%); 0.0 associated protein NTRAN21..1405 1361/1413 (95%) -Homo sapiens, 1422 aa.

[WO200266646-A2, 29-AUG-2002]

ABU04094 Human expressed protein 21..1495819/1610 (50%)0.0 tag (EPT) #760 - Homo Sapiens,58..16341040/1610 (63%) aa. [W0200278524-A2, 10-OCT-2002]

ABU04093 Human expressed protein 21..1495819/1610 (50%)0.0 tag (EPT) #759 - Homo Sapiens,58..16341040/1610 (63%) aa. [W0200278524-A2, 10-OCT-2002]

ABU04092 ' Human expressed protein21..1495819/1610 (50%)0.0 tag (EPT) #758 - Homo sapiens,' 58..16341040/1610 (63%) aa. [W0200278524-A2, 10-OCT-2002]

In a BLAST search of public sequence databases, the NOV6a protein was found to have homology to the proteins shown in the BLASTP data in Table 6D.

Table 6D. Public BLASTP Results for NOV6a Protein NOV6a Identities/
Residues! Expect Accession ProteinlOrganism/Length Match ' Similarities for the Value Number Residues '; Matched Portion Q9HCK4 Hypothetical protein KIAA15681..1400 1316/1408 (93%)0.0 -Homo sapiens (Human), 3..1361 1321/1408 (93%) 1380 as (fragment).

Q90Z70 Roundabout2 - Brachydanio7..1509 1152/1525 (75%)0.0 rerio (Zebrafish) (Danio rerio),5..1 1294/1525 (84%) 1513 aa. S 13 Q9QZI3 ' Robo2 - Rattus norvegicus1..1053 974/1056 (92%) 0.0 (Rat), 1060 as (fragment). 1..1050 100111056 (94%) Q8UVD7 Roundabout-1 - Xenopus 10..1495826/1620 (50%) 0.0 laevis (African clawed frog), 10..15981060/1620 (64%) 1614 aa.

Q9Y6N7 Roundabout 1 - Homo sapiens21..1495819/1610 (50%) 0.0 (Human), 1651 aa. 58..16341040/1610 (63%) PFam analysis predicts that the NOV6a protein contains the domains shown in the Table 6E.
Table 6E. Domain Analysis of NOV6a Identities/

Pfam DomainNOV6a Match RegionSimilarities Expect Value for the Matched Region lg 45..112 17/71 (24%) 6.2e-06 52171 (73%) ig 147..205 16/61 (26%) 8.2e-06 42/61 (69%) ig 239..295 17/60 (28%) 1.6e-08 44/60 (73%) ig 328..393 17/69 (25%) 7.6e-09 51/69 (74%) ig 432..490 17/62 (27%) 1.8e-08 46/62 (74%) fn3 522..607 33/88 (38%) 3.2e-I7 64/88 (73%) fn3 638..724 24/90 (27%) 0.0086 63/90 (70%) fn3 736..826 33/93 (35%) 3.Se-I4 64/93 (69%) Example 7.
The NOV7 clone was analyzed, and the nucleotide and encoded pOlypeptide sequences are shown in Table 7A.
Table 7A. NOV7 ~a, CG183585-O1 ~SEQ ID NO. 73 X1385 by , Sequence ORF Start: ATG at 145 ORF Stop: TAG at 1264 AGACTGTAAAGGGTACCTTCCC
GGTGGAGATTGCGACTTCTTTTTCCTTAGCAGAGCCAAGCTCCATTCAGCT_GG_TTACCACTTTGTGGG
TGTCTTTAATGAAGCTTATAAATGGCAGGAAGCAAACATTCCCGTGGTTTGGCATGGATATTGGTGGA
ACCCTGGTTAAGTTGGTTTACTTTGAACCGAAGGATATCACGGCAGAAGAAGAGCAGGAAGAAGTGGA
GAACCTGAAGAGCATCCGGAAGTATTTGACTTCTAATACTGCTTATGGGAAAACTGGGATCCGAGACG
TCCACCTGGAACTGAAAA.ACCTGACCATGTGTGGACGCAAAGGGAACCTGCACTTCATCCGCTTTCCC
~AGCTGTGCCATGCACAGGTTCATTCAGATGGGCAGCGAGAAGAACTTCTCTAGCCTTCACACCACCCT
TGAACTGGACTGTCTGATTCAGGGCCTGCTTTATGTCGACTCTGTTGGCTTCAACGGC
GTTACTATTTTGAAAATCCCACAAATCCTGAATTGTGTCAAA.AAAAGCCGTACTGCCT
TACCCTATGTTGCTGGTTAACATGGGCTCAGGTGTCAGCATTCTAGCCGTGTACTCCA
CTGACTGGTTGTGAGACCTTTGAAGAAGCTCTGGAAATGGCAGCTAAAGGCGACAGCACCAATGTTGA
TAAACTGGTGAAGGACATTTACGGAGGAGACTATGAACGATTTGGCCTTCAAGGATCTGCTGTAGCAT
.TTGGTCACCATCACCAACAACATTGGCTCCATTGCTCGGATGTGTGCGTTGAATGAGAACATAGA
AGTTGTGTTTGTTGGAAATTTTCTCAGAATCAATATGGTCTCCATGAAGCTGCTGGCATATGCCA
ATTTTTGGTCCAAAGGACAACTGAAAGCTCTGTTTTTGGAACATGAGGGTTATTTTGGAGCCGTT
GCACTGTTGGAACTGTTCAAAATGACTGATGATAAGTAGAGACGAGCAGTGGAGGAAACAGCCTC
OV7a, CG183585-O1 ~SEQ ID NO: 74 373 as ~MW at 41664.6kD
uence INGRKQTFPWFGMDIGGTLVKLVYFEPKDITAEEEQEEVENLKSIRKYLTSNTAYGKTGIRDVHL
NLTMCGRKGNLHFIRFPSCAMHRFIQMGSEKNFSSLHTTLCATGGGAFKFEEDFRMIADLQLHKL
DCLIQGLLYVDSVGFNGKPECYYFENPTNPELCQKKPYCLDNPYPMLLVNMGSGVSILAVYSKDN
VTGTSLGGGTFLGLCCLLTGCETFEEALEMAAKGDSTNVDKLVKDIYGGDYERFGLQGSAVASSL
MSKEKRDSISKEDLARATLVTITNNIGSIARMCALNENIDRVVFVGNFLRINMVSMKLLAYAMDF
GQLKALFLEHEGYFGAVGALLELFKMTDDK
Further analysis of the NOV7a protein yielded the following properties shown in Table 7B.
Table '7S. Protein Sequence Properties NOV7a SignalP analysis: No Known Signal Sequence Predicted PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length 8~ pos.chg 3; neg.chg 0 H-region: length 8~ peak value 5.54 PSG score: 1.14 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -10.98 possible cleavage site: between 59 and 60 »> Seems to have no N-terminal signal peptide ALOM: Klein et al's method for TM region allocation Init position for calculation: l Tentative number of TMS(s) for the threshold 0.5: 1 Number of TMS(s) for threshold 0.5: 0 PERIPHERAL Likelihood = 0.95 (at 212) ALOM score: 0.42 (number of TMSs: 0) 'MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 6 Charge difference: -2.0 C( 1.0) - N( 3.0) N >= C: N-terminal side will be inside MITDISC: discrimination of mitochondria) targeting seq ', R content: 1 Hyd Moment(75): 10.35 Hyd Moment(95): 1.52 G content: 2 D/E content: 1 S/T content: 1 Score: -4.69 'Gavel: prediction of cleavage sites for mitochondria) preseq R-2 motif at 17 GRK~QT
NUCDTSC: discrimination of nuclear localization signals j pat4: none pat7: none bipartite: none content of basic residues: 11.3%
NLS Score: -0.47 iKDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: none f 'SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif:
type 1: none type 2: none ~NMYR: N-myristoylation pattern : none ~Prenylation motif: none ~memYQRL: transport motif from cell surface to Golgi: none ~Tyrosines in the tail: none 'Dileucine motif in the tail: none 'checking 63 PROSTTE DNA binding motifs: none 'checking 71 PROSITE ribosomal protein motifs: none i ichecking 33 PROSITE prokaryotic DNA binding motifs: none i ~NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination j Prediction: cytoplasmic Reliability: 94.1 COIL: Zupas's algorithm to detect coiled-coil regions i total: 0 residues Final Results (k = 9/23):
60.9 0: cytoplasmic 21.7 %: nuclear 17.4 %: mitochondrial » prediction for CG183585-01 is cyt (k=23) A search of the NOV7a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 7C.

Table 7C. Geneseq Results for NOV7a ~.",.,.~~...,~..u.. ..,~....~...~,...~~~,~."~~...,.....~""..~.

~....,~.....~.~..... ~"..~. ~,.~,..~,.-...~",.~,..

NOV7a Identities/

Geneseq Protein/Organistn/Length Residues/Similarities ' Expect [Patent for Identifier #, Date] Match the Matched Value Residues Region AAE24134 Human kinase (PK1N)-5 protein1..373 371/373 (99%)0.0 -Homo Sapiens, 373 aa. 1..373 372/373 (99%) [W0200233099-A2, 25-APR-2002] .

AAE21720 Human PKIN-15 protein - 7..369 297/363 (81%)e-178 Homo Sapiens, 447 aa. [WO2002I8557- 84..446 332/363 (90%) A2, 07-MAR-2002]

AAM40613 Human polypeptide SEQ ID 7..369 297/363 (81%)e-178 NO

5544 - Homo Sapiens, 460 aa. 97..459 332/363 (90%) [W0200I53312-A1, 26-JITL-2001]

AAM38827 Human polypeptide SEQ ID 7..369 296/363 (81 e-178 NO %) 1972 - Homo sapiens, 447 aa. 84..446 331/363 (90%) [WO200153312-A1, 26-JUL-2001]

AAB94366 ~ Human protein sequence 1..366 291/366 (79%)e-173 SEQ ID

N(~_14R99 - H~m~ ca.iens_ 37O aa.. I..366 330/366 (89%) [EP 1074617-A2, 07-FEB-2001 In a BLAST search of public sequence databases, the NOV7a protein was found to have homology to the proteins shown in the BLASTP data in Table 7D.
Table 7D. Public BLASTP Results for NOV7a NOV7a Identities/
Protein Residues/SimilaritiesExpect Accession Protein/Organism/Length for Match the Matched Value Number Residues Portion BAC34132 . Adult male liver tumor 1..371 365/371 (98%)0.0 cDNA, RIKEN

full-length enriched library,1..371 369/371 (99%) clone:C7300270 l7 product:hypothetical protein, full insert sequence - Mus musculus (Mouse), 373 aa.

Q8TE04 ' Pantothenate kinase 1 2..373 365/374 (97%)0.0 (EC 2.7.1.33) (Pantothenic acid kinase 225..598 368/374 (97%)' 1) (hPanKl) (hPanK) - Homo sapiens (Human), 598 aa.

Q8K4K6 Pantothenate kinase 1 (EC 2..371 359/372 (96%)0.0 2.7.1.33) (Pantothenic acid kinase 175..546 365/372 (97%) 1) (mPankl) (mPank) - Mus musculus (Mouse), 548 aa.
.. .
..

Q9BZ23 Pantothenate kinase 2 (EC 7..369 ~ 297/363 e-178 2.7.1.33) (81%) (Pantothenic acid kinase 207..569 332/363 (90%) 2) (hPANK2) - Homo Sapiens (Human), 570 aa.

Q9H999 Pantothenate kinase 3 (EC 1..366 291/366 (79%)' e-173 2.7.1.33) (Pantothenic acid kinase 1..366 330/366 (89%) 3) (hPanK3) -Homo sapiens (Human), 370 aa.

PFam analysis predicts that the NOV7a protein contains the domains shown in the Table 7E.
Table 7E. Domain Analysis of NOV7a Identities/
Pfam Domain ' NOV7a Match Region Similarities Expect Value for the Matched Region Fumble 12..367 196/401 (49%) 2.3e-234 346/401 (86%) Example 8.
The NOVB clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 8A.
_ _ Table 8A. NOV8 Sequence Analysis OVBa, CG183860-01 SEQ ID NO 75 , _, 4,1,858 bp~ Y ~ ,~ '~4 y ..
NA Sequence ORF Start: ATG at 72 ' ORF Stop: TGA at 786 CGGAGAGTACTGCCACGGCTGGGTGGACGTGCAGGGCAACTACCACGAGGGCTTCCAGTGCC
T
CTATTTATTGTTGCACCTGTTTGAGACCCAAGGAGCCCTCGCAGCAGCCAATCCGC
AGCTATCAGACAGAGACCCTGCCCATGATCCTGACCTCCACCAGCCCCAGGGCACC
CAGCACAGCCACGAGCTCCAGC'TCCACAGGCGGCTCCATCCGCAGGTTCTCCTTTG
CGGGCTGCCTGGTGCCCTCACCGCCCCCGCCATACACCACCAGCCACTCAATCCAC
TCTGGTTTCCTGGTGTCACCCCAGTATTTCGCTTACCCCCTCCAGCAGGAGCCCCC
.TTTCCCTTGTA
ACTGATCAGTGTCATGGAGGAGCATGCTAGGAAAACACAGCACCTTCTAATTTGAAAGTTCCTGTCTC
CAATCACAGAAAGGCTAAACCAGAGAACTGTTTTCTGGTTTTGCAAACATGTGATCATTACATTTCAA
~TACTGGACATTCAGCTATATTGCTTAGAAAAGGGCTACATGTTTCTTTTTCATATAAGTTGTTCATTG
~CCTAACCATGAATAATATTAGCATAATGAGAACATTTACTTTTTAAATAAATAACTAAATTTTGTTTA
CTCAGACATTCATTGTAACACAGAGTGTATGTAAAATCATTTCCCCCACTCACTGGAGGGAGTATTTA
ATTTGTAAAA
CTAATTATTTAGTAGTCATACTGTAATTTTTATGTTAATAATAACTGGAGTTCAAAGTCTAGCTATTG
GTATAATCATCTAATATTATATATATCTCCAGTGCCCCTGAATTTTATGTTTGATGACTATATATTTG
CG 183860-Ol SEO ID NO: 76 X238 as BMW at 25860.1kD
~CLLLGWLRWGPAGAQQSGEYCHGWVDVQGNYHEGFQCPEDFDTLDATICCGSCALRYCCAA
iGGCTNDRRELEHPGITAQPVYVPFLIVGSIFIAFIILGSVVAIYCCTCLRPKEPSQQPIRF
'ETLPMILTSTSPRAPSRQSSTATSSSSTGGSIRRFSFARAEPGCLVPSPPPPYTTSHSIHL
~VSPQYFAYPLQQEPPLPGKSCPDFSSS
Further analysis of the NOV8a protein yielded the following properties shown in Table 8B.
Table 8B. Protein Sequence Properties NOVBa SignalP analysis: , ~ Cleavage site between residues 22 and 23 PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length 2~ pos.chg 1; neg.chg 0 H-region: length 12; peak value 10.66 PSG score: 6.26 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 1.27 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: 1 Number of TMS(s) for threshold 0.5: I
INTEGRAL ~ Likelihood =-11.04 Transmembrane 100 - 116 PERIPHERAL Likelihood = 1.27 (at 53) ALOM score: -11.04 (number of TMSs: 1) MTOP: Prediction of membrane topology (Hartmann et al.) Center position fox calculation: 10 Charge difference: -1.5 C( 0,5) - N( 2.0) N >= C: N-terminal side will be inside »> membrane topology: type la (cytoplasmic tail 117 to 238) MITDISC: discrimination of mitochondrial targeting seq i R content: 2 Hyd Moment(75): 6.25 ' Hyd Moment(95): 8.60 G content: 4 D/E content: 1 S/T content: 1 Score: -4.46 Gavel: prediction of cleavage sites for mitochondrial preseq i R-2 motif at 25 LRW~GP
NUCDISC: discrimination of nuclear localization signals i pat4: none pat7: none bipartite: none content of basic residues: 6.7s NLS Score: -0.47 'KDEL: ER retention motif in the C-terminus: none ~ER Membrane Retention Signals:
XXRR-like motif in the N-terminus: RALL
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 l: none type 2: none 'NMYR: N-myristoylation pattern : none ~Pren lation motif~ a y . non memYQRL: transport motif from cell surface to Golgi: none ~Tyrosines in the tail: too long tail a IDileucine motif in the tail: none Echecking 63 PROSITE DNA binding motifs: none 'checking 71 PROSTTE ribosomal protein motifs: none "checking 33 PROSTTE prokaryotic DNA binding motifs: none i 'NNCN: Reinhardt's method for CytoplasmicJNuclear discrimination Prediction: nuclear Reliability: 89 'COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues ';Final Results (k = 9/23):
44.4 %: extracellular, including cell wall 22.2 s: Golgi 22.2 ~: endoplasmic reticulum 11.1 ti: plasma membrane » prediction for CG183860-01 is exc (k=9) A search of the NOVBa protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 8C.

Table 8C. Geneseq Results for NOVBa NOVBa Identities/

Geneseq Protein/Organism/Length Residues/ SimilaritiesExpect [Patent for Identifier' #, Date] Match the MatchedValue Residues Region AAY86234 ' Human secreted protein 1..195 179/195 e-103 HNTNC20, (91%) ' SEQ ID NO:149 - Homo sapiens,1..195 179/195 219 (91%) aa. [W09966041-Al, 23-DEC-1999]

ABU56619 Lung cancer-associated polypeptide19..212 107/204 2e-51 (52%) #212 - Unidentified, 295 29..226 136/204 aa. (66%) [WO200286443-A2, 31-OCT-2002]

ABB85001 Human PR028631 protein sequence19..212 107/204 2e-51 (52%) SEQ ID NO:370 - Homo sapiens,29..226 136/204 295 (66%) aa. [W0200200690-A2, 03-JAN-2002]

ABB95607 Human angiogenesis related 19..212 107/204 : 2e-51 protein (52%) PRO28631 SEQ ID NO: 370 29..226 1361204 - Homo ' (66%) sapiens, 295 aa. [WO200208284-A2, 31-JAN-2002]

ABG61896 Prostate cancer-associated 13..84 48/72 (66%)3e-25 protein #97 ' j - Mammalia, 582 aa. 243..314 56/72 (77%) [W0200230268-A2, 18-APR-2002]

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

Table 8D. Public BLASTP
Results for NOVBa Protein NOYBa Identities/

Accession Protein/Organism/Length Residues/ Similarities Expect for Number Match the Matched Value Residues Portion Q8CSV3 ~ Hypothetical protein 79..238 149/I60 (93%)4 4e-83 - Mus~ ;

musculus (Mouse), 160 1..160 153/160 (9S%) as (fragment).

Q96EQS Hypothetical protein 96..238 143/143 (100%)3e-79 - Homo ~

sapiens (Human), 144 2..144 143/143 (100%) as (fragment).
_ H

Q8QZV2 Hypothetical pxotein 2..212 114/221 (51%)~ le-Sl - Mus musculus (Mouse), 295 1 S..226 142/221 (63%) aa.

_ _ Q8BN61 Hypothetical pxotein 2,.212 1131221 (S1%)le-SO
- Mus musculus (Mouse), 295 15..226 141/221 (63%) aa.

CACS 11 Sequence 26 from Patent 24..196 44/183 (24%) 6e-06 SO ~

W00149728 - Homo sapiensl 27..187 76/183 (41 %) (Human), 197 aa.

Example 9.
The NOV9 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 9A.
Table 9A. NOV9 OV9a, CG184416-01 SEQ ID NO T7 1321 by NA Sequence p~ Start: ATG at 39 ORF Stop: TGA at ~I284 TCCTCTCCTTCCC
AGCTGCCGCCTTCCGCATGTGGAGCGACG
TCGGC
TGCACCTGAACGCCACGCTGCGCGG
TTCCCCTTCCCCACGGTGGCCACCACCCCACCGC
ACTCCTGGCGAGTCCGTGTGCGGGGCTGAGCCCGG

CG184416-O1 ~SEQ ID NO: 78 X415 as BMW at 46304.OkD
fGRGARVPSEAPGAGVERRWLGAALVALCLLPALVLLARLGAPAVPAWSAAQGDVAALGLSAVPPTRV
~GPLAPRRRRYTLTPARLRWDHFNLTYRILSFPRNLLSPRETRRALAAAFRMWSDVSPFSFREVAPEQ
~SDLRIGFYPINHTDCLVSALHF3CFDGPTGELAHAFFPPHGGIHFDDSEYWVLGPTRYSWKKGVWLTD
~VHVAAHEIGHALGLMHSQHGRALMHLNATLRGWKALSQDELWGLHRLYGESLCRAGGRGPGGPEPGV
~PTLPIGCLDRLFVCASWARRGFCDARRRLMKRLCPSSCDFCYEFPFPTV'ATTPPPPRTKTRLVPEGR
!!VTFRCGQKILHKKGKVYWYKDQEPLEFSYPGYLALGEAHLSIIANAVNEGTYTCVVRRQQRVLTTYS
Further analysis of the NOV9a protein yielded the following properties shown in Table 9B.
Table 9B. Protein Sequence Properties NOV9a SignalP analysis: Cleavage site between residues 45 and 46 PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length Z0; pos.chg 2; neg.chg 1 H-region: length 6; peak value -6.74 PSG score: -11.14 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 1.31 possible cleavage site: between 37 and 38 »> Seems to have no N-terminal signal peptide ALOM: IClein et al's method for TM region allocation Init position for calculation: 1 Tentative number of TMS(s) for the threshold 0.5: 1 Number of TMS(s) for threshold 0.5: 1 INTEGRAL Likelihood =-10.40 Transmembrane 21 - 37 PERIPHERAL Likelihood = 0.79 (at 272) ALOM score: -10.40 (number of TMSs: 1) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 28 Charge difference: 0.0 C( 1.0) - N( 1.0) N >= C: N-terminal side will be inside »> membrane topology: type 2 (cytoplasmic tail 1 to 21) MITDISC: discrimination of mitochondrial targeting seq R content: 2 Hyd Moment(75): 4.37 Hyd Moment(95): 11.61 G content: 4 D/E content: 2 S/T content: 1 Score: -6.42 Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 16 ARV~PS

NUCDISC: discrimination of nuclear localization signals pat4: PRRR (4) at 74 pat4: RRRR (5) at 75 pat7: PLAPRRR (3) at 71 pat7: PRRRRYT (5) at 74 bipartite: RRQQRVLTTYSWRVRVR at 398 content of basic residues: 12.8%
NLS Score: 1.27 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals:
XXRR-like motif in the N-terminus: GRGA
none SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: found TLPI at 275 ,RNA-binding motif: none Actinin-type actin-binding motif:
type 1: none type 2: none ';NMYR: N-myristoylation pattern : none 'Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none ~Tyrosines in the tail: none 'Dileucine motif in the tail: none ;checking 63 PROSITE DNA binding motifs: none ;checking 71 PROSITE ribosomal protein motifs: none ;checking 33 PROSITE prokaryotic DNA binding motifs: none ~NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: cytoplasmic Reliability: 94.1 COIL: Lupas's algorithm to detect coiled-coil regions [ total: 0 residues ,_____-_____-______________ Final Results (k = 9/23):
39.1 %: mitochondrial 30.4 0: cytoplasmic 8.7 0: vacuolar 8.7 ~: endoplasmic reticulum 4.3 %: Golgi 4.3 ~: vesicles of secretory system 4.3 0: nuclear » prediction for CG184416-OZ is mit (k=23) A search of the NOV9a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 9C.
Table 9C. Geneseq Results for NOV9a NOV9a Identities/

Geneseq Protein/Organism/Length Residues/Similarities Expect [Patent for Identifier#, Date] Match the Matched Value ' ResiduesRegion ABG72777 Human matrix metalloproteinase1..415 390!415 (93%)0.0 (MMP23) protein - Homo sapiens,1..390 390/41 S (93%) 390 aa. [W020028S28S-A2, AAB84622 Amino acid sequence of matrix1..41 3 90/41 S 0.0 S (93 /o) metalloproteinase-21 - Homo1..390 390/415 (93%) sapiens, 390 aa. [W0200149309-A2, 2001]

AAE10430 Human matrix metalloprotinase-22P1 .4I 390141 S (93%)' 0.0 ' S

(MMP-22P) protein - Homo 1..390 390/415 (93%) sapiens, 390 aa. [W0200166766-A2, 2001 ]
..

AAY78S8S __ 1..415 390/415 (93%)0.0 Metalloprotease in the female reproductive tract protein 1..390 390/41 S (93%) sequence -Homo Sapiens, 390 aa.

[JP2000014387-A, 18-JAN-2000]

AAY783S3 Rat metalloproteinase protein1..414 327/417 (78%)0.0 sequence SEQ ID N0:2 - Rattus1..390 3_44/417 (82%) norvegicus, 391 aa. [JP20000I4386-A, 18-JAN-2000]

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

Table 9D. Public BLASTP Results for NOV9a Protein NOV9a Identities/

AccessionProtein/Organism/Length Residues/Similarities Expect for Number Match the Matched Value Residues Portion Q9UBR9 ' MMP-23 (MIFR/FEMALYSIN) 1..41 390/41 S (93%)0.0 S

(DJ283E3.2.1) (Matrix 1..390 390/415 (93%) metalloproteinase MMP21/22A

(MIFRI)) (Matrix metalloproteinase 23B) - Homo Sapiens (Human), v 075900 Metalloprotease mmp21/22A 1..415 389/415 (93%)0.0 - Homo Sapiens (Human), 390 aa. 1..390 389/415 (93%) a 088676 cAMP metalloproteinase - 1..414 328/416 (78%)0.0 Mus musculus (Mouse), 391 aa. 1..390 345/416 (82%) 088272 MIFR - Rattus norvegicus 1..414 327/417 (78%)0.0 (Rat), 391 aa. 1..390 344/417 (82%) ~

075894 Metalloprotease isoform 149..398 250/250 (100%)e-156 C

(Metalloprotease MMP21/22C)1..250 250/250 (100%) -Homo sapiens (Human), 250 as (fragment).

PFam analysis predicts that the NOV9a protein contains the domains shown in the Table 9E.
Table 9E. Domain Analysis of NOV9a Identities/

Pfam Domain NOV9a Match RegionSimilarities Expect Value for the Matched Region Peptidase 81..192 43/115 (37%) 1.9e-23 74/1 I S (64%) ShTK 279..315 16/44 (36%) 3.4e-09 27/44 (61 %) ig 339..397 17/61 (28%) 0.00051 39/61 (64%) Example 10.
The NOV 10 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 10A.
T_ able_10A. N_OVl_0 Sequence Analysis OV 10a, CGl 85200-01 ~SEQ ID NO 79_~_~ ~ 2050.bp _~
NA Sequence ' p~ Start: ATG at 66~~ ORF Stop TAA at 918 TGTTGGTGGGATGTTACGTGGCCGGAATCAT
GTGGAACTGCTCTGGCAGTCATCGTGCCTGAAGGAGTACATGCCCTTTATGAAGATATTCTTGAGGGA
AAACACCACCAAGCAAGTGAAACACATAA'PGTGATTGCATCAGACAAAGCAGCAGAAAAATCAGTTGT
CCATGAACATGAGCACAGCCACGACCACACACAGCTGCATGCCTATATTGGTGTTTCCCTCGTTCTGG
GTTGCTTTGGGAGCAGCAGCATCTACTTCACAGACCAGTGTCCAGTTAATTGTGTTTGTGGCAATCAT
GCTACATAAGGCACCAGCTGCTTTTGGACTGGTTTCCTTCTTGATGCATGCTGGCTTAGAGCGGAATC
GAATCAGAAAGCACTTGCTGGTCTTTGCATTGGCAGCACCAGTTACGTCCATGGTGACATACTTAGGA
CTGAGTAAGAGCAGTAAAGAAGCCCTTTCAGAGGTGAACGCCACGGGAGTGGCCATGCTTTTCTCTGC
CGGGACATTTCTTTATGTTGCCACAGTACATGTCCTCCCTGAGGTGGGCGGAATAGGGCACAGCCACA
A
TCCAGCCTGC
ACCAAAA
10a, CG185200-01 SEQ ID NO: 80 X284 as MVV at 29900.4kD
DFISTSLLSLAMLVGCYVAGIIPLAVNFSEERLKLVTVLGAGLLCGTALAVIVPEGVHALYEDILE

ALGAAASTSQTSVQLIVFVAIMLHKAPAAFGLVSFLMHAGLERNRIRKHLLVFALAAPVTSMVTYL
SKSSKEALSEVNATGVAMLFSAGTFLYVATVHVLPEVGGIGHSHKPDATGGRGLSRLEVAALVLGC
PLTLSVGHQH
OV l Ob, CG185200-OZ SEQ ID NO 81 _ 1120 by NA Sequence ORF Start: ATG at 94 ~ORF Stop: TAA at 1015 GAATAAAGGAGGGCAGAATGGATGATTTCATCTCCATTAGCCTGCTGTCTCTGGCTATGT
TGTTACGTGGCCGGAATCATTCCCTTGGCTGTTAATTxCTCAGAGGAACGACTGAAGCTG
TCAGTTGTCCATGAACATGAGCACAGCCACGACCACACACAGCTGCAT

TGTTGCTGGTGGACCAGATTGGTAACTC
..TGCATGCTGGCTTAGAGCGGAATCGAATCAGAAAGCACTTGCTGGTCTTTGCATTGGCAGCAC
ATGTCCATGGTGACATACTTAGGACTGAGTAAGAGCAGTAA.AGAAGCCCTTTCAGAGGTGAAC
CTCATCCCTCTCATCCTGTCAGTAGGACACCAGCATTAAATG
OVlOb, CG185200-02 SEQ ID NO: 82 307 as ~MW at 32221.OkD
rotein Seauence ISLLSLAMLVGCWAGIIPLAVNFSEERLKLVTVLGAGLLCGTALAVIVPEGVHALYEDILE
SETHNVIASDKAAEKSVVHEHEHSHDHTQLHAYIGVSLVLGFVFMLLVDQIGNSHVHSTDDP
NSKITTTLGLVVFIA.AADGVALGAAASTSQTSVQLIVFVAIMLHKAPAAFGLVSFLMHAGLER
LLVFALAAPVMSMVTYLGLSKSSKEALSEVNATGVAMLFSAGTFLWATVHVLPEVGGIGHS
A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table IOB.
Table lOS. Comparison of the NOV10 protein sequences.
NOVlOa MDDFISISLLSLAMLVGCYVAGIIPLAVNFSEERLKLVTVLGAGLLCGTALAVIVPEGVH
NOVlOb MDDFISISLLSLAMLVGCYVAGIIPLAVNFSEERLKLVTVLGAGLLCGTALAVIVPEGVH
NOVlOa ALYEDILEGKHHQASETHNVIASDKAAEKSWHEHEHSHDHTQLHAYIGVSLVLGFVFML
NOVlOb ALYEDILEGKHHQASETHNVIASDKAAEKSWHEHEHSHDHTQLHAYIGVSLVLGFVFML
NOVlOa LVDQIGNSHVHSTD-----------------------ADGVALGAAASTSQTSVQLIVFV
NOVlOb LVDQIGNSHVHSTDDPEAARSSNSKITTTLGLWHAAADGVALGAAASTSQTSVQLIVFV
NOVlOa AIMLHKAPAAFGLVSFLMHAGLERNRIRKHLLVFALAAPVTSMVTYLGLSKSSKEALSEV
NOVlOb AIMLHKAPAAFGLVSFLMHAGLERNRIRKHLLVFALAAPVMSMVTYLGLSKSSKEALSEV
NOVlOa NATGVAMLFSAGTFLYVATVHVLPEVGGIGHSHKPDATGGRGLSRLEVAALVLGCLIPLI
NOVlOb NATGVAMLFSAGTFLYVATVHVLPEVGGIGHSHKPDAAGGRGLSRLEVAALVLGCLIPLI
NOVlOa LSVGHQH
NOVlOb LSVGHQH
NOVlOa (SEQ ID NO: 80) ~NOVlOb (SEQ ID N0: 82) Further analysis of the NOV l0a protein yielded the following properties shown in Table 10C.
Table 10C. Protein Sequence Properties NOVlOa SignalP analysis: Cleavage site between residues 62 and 63 PSORT II analysis:
1~1 PSG: a new signal peptide prediction method N-region: length 3; pos.chg 0; neg.chg 2 H-region: length 28; peak value 0.00 PSG score: -4.40 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -1.40 possible cleavage site: between 52 and 53 »> Seems to nave no N-terminal signal peptide 'ALOM: Klein s method TM
et al' for region allocation Tnit position 1 for calculation:

Tentative number the threshold0.5: 7 of TMS(s) for TNTEGRAL Likelihood -6.48Transmembrane12 -= 28 INTEGRAL Likelihood -5.68Transmembrane38 -= 54 TNTEGRAL Likelihood -8.49Transmembrane106 -= 122 TNTEGRAL Likelihood -1.97Transmembrane153 -= 169 TNTEGRAL Likelihood -3.13Transmembrane188 -i = 204 TNTEGRAL Likelihood -1.01Transmembrane221 -i = 237 INTEGRAL Likelihood -8.81Transmembrane265 -= 281 PERIPHERAL Likelihood 9.18 (at 135) =

[ ALOM score; -8.81 (number TMSs: 7) of MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 19 Charge difference: 1.0 C( 0.0) - N(-1.0) C > N: C-terminal side will be inside »> membrane topology: type 3b MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment(75): 8.48 Hyd Moment(95): 7.98 G content: 0 D/E content: 2 S/T content: 0 Score: -6.50 Gavel: prediction of cleavage sites for mitochondrial preseq cleavage site motif not found NUCDTSC: discrimination of nuclear localization signals pat4: none pat7: none bipartite: none content of basic residues: 5.3%
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:
j type 1: none type 2: none ~NMYR: N-myristoylation pattern : none ~Prenylation motif: none ~memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none ~Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: none checking 7I 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 ~COTL: Lupas's algorithm to detect coiled-coil regions I total: 0 residues Final Results (k = 9/23):
55.6 0: endoplasmic reticulum 11.1 0: Golgi 11.1 %: vacuolar 11.1 s: vesicles of secretory system 11.1 0: mitochondrial » prediction for CG105200 01 is end (k 9) _ . ~.,_., ."_. , .x~ . . ~,~., ~ ., ~ .,." .~. .. ....
A search of the NOV l0a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table l OD.

Table lOD. Geneseq Results for NOVlOa NOVlOa Identities!
Geneseq Protein/Organism/Length [Patent ' Residues/ Similarities for . Erect Identifier #, Date] Match the Matched Value Residues Region AAB93646 ' Human protein sequence SEQ ID 1..284 284/307 (92%) e-154 NO:13148 - Homo sapiens, 307 aa. 1..307 284/307 (92%) [EP1074617-A2, 07-FEB-2001) ABU57061 ' Human PRO polypeptide #131 - 1..284 283/307 (92%) e-153 Homo Sapiens, 307 aa. 1..307 283/307 (92%) [US2003027280-A1, 06-FEB-2003]
ABU56066 ' Human secreted/transmembrane 1..284 283/307 (92%) e-153 protein, PR01377 - Homo sapiens, 1..307 283/307 (92%) 307 aa. [US2003022298-Al, 30-JAN-2003]
ABU10640 ' Human secreted/transmembrane 1..284 283/307 (92%) e-153 protein #131 - Homo Sapiens, 307 aa. 1..307 283/307 (92%) [US2002127584-A1, 12-SEP-2002]
AAB66116 Protein of the invention #28 - 1..284 283/307 (92%) e-153 Unidentified, 307 aa. 1..307 283/307 (92%) [W0200078961-A1, 28-DEC-2000) In a BLAST search of public sequence databases, the NOV l0a protein was found to have homology to the proteins shown in the BLASTP data in Table 10E.

Table 10E. Public BLASTP Results for NOVlOa Protein NOVlOa Identities/

Accession Protein/Organism/Length Residues/Similarities Expect for Number Match the Matched Value Residues Portion Q9NUM3 Hypothetical protein FLJI 1..284 284/307 (92%)e-154 Homo Sapiens (Human), 307 1..307 284/307 (92%) aa.

AAH47682 Hypothetical protein - I ..284 283/307 (92%)e-153 Homo sapiens (Human), 307 aa. . 1,..307283/307 (92%) Q8BFU1 CDNA FLJ11274 FIS - Mus 1..284 266/308 (86%)e-143 musculus (Mouse), 308 aa. 1..308 275/308 (88%) Q95JP5 Hypothetical 25.0 kDa protein130..284 149/155 (96%)2e-76 -Macaca fascicularis (Crab 82..235 152/155 (97%) eating macaque) (Cynomolgus monkey), 23 S aa.

AAH44279 Hypothetical protein - 1..281 154/308 (50%)7e-69 Xenopus laevis (Afncan clawed frog),1. 299 197/308 (63%) 303 as PFam analysis predicts that the NOVlOa protein contains the domains shown in the Table lOF.
Table lOF. Domain Analysis of NOVlOa Example 11.
The NOV I 1 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 11 A.
Table 11A. NOVIl Vlla, CG50513-O1 SE ID_NO 83 1598 b A Sequence ORF Start: at l ~ORF Stop TGA at 1=354 TTAGGTTGACTTCAAAGATGCCTCAGTTACTGCAAAA
TCATCGAGGCCTTCAGGCGCTATGCAAGGACGGAGGGCAACTGCACAGCGCTCACCC

ACTGAAGTGGGAAGGGCGGGGAAAGGGCAGCATTATGAGGGGAGCAGCCACAGACAGAGCCAGCAGGG
TTCCAGAGGGCAGAACAGGCCTGGGGTTCAGACCCAGGGTCAGGCCACTGGCTCTGCGTGGGTCAGCA
GCTATGACAGGCAAGCTGAGTCCCAGAGCCAGGAAAGAATAAGCCCGCAGATACAACTCTCTGGGCAG
ACAGAGCAGACCCAGAAAGCTGGAGAAGGCAAGAGGAATCAGACAACAGAGATGAGGCCAGAGAGACA
GCCACAGACCAGGGAACAGGACAGAGCCCACCAGACAGGTGAGACTGTGACTGGATCTGGAACTCAGA
CCCAGGCAGGTGCCACCCAGACTGTGGAGCAGGACAGCAGCCACCAGACAGGAAGCACCAGCACCCAG
ACACAGGAGTCCACCAATGGCCAGAACAGAGGGACTGAGATCCACGGTCAAGGCAGGAGCCAGACCAG
CCAGGCTGTGACAGGAGGACACACTCAGATACAGGCAGGGTCACACACCGAGACTGTGGAGCAGGACA
GAAGCCAAACTGTAAGCCACGGAGGGGCTAGAGAACAGGGACAGACCCAGACGCAGCCAGGCAGTGGT
CAAAGATGGATGCAAGTGAGCAACCCTGAGGCAGGAGAGACAGTACCGGGAGGACAGGCCCAGACTGG
GGCAAGCACTGAGTCAGGAAGGCAGGAGTGGAGCAGCACTCACCCAAGGCGCTGTGTGACAGAAGGGC
AGGGAGACAGACAGCCCACAGTGGTTGGTGAGGAATGGGTTGATGACCACTCAAGGGAGACAGTGATC
CTCAGGCTGGACCAGGGCAACTTGCATACCAGTGTTTCCTCAGCACAGGGCCAGGATGCAGCCCAGTC
AGAAGAGAAGCGAGGCATCACAGCTAGAGAGCTGTATTCCTACTTGAGAAGCACCAAGCCATGACTTC
CCCGACTCCAATGTCCAGTACTGGAAGAAGACAGCTGGAGAGAGTTTGGCTTGTCCTGCATGGCCAAT
CCAGTGGGTGCATCCCTGGACATCAGCTCTTCATTATGCAGCTTCCCTTTTAGGTCTTTCTCAATGAG
ATAATTTCTGCAAGGAGCTTTCTATCCTGAACTCTTCTTTCTTACCTG_CTTTGCGGTGCAGACCCTCT
CAGGAGCAGGAAGACTCAGAACAAGTCACCCCTT
~;~v;~,~"~, _ NOVl la, CG50513-O1 SEQ ID NO: 84 451 as MW at 48908.6kD
Protein Sequence . _ . . . .. _ . _. .
KQPLVSSHLGIRLTSKMPQLLQNINGIIEAFRRYARTEGNCTALTRGELKRLLEQEFADVIVKPHDPA
TVDEVLRLLDEDHTGTVEFKEFLVLVFKVAQACFKTLSESAEGACGSQESGSLHSGASQELGEGQRSG
TEVGRAGKGQHYEGSSHRQSQQGSRGQNRPGVQTQGQATGSAWVSSYDRQAESQSQERISPQIQLSGQ
TEQTQKAGEGKRNQTTEMRPERQPQTREQDRAHQTGETVTGSGTQTQAGATQTVEQDSSHQTGSTSTQ
TQESTNGQNRGTEIHGQGRSQTSQAVTGGHTQIQAGSHTETVEQDRSQTVSHGGAREQGQTQTQPGSG
QRWMQVSNPEAGETVPGGQAQTGASTESGRQEWSSTHPRRCVTEGQGDRQPTWGEEWVDDHSRETVI
LRLDQGNLHTSVSSAQGQDAAQSEEKRGITARELYSYLRSTKP
NOVllb, 273654175 SEQ ID NO 85 151 by _. _ _ . .... .. .. _ _.. . _ ..~ .._.__. __ __ ..... . ._._ _m__.~_ DNA Sequence OgE Start: at 2 ORF Stop: at End of Sequence _ ACCGGATCCTTACTGCAAAACATTAATGGGATCATCGAGGCCTTCAGGCGCTATGCAAGGACGGAGG
GCAACTGCACAGCGCTCACCCGAGGGGAGCTGAAAAGACTCTTGGAGCAAGAGTTTGCCGATGTGATT
GTGAAACTCGAGGGC
NOVI lb, 273654175 SEQ ID NO 86 50 as MW at 5608 3kD
Protein Sequence _ TGSLLQNINGIIEAFRRYARTEGNCTALTRGELKRLLEQEFADVIVKLEG
s._..
NOVl lc, CG50513-02 SE ID NO 87 ' 1039 by _ _ ........ ... .._ ..... _...- _.
DNA Sequence ~ ORF Start at 1 ' ORF Stop end of sequence . .. . ... _.W.~. ._... ... . . .. ...... .._ . . W .. _ . _.._... . . .... _ __...... .
GTCAATGACAGCTTGTGTGATATGGTCCACCGTCCTCCAGCCATGAGCCAGGCCTGTAACACAGAGCC
CTGTCCCCCCAGGTGGCATGTGGGCTCTTGGGGGCCCTGCTCAGCTACCTGTGGAGTTGGAATTCAGA
CCCGAGATGTGTACTGCCTGCACCCAGGGGAGACCCCTGCCCCTCCTGAGGAGTGCCGAGATGAAAAG
CCCCATGCTTTACAAGCATGCAATCAGTTTGACTGCCCTCCTGGCTGGCACATTGAAGAATGGCAGCA

i GCAGCTTTTTGAATCTCTCAGATGAATTGTGCCAAGGACCCAAGGCATCGTCTCACAAGTCCTGTGCC
AGGACAGACTGTCCTCCACATTTAGCTGTGGGAGACTGGTCGAAGTGTTCTGTCAGTTGTGGTGTTGG
AATCCAGAGAAGAAAGCAGGTGTGTCAAAGGCTGGCAGCCAAAGGTCGGCGCATCCCCCTCAGTGAGA
TGATGTGCAGGGATCTACCAGGGTTCCCTCTTGTAAGATCTTGCCAGATGCCTGAGTGCAGTAAAATC
AAATCAGAGATGAAGACAAA.ACTTGGTGAGCAGGGTCCGCAGATCCTCAGTGTCCAGAGAGTCTACAT
TCAGACAAGGGAAGAGAAGCGTATTAACCTGACCATTGGTAGCAGAGCCTATTTGCTGCCCAACACAT
CCGTGATTATTAAGTGCCCAGTGCGACGATTCCAGAAATCTCTGATCCAGTGGGAGAAGGATGGCCGT I, TGCCTGCAGAACTCCAAACGGCTTGGCATCACCAAGTCAGGCTCACTAAAAATCCACGGTCTTGCTGC
CCCCGACATCGGCGTGTACCGGTGCATTGCAGGCTCTGCACAGGAAACAGGTGTGCTCAAGCTCATTG
GTACTGACAACCGGCTCATTGCACGCCCAACCCTCANGGAGCCTATGAGGGAATATCCTGGGATGGAC
CACAACGAAGCCAATAGTT
NOVl lc, CG50513-02 SEQ ID NO: 88 346 as MW at 38248.6kD

Protein Sequence ~" .
VNDSLCDMVHRPPAMSQACNTEPCPPRWHVGSWGPCSATCGVGIQTRDVYCLHPGETPAPPEECRDEK
PHALQACNQFDCPPGWHIEEWQQCSRTCGGGTQNRRVTCRQLLTDGSFLNLSDELCQGPKASSHKSCA
RTDCPPHLAVGDWSKCSVSCGVGIQRRKQVCQRLAAKGRRIPLSEMMCRDLPGFPLVRSCQMPECSKI
KSEMKTKLGEQGPQTLSVQRVYIQTREEKRINLTIGSRAYLLPNTSVIIKCPVRRFQKSLIQWEKDGR
CLQNSKRLGITKSGSLKIHGLAAPDIGVYRCIAGSAQETGVLKLIGTDNRLIARPTLXEPMREYPGMD
HNEANS
NOVlld, CG50513-03 SEQ ID NO: 89 6303 by DNA Sequence ;ORF Start ATG at 425 ~ORF Stop TAA at 4268 TATAATTATTAATAGAGACCTTTCAAAGGACAAATTCTGTGAAATAAAGTGGTTTTCTGAAGAGCCTA
CTAATAGGACAGTGTGTTAATATCACTAATAAGAGAGTAATGATTATAAAAAGGAATAAATTTATTGA
AATTGCAAGATACTTTTCTCCTTTGATTAATATACTGCTAGTTTAGTTTTCTACATTTTCAAATAGAA
CTGGGGAATTTGTGTCGTAGATATTCTTGACAACTAAAGAGATGGTGGCTGAATTTTTGGGAATGGTT
GATAACACTTGATATTTTTAGTTTCCAATTTGGAAGAGCTCTGTCTCTTGGGATGTCAAATATTATAT
TCGTCAATTAATGAATGTGTTAATTTATTATAGAAATGATATTCTCACAATGATTTCATTTGTAGTGA l TGGATTTAAAGAGATAATGCCCTATGACCACTTCCAACCTCTTCCTCGCTGGGAACATAATCCTTGGA

CTGCATGTTCCGTGTCCTGTGGAGGAGGGATTCAGAGACGGAGCTTTGTGTGTGTAGAGGAATCCATG i CATGGAGAGATATTGCAGGTGGAAGAATGGAAGTGCATGTACGCACCCAAACCCAAGGTTATGCAAAC
TTGTAATCTGTTTGATTGCCCCAAGTGGATTGCCATGGAGTGGTCTCAGTGCACAGTGACTTGTGGCC
GAGGGTTACGGTACCGGGTTGTTCTGTGTATTAACCACCGCGGAGAGCATGTTGGGGGCTGCAATCCA
CAACTGAAGTTACACATCAAAGAAGAATGTGTCATTCCCATCCCGTGTTATAAACCAAAAGAAAAAAG
TCCAGTGGAAGCAAAATTGCCTTGGCTGAAACAAGCACAAGAACTAGAAGAGACCAGAATAGCAACAG
AAGAACCAACGTTCATTCCAGAACCCTGGTCAGCCTGCAGTACCACGTGTGGGCCGGGTGTGCAGGTC
CGTGAGGTGAAGTGCCGTGTGCTCCTCACATTCACGCAGACTGAGACTGAGCTGCCCGAGGAAGAGTG
TGAAGGCCCCAAGCTGCCCACCGAACGGCCCTGCCTCCTGGAAGCATGTGATGAGAGCCCGGCCTCCC
GAGAGCTAGACATCCCTCTCCCTGAGGACAGTGAGACGACTTACGACTGGGAGTACGCTGGGTTCACC
CCTTGCACAGCAACATGCGTGGGAGGCCATCAAGAAGCCATAGCAGTGTGCTTACATATCCAGACCCA
GCAGACAGTCAATGACAGCTTGTGTGATATGGTCCACCGTCCTCCAGCCATGAGCCAGGCCTGTAACA
CAGAGCCCTGTCCCCCCAGGTGGCATGTGGGCTCTTGGGGGCCCTGCTCAGCTACCTGTGGAGTTGGA
ATTCAGACCCGAGATGTGTACTGCCTGCACCCAGGGGAGACCCCTGCCCCTCCTGAGGAGTGCCGAGA
TGAAAAGCCCCATGCTTTACAAGCATGCAATCAGTTTGACTGCCCTCCTGGCTGGCACATTGAAGAAT
GGCAGCAGTGTTCCAGGACTTGTGGCGGGGGAACTCAGAACAGAAGAGTCACCTGTCGGCAGCTGCTA
ACGGATGGCAGCTTTTTGAATCTCTCAGATGAATTGTGCCAAGGACCCAAGGCATCGTCTCACAAGTC
CTGTGCCAGGACAGACTGTCCTCCACATTTAGCTGTGGGAGACTGGTCGAAGTGTTCTGTCAGTTGTG
GTGTTGGAATCCAGAGAAGAAAGCAGGTGTGTCAAAGGCTGGCAGCCAAAGGTCGGCGCATCCCCCTC
AGTGAGATGATGTGCAGGGATCTACCAGGGTTCCCTCTTGTAAGATCTTGCCAGATGCCTGAGTGCAG
TAAAATCAAATCAGAGATGAAGACAAAACTTGGTGAGCAGGGTCCGCAGATCCTCAGTGTCCAGAGAG
TCTACATTCAGACAAGGGAAGAGAAGCGTATTAACCTGACCATTGGTAGCAGAGCCTATTTGCTGCCC
AACACATCCGTGATTATTAAGTGCCCCGTGCGACGATTCCAGAAATCTCTGATCCAGTGGGAGAAGGA
TGGCCGTTGCCTGCAGAACTCCAAACGGCTTGGCATCACCAAGTCAGGCTCACTAAAAATCCACGGTC
TTGCTGCCCCCGACATCGGCGTGTACCGGTGCATTGCAGGCTCTGCACAGGAAACAGTTGTGCTCAAG
CTCATTGGTACTGACAACCGGCTCATCGCACGCCCAGCCCTCAGGGAGCCTATGAGGGAATATCCTGG
GATGGACCACAGCGAAGCCAATAGTTTGGGAGTCACATGGCACAAAATGAGGCAAATGTGGAATAACA
AAAATGACCTTTATCTGGATGATGACCACATTAGTAACCAGCCTTTCTTGAGAGCTCTGTTAGGCCAC
TGCAGCAATTCTGCAGGAAGCACCAACTCCTGGGAGTTGAAGAATAAGCAGTTTGAAGCAGCAGTTAA
ACAAGGAGCATATAGCATGGATACAGCCCAGTTTGATGAGCTGATAAGAAACATGAGTCAGCTCATGG
AAACCGGAGAGGTCAGCGATGATCTTGCGTCCCAGCTGATATATCAGCTGGTGGCCGAATTAGCCAAG
GCACAGCCAACACACATGCAGTGGCGGGGCATCCAGGAAGAGACACCTCCTGCTGCTCAGCTCAGAGG
GGAAACAGGGAGTGTGTCCCAAAGCTCGCATGCAAHAAACTCAGGCAAGCTGACATTCAAGCCGAAAG
GACCTGTTCTCATGAGGCAAAGCCAACCTCCCTCAATTTCATTTAATAAAACAATAAATTCCAGGATT
GGAAATACAGTATACATTACAAAAAGGACAGAGGTCATCAATATACTGTGTGACCTTATTACCCCCAG
TGAGGCCACATATACATGGACCAAGGATGGAACCTTGTTACAGCCCTCAGTAAAAATAATTTTGGATG
GAACTGGGAAGATACAGATACAGAATCCTACAAGGAAAGAACAAGGCATATATGAATGTTCTGTAGCT
AATCATCTTGGTTCAGATGTGGAAAGTTCTTCTGTGCTGTATGCAGAGGCACCTGTCATCTTGTCTGT
TGAAAGAAATATCACCAAACCAGAGCACAACCATCTGTCTGTTGTGGTTGGAGGCATCGTGGAGGCAG
CCCTTGGAGCAAACGTGACAATCCGATGTCCTGTAAAAGGTGTCCCTCAGCCTAATATAACTTGGTTG
AAGAGAGGAGGATCTCTGAGTGGCAATGTTTCCTTGCTTTTCAATGGATCCCTGTTGTTGCAGAATGT

TTCCCTTGAAAATGAAGGAACCTACGTCTGCATAGCCACCAATGCTCTTGGAAAGGCAGTGGCAACAT
CTGTACTCCACTTGCTGGAACGAAGATGGCCAGAGAGTAGAATCGTATTTCTGCAAGGACATAAAAAG
TACATTCTCCAGGCAACCAACACTAGAACCAACAGCAATGACCCA.ACAGGAGAACCCCCGCCTCAAGA
GCCTTTTTGGGAGCCTGGTAACTGGTCACATTGTTCTGCCACCTGTGGTCATTTGGGAGCCCGCATTC
'AGAGACCCCAGTGTGTGATGGCCAATGGGCAGGAAGTGAGTGAGGCCCTGTGTGATCACCTCCAGAAG
'CCACTGGCTGGGTTTGAGCCCTGTAACATCCGGGACTGCCCAGCGAGGTGGTTCACAAGTGTGTGGTC
~ACAGTGCTCTGTGTCTTGCGGTGAAGGATACCACAGTCGGCAGGTGACGTGCAAGCGGACAAAAGCCA
ATGGAACTGTGCAGGTGGTGTCTCCAAGAGCATGTGCCCCTAAAGACCGGCCTCTGGGAAGAAAACCA
TGTTTTGGTCATCCATGTGTTCAGTGGGAACCAGGGAACCGGTGTCCTGGACGTTGCATGGGCCGTGC
TGTGAGGATGCAGCAGCGTCACACAGCTTGTCAACACAACAGCTCTGACTCCAACTGTGATGACAGAA
AGAGACCCACCTTAAGAAGGAACTGCACATCAGGGGCCTGTGATGTGTGTTGGCACACAGGCCCTTGG
AAGCCCTGTACAGCAGCCTGTGGCAGGGGTTTCCAGTCTCGGAAAGTCGACTGTATCCACACAAGGAG
TTGCAAACCTGTGGCCAAGAGACACTGTGTACAGAAAAAGAAACCAATTTCCTGGCGGCACTGTCTTG
GGCCCTCCTGTGATAGAGACTGCACAGACACAACTCACTACTGTATGTTTGTAAAACATCTTAATTTG
TGTTCTCTAGACCGCTACAAACAAAGGTGCTGCCAGTCATGTCAAGAGGGATAAACCTTTGGAGGGGT
CATGATGCTGCTGTGAAGATAAAAGTAGAATATAAAAGCTCTTTTCCCCATGTCGCTGATTCAAAA.AC
ATGTATTTCTTAAAAGACTAGATTCTATGGATCAAACAGAGGTTGATGCAAAAACACCACTGTTAAGG
TGTAAAGTGAAATTTTCCAATGGTAGTTTTATATTCCAATTTTTTAAAATGATGTATTCAAGGATGAA
CAAAATACTATAGCATGCATGCCACTGCACTTGGGACCTCATCATGTCAGTTGAATCGAGAAATCACC
AAGATTATGAGTGCATCCTCACGTGCTGCCTCTTTCCTGTGATATGTAGACTAGCACAGAGTGGTACA
TCCTAAAAACTTGGGAAACACAGCAACCCATGACTTCCTCTTCTCTCAAGTTGCAGGTTTTCAACAGT
TTTATAAGGTATTTGCATTTTAGAAGCTCTGGCCAGTAGTTGTTAAGATGTTGGCATTAATGGCATTT
TCATAGATCCTTGGTTTAGTCTGTGAAAAAGAAACCATCTCTCTGGATAGGCTGTCACACTGACTGAC
CTAAGGGTTCATGGAAGCATGGCATCTTGTCCTTGCTTTTAGAACACCCATGGAAGAAAACACAGAGT
AGATATTGCTGTCATTTATACAACTACAGAAATTTATCTATGACCTAATGAGGCATCTCGGAAGTCAA
AGAAGAGGGAAAGTTAACCTTTTCTACTGATTTCGTAGTATATTCAGAGCTTTCTTTTAAGAGCTGTG
AATGAAACTTTTTCTAAGCACTATTCTATTGCACACAAACAGAAAACCAAAGCCTTATTAGACCTAAT.
TTATGCATAAAGTAGTATTCCTGAGAACTTTATTTTGGAAAATTTATAAGAAAGTAATCCAAATAAGA
AACACGATAGTTGAAAATAATTTTTATAGTAAATAATTGTTTTGGGCTGATTTTTCAGTAAATCCAAA
GTGACTTAGGTTAGAAGTTACACTAAGGACCAGGGGTTGGAATCAGAATTTAGTTTAAGATTTGAGGA
AAAGGGTAAGGGTTAGTTTCAGTTTTAGGATTAGAGCTAGAATTGGGTTAGGTGAGAAAGAAAGTTAA
GGTTAAGGCTAGAGTTGTCTTTAAGGGTTAGGGTTAGGACCAGGTTAGGTCAGGGTTGGATTGGGTTT
AGATTGGGGCCAGTGCTGGTGTTAGTGATAGTGTCAGGATGGAGGTTAGGTTTGGAGTAAGCGTTGTT
GCTGAAGTGAGTTCAGGCTAGCATTAAATTGTAAGTTCTGAAGCTGATTTGGTTATGGGGTCTTTCCC
CTGTATACTACCAGTTGTGTCTTTAGATGGCACACAAGTCCAAATAAGTGGTCATACTTCTTTATTCA
GGGTCTCAGCTGCCTGTACACCTGCTGCCTACATCTTCTTGGCAACAAAGTTACCTGCCACAGGCTCT
GCTGAGCCTAGTTCCTGGTCAGTAATAACTGAACAGTGCATTTTGGCTTTGGATGTGTCTGTGGACAA
GCTTGCTGAGTTTCTCTACCATATTCTGAGCACACGGTCTCTTTTGTTCTAATTTCAGCTTCACTGAC
ACTGGGTTGAGCACTACTGTATGTGGAGGGTTTGGTGATTGGGAATGGATGGGGGACAGTGAGGAGGA
CACACCAGCCCATTAGTTGTTAP.TCATCAATCACATCTGATTGTTGAAGGTTATTAAATTAAAAGAAA
GATCATTTGTAACATACTCTTTGTATATATTTATTATATGAAAGGTGCAATATTTTATTTTGTACAGT
ATGTAATAAAGACATGGGACATATATTTTTCTTATTAACAAAATTTCATATTAAATTGCTTCACTTTG
TATTTAAAGTTAAAAGTTACTATTTTTCATTTGCTATTGTACTTTCATTGTTGTCATTCAATTGACAT
TCCTGTGTACTGTATTTTACTACTGTTTTTATAACATGAGAGTTAATGTTTCTGTTTCATGATCCTTA
TGTAATTCAGAAATAAATTTACTTTGATTATTCA.GTGGCATCCTTAT
NOVIId, CG50513-03 SEQ ID NO: 90.1281 as MW at 142825.9kD
Protein Sequence MPYDHFQPLPRWEHNPWTACSVSCGGGIQRRSFVCVEESMHGEILQVEEWKCMYAPKPKVMQTCNLFD
CPKWIAMEWSQCTVTCGRGLRYRVVLCINHRGEHVGGCNPQLKLHIKEECVIPIPCYKPKEKSPVEAK
LPWLKQAQELEETRIATEEPTFIPEPWSACSTTCGPGVQVREVKCRVLLTFTQTETELPEEECEGPKL
PTERPCLLEACDESPASRELDIPLPEDSETTYDWEYAGFTPCTATCVGGHQEAIAVCLHIQTQQTVND
SLCDMVHRPPAMSQACNTEPCPPRWHVGSWGPCSATCGVGIQTRDVYCLHPGETPAPPEECRDEKPHA

CPPHLAVGDWSKCSVSCGVGIQRRKQVCQRLAAKGRRIPLSEMMCRDLPGFPLVRSCQMPECSKIKSE
MKTKLGEQGPQILSVQRVYIQTREEKRINLTIGSRAYLLPNTSVIIKCPVRRFQKSLIQWEKDGRCLQ ', NSKRLGITKSGSLKIHGLAAPDIGVYRCIAGSAQETVVLKLIGTDNRLIARPALREPMREYPGMDHSE ' ANSLGVTWHKMRQMWNNKNDLYLDDDHTSNQPFLRALLGHCSNSAGSTNSWELKNKQFEAAVKQGAYS
MDTAQFDELIRNMSQLMETGEVSDDLASQLIYQLVAELAKAQPTHMQWRGIQEETPPAAQLRGETGSV

SHAKNSGKLTFKPKGPVLMRQSQPPSISFNKTINSRIGNTVYITKRTEVINILCDLITPSEATYT
DGTLLQPSVKIILDGTGKIQIQNPTRKEQGIYECSVANHLGSDVESSSVLYAEAPVILSVERNIT
HNHLSVVVGGIVEAALGANVTIRCPVKGVPQPNITWLKRGGSLSGNVSLLFNGSLLLQNVSLENE
VCIATNALGKAVATSVLHLLERRWPESRIVFLQGHKKYILQATNTRTNSNDPTGEPPPQEPFWEP
SHCSATCGHLGARIQRPQCVMANGQEVSEALCDHLQKPLAGFEPCNIRDCPARWFTSVWSQCSVS
VTCKRTKANGTVQVVSPRACAPKDRPLGRKPCFGHPCVQWEPGNRCPGRCMGRAVRMQQ
SDSNCDDRKRPTLRRNCTSGACDVCWHTGPWKPCTAACGRGFQSRKVDCTHTRSCKPVA
SWRHCLGPSCDRDCTDTTHYCMFVKHLNLCSLDRYKQRCCQSCQEG
i.le, CG50513-04 SEQ ID NO 91 _ _ " 7260 by _ Sequence aORF Start:'ATG at 136 ORF Stop: TAA at 5209 ~r_r_rrmr_rnrrmrrrnrAmArmmrmGCGGCGCAAGGCTACAACTGAGACCCGGAGGAGACTAGACCCCA
CTCCCGCAG
CTGGAGCCTATTTCCTTCCCGAGTTTGCACTTTCTCCTCAGGGAAGTTT
GAGCAGTTCCTCACTTATCGCTATGATGACCAGACCTCAAGAAACACTC
.TGAAGACAAAGATGGCAACTGGGATGCTTGGGGCGACTGGAGTGACTGCTCCCGGACCTGT
CAGCAGTGCTCAG
ACAATGATGTCCAGTATCAGGGGCATTACTATGAATGGCTTCCACGATATAATGATCCTGCTGCC
TGTGCACTCAAGTGTCATGCACAAGGACAAAACTTGGTGGTGGAGCTGGCACCTAAGGTACTGGA
TCAGTGGCATCTGTCAGGCAGTGGGCTGCG
CAAAGTCACACGTTTCTCCTGAAAAAAGAGAAGAAAATGTAATTGCTGT
AACAGCCCCGGCGTCTTTGTCGTAGAAAACACAACA
CTTCAAGACCAGGTACACTGCAGCCAAAGACAGCGTGGTTCAGTTCTTCTTTTACCAGCCCATCAGTC
ATCAGTGGAGACAAACTGACTTCTTTCCCTGCACTGTGACGTGTGGAGGAGGTTATCAGCTCAATTCT
ATCCGCTTGAAGAGGGTAGTTCCTGACCATTATTGTCACTACTACCCTGAAAA
.TGCAGCATGGATCCCTGCCCATCAAGTGATGGATTTAAAG
TGCCCTATGACCACTTCCAACCTCTTCCTCGCTGGGAACATAATCCTTGGACTGCATGTTCC
AATCTGT
CCAAGTGGATTGCCATGGAGTGGTCTCAGTGCACAGTGACTTGTGGCCGAGGGTTACGG
TTAACCACCGCGGAGAGCATGTTGGGGGCTGCAATCCACAACTGAAGTT
GTCATTCCCATCCCGTGTTATAAACCAAAAGAAAAAAGTCCAGTGGAAG
CAAAATTGCCTTGGCTGAAACAAGCACAAGAACTAGAAGAGACCAGAATAGCAACAGAAGAACCAACG
CGAACGGCCCTGCCTCCTGGAAGCATGTGATGAGAGCCCGGCCTCCCGAGAGCTAGAC
TGCGTGGGAGGCCATCAAGAAGCCATAGCAGTGTGCTTACATATCCAGACCCAGCAGACAGTCA
CAGCTTGTGTGATATGGTCCACCGTCCTCCAGCCATGAGCCAGGCCTGTAACACAGAGCCCTGT
TGTGTACTGCCTGCACCCAGGGGAGACCCCTGCCCCTCCTGAGGAGTGCCGAGATGAAAAGCCCC
TGCAATCAGTTTGACTGCCCTCCTGGCTGGCACATTGAAGAATGGCAGCAGTGT
CAGACTGTCCTCCACATTTAGCTGTGGGAGACTGGTCGAAGTGTTCTGTCAGTTGTGGTGTTGGAATC
rnr_nrn,ArAAArrnrrmrmrmrAAArrr_mrrrArrrApAGGTCGGCGCATCCCCCTCAGTGAGATGAT
AAGATCTTGCCAGATGCCTGAGTGCAGTAAAATCAAAT
GGGAAGAGAAGCGTATTAACCTGACCATTGGTAGCAGAGCCTATTTGCTGCCCAACACATCCGT
ATTAAGTGCCCCGTGCGACGATTCCAGAAATCTCTGATCCAGTGGGAGAAGGATGGCCGTTGCC
CGGTGCATTGCAGGCTCTGCACAGGAAACAGTTGTGCTCAAGCTCATTGGTAC
CGCACGCCCAGCCCTr_AGGGAGCCTATGAGGGAATATCCTGGGATGGACCACA

GAAGCCAATAGTTTGGGAGTCACATGGCACAAAATGAGGCAAATGTGGAATAACAAAAATGACCTT
TCTGGATGATGACCACATTAGTAACCAGCCTTTCTTGAGAGCTCTGTTAGGCCACTGCAGCAATTC
ATGGATACAGCCCAGTTTGATGAGCTGATAAGAA.ACATGAGTCAGCTCATGGAA.ACCGGAGAG
CGATGATCTTGCGTCCCAGCTGATATATCAGCTGGTGGCCGAATTAGCCAAGGCACAGCCA.AC
TGCAGTGGCGGGGCATCCAGGAAGAGACACCTCCTGCTGCTCAGCTCAGAGGGGAAACAGGGA
TCCCAAAGCTCGCATGCAAAAAACTCAGGCAAGCTGACATTCAAGCCGAA.AGGACCTGTTCTC
GCAAAGCCAACCTCCCTCAATTTCATTTAATAAAACAATAAATTCCAGGATTGGAAATACAGT
TTACAAAAAGGACAGAGGTCATCAATATACTGTGTGACCTTATTACCCCCAGTGAGGCCACAT
TGGACCAAGGATGGAACCTTGTTACAGCCCTCAGTAAAAATAATTTTGGATGGAACTGGGAAG
GATACAGAATCCTACAAGGAAA.GAACAAGGCATATATGAATGTTCTGTAGCTAATCATCTTGG
ATGTGGAAAGTTCTTCTGTGCTGTATGCAGAGGCACCTGTCATCTTGTCTGTTGAAAGAAATA
TCCGATGTCCTGTAAAAGGTGTCCCTCAGCCTAATATAACTTGGTTGAAGAGAGGAGG
AGCCTGGTAACTGGTCACATTGTTCTGCCACCTGTGGTCATTTGGGAGCCCGCA'T'TCAGAGACCCCAG
TGTGTGATGGCCAATGGGCAGGAAGTGAGTGAGGCCCTGTGTGATCACCTCCAGAAGCCACTGGCTGG
GTTTGAGCCCTGTAACATCCGGGACTGCCCAGCGAGGTGGTTCACAAGTGTGTGGTCACAGTGCTCTG
TGTCTTGCGGTGAAGGATACCACAGTCGGCAGGTGACGTGCAAGCGGACAAAAGCCAATGGAACTGTG
CAGGTGGTGTCTCCAAGAGCATGTGCCCCTAAAGACCGGCCTCTGGGAAGAAAACCATGTTTTGGTCA
AC
CAATTTCCTGGCGGCACTGTCTTGGGCCCTCCTGT
ATGTTTGTAAAACATCTTAATTTGTGTTCTCTAGA
AGAGGGATAAACCTTTGGAGGGGTCATGATGCTGC
AAAAGACTAGATTCTATGGATCAAACAGAGGTTGATGCAAAAACACCA.CTGTTAAGGTGTAAAGTGAA
ATTTTCCAATGGTAGTTTTATATTCCAATTTTTTAAAATGATGTATTCAAGGATGAACAAAATACTAT
~TGCATCCTCACGTGCTGCCTCTTTCCTGTGATATGTAGACTAGCACAGAGTGGTACATCCTAAAAACT
~TTTGCATTTTAGAAGCTCTGGCCAGTAGTTGTTAAGATGTTGGCATTAATGGCATTTTCATAGATCCT
TTCTAAGCACTATTCTATTGCACACAAACAGAAAACCAAAGCCTTATTAGACCTAATTTATGCATAAA
GTAGTATTCCTGAGAACTTTATTTTGGAAAATTTATAAGAAAGTAATCCAAATAAGAAACACGATAGT
GAGTTGTCTTTAAGGGTTAGGGTTAGGACCAGGTTAGGTCAGGGTTGGATTGGGTTTAGATTGGGGCC
AGTGCTGGTGTTAGTGATAGTGTCAGGATGGAGGTTAGGTTTGGAGTAAGCGTTGTTGCTGAAGTGAG
~CAGTTGTGTCTTTAGATGGCACACAAGTCCAAATAAGTGGTCATACTTCTTTATTCAGGGTCTCAGCT
ACATACTCTTTGTATATATTTATTATATGAA_AGGTGCAATATTTTATTTTGTACAGTATGTAATAAAG
ACATGGGACATATATTTTTCTTATTAACAAAATTTCATATTAAATTGCTTCACTTTGTATTTAAAG_TT
'AAAAGTTACTATTTTTCATTTGCTATTGTACTTTCATTGTTGTCATTCAATTGACATTCCTGTGTACT

OV 11 e, CG50513-04 ~SEQ ID NO: 92 ~ 1691 as BMW at 188743.8kD
WTSPWWVLIGMVFMHSPLPQTTAEKSPGAYFLPEFALSPQGSFLEDTTGEQFLTYRYDDQTSRNT
PAAPCALKCHAQGQNLWELAPKVLDGTRCNTDSLDMCISGTCQAVGC
STCRLVRGQSKSHVSPEKREENVIAVPLGSRSVRITVKGPAHLFIESK
EHSFNSPGVFWENTTVEFQRGSERQTFKIPGPLMADFIFKTRYTAAKDSWQFFFYQPIS
FFPCTVTCGGGYQLNSAECVDIRLKRWPDHYCHWPENVKPKPKLKECSMDPCPSSDGFK
FQPLPRWEHNPWTACSVSCGGGIQRRSFVCVEESMHGEILQVEEWKCMYAPKPKVMQTCNL
AMEWSQCTVTCGRGLRYRWLCINHRGEHVGGCNPQLKLHIKEECVIPIPCYKPKEKSPVE
QAQELEETRIATEEPTFIPEPWSACSTTCGPGVQVREVKCRVLLTFTøTETELPEEECEGP
CLLEACDESPASRELDTPLPEDSETTYDWEYAGFTPCTATCVGGHQEAIAVCLHIQTQQTV
H.Hly~c:~uc~rwc:Y~GWHZEEWQQCSRTCGGGTQNRRVTCRQLLTDGS FLNLSDELCQGPKASSHKSCAR
TDCPPHLAVGDWSKCSVSCGVGIQRRKQVCQRLAAKGRRIPLSEMMCRDLPGFPLVRSCQMPECSKIK

LQNSKRLGITKSGSLKTHGLAAPDIGVYRCIAGSAQETWLKLIGTDNRLIARPALREPMREYPGMDH
SEANSLGVTWHKMRQMWNNKNDLXLDDDHISNQPFLRALLGHCSNSAGSTNSWELKNKQFEAAVKQGA
YSMDTAQFDELIRNMSQLMETGEVSDDLASQLIYQLVAELAKAQPTHMQWRGIQEETPPAAQLRGETG
SVSQSSHAKNSGKLTFKPKGPVLMRQSQPPSISFNKTINSRIGNTWITKRTEVINILCDLITPSEAT
YTWTKDGTLLQPSVKIILDGTGKIQIQNPTRKEQGIYECSVANHLGSDVESSSVLYAEAPVILSVERN
ITKPEHNHLSVWGGIVEAALGANVTIRCPVKGVPQPNITWLKRGGSLSGNVSLLFNGSLLLQNVSLE
N'EGTWCIATNALGKAVATSVFHLLERRWPESRIVFLQGHKKYILQATNTRTNSNDPTGEPPPQEPFW
EPGNWSHCSATCGHLGARIQRPQCVMANGQEVSEALCDHLQKPLAGFEPCNIRDCPARWFTSWSQCS
VSCGEGYHSRQVTCKRTKANGTVQWSPRACAPKDRPLGRKPCFGHPCVQWEPGNRCPGRCMGRAVRM
QQRHTACQHNSSDSNCDDRKRPTLRRNCTSGACDVCWHTGPWKPCTAACGRGFQSRKVDCIHTRSCKP
VAKRHCVQKKKPISWRHCLGPSCDRDCTDTTHYCMFVKHLNLCSLDRYKQRCCQSCQEG
OV 11 f, CGSOS 13-OS SEQ.ID NO: 93 , 6294, by NA Sequence ORF Start: ATG at 416 ORF Ston: TAA at 4259 TAAAGTGGTTTTCTGAAGAGCCTACTAATAGGA
~CAGTGTC7TTAATATCACTAATAAGAGAGTAATGATTATAAAAAGGAATAAATTTATTGAAATTGCAAG
~ATACTTTTCTCCTTTGATTAATATACTGCTAGTTTAGTTTTCTACATTTTCAAATAGAACTGGGGAAT
~TTGTGTCGTAGATATTCTTGACAACTAAAGAGATGGTGGCTGAATTTTTGGGAATGGTTGATAACACT
TTTGGAAGAGCTCTGT
TAATGCCCTATGACCACTTCCAACCTCTTCCTCGCTGGGAACATAATCCTTGGACTGCATGTT
TTGCAGGTGGAAGAATGGAAGTGCATGTACGCACCCAAACCCAAGGTTATGCAAACTTGTAATCT
TTGCCATGGAGTGGTCTCAGTGCACAGTGACTTGTGGCCGAGGGTTAC
ATTAACCACCGCGGAGAGCATGTTGGGGGCTGCAATCCACAACTGAAG
ACACATCAAAGAAGAATGTGTCATTCCCATCCCGTGTTATAAACCAAAAGAAAAAAGTCCAGTGGA
TAGCAACAGAAGAACCAA
TTCACGCAGACTGAGACTGAGCTGCCCGAGGAAGAGTGTGAAGGCCC
TGTGATGAGAGCCCGGCCTCCCGAGAGCTAG
CTTGCACA
TGCGTGGGAGGCCATCAAGAAGCCATAGCAGTGTGCTTACATATCCAGACCCAGCAGACAGT
CTGCACCCAGGGGAGACCCCTGCCCCTCCTGAGGAGTGCCGAGATGAA.AAGCC
TTGAAGAATGGCAGCAGT
TCTCTCAGATGAATTGTGCCAAGGACCCAAGGCATCGTCTCACAAGTCCTGTGCCAG

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

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Claims (45)

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 174.

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 174.

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 174.

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 174.

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

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

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

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

9. A method for determining the presence or amount of the polypeptide of claim 1 in a sample, the method comprising:
(a) providing said sample;

(b) introducing said sample to an antibody that binds immunospecifically to the polypeptide; and (c) determining the presence or amount of antibody bound to said polypeptide, thereby determining the presence or amount of polypeptide in said sample.

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

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

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

13. A method for identifying a potential therapeutic agent for use in treatment of a pathology, wherein the pathology is related to aberrant expression or aberrant physiological interactions of the polypeptide of claim 1, the method comprising:
(a) providing a cell expressing the polypeptide of claim 1 and having a property or function ascribable to the polypeptide;
(b) contacting the cell with a composition comprising a candidate substance;
and (c) determining whether the substance alters the property or function ascribable to the polypeptide;

whereby, if an alteration observed in the presence of the substance is not observed when the cell is contacted with a composition in the absence of the substance, the substance is identified as a potential therapeutic agent.

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

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

16. A method for modulating the activity of the polypeptide of claim l, 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 174 or a biologically active fragment thereof.

20. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2n-1, wherein n is an integer between 1 and 174.

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

22. A nucleic acid molecule, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NO:
2n-1, wherein n is an integer between 1 and 174.

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 174.

24. An isolated nucleic acid molecule comprising a nucleic acid selected from the group consisting of 2n-1, wherein n is an integer between 1 and 174.

25. The nucleic acid molecule of claim 20, wherein said nucleic acid molecule hybridizes under stringent conditions to the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 174, or a complement of said nucleotide sequence.

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

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

28. A cell comprising the vector of claim 26.

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

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

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

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

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

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

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

wherein an alteration in the level of expression of the nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.

36. A method of producing the polypeptide of claim 1, the method comprising culturing a cell under conditions that lead to expression of the polypeptide, wherein said cell comprises a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2n-1, wherein n is an integer between 1 and 174.

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

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

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

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

41. A method of producing the polypeptide of claim 2, the method comprising culturing a cell under conditions that lead to expression of the polypeptide, wherein said cell comprises a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2n-1, wherein n is an integer between 1 and 174.

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.