CA2486490A1 - Therapeutic polypeptides, nucleic acids encoding same, and methods of use - Google Patents

Abstract

Disclosed herein are nucleic acid sequences that encode novel polypeptides.
Also disclosed are polypeptides encoded by these nucleic acid sequences, and antibodies that immunospecifically bind to the polypeptide, as well as derivatives, variants, mutants, or fragments of the novel polypeptide, polynucleotide, or antibody specific to the polypeptide. 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

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

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THERAPEUTIC POLYPEPTIDES, NUCLEIC ACIDS ENCODING
SAME, AND METHODS OF USE
FIELD OF THE INVENTION
The present invention relates to novel polypeptides, and the nucleic acids encoding S them, having properties related to stimulation of biochemical or physiological responses in a cell, a tissue, an organ or an organism. More particularly, the novel polypeptides are gene products of novel genes, or are specified biologically active fragments or derivatives thereof. Methods of use encompass diagnostic and prognostic assay procedures as well as methods of treating diverse pathological conditions.

BACKGROUND OF THE INVENTION
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 Ievel of synthesis and secretion of protein effectors. In other classes of pathologies the dysregulation is manifested as increased or up-regulated level of synthesis and secretion of protein effectors. In a clinical setting a subject may be suspected of suffering from a condition brought on by altered or mis-regulated levels of a protein effector of interest. Therefore there is a need to assay for the level of the protein effector of interest in a biological sample from such a subject, and to compare the level with that characteristic of a nonpathological condition. There also is a need to provide the protein effector as a product of manufacture. Administration of the effector to a subject in need thereof is useful in treatment of the pathological condition.
Accordingly, there is a need for a method of treatment of a pathological condition brought on by a diminished or suppressed levels of the protein effector of interest. In addition, there is a need for a method of treatment of a pathological condition brought on by a increased or up-regulated levels of the protein effector of interest.
Antibodies are multichain proteins that bind specifically to a given antigen, and bind poorly, or not at all, to substances deemed not to be cognate antigens.
Antibodies are comprised of two short chains termed light chains and two long chains termed heavy 15. 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.

S~JIVE1~IARY 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 N0:2n, wherein n is an integer between 1 and I41. The novel nucleic acids and polypeptides are referred to herein as NOVX, or NOVl, NOV2, NOV3, etc., nucleic acids and polypeptides. These nucleic acids and polypeptides, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as "NOVX" nucleic acid or polypeptide sequences.
The invention also is based in part upon variants of a mature form of the amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141, wherein any amino acid in the mature form is changed to a different amino acid, provided that no more than I 5% 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 141. 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 I and 141, wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15%
of the amino acid residues in the sequence are so changed. The invention also involves fragments of any of the mature forms of the amino acid sequences selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 141, or any other amino acid sequence selected from this group. The invention also comprises fragments from these groups in which up to I S% of the residues are changed.
In another embodiment, the invention encompasses polypeptides that are naturally occurring allelic variants of the sequence selected from the group consisting of SEQ ID
NO:2n, wherein n is an integer between 1 and 141. These allelic variants include amino acid sequences that are the translations of nucleic acid sequences differing by a single nucleotide from nucleic acid sequences selected from the group consisting of SEQ ID
NOS: 2n-1, wherein n is an integer between 1 and 141. The variant polypeptide where any amino acid changed in the chosen sequence is changed to provide a conservative substitution.

In another embodiment, the invention comprises a pharmaceutical composition involving a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 141, 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 141, 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 I and 141, in a sample, the method involving providing the sample; introducing the sample to an antibody that binds immunospecifically to the polypeptide; and determining the presence or amount of antibody bound to the polypeptide, thereby determining the presence or amount of polypeptide in the sample.
In another embodiment, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID
NO:2n, wherein n is an integer between 1 and 141, 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 141, 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 fox 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 141, 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 1 and 141, 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 N0:2n, wherein n is an integer between 1 and 141, 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 141, the method including administering the polypeptide to a subject in which such treatment or prevention is desired in an amount sufficient to treat or prevent the pathology in the subject. The subject could be human.
In another embodiment, the invention involves a method of treating a pathological state in a mammal, the method including administering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95% identical to a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 141, or a biologically active fragment thereof.
In another embodiment, the invention involves an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 141; a variant of a mature I S form of the amino acid sequence selected from the group consisting of SEQ
ID NO:2n, wherein n is an integer between 1 and 141, 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 Land 141, 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 141, in which any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed; a nucleic acid fragment encoding at least a portion of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between 1 and 141, 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 141, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally occurring allelic nucleic acid variant.
In another embodiment, the invention involves an isolated nucleic acid molecule including a nucleic acid sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 141, that encodes a variant polypeptide, wherein the variant polypeptide has the polypeptide sequence of a naturally occurring polypeptide variant.
In another embodiment, the invention comprises an isolated nucleic acid molecule having a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID NO:2n, wherein n is an integer between 1 and 141, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 2n-1, wherein n is an integer between 1 and 141.
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 141, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence selected from the group consisting of SEQ ID NO:2n-1, wherein n is an integer between 1 and 141, 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 141, 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 141, and a nucleic acid fragment 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 141, 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 I41, 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 14I, 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 141, 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 1 S% 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 1 and 141. This vector can have a promoter operably linked to the nucleic acid molecule. This vector can be located within a cell.
In another embodiment, the invention involves a method for determining the presence or amount of a nucleic acid molecule having a nucleic acid sequence encoding a polypeptide including an amino acid sequence selected from the group consisting of a mature form of the amino acid sequence given SEQ ID N0:2n, wherein n is an integer between 1 and 141, 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 141, 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.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel nucleotides and polypeptides encoded thereby. Included in the invention are the novel nucleic acid sequences, their encoded polypeptides, antibodies, and other related compounds. The sequences are collectively referred to herein as "NOVX nucleic acids" or "NOVX polynucleotides" and the corresponding encoded polypeptides are referred to as "NOVX polypeptides" or "NOVX
proteins." Unless indicated otherwise, "NOVX" is meant to refer to any of the novel sequences disclosed herein. Table A provides a summary of the NOVX nucleic acids and their encoded polypeptides.

TABLE A. Sequences and Corresponding SEQ ID Numbers SEQ SEQ ID
ID

NOVX Internal NO NO gomology AssignmentIdentification(nucleic(amino acid) acid) NOVla CG103945-021 2 Semaphorin sem2 (FLJ00014 protein) -Homo Sapiens _....._...._.... _......._..__.._ _....___...___......__.........._...___.
._.._.......___...
.. ....._.._.__..___ ....._ ____......_.
._..____..__.._.._._._......_.._._..._..
..__....__ ... ._...........__.._._._._.._.
....... _ NOVlb CG103945-O13 4 Semaphorin sem2 (FLJ00014 protein) -Homo Sapiens , NOV2a CG106951-Ol5 6 Human semaphorin G-like ~ ~ ~ NHP protein ~

_ CG106951-047 _ 8 H__uman__se_maph_orin_G-like NOV2b Y NH_P protein OV2c 209829549 9 10 Human semaphorin G-like NHPprotein NOV2d _ 20982955311 12 Human semaphorin G-like NHP protein 2e _ 209829 13_ 14 _ Human se_map_horin_G-like NO 642 ~ -~ NHP protein V

_ _ 15 x16 ;Human semaphorin G-like _ 209829670 _ _ ~._. NHP protein NOV2f~ ~ ., _ ~, =, _ ._~. __._.~..,"_..._ ._,_.

NOV2g _: ~,~ , ....3 Human semaphorin . ,. . , CG106951-02.. ' 18 ,... G-like,NHP protein, ,. .. .,.. 17. . ........
. ., ., .

NOV2h CG_ 106951-03_ 19 20 Human semaphorin G-like _ -~ ~ NHP protein n NOV2i SNP13382456_ ~2- ~ Human semaphorin G-like _21 NHP protein NOV3a CG121295-01~ 24 Endothelia-1 precursor 23 (ET-1) -Homo _ .. _. ._.. _ .. saPiens,_. .. . ...
. _ ....
.

NOV4a CG124756-O1 26 complement subcomponent Clq chain 25 _B precursor [validated]
_ NOV4b CG124756-0227 28 complement subcomponent Clq chain B precursor [validated]

NOV4c SNP1338247529 30 complement subcomponent Clq chain _ _ - B precursor [validated]
~

OV4d SNP13382476,31 32 complement subcomponent 4 Clq chain _ .._ .... . . _............ B. precursor. [validated]...]...
_ .. _ .... ...... . . . .
. ~~ ~

OVSa CG50353-Ol 33 34 Wnt-7a protein precursor - Homo Sapiens ~c NOVSb 228753443 35 36 Wnt-7a protein precursor ~ - Homo Sapiens NOVSc 169475673 37 38 Wnt-7a protein precursor - Homo Sapiens _ _ _ ~

NOVSd 228753459 39 40 Wnt-7a protein precursor - Homo _... ....~.........__.__._...._,._,......_..__......,.....__...~ _ ... _ Sapiens ......_ ._.._....._.._.._......._..._..,.....,__..__..._......_._.., .._ . . . ...
..
...._...

OVSe 228753462 ~ 41 42 Wnt-7a protein precursor - Homo Sapiens _ NOVSf 228753446 43 44 Wnt-7a protein precursor - Homo Sapiens NOVSg 228753465 45 46 Wnt-7a protein precursor - Homo _ _ Sapiens _ _ _ - ~V

NOVSh 228753438 47 48 Wnt-7a protein precursor - Homo Sapiens NOVSi 228753449 49 50 Wnt-7a protein precursor - Homo sapiens _......_._.._..._.._.__..._.._._.....

NOVSj CG50353-02 51 52 Wnt-7a protein precursor - Homo Sapiens OVSk CG50353-03 53 54 Wnt-7a protein precursor - Homo v Sapiens NOV51 SNP13382474 55 56 Wnt-7a protein precursor ~ - Homo _ _ __ _ sapie_ns _ _ - v ~ ~
~

OV6a CG50709-03 57 58 Wnt-9b protein precursor (Wnt-15) (Wnt-14b) - Homo Sapiens NOV6b 282997951 59 60 Wnt-9b protein precursor (Wnt-15) (Wnt-14b) - Homo Sapiens NOV6c CG50709-OS 61 62 Wnt-9b protein precursor (Wnt-15) (Wnt-14b) - Homo Sapiens OV6d 277582109 63 64 Wnt-9b protein precursor ~ (Wnt-15) _.. _. . . ........_.. . (Wnt-14b).-Homo Sapiens.
_ NOV6e 277582117 65 66 Wnt-9b protein precursor (Wnt-15) (Wnt-14b) - Horno Sapiens NOV6f CG50709-O1 67 68 Wnt-9b protein precursor (Wnt-15) _ ; (Wnt-14b) - Homo Sapiens ~

OV6g CG50709-02 69 70 Wnt-9b protein precursor . (Wnt-15) (Wnt-14b) - Homo sapiens NOV6h CG50709-04 71 72 Wnt 9b protein precursor (Wnt-15) (Wnt 14b) Homo Sapiens ~

NOV6i CG50709-06 73 74 Wnt-9b protein precursor (Wnt-15) (Wnt-14b) - Homo sapiens NOV6j CG50709-07 75 76 Wnt-9b protein precursor (Wnt-15) ~ . ~ (Wnt-14b) - Homo sapien_s ~~

OV6k SNP13381605 77 78 a Wnt-9b protein precursor (Wnt-15) (Wnt-14b) - Homo Sapiens Y

OV61 SNP13381606 79 80 Wnt-9b protein precursor (Wnt-15) _ (Wnt-14b) - Homo Sapiens ~

NOV6m SNP13378337 81 82 Wnt-9b protein precursor (Wnt-15) (Wnt-14b) - Homo sapiens NOV6n SNP13381607 83 84 Wnt-9b protein precursor (Wnt-15) (Wnt-14b) - Homo sapiens NOV6o SNP13378336 85 86 Wnt-9b protein precursor (Wnt-15) (Wn_t-14b) -_Homo Sapiens NOV6p SNP13378335 87 88 Wnt-9b protein precursor (Wnt-15) (Wnt-14b) - Homo sapiens ~

NOV7a CG53054-02 89 90 Wnt-9a protein precursor (Wnt-14) -Homo Sapiens NOV7b 170251039 91 92 Wnt-9a protein precursor (Wnt-14) -Homo Sapiens NOV7c 170251076 93 94 Wnt-9a protein precursor (Wnt-14) -Homo sapiens NOV7d CG53054-Ol 95 96 Wnt-9a protein precursor (Wnt-14) -Homo sapiens NOV7e CG53054-03 , 97 98 Wnt-9a proteinprecursor (Wnt-14) -H_omo Sapiens NOV7f CG53054-04 99 100 Wnt-9a protein precursor (Wnt-14) -Homo sapiens ~ F

OVBa CG53473-02 101 102 Neuromedin B-32 precursor [Contains:

euromedin B] - Homo Sapiens NOV8b CG53473-Ol 103 104 Neuromedin B-32 precursor [Contains:

_ __ _ N_euromedin B] - Homo sapiens T ~ _ NOV8c CG53473-03 105 106 euromedin B-32 precursor ~~ [Contains:

Neuromedin B] - Homo Sapiens OV8d SNP13376396 107 108 Neuromedin B-32 precursor [Contains:

_ Neuromedin B] - Homo Sapiens ~

NOV8e SNP13376395 109 110 Neuromedin B-32 precursor [Contains:

Neuromedin B] - Homo Sapiens NOV8f SNP13376394 111 112 Neuromedin B-32 precursor [Contains:

euromedin B] - Homo Sapiens .

NOV9a CG55184-03 113 114 Cerebellin-like glycoprotein l precursor - Homo Sapiens NOV9b CG55184-O1 115 116 Cerebellin-like glycoprotein 1 precursor - Homo Sapiens N

NOV9c CGSSI84-02 117 118 Cerebellin-like glycoprotein 1 precursor - Homo Sapiens NOV9d CG55184-04 119 120 Cerebellin-like glycoprotein 1 precursor - Homo Sapiens y NOV9e CG55184-OS 121 122 Cerebellin-like glycoprotein 1 precursor - Homo Sapiens NOVlOa CG55274-OS 123 124 Human endozepine-like ENDOS

NOVlOb CG55274-01 125 126 Human endozepine-like ENDOS
_. ... .....~_ ~.._..................._..............._.........._...._................ _ ......_...._..._........_...._........_..._._....._....
...... .. .....
....,....._......_.............._........._............_.__..,_...v............

.... _.... .........~..._.._ _.

NOVlOc CG55274_-02 3_127 128 Human e_ndozepi_n_e-Like E_NDO_5 --NOVlOd CG55274-03 '_129 ' 130 Human endozepine-like ENDOS

NOVlOe CG55274-04 131 132 Human endozepine-like ENDOS
_ _ A -~

NOVl CG55379-04 ;133 ' 134 HDDM36 -_Hom_o s_apiens la ~
~ i NOVl CG55379-O1 135 _136 HDDM36 - Homo Sapiens lb A

NOVllc 258065951 137 138 HDDM36 -Homo Sapiens OVl_ld 209886264 139 140 _HDDM36 - Homo Sapiens ~

NOVl 209886345 141 142 HDDM36 - Homo sapiens le OVl 209886357 143 144 HDDM36 - Homo Sapiens if NOVllg C_G55379-02 145 146 HDDM36 -Homo Sapiens _ _ - y ~

NOVllh CG55379-03 147 148 HDDM36 -Homo Sapiens ' NOVl2a CG55688-Ol 150 CYR61 protein precursor (Cysteine-rich, angiogenic inducer, 61) 149 (Insulin-like growth factor-binding protein 10) (GIGl protein) - Homo Sapiens NOVl2b 254087906 152 CYR61 protein precursor (Cysteine-rich, angiogenic inducer, 61) 151 (Insulin-like growth factor-binding protein 10) (GIGl protein) - Homo _ Sapiens NOVI2c 259278648 154 CYR61 protein precursor (Cysteine-rich, angiogenic inducer, 61) 153 (Insulin-like growth factor-binding protein 10) (GIGl protein) - Homo __ Sapiens ~

NOVl2d 259280032 CYR61 protein precursor (Cysteine-rich, angiogenic inducer, 61) 155 (Insulin-like growth factor-binding protein 10) (GIGl protein) - Homo Sapiens NOVl2e 254756530 158 CYR61 protein precursor (Cysteine-rich, angiogenic inducer, 61) I57 (Insulin-like growth factor-binding protein 10) (GIGl protein) - Homo Sapiens NOVl2f 229509618 160 CYR61 protein precursor (Cysteine-rich, angiogenic inducer, 61) 159 (Insulin-like growth factor-binding protein 10) (GIGl protein) - Homo Sapiens NOVl2g 229509658 162 CYR61 protein precursor (Cysteine-rich, angiogenic inducer, 61) 161 (Insulin-like growth factor-binding protein 10) (GIGl protein) - Homo Sapiens _ J

NOVl2h CG55688-02 164 CYR61 protein precursor (Cysteine-rich, angiogenic inducer, 61) 163 (Insulin-like growth factor-binding protein 10) (GIGl protein) - Homo Sapiens NOVl2i CG55688-03 166 CYR61 protein precursor (Cysteine-rich, angiogenic inducer, 61) 165 (Insulin-like growth factor-binding protein 10) (GIGl protein) - Homo Sapiens NOVl2j CG55688-04 168 CYR61 protein precursor (Cysteine-rich, angiogenic inducer, 61) 167 (Insulin-like growth factor-binding protein 10) (GIGl protein) - Homo Sapiens NOVl2k CG55688-05 170 CYR61 protein precursor (Cysteine-rich, angiogenic inducer, 61) 169 (Insulin-like growth factor-binding protein 10) (GIGl protein) - Homo Sapiens :VOV121 CG55688-06 172 CYR61 protein precursor ~ ~~

( Cysteine-rich, angiogenic inducer, 6I) 1 71 ( Insulin-like growth factor-binding p rotein 10) (GIGl protein) ~ - Homo _ _ _ _ ~_ ____._...apiens ~ _~_.~___.,~..~
__ __._.._.._~,_ .s .___ __....___.__- _._. 174 CYR61 protein precursor . ..__ lVOVl2m SNP13376428 ( Cysteine-rich, angiogenic inducer, 61) 173 (Insulin-like growth factor-binding protein 10) (GIGl protein) - Homo Sapiens _ NOVl3a CG56768-O1 176 Wnt-5a protein precursor-Homo Sapiens NOVlab CG56768-02 178 Wnt-Saproteinprecursor-Homo __ _. . __._..__ .._.._Sa1?iens_.._..._...__.._..
......_._...._.__. ... .....___.._ ____.__. ....._...__......
.____._. ... .._._ .. . ._.......__._._._.
. ..

_..__. _ 180 Wnt-Sa protein precursor . . ___..._CG56768-03 - Homo ___ NOV 13c Sapiens NOVl4a CG57054-03 182 rotein precursor (Wnt-12) -Wnt-lOb 181 p ,_._.__.. ..._._.__.._Homo sa.....lens,...._._..._._ _.__ _.._._.___..___CG57054-Ol 184 Wnt-lOb protein precursor NOV 14b (Wnt-12) -183 Homo Sapiens NOV l4c CG57054-02 186 Wnt-l Ob protein precursor (Wnt-12) -__.._...______._Homo Sapiens .._ ...........__.........
__ _._.
_.... ___ ...........____. _...
_._..___ NOVlsa _ ___. ._._....._.. >
7431 03 .. ( CGS - 188 p m-2 recursor ET-2 - Homo Endothel-sapiens NOV l CG57431-OZ 190 Endothelin-2 precursor Sb (ET-2) - Homo Sapiens J

NOVlsc CG57431-O1 192 (ET-2) - Homo Endothelin-2 precursor Sapiens NOVlsd CG57431-04 194 Endothelin-2 precursor (ET-2) - Homo Sapiens _......._.....__.._._._._......_._.__._......._....__.._....._........._._..._.
_..._.__.._____.._...._....._....._...._........____._......
....... .__.._..._. ............
_..__....__..._.........__ _.

_.._ ......_........._..._..._.._..___.__ ... 196 Semaphorin 6D
isofonn NOVl6a ... 2 - Homo CG59253-Ol Sapiens NOVl6b 194877881 198 Semaphorin 6D isoform 2 - Homo Sapiens NOVl6c CG59253-02 200 Semaphorin 6D isoform 2 - Homo Sapiens NOVl6d 191815765 202 Semaphorin 6D isoform 2 - Homo _.. . _ .. .. _..... Sapiens w.._. .
_..._...._............_.........,...._..
. . . . _ .......__..._...__..._......._......_..............
.. . . . .. .

OV 16e CG59253-03 204 3 Semaphorin 6D isofonn 2 - Homo ' Sapiens OV 16f CG59253-04 206 Semaphorin 6D isoform 2 - Homo _._... Sapiens _ .. . _ .. _ _ _.. .

- CG59253-OS 208 Semaphorin 6D isoform NOV 16g 2 - Homo Sapiens NOV 16h CG59253-06 210 Semaphorin 6D isoform 2 - Homo sa iens . .. _ _. ..... __...._......_._....__..._.p . . . . . . .. ..__..-_......_._....-...._ _.,...._.......
___. __ ..~, _.........._.....,._.._ . . ..... _.._..__~...._.
_ _.
___...

_._:..._ - 2 i i ., NOVl6i CG59253 212 p 07 Sema horm 6D isoform 2 Homo -Sapiens NOVI6j CG59253-08 214 Semaphorin 6D isoform 2 - Homo Sapiens NOV CG59253-09 216 Semaphorin 6D isoform 2 16k - Homo Sapiens NOV161 CG59253-10 218 Semaphorin 6D isoform 2 - Homo _ Sapiens NOVl6m SNP13381547 220 Semaphorin 6D isoform 2 -Homo ~_. _ ____.._.__._. .._._ ___.... _Sapiens.__.._.__.._..__._...
._._ _. _..__ __ _ _. _.__..__... ..,,~,.~___.._.__._ .__ ... ... .. ._.._~
.. ._ _..._.____ _._ _ NOV SNP13378936 222 Semaphorin 6D isoform 2 16n - Homo Sapiens f NOVl6o SNP13378935 224 Semaphorin 6D isoform 2 -Homo sapiens _ ~

NOVl6p SNP13381569 226 ~Semaphorin 6D isoform 2 -Homo Sapiens NOVl6q SNP13382528 228 Semaphorin 6D isoform 2 - Homo Sapiens NOV CG_95430-02 229 230 Energen-related secreted 17a protein - C2P

NOV CG95430-04 231 232 Energen-related secreted 17b protein - C2P

NOVl7c CG95430-Ol 233 234 Energen-related_secreted protein - C2P

NOV 319194717 235 236 _ Energen-related secreted 17d protein - C2P

NOVl7e CG95430-03 237 238 Energen-related secreted protein - C2P

NOV CG95430-05 239 240 Energen-related secreted 17f ~ protein - C2P

NOVl7g CG95430-06 241 242 Energen-related secreted _ _ protein - C2P
~ ~ ~
y4 NOV CG95430-07 24 244_,~~sec_reted protein - C2P
17h i 3 , Energen-relate_d NOV CG95430-08 _ _ 246 Energen-related secreted 17i , protein - C2P

NOVl7j CG95430-09 247 248 Energen-related_secreted protein - C2P
~
~

NOVI7k CG95430-10 249 250 '_Ener_g_en-rela_t_ed secreted X ~~ protein - C2P
_ NOV CG95430-11 251 252 1 Energen-related secreted 171 protein - C2P
~

OV 17m CG95430-12 253 254 E_nergen-re_lat_ed_secreted ~ ~ protein - C2P

NOVl7n RCG95430-13 255 256_~ ~Energen-related secreted p_rot_ein - C2P

NOV SNP13379412 257 258 ~ Energen-related secreted 17o protein - C2P

OVl7p SNP13381828 259 260 Energen-related secreted protein - C2P

NOVl7q SNP13379125 261 262 Energen-related secreted protein - C2P
~

NOVl7r SNP13381827 263 264 Energen-related secreted protein - C2P

OV 17s SNP13381822 265 266 Energen-related secreted protein - C2P

NOVl7t ~SNP13381826 267 268 Energen-related secreted ~ protein - C2P

NOVlBa CG97111-Ol 269 270 Human IL-1 receptor antagonist protein NOVlBb CG97111-02 271 272 Human IL-1 receptor antagonist protein NOVl8c CG9711 I-03 273 274 Human IL-1 receptor antagonist _.. ~., . _. _.... ......._.__.protein _. __ ._ ....._..... _..... ._ _ . _.. . _.._.. . _._..
_ . .
...

NOVl8d SNP13382516 275 276 Human IL-1 receptor antagonist protein NOVl8e SNP13382517 277 278 Human IL-1 receptor antagonist _ protein NOVlBf SNP13382518 279 280 Human IL-1 receptor antagonist protein NOVl9a 10132038Ø67281 282 Domain of CG50513-05 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, conditions and the like that are associated with NOVX sequences include, but are not limited to, e.g., cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, metabolic disturbances associated with obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer, diabetes, metabolic disorders, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, Crohn's disease; multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers, as well as conditions such as transplantation and fertility.
NOVA nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various NOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
Additionally, NOVX nucleic acids and polypeptides can also be used to identify proteins that are members of the family to which the NOVX polypeptides belong.
Consistent with other known members of the family of proteins, identified in column 5 of Table A, the NOVX polypeptides of the present invention show homology to, and contain domains that are characteristic of, other members of such protein families.
Details of the sequence relatedness and domain analysis for each NOVX are presented in Example A.

The NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit diseases associated with the protein families listed in Table A.
The NOVX nucleic acids and polypeptides are also useful for detecting specific cell types. Details of the expression analysis for each NOVX are presented in Example C.
Accordingly, the NOVX nucleic acids, polypeptides, antibodies and related compounds according to the invention will have diagnostic and therapeutic applications in the detection of a variety of diseases with differential expression in normal vr.
diseased tissues, e.g., detection of a variety of cancers.
Additional utilities for NOVX nucleic acids and polypeptides according to the invention are disclosed herein.
NOVX clones NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various NOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
Additionally, NOVX nucleic acids and polypeptides can also be used to identify proteins that are members of the family to which the NOVX polypeptides belong.
The NOVX genes and their corresponding encoded proteins are useful for preventing, treating or ameliorating medical conditions, e.g., by protein or gene therapy.
Pathological conditions can be diagnosed by determining the amount of the new protein in a sample or by determining the presence of mutations in the new genes.
Specific uses are described for each of the NOVX genes, based on the tissues in which they are most highly expressed. Uses include developing products for the diagnosis or treatment of a variety of diseases and disorders.
The NOVX nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and ira 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
N0:2n, wherein n is an integer between 1 and 141, (b) a variant 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 141, 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 N0:2n, wherein n is an integer between 1 and 141, (d) 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 141, 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 N0:2n, wherein n is an integer between 1 and 141; (b) a variant 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 141, 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
N0:2n, wherein n is an integer between 1 and 141; (d) a variant of the amino acid sequence selected from the group consisting of SEQ ID N0:2n, wherein n is an integer between I and 141, 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 N0:2n, wherein n is an integer between 1 and 141, 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 N0:2n-l, wherein n is an integer between 1 and 141; (b) 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 141, 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
N0:2n-I, wherein n is an integer between I and 141; and (d) 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 141, 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 poIypeptide. As used herein, a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide, precursor form, or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF
described herein.
The product "mature" form arises, by way of nonlimiting example, as a result of one or more naturally occurnng 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 post-translational modification step 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-or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
The term "isolated" nucleic acid molecule, as used herein, is a nucleic acid that is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NOVX nucleic acid molecules can contain less than about 5 kb, about 4 kb, about 3 kb, about 2 kb, about 1 kb, about 0.5 kb, or about 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 NOS: 2n-l, wherein n is an integer between 1 and 141, 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 NOS:2n-l, wherein n is an integer between 1 and 141, as a hybridization probe, NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2"d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al., (eds.), CURRENT
PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993).
A nucleic acid of the invention can be amplified using cDNA, mRNA or, alternatively, genomic DNA as a template with appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA
synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides comprise a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment of the invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEQ ID NOS:2n-l, wherein n is an integer between 1 and 141, 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 NOS:2n-l, wherein n is an integer between 1 and 141, 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 NOS:2n-l, wherein n is an integer between 1 and 141, is one that is su~ciently complementary to the nucleotide sequence of SEQ ID NOS:2n-l, wherein n is an integer between 1 and 141, that it can hydrogen bond with few or no mismatches to a nucleotide sequence of SEQ ID
NOS:2n-1, wherein n is an integer between 1 and 141, thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding"
means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
A "fragment" provided herein is defined as a sequence of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, and is at most some portion less than a full length sequence.
Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice.
A full-length NOVX clone is identified as containing an ATG translation start codon and an in-frame stop codon. Any disclosed NOVX nucleotide sequence lacking an ATG start codon therefore encodes a truncated C-terminal fragment of the respective NOVX polypeptide, and requires that the corresponding full-length cDNA extend in the 5' direction of the disclosed sequence. Any disclosed NOVX nucleotide sequence lacking an in-frame stop codon similarly encodes a truncated N-terminal fragment of the respective NOVX polypeptide, and requires that the corresponding full-length cDNA
extend in the 3' direction of the disclosed sequence.

A "derivative" is a nucleic acid sequence or amino acid sequence formed from the native compounds either directly, by modification or partial substitution. An "analog" is a nucleic acid sequence or amino acid sequence that has a structure similar to, but not identical to, the native compound, e.g. they differs from it in respect to certain components or side chains. Analogs may be synthetic or derived from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. A "homolog" is a nucleic acid sequence or amino acid sequence of a particular gene that is derived from different species.
Derivatives and analogs may be full length or other than full length.
Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95%
identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the proteins under stringent, moderately stringent, or low stringent conditions. See e.g.
Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. homologous nucleotide sequences include those sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for a NOVX
polypeptide of species other than humans, including, but not limited to:
vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms.
Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein.
A
homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ )D NO:2n-l, wherein n is an integer between 1 and 141, 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
N0:2n-l, wherein n is an integer between 1 and 141; or an anti-sense strand nucleotide sequence of SEQ ID N0:2n-l, wherein n is an integer between 1 and 141; or of a naturally occurring mutant of SEQ ID N0:2n-1, wherein n is an integer between 1 and 141.
Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe has a detectable label attached, e.g. the label can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express a NOVX protein, such as by measuring a level of a NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.

"A polypeptide having a biologically-active portion of a NOVX polypeptide"
refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically-active portion of NOVX" can be prepared by isolating a portion of SEQ ID N0:2n-1, wherein n is an integer between 1 and 141, that encodes a poIypeptide 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 irz vitro) and assessing the activity of the encoded portion of NOVX.
NOVX Nucleic Acid and Polypeptide Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences of SEQ ID N0:2rz-l, wherein n is an integer between 1 and 141, 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-l, wherein n is an integer between 1 and 141. 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 N0:2n, wherein n is an integer between 1 and 141.
In addition to the human NOVX nucleotide sequences of SEQ ID N0:2n-1, wherein n is an integer between 1 and I41, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the NOVX polypeptides may exist within a population (e.g., the human population).
Such genetic polymorphism in the NOVX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene"
and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF) encoding a NOVX protein, preferably a vertebrate NOVX protein. Such natural allelic variations can typically result in 1-5°!° variance in the nucleotide sequence ofthe NOVX genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the NOVX
polypeptides, are intended to be within the scope of the invention.

Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from a human SEQ ID
N0:2n-l, wherein n is an integer between 1 and 141, are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the NOVX cDNAs of the invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID N0:2n-1, wherein n is an integer between 1 and 141. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length.
' In yet another embodiment, an isolated nucleic acid molecule of the invention hybridizes 1 S to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 65% homologous to each other typically remain hybridized to each other.
Homologs (i. e., nucleic acids encoding NOVX proteins derived from species other than human) or other related sequences (e.g., paraIogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at eaccess, 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 I .0 M sodium ion, typically about O.OI 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 oligonucIeotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &
Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X
SSC, 50 mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C, followed by one or more washes in 0.2X
SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a sequence of SEQ ID N0:2n-l, wherein n is an integer between 1 and 141, corresponds to a naturally-occurring nucleic acid molecule.
As used herein, a "naturally-occurnng" 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 N0:2n-l, wherein n is an integer between 1 and 141, 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 mg/ml denatured salmon sperm DNA at 55 °C, followed by one or more washes in 1X SSC, 0.1% SDS at 37 °C. Other conditions of moderate stringency that may be used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Krieger, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences of SEQ ID N0:2ra-l, wherein n is an integer between l and 141, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCI (pH
7.5), 5 mM EDTA, 0.02% PVP, 0.02°!° Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCI (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50 °C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1951. Proc Natl Acad Sci USA 78: 6759-6792.
Conservative Mutations In addition to naturally-occurring allelic variants of NOVX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID N0:2n-1, wherein n is an integer between 1 and 141, thereby leading to changes in the amino acid sequences of 1 S the encoded NOVX protein, without altering the functional ability of that NOVX protein.
For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID
NO:2n, wherein n is an integer between 1 and 141. 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 N0:2n-1, wherein n is an integer between 1 and 141, 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 N0:2n, wherein n is an integer between 1 and 141. Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID N0:2n, wherein n is an integer between 1 and 141; more preferably at least about 70% homologous to SEQ ID N0:2n, wherein n is an integer between 1 and 141; still more preferably at least about 80% homologous to SEQ
ID N0:2n,.wherein n is an integer between 1 and I41; even more preferably at least about 90% homologous to SEQ ID N0:2n, wherein n is an integer between 1 and 141;
and most preferably at least about 95% homologous to SEQ ID N0:2n, wherein n is an integer between 1 and 14I .
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 141, 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 141, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
Mutations can be introduced any one of SEQ ID NO:2n-1, wherein n is an integer between 1 and 141, by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g:, threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity. Following mutagenesis of a nucleic acid of SEQ ID
N0:2n-I, wherein n is an integer between 1 and 14I, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
The relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved "strong" residues or fully conserved "weak" residues. The "strong" group of conserved amino acid residues may be any one of the following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other. Likewise, the "weak" group of conserved residues may be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, HFY, wherein the letters within each group represent the single letter amino acid code.
In one embodiment, a mutant NOVX protein can be assayed for (i) the ability to form protein:protein interactions with other NOVX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant NOVX
protein and a NOVX ligand; or (iii) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g.
avidin proteins).
In yet another embodiment, a mutant NOVX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).
Interfering RNA
In one aspect of the invention, NOVX gene expression can be attenuated by RNA
interference. One approach well-known in the art is short interfering RNA
(siRNA) mediated gene silencing where expression products of a NOVX gene are targeted by specific double stranded NOVX derived siRNA nucleotide sequences that are complementary to at least a 19-25 nt long segment of the NOVX gene transcript, including the 5' untranslated (UT) region, the ORF, or the 3' UT region. See, e.g., PCT
applications WO00/44895, WO99/32619, WO01/75164, WO01/92513, WO01/29058, WO01/89304, W002/16620, and W002/29858, each incorporated by reference herein in their entirety.
Targeted genes can be a NOVX gene, or an upstream or downstream modulator of the NOVX gene. Nonlimiting examples of upstream or downstream modulators of a NOVX
gene include, e.g., a transcription factor that binds the NOVX gene promoter, a kinase or phosphatase that interacts with a NOVX polypeptide, and polypeptides involved in a NOVX regulatory pathway.

According to the methods of the present invention, NOVX gene expression is silenced using short interfering RNA. A NOVX polynucleotide according to the invention includes a siRNA polynucleotide. Such a NOVX siRNA can be obtained using a NOVX
polynucleotide sequence, for example, by processing the NOVX
ribopolynucleotide sequence in a cell-free system, such as but not limited to a Drosophila extract, or by transcription of recombinant double stranded NOVX RNA or by chemical synthesis of nucleotide sequences homologous to a NOVX sequence. See, e.g., Tuschl, Zamore, Lehmann, Bartel and Sharp (1999), Genes & Dev. 13: 3191-3197, incorporated herein by reference in its entirety. When synthesized, a typical 0.2 micromolar-scale RNA synthesis provides about 1 milligram of siRNA, which is sufficient for 1000 transfection experiments using a 24-well tissue culture plate format.
The most efficient silencing is generally observed with siRNA duplexes composed of a 21-nt sense strand and a 21-nt antisense strand, paired in a manner to have a 2-nt 3' overhang. The sequence of the 2-nt 3' overhang makes an additional small contribution to the specificity of siRNA target recognition. The contribution to specificity is localized to the unpaired nucleotide adjacent to the first paired bases. In one embodiment, the nucleotides in the 3' overhang are ribonucIeotides. In an alternative embodiment, the nucleotides in the 3' overhang are deoxyribonucleotides. Using 2'-deoxyribonucleotides.
in the 3' overhangs is as efficient as using ribonucleotides, but deoxyribonucleotides are often cheaper to synthesize and are most likely more nuclease resistant.
A contemplated recombinant expression vector of the invention comprises a NOVX DNA molecule cloned into an expression vector comprising operatively-linked regulatory sequences flanking the NOVX sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands. An RNA molecule that is antisense to NOVX mRNA is transcribed by a first promoter (e.g., a promoter sequence 3' of the cloned DNA) and an RNA molecule that is the sense strand for the NOVX
mRNA
is transcribed by a second promoter (e.g., a promoter sequence 5' of the cloned DNA).
The sense and antisense strands may hybridize in vivo to generate siRNA
constructs for silencing of the NOVX gene. Alternatively, two constructs can be utilized to create the sense and anti-sense strands of a siRNA construct. Finally, cloned DNA can encode a construct having secondary structure, wherein a single transcript has both the sense and complementary antisense sequences from the target gene or genes. In an example of this embodiment, a hairpin RNAi product is homologous to all or a portion of the target gene.

In another example, a hairpin RNAi product is a siRNA. The regulatory sequences flanking the NOVX sequence may be identical or may be different, such that their expression may be modulated independently, or in a temporal or spatial manner.
In a specific embodiment, siRNAs are transcribed intracellularly by cloning the NOVX gene templates into a vector containing, e.g., a RNA pol III
transcription unit from the smaller nuclear RNA (snRNA) U6 or the human RNase P RNA H I . One example of a vector system is the GeneSuppressorTM RNA Interference kit (commercially available from Imgenex). The U6 and Hl promoters are members of the type III class of Pol III
promoters. The +I nucleotide of the U6-like promoters is always guanosine, whereas the I O +1 for H1 promoters is adenosine. The termination signal for these promoters is defined by five consecutive thyrnidines. The transcript is typically cleaved after the second uridine.
Cleavage at this position generates a 3' UU overhang in the expressed siRNA, which is similar to the 3' overhangs of synthetic siRNAs. Any sequence less than 400 nucleotides in length can be transcribed by these promoter, therefore they are ideally suited for the expression of around 21-nucleotide siRNAs in, e.g., an approximately 50-nucleotide RNA
stem-loop transcript.
A siRNA vector appears to have an advantage over synthetic siRNAs where long term knock-down of expression is desired. Cells transfected with a siRNA
expression vector would experience steady, long-term mRNA inhibition. In contrast, cells transfected with exogenous synthetic siRNAs typically recover from mRNA suppression within seven days or ten rounds of cell division. The long-term gene silencing ability of siRNA
expression vectors may provide for applications in gene therapy.
In general, siRNAs are chopped from longer dsRNA by an ATP-dependent ribonuclease called DICER. DICER is a member of the RNase III family of double-stranded RNA-specific endonucleases. The siRNAs assemble with cellular proteins into an endonuclease complex. In vitro studies in Drosophila suggest that the siRNAs/protein complex (siRNP) is then transferred to a second enzyme complex, called an RNA-induced silencing complex (RISC), which contains an endoribonuclease that is distinct from DICER. RISC uses the sequence encoded by the antisense siRNA
strand to find and destroy mRNAs of complementary sequence. The siRNA thus acts as a guide, restricting the ribonuclease to cleave only mRNAs complementary to one of the two siRNA strands.

A NOVX mRNA region to be targeted by siRNA is generally selected from a desired NOVX sequence beginning 50 to 100 nt downstream of the start codon.
Alternatively, S' 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 CI/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 (Nl9)TT or N21, respectively. In the latter case, conversion of the 3' end of the sense siRNA to TT can be performed if such a sequence does not naturally occur in the NOVX polynucleotide. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3' overhangs. Symmetric 3' overhangs may help to ensure that the siRNPs are formed with approximately equal ratios of sense and antisense target RNA-cleaving siRNPs. See, e.g., Elbashir, Lendeckel and Tuschl (2001). Genes &
Dev.
15: 188-200, incorporated by reference herein in its entirely. The modification of the overhang of the sense sequence of the siRNA duplex is not expected to affect targeted mRNA recognition, as the antisense siRNA strand guides target recognition.
Alternatively, if the NOVX target mRNA does not contain a suitable AA(N21) sequence, one may search for the sequence NA(N21). Further, the sequence of the sense strand and antisense strand may still be synthesized as 5' (Nl9)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 ~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 ~g single-stranded sense NOVX
siRNA will have no effect on NOVX silencing, and 0.84 ltg antisense siRNA has a weak silencing effect when compared to 0.84 ~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 cases where no NOVX knock-down phenotype is observed, depletion of the NOVX polynucleotide may be observed by immunofluorescence or Western blotting.
If the NOVX polynucleotide is still abundant after 3 days, cells need to be split and transferred to a fresh 24-well plate for re-transfection. If no knock-down of the targeted protein is observed, it may be desirable to analyze whether the target mRNA
(NOVX or a NOVX upstream or downstream gene) was effectively destroyed by the transfected siRNA
duplex. Two days after transfection, total RNA is prepared, reverse transcribed using a target-specific primer, and PCR-amplified with a primer pair covering at least one exon-exon junction in order to control for amplification of pre-mRNAs. RT/PCR
of a non-targeted mRNA is also needed as control. Effective depletion of the mRNA
yet undetectable reduction of target protein may indicate that a large reservoir of stable NOVX protein may exist in the cell. Multiple transfection in sufficiently long intervals may be necessary until the target protein is finally depleted to a point where a phenotype may become apparent. If multiple transfection steps are required, cells are split 2 to 3 days after transfection. The cells may be transfected immediately after splitting.
An inventive therapeutic method of the invention contemplates administering a NOVX siRNA construct as therapy to compensate for increased or aberrant NOVX
expression or activity. The NOVX ribopolynucleotide is obtained and processed into siRNA fragments, or a NOVX siRNA is synthesized, as described above. The NOVX
siRNA is administered to cells or tissues using known nucleic acid transfection techniques, as described above. A NOVX siRNA specific for a NOVX gene will decrease or knockdown NOVX transcription products, which will lead to reduced NOVX
polypeptide production, resulting in reduced NOVX polypeptide activity in the cells or tissues.

The present invention also encompasses a method of treating a disease or condition associated with the presence of a NOVX protein in an individual comprising administering to the individual an IRNAi construct that targets the mRNA of the protein (the mRNA that encodes the protein) for degradation. A specific RNAi construct includes a siRNA or a double stranded gene transcript that is processed into siRNAs. Upon treatment, the target protein is not produced or is not produced to the extent it would be in the absence of the treatment.
Where the NOVX gene function is not correlated with a known phenotype, a control sample of cells or tissues from healthy individuals provides a reference standard for determining NOVX expression levels. Expression levels are detected using the assays described, e.g., RT-PCR, Northern blotting, Western blotting, ELISA, and the like. A
subject sample of cells or tissues is taken from a mammal, preferably a human subject, suffering from a disease state. The NOVX ribopolynucleotide is used to produce siRNA
constructs, that are specific for the NOVX gene product. These cells or tissues are treated by administering NOVX siItNA'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 ~.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 rnin. 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 K buffer and deproteinized as described previously (Tuschl et al., Genes Dev., 13:3191-3197 (1999)). Products are analyzed by electrophoresis in 15% or 18%
polyacrylamide sequencing gels using appropriate RNA standards. By monitoring the gels 1 S 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-purred (Elbashir, Lendeckel, & Tuschl, Genes & Dev. 15, 188-200 (2001)), followed by Sep-Pak C18 cartridge (Waters, Milford, Mass., USA) purification (Tuschl, et al., Biochemistry, 32:11658-11668 (1993)).
These RNAs (20 pM) single strands are incubated in annealing buffer (100 mM
potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90° C followed by 1 h at 37° C.

Cell Culture A cell culture known in the art to regularly express NOVX is propagated using standard conditions. 24 hours before transfection, at approx. ~0% confluency, the cells are trypsinized and diluted 1:5 with fresh medium without antibiotics (1-3 X 105 cellslml) and transferred to 24-well plates (500 mllwell). Transfection is performed using a commercially available lipofection kit and NOVX expression is monitored using standard techniques with positive and negative control. A positive control is cells that naturally express NOVX while a negative control is cells that do not express NOVX. Base-paired 21 and 22 nt siRNAs with overhanging 3' ends mediate efficient sequence-specific mRNA
degradation in lysates and in cell culture. Different concentrations of siRNAs are used.
An efficient concentration for suppression in vitro in mammalian culture is between 25 nM to 100 nM final concentration. This indicates that siRNAs are effective at concentrations that are several orders of magnitude below the concentrations applied in conventional antisense or ribozyme gene targeting experiments.
The above method provides a way both for the deduction of NOVX siRNA
sequence and the use of such siRNA for in vitro suppression. In vivo suppression may be performed using the same siRNA using well known in vivo transfection or gene therapy transfection techniques.
Antisense Nucleic Acids Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID N0:2ra-l, wherein n is an integer between 1 and 141, 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 N0:2ra, wherein n is an integer between 1 and 141, or antisense nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ID NO:2n-1, wherein n is an integer between 1 and I41, are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a NOVX protein.
The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding the NOVX protein. The term "noncoding region"
refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding the NOVX protein disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of ' NOVX mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-carboxymethylaminomethyl-2-thiouridine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 5-methoxyuracil, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 2-thiouracil, 4-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, S-methyluracil, uracil-S-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 andlor translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens). The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II
or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an 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., moue, et al. 1987. Nucl. Acids Res. 15:
6131-6148) or a chimeric RNA DNA analogue (See, e.g., moue, et al., 1987. FEBSLett. 215:
327-330.
Ribozymes and PNA Moieties Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized.
These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave NOVX mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme having specificity for a NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of a NOVX cDNA disclosed herein (i.e., SEQ ID N0:2n-1, wherein n is an integer between 1 and 141). For example, a derivative of a Tetrahyrnena 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. Anra. 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 Claem 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 polyrnerases) 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 S' 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 S such as peptides (e.g., for targeting host cell receptors izz vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al., 1989. Proc.
Natl. Acad.
~'ci. U.S.A. 86: 6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84:
648-652;
PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT
Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al., 1988. BioTeclzniques 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 N0:2zz, wherein n is an integer between 1 and 141. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in any one of SEQ ID NO:2n, wherein n is an integer between 1 and 141, while still encoding a protein that maintains its NOVX
activities and physiological functions, or a functional fragment thereof.
In general, a NOVX variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
One aspect of the invention pertains to isolated NOVX proteins, and biologically-active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX antibodies. In one embodiment, native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, NOVX proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a NOVX
protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An "isolated" or "purified" polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of NOVX
proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the language "substantially free of cellular material" includes preparafiions 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 N0:2ra, wherein n is an integer between 1 and 141) that include fewer amino acids than the full-length NOVX
proteins, and exhibit at least one activity of a NOVX protein. Typically, biologically-active portions comprise a domain or motif with at least one activity of the NOVX protein. A biologically-active portion of a NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.
Moreover, other biologically-active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native NOVX protein.
In an embodiment, the NOVX protein has an amino acid sequence of SEQ ID
NO:2n, wherein n is an integer between 1 and 141. In other embodiments, the NOVX
protein is substantially homologous to SEQ ID N0:2n, wherein n is an integer between 1 and 141, and retains the functional activity of the protein of SEQ ID NO:2n, wherein n is an integer between 1 and 141, yet differs in amino acid sequence due to natu~'al allelic variation or mutagenesis, as described in detail, below. Accordingly, in another embodiment, the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO:2n, wherein n is an integer between 1 and 141, and retains the functional activity of the NOVX
proteins of SEQ ID N0:2rz, wherein n is an integer between 1 and 141.
Determining Homology Between Two or More Sequences To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package.
See, Needleman and Wunsch, 1970. ,7Mol 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 N0:2n-1, wherein n is an integer between 1 and 141.
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 1 S 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 141, 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 heteroIogous 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-imriiunoglobulin 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 irz 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 Iigation, 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, Tohn Wiley &
Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.
NOVX Agonists and Antagonists 'The invention also pertains to variants of the NOVX proteins that function as either NOVX agonists (i. e., mimetics) or as NOVX antagonists. Variants of the NOVX
protein can be generated by mutagenesis (e.g., discrete point mutation or truncation of the NOVX protein). An agonist of the NOVX protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the NOVX protein.
1 S 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 occurnng form of the protein has fewer side effects in a subject relative to treatment with the naturally occurnng 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., forphage display) containing the set of NOVX sequences therein. There are a variety of methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential NOVX
sequences. Methods for synthesizing degenerate oligonucleotides are well-known within the art. See, e.g., Narang, 1983. Tetrahedron 39: 3; Itakura, et al., 1984.
Annu. Rev.
Bioclaern. 53: 323; Itakura, et al., 1984. Science 198: 1056; Ike, et al., 1983. Nucl. Acids Res. 11: 477.
Polypeptide Libraries In addition, libraries of fragments of the NOVX protein coding sequences can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of a NOVX protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR
fragment of a NOVX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA
to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
By this method, expression libraries can be derived which encodes N-terminal and internal fragments of various sizes of the NOVX proteins.
Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA
libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of NOVX proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify NOVX variants. See, e.g., Arkin and Yourvan, 1992. Proc. IVatl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al., 1993.
Protein Engineering 6:327-331.
NOVX Antibodies The term "antibody" as used herein refers to immunoglobulin molecules and imrnunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab' and F~ab~z fragments, and an Fab expression library. In general, antibody molecules obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGI, IgG2, and others.
Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.
Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
An isolated protein of the invention intended to serve as an antigen, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens.
An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence of SEQ ID
N0:2n, wherein n is an integer between 1 and I4I, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of NOVX that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of the human NOVX protein sequence will SI

indicate which regions of a NOVX polypeptide are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.
As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Pros. Nat.
Acad.
Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each incorporated herein by reference in their entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. A NOVX polypeptide or a fragment thereof comprises at least one antigenic epitope. An anti-NOVA antibody of the present invention is said to specifically bind to antigen NOVX when the equilibrium binding constant (KD) is __<1 ~.M, preferably < 100 nM, more preferably <_ 10 nM, and most preferably < 100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.
A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 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 parwm, or similar immunostimulatory agents. Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as amity 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 axe desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to .a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, fox 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 Technigues and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimrnunoassay (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 murine antibodies). 'The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, Nature 368, (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of I O 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 I S 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 irnmunoglobulin and all or substantially all of the framework regions are 20 those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).
Human Antibodies 25 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 30 Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.
Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote? et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES
AND
CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.
This approach is described, for example, in U.S. Patent Nos. 5,545,807;
5,545,806;
5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.
(Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-13 (1994)); Fishwild et al,( Nature BiotechnoloQV 4 845-51 (1996));
Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev.
Immunol.
13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT
publication W094/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse~'' as disclosed in PCT publications WO 96/33735 and WO 96134096.
This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.

Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S.
Patent No. 5,939,598. It can be obtained by a method including deleting the J
segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.
In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT
publication WO 99/53049.
Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S.
Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F~ab')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F~~b72 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F" fragments.
Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispeciBc 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 1 S correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO
93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., I0:3655-(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 brst 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 Enzymolo~y, 121:210 (1986).
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')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., 3. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')a molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T
cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et aL, J.
Immunol.
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc.
Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL
domains of one fragment are forced to pair with the complementary VL and VH
domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.
CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcYR), such as FcyRI (CD64), FcyRII
(CD32) and FcyRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such antibodies 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 IT.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 Desi~,n, 3: 219-230 (1989).
Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A
chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 2~ZBi, i3il, l3iln, 9oY, and I$6Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.
See 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 I S 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 Antibodies directed against a protein of the invention may be used in methods known within the art relating to the localization and/or quantitation of the protein (e.g., for use in measuring levels of the protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies against the proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antigen binding domain, are utilized as pharmacologically-active compounds (see below).
An antibody specific for a protein of the invention can be used to isolate the protein by standard techniques, such as immunoaffinity chromatography or immunoprecipitation. Such an antibody can facilitate the purification of the natural protein antigen from cells and of recombinantly produced antigen expressed in host cells.
Moreover, such an antibody can be used to detect the antigenic protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the antigenic protein. Antibodies directed against the 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 ~ZSI, lsih ssS
or 3H.
Antibody Therapeutics Antibodies of the invention, including polyclonal, monoclonal, humanized and fully human antibodies, may used as therapeutic agents. Such agents will generally be employed to treat or prevent a disease or pathology in a subject. An antibody preparation, preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target. Such an effect may be one of two kinds, depending on the specific nature of the interaction between the given antibody molecule and the target antigen in question. In the first instance, administration of the antibody may abrogate or inhibit the binding of the target with an endogenous ligand to which it naturally binds. In this case, the antibody binds to the target and masks a binding site of the naturally occurring ligand, wherein the ligand serves as an effector molecule. Thus the receptor mediates a signal transduction pathway for which ligand is responsible.
Alternatively, the effect may be one in which the antibody elicits a physiological result by virtue of binding to an effector binding site on the target molecule. In this case the target, a receptor having an endogenous Iigand which may be absent or defective in the disease or pathology, binds the antibody as a surrogate effector ligand, initiating a receptor-based signal transduction event by the receptor.
A therapeutically effective amount of an antibody of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target, and in other cases, promotes a physiological response. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.
Pharmaceutical Compositions of Antibodies Antibodies specifically binding a protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions.
Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington : The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa. : 1995;
Drug Absorption Enhancement : Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

If the antigenic protein is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci.
USA, 90:
7889-7893 (1993). The formulation herein can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (LT.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 'y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
ELISA Assay An agent for detecting an analyte protein is an antibody capable of binding to an analyte protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab)2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term "biological sample", therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, ira vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations.
In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations.
Procedures for conducting immunoassays are described, for example in "ELISA:
Theory and Practice: Methods in Molecular Biology", Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P.
Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-an analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

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

METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NOVX proteins, mutant forms of NOVX proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX
proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated izz vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;
Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and 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 (Amrann 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).
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS
IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. c~li (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the invention can be carned out by standard DNA
synthesis 1 S techniques.
In another embodiment, the NOVX expression vector is a yeast expression vector.
Examples of vectors for expression in yeast Sacclzaromyces cerivisae include pYepSec 1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
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 pCDMB (Seed, 1987. Nature 329: 840) and pMT2PC
(Kaufinan, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MAt~ItlAZ.. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements axe used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specif c promoters include the albumin promoter (liver-specific;
Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-speciEc promoters (Calame and Eaton, 1988.
Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neuroflament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad.
Sci. USA 86:
5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230:
9I2-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,3I6 I S and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.
Science 249: 374-379) and the oc-fetoprotein promoter (Campes and Tilghman, 1989.
Genes Dev. 3: 537-546).
The invention further provides a recombinant expression vector comprising a DNA
molecule of the invention cloned into the expression vector in an antisense orientation.
That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA
molecule that is antisense to NOVX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub, et al., "Antisense RNA as a molecular tool for genetic analysis,"
Reviews-Trends in Genetics, Vol. 1(I) 1986.

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell"
and "recombinant host cell" are used interchangeably herein. It is~understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, NOVX
protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAF-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A
LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
Various selectable markers include those that confer resistance to drugs, such as 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 r 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 NOS:2n-l, wherein n is an integer between 1 and I41, 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 andlor 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 NOS:2n-1, wherein n is an integer between I and 141), 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
NOS:2n-1, wherein n is an integer between 1 and 141, 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 ANn EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp.
113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991.
Curr. Opirz. Biotechnol. 2: 823-829; PCT International Publication Nos.: WO
90/I 1354;
WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene.
One example of such a system is the cre/IoxP 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. ZISA 89: 6232-6236. Another example of a recombinase system is the FLP
recombinase system of Sacclzarornyces 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 3 ~5: ~ 10-~ 13. 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), transdermaI
(i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA);
buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable Garners include physiological saline, bacteriostatic water, Cremophor EL~' (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
The Garner 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 injectabIe 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 Garner 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 barner to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds-are prepared with Garners that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
Biodegradable, 7~

biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable Garners. 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 Garner. The specification for the dosage unit forms of the invention are dictated by and 1 S 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.
A,cad. 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 NOVVX mRNA (e.g., in a biological sample) or a genetic lesion in a NOVX gene, and to modulate NOVX activity, as described further, below. In addition, the NOVX proteins can be used to screen drugs or compounds that modulate the NOVX
protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of NOVX protein or production of NOVX protein forms that have decreased or aberrant activity compared to NOVX wild-type protein (e.g.;
diabetes (regulates insulin release); obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias. In addition, the anti-NOVX antibodies of the invention can be used to detect and isolate NOVX
proteins and modulate NOVX activity. In yet a further aspect, the invention can be used in methods to influence appetite, absorption of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.
The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.
Screening Assays The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOVX
protein activity. The invention also includes compounds identified in the screening assays described herein.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a NOVX protein or polypeptide or biologically-active portion thereof. The test compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997.
Anticarzcer Drug Design 12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90:
6909; Erb, et al., 1994. Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al., 1994. J.
Med. Chezn.
37: 2678; Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew.
Clzem. Irzt. Ed.
Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061;
and Gallop, et al., 1994. J. Med. Chem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992.
Bioteclzzziques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No.
5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl.
Acad. Sci. USA
89: 1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390;
Devlin, 1990.
Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87:
6378-6382;
Felici, 1991. J. Mol. Biol_ 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to a NOVX protein determined. The cell, for example, can of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the NOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the NOVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with ~ZSI, 3sS, iaC, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX protein or a biologically-active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the NOVX
protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the NOVX protein to bind to or interact with a NOVX target molecule. As used herein, a "target molecule" is a molecule with which a NOVX protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a NOVX interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. a NOVX target molecule can be a non-NOVX molecule or a NOVX protein or polypeptide of the invention.
In one embodiment, a NOVX target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound NOVX molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with NOVX.
Determining the ability of the NOVX protein to bind to or interact with a NOVX
target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the NOVX
protein to bind to or interact with a NOVX target molecule can be accomplished by determining_the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i.e. intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalyticlenzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a NOVX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay comprising contacting a NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to bind to the NOVX
protein or biologically-active portion thereof. Binding of the test compound to the NOVX protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX or biologically-active portion thereof as compared to the known compound.
In still another embodiment, an assay is a cell-free assay comprising contacting NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof.
Determining the ability of the test compound to modulate the activity of NOVX can be accomplished, for example, by determining the ability of the NOVX protein to bind to a NOVX
target molecule by one of the methods described above for determining direct binding.
In an alternative embodiment, determining the ability of the test compound to modulate the activity of NOVX protein can be accomplished by determining the ability of the NOVX
protein further modulate a NOVX target molecule. For example, the catalytic%nzy~natic 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-dodecylinaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton~ X-I00, Triton~ X-114, Thesit~, Isotridecypoly(ethylene glycol ether)n, N-dodecyl--N,N-dimethyl-3-ammonio-1-propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
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 deriyatized 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 ofNOVX 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. Claem.
268:
12046-12054; Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993.
Oncogene 8: 1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with NOVX ("NOVX-binding proteins" or "NOVX-by") and modulate NOVX
activity. Such NOVX-binding proteins are also likely to be involved in the propagation of signals by the NOVX proteins as, for example, upstream or downstream elements of the NOVX pathway.
The two-hybrid system is based on the modular nature of most transcription factors, Which consist of separable DNA-binding and activation domains.
Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for NOVX is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA
sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a NOVX-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor.
Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with NOVX.
The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
Detection Assays Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Some of these applications are described in the subsections, below.
Chromosome Mapping Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of a NOVX
sequence, i. e., of SEQ ID NOS:2n-1, wherein n is an integer between 1 and 141, 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., 193. Seience 220: 919-924.
Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the NOVX sequences to design oligonucleotide primers, sub-localization can be achieved with panels of fragments from specific chromosomes.
Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa.
A pattern ~7 of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see, Verma, et al., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988).
Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes.
Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data.
Such data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland, et al., 1987. Nature, 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NOVX gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease.
Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA
sequence.
Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

Tissue Typing The NOVX sequences of the invention can also be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA
is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences of the invention are useful as additional DNA markers for RFLP ("restriction fragment length polymorphisms," described in U.S.
Patent No. 5,272,057).
Furthermore, the sequences of the invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the NOVX sequences described herein can be used to prepare two PCR primers from the 5'- and 3'-termini of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the invention can be used to obtain such identification sequences from individuals and from tissue. The NOVX sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (RFLPs).
Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes.
Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If coding sequences, such as those of SEQ ID NOS:2n-l, wherein n is an integer between 1 and 141, 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 andlor 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 NOS:2n-1, wherein n is an integer between 1 and 141, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.
An agent for detecting NOVX protein is an antibody capable of binding to NOVX
protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.. That is, the detection method of the invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of NOVX mRNA
include Northern hybridizations and izz situ hybridizations. In vitro techniques for detection of NOVX protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of NOVX genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of NOVX protein include introducing into a subject a labeled anti-NOVX

antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting NOVX protein, mRNA, or genomic DNA, such that the presence of NOVX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of NOVX protein, mRNA or genomic DNA in the control sample with the presence of NOVX protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of NOVX in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting NOVX protein or mRNA in a biological sample; means for determining the amount of NOVX in the sample; and means for comparing the amount of NOVX in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect NOVX protein or nucleic acid.
Prognostic Assays The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant NOVX expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with aberrant NOVX
expression or activity in which a test sample is obtained from a subject and NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of NOVX
protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant NOVX expression or activity. As used herein, a "test sample"

refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein,~peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant NOVX expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder. Thus, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant NOVX expression or activity in which a test sample is obtained and NOVX
protein or nucleic acid is detected (e.g., wherein the presence of NOVX
protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant NOVX expression or activity).
The methods of the invention can also be used to detect genetic lesions in a NOVX
gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding a NOVX-protein, or the misexpression of the NOVX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from a NOVX gene;
(ii) an addition of one or more nucleotides to a NOVX gene; (iii) a substitution of one or more nucleotides of a NOVX gene, (iv) a chromosomal rearrangement of a NOVX
gene;
(v) an alteration in the level of a messenger RNA transcript of a NOVX gene, (vi) aberrant modification of a NOVX gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild-type splicing pattern of a messenger RNA
transcript of a NOVX gene, (viii) a non-wild-type level of a NOVX protein, (ix) allelic loss of a NOVX
gene, and (x) inappropriate post-translational modification of a NOVX protein.
As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in a NOVX gene. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.

In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et al., 1988. Science 241: 1077-1080;
and Nakazawa, et al., 1994. Pr~c. Natl. Acad. Sei. 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. Aead. Sci. USA 87: 1874-1878), transcriptional amplification system (see, Kwoh, et al., 1989. Prac. Natl. Acad. Sci. USA 86:
1173-1177);
Q(3 Replicase (see, Lizardi, et al, 1988. BioTechn~logy 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 sample and control nucleic acids, e.g., DNA or RNA to high-density arrays containing hundreds or thousands of oligonucleotide 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. Aead: Sci. LISA 74: 560 or Sanger, 1977. Proc.
Natl. Acad.
Sci. TISA 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. Adu Chromatography 36: 127-162; and Grin, 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 RNAlRNA
or RNA/DNA heteroduplexes. See, e.g., Myers, et al., 1985. Scierzce 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 SI 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. Geraet.
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. Bioplzys. Chem. 265:
12753.
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, et al., 1986.
Nature 324: 163; Saiki, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 6230.
Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention.
Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al., 1989. Nuel. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech.
11: 238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection. See, e.g., Gasparini, et al., 1992. Mol. Cell Probes 6: 1. It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification. See, e.g., Barany, 1991. Proc.
Natl. Acad.
Sci. USA 88: 189. In such cases, ligation will occur only if there is a perfect match at the 3'-terminus of the 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which 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 NOVA is expressed may be utilized in the prognostic assays described herein.
However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
Pharmacogenomics Agents, or modulators that have a stimulatory or inhibitory effect on NOVX
activity (e.g., NOVX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (The disorders include 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.) 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. Clirt. Exp. Pharrraacol. Physiol., 23: 983-985;
Linder, 1997.
Clin. 'Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deftciency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT
2) and cytochrome Pregnancy Zone Protein Precursor enzymes CYP2D6 and CYP2C19) .
has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
Thus, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agents) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a NOVX modulator, such as a modulator identified by one of the exemplary screening assays described herein.

Monitoring of Effects During Clinical Trials Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials.
For example, the effectiveness of an agent determined by a screening assay as described herein to increase NOVX gene expression, protein levels, or upregulate NOVX
activity, can be monitored in clinical trails of subjects exhibiting decreased NOVX gene expression, protein levels, or downregulated NOVX activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease NOVX
gene expression, protein levels, or downregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting increased NOVX gene expression, protein levels, or upregulated NOVX activity. In such clinical trials, the expression or activity of NOVX
and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a "read out" or markers of the immune responsiveness of a particular cell.
By way of example, and not of limitation, genes, including NOVX, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates NOVX activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of NOVX and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of NOVX or other genes. In this manner, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent.
Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent;
(ii) detecting the level of expression of a NOVX protein, mltNA, 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, mIRNA, 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 cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hypercoaguIation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, Crohn's disease;
multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy, and other diseases, disorders and conditions of the like.
These methods of treatment will be discussed more fully, below.
Diseases and Disorders Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner.
Therapeutics that may be utilized include, but are not limited to: (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to an aforementioned peptide;
(iii) nucleic acids encoding an aforementioned peptide; (iv) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to "knockout" endogenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989. Science 244: 1288-1292); or (v) modulators i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between an aforementioned peptide and its binding partner.
Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (i. e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner.
Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of an aforementioned peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like).
Prophylactic Methods In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NOVX expression or activity, by administering to the subject an agent that modulates NOVX expression or at least one NOVX activity. Subjects at risk for a disease that is caused or contributed to by aberrant NOVX expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the NOVX

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

Determination of the Biological Effect of the Therapeutic In various embodiments of the invention, suitable irz vitro or irz vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue.
In various specific embodiments, in vitro assays may be performed with representative cells of the 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 including, but not limited to: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, 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.
As an example, a cDNA encoding the NOVX protein of the invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the invention will have efficacy for treatment of patients suffering from: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias.
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.
S E~~AMPLES
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 lA.
lA. NOV1 .a, CG103945-02 SEQ ID NO: 1 2414 by Sequence ORF Start: ATG at l~. ~Y ' ORF Stop: TAG at 2401 AGGGGGCCTCCTGCTCCATGGGGGTAGCTCTGGCCCCA
TACCGAGGAGCCGTGGTCCGAAAGCCTTCCAGCACCAT
CAGGCCATGTACCTAGATGAGTACCGAGACCGCCTCTTTCTGGGTGGCCTGGACGCCCTCTACT
CACCTTCATAGACGGGGAGCTGTACACGGGTCTCACTGCTGACTTCCTGGGGCGAGAGGCCATG
GATGGCCGCCCGGATCCCTGAGAACTCTGACCAGGACAATGACAAGGTGTACTTCTTCTTCTCGGAGAC
CCCTCGCCCGATGGTGGCTCGAACCATGTCACTGTCAGCCGCGTGGGCCGCGTCTGCGTGAATGATGCT
CTGGTGGTGCCGAGACCCACTTTGACCAGCTAGAGGATGTGTTCCTGCTGTGGCCCAAGGCCGGGAAGAGCCT
GACATCTGGGAGGTTTTCAACGGGCCCTTTGCCCACCGAGATGGGCCTCAGCACCAGTGGGGGCCCTATGGGG
GCAAGGTGCCCTTCCCTCGCCCTGGCGTGTGCCCCAGCAAGATGACCGCACAGCCAGGACGGCCTTTTGGCAG
CACCAAGGACTACCCAGATGAGGTGCTGCAGTTTGCCCGAGCCCACCCCCTCATGTTCTGGCCTGTGCGGCCT
CGACATGGCCGCCCTGTCCTTGTCAAGACCCACCTGGCCCAGCAGCTACACCAGATCGTGGTGGACCGCGTGG
AGGCAGAGGATGGGACCTACGATGTCATTTTCCTGGGGACTGACTCAGGGTCTGTGCTCAAAGTCATCGCTCT
CCAGGCAGGGGGCTCAGCTGAACCTGAGGAAGTGGTTCTGGAGGAGCTCCAGGTGTTTAAGGTGCCAACACCT
ATCACCGAAP.TGGAGATCTCTGTCAAAAGGCAAATGCTATACGTGGGCTCTCGGCTGGGTGTGGCCCAGCTGC
ACTGTGCCTG
CTTGCAGAGGCCAGGGGATGAGGGGCCTGACCAGGTGAAGACGGACGAGCGAGTCTTGCACACGGAGCGGGGG
TCCTGCAG
CACGGAAT

CG103945-02 ~SEQ ID NO: 2 X800 as BMW at 88800.3kD
MAPSAWAICWLLGGLLLHGGSSGPSPGPSVPRLRLSYRGAVVRKPSSTMWMETFSRYLLSANRSAIFLGPQGS
LNLQAMYLDEYRDRLFLGGLDALYSLRLDQAWPDPREVLWPPQPGQREECVRKGRDPLTECANFVRVLQPHNR
THLLACGTGAFQPTCALITVGHRGEHVLHLEPGSVESGRGRCPHEPSRPFASTFIDGELYTGLTADFLGREAM
IFRSGGPRPALRSDSDQSLLHDPRFVMAARIPENSDQDNDKVYFFFSETVPSPDGGSNHVTVSRVGRVCVNDA
GGQRVLVNKWSTFLKARLVCSVPGPGGAETHFDQLEDVFLLWPKAGKSLEVYALFSTVSAVFQGFAVCVYHMA
DIWEVFNGPFAHRDGPQHQWGPYGGKVPFPRPGVCPSKMTAQPGRPFGSTKDYPDEVLQFARAHPLMFWPVRP
RHGRPVLVKTHLAQQLHQIVVDRVEAEDGTYDVIFLGTDSGSVLKVIALQAGGSAEPEEVVLEELQVFKVPTP
ITEMEISVKRQMLYVGSRLGVAQLRLHQCETYGTACAECCLARDPYCAWDGASCTHYRPSLGKRRFRRQDIRH
GNPALQCLGQSQEEEAVGLVAATMVYGTEHNSTFLECLPKSPQAAVRWLLQRPGDEGPDQVKTDERVLHTERG
LLFRRLSRFDAGTYTCTTLEHGFSQTVVRLALVVIVASQLDNLFPPEPKPEEPPARGGLASTPPKAWYKDILQ
LIGFANLPRVDEYCERVWCRGTTECSGCFRSRSRGKQARGKSWAGLELGKKMKSRVHAEHNRTPREVEAT
dOVlb, CG103945-Ol SEQ ID NO 3 _ 4700 by _ _ _ >NA Sequence ' ORF Start ATG at 1 ~~Y ORF Sto-:-TAG at 2347 _.___ _~__.___.._._~.__. __~__-_~~re___.~~ ,r.:~~:.-.,~:~.~~_. _.
.TGGCCCCCTCGGCCTGGGCCATTTGCTGGCTGCTAGGGGGCCTCCTGCTCCATGGGGGTAGCTCTGGCCCCA
~CCCCGGCCCCAGTGTGCCCCGCCTGCGGCTCTCCTACCGAGACCTCCTGTCTGCCAACCGCTCTGCCATCTT
'CTGGGCCCCCAGGGCTCCCTGAACCTCCAGGCCATGTACCTAGATGAGTACCGAGACCGCCTCTTTCTGGGT
TCACAACCGGACCCACCTGCTAGCCTGTGGCACTGGGGCCTTCCAGCCCACCTGTGCCCTCATC
CACCGTGGGGAGCATGTGCTCCACCTGGAGCCTGGCAGTGTGGAAAGTGGCCGGGGGCGGTGCC
CCAGCCGTCCCTTTGCCAGCACCTTCATAGACGGGGAGCTGTACACGGGTCTCACTGCTGACTT
AGAGGCCATGATCTTCCGAAGTGGAGGTCCTCGGCCAGCTCTGCGTTCCGACTCTGACCAGAGT
.TGCTGGGGGCCAGCGGGTGCTGGTGAACAAATGGAGCACTTTCCTCAAGGCCAGGCTG
GTGTACGCGCTGTTCAGCACCGTCAGTGCCGTGTTCCAGGGCTTCGCCGT
TCTGGGAGGTTTTCAACGGGCCCTTTGCCCACCGAGATGGGCCTCAGCAC
GCGGCCTCGACATGGCCGCCCTGTCCTTGTCAAGACCCACCTGGCCCAGCAGCTACACCAG
CGCGTGGAGGCAGAGGATGGGACCTACGATGTCATTTTCCTGGGGACTGACTCAGGGTCTG
TCGCTCTCCAGGCAGGGGGCTCAGCTGAACCTGAGGAAGTGGTTCTGGAGGAGCTCCAGGT
AACACCTATCACCGAAATGGAGATCTCTGTCAAAAGGCAAATGCTATACGTGGGCTCTCGG
TGGTGCCTCCTGTACCCACTACCGCCCCAGCCTTGGCAAGCGCCGGTTCCG
TGGTCTACGGCACGGAGCACAATAGCACCTTCCTGGAGTGCCTGCCCAAGTCTCCCC
GCTCTTGCAGAGGCCAGGGGATGAGGGGCCTGACCAGGTGAAGACGGACGAGCGAGT
GGGCTGCTGTTCCGCAGGCTTAGCCGTTTCGATGCGGGCACCTACACCTGCACCACT
AGCAGCCCCCAGCAT
ACACCGCTACTGGGGTCTAATGGAGGGGCTGGGTTCTTGAAGCCTGTTCCCTGCCCTTCTCTGTGCTCTTAGA
CCCAGCTGGAGCCAGCACCCTCTGGCTGCTGGCAGCCCCAAGGGATCTGCCATTTGTTCTCAGAGATGGCCTG
(GCTTCCGCAACACATTTCCGGGTGTGCCCAGAGGCAAGAGGGTTGGGTGGTTCTTTCCCAGCCTACAGAACAA
~TGGCCATTCTGAGTGACCCTCAGAGTGGGTGTGTGGGTGCGTCTAGGGGGTATCCCGGTAGGGGGCCTGCAGG

~ACACAGAGGTGTTGGGAAGGTGGAGCAACAATGCACCTCCCCTCCTGTCGCGCCGTGATATCTTGGTGGCTCC
CTGCCACTGCCCACCGCCTCTTCTCCATCTGAGAATCACGGAGAGGTGTAGATAATCTAGAGGCATAGACTGC
TGCCG
AGAGGGCAGGAGACCCTTAGGAT
AAGGGGGAAACAAGGTAGAGAAAAGGACGAAGAAGTGTAAGTCCCGCTGATTCTCGGGGGTAAGGCTCGGATG
~GCAAGGACGCGTTCTGCCTGGGCATGTAGGGGAGGTGTTTTTGCCATCACCAGTTTCTCAGGCTGGGGAGCAC
AGAGGGGAGGAGGAGGACTAAATGAAAAGTTGTTCCCAGCCTGCACATGAACACATTCATGACACACAAAACT
CAGAGGGGATTAAAGAGGGGAGGAGAGAGTGCAGAGCTCCAGGAAAGGGTATCAGAGCTGCAGCCAGCTCTGC
CCTCTACCCTAGGGAGGCCAGAAAGACACAAACAGCCCTCCGGGCCTTTACGCTGGACTCTGGCTTGGCAGGC
TCCAGGCAGGGTCCTCTGGGAAGTTACTCTAGAAAACGAAGGGAGGAGGAGCACAAGATCCTCAGCAACGAAC
ACCTGCACTTAGAAAAAGTGGACAGCTTCTGCCAACCACACCCTACCCATGGTACTGTATGCTATTAACTCCT
~GAGCACATTTCTTGTAATTACTATTGTTATTTTTATTGTCATGACTGCCCCTGAGCTCTGGTGAGAAAAGCTG
AATTTACAAGGAAAGGGATGAAGTTAATATTTGCATCACATAATTATATCATTACTGTGTATCTGTGTATTGT
ACTAAATGGACTGATGCTGCGCACATGAGCTGAAAATGAAGAGCCCTCCCATCC
b, CG103945-Ol ~SEQ ID NO: 4 782 as 1MW at 86699.9kD
iSequence APSAWAICWLLGGLLLHGGSSGPSPGPSVPRLRLSYRDLLSANRSAIFLGPQGSLNLQAMYLDEYRDRLFLG
LDALYSLRLDQAWPDPREVLWPPQPGQREECVRKGRDPLTECANFVRVLQPHNRTHLLACGTGAFQPTCALI
VGHRGEHVLHLEPGSVESGRGRCPHEPSRPFASTFIDGELYTGLTADFLGREAMIFRSGGPRPALRSDSDQS
LHDPRFVMAARIPENSDQDNDKWFFFSETVPSPDGGSNHWVSRVGRVCVNDAGGQRVLVNKWSTFLKARL
CSVPGPGGAETHFDQLEDVFLLWPKAGKSLEWALFSTVSAVFQGFAVCWHMADIWEVFNGPFAHRDGPQH
WGPYGGKVPFPRPGVCPSKMTAQPGRPFGSTKDYPDEVLQFARAHPLMFWPVRPRHGRPVLVKTHLAQQLHQ
VVDRVEAEDGTYDVIFLGTDSGSVLKVIALQAGGSAEPEEVVLEELQVFKVPTPITEMEISVKRQMLYVGSR
GVAQLRLHQCETYGTACAECCLARDPYCAWDGASCTHYRPSLGKRRFRRQDIRHGNPALQCLGQSQEEEAVG
VAATMVYGTEHNSTFLECLPKSPQAAVRWLLQRPGDEGPDQVKTDERVLHTERGLLFRRLSRFDAGTYTCTT
EHGFSQTVVRLALWIVASQLDNLFPPEPKPEEPPARGGLASTPPKAWYKDILQLIGFANLPRVDEYCERVW
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 MAPSAWAICWLLGGLLLHGGSSGPSPGPSVPRLRLSYRGAVVRKPSSTMWMETFSRYLLS
NOVlb MAPSAWAICWLLGGLLLHGGSSGPSPGPSVPRLRLSYR-------------D-----LLS
NOVla ANRSAIFLGPQGSLNLQAMYLDEYRDRLFLGGLDALYSLRLDQAWPDPREVLWPPQPGQR
NOVlb ANRSAIFLGPQGSLNLQAMYLDEYRDRLFLGGLDALYSLRLDQAWPDPREVLWPPQPGQR
~!NOVla EECVRKGRDPLTECANFVRV.LQPHNRTHLLACGTGAFQPTCALITVGHRGEHVLHLEPGS
',NOVlb EECVRKGRDPLTECANFVRVLQPHNRTHLLACGTGAFQPTCALITVGHRGEHV.LHLEPGS
',NOVla VESGRGRCPHEPSRPFASTFIDGELYTGLTADFLGREAMIFRSGGPRPALRSDSDQSLLH
'NOVlb VESGRGRCPHEPSRPFASTFIDGELYTGLTADFLGREAMIFRSGGPRPALRSDSDQSLLH
NOVla DPRFVMAARIPENSDQDNDKWFFFSETVPSPDGGSNHVTVSRVGRVCVNDAGGQRVLVN
NOVlb DPRFVMAARIPENSDQDNDKWFFFSETVPSPDGGSNHVTVSRVGRVCVNDAGGQRVLVN
NOVla KWSTFLKARLVCSVPGPGGAETHFDQLEDVFLLWPKAGKSLEWALFSTVSAVFQGFAVC
NOVlb KWSTFLKARLVCSVPGPGGAETHFDQLEDVFLLWPKAGKSLEWALFSTVSAVFQGFAVC
NOVIa VYHMADIWEVFNGPFAHRDGPQHQWGPYGGKVPFPRPGVCPSKMTAQPGRPFGSTKDYPD
NOVlb VYHMADIWEVFNGPFAHRDGPQHQWGPYGGKVPFPRPGVCPSKMTAQPGRPFGSTKDYPD
NOVla EVLQFARAHPLMFWPVRPRHGRPVLVKTHLAQQLHQIVVDRVEAEDGTYDVIFLGTDSGS
NOVlb EVLQFARAHPLMFWPVRPRHGRPVLVKTHLAQQLHQIVVDRVEAEDGTYDVIFLGTDSGS
NOVla VLKVIALQAGGSAEPEEVVLEELQVFKVPTPITEMEISVKRQMLWGSRLGVAQLRLHQC
NOVlb VLKVIALQAGGSAEPEEVVLEELQVFKVPTPITEMEISVKRQMLWGSRLGVAQLRLHQC
NOVla ETYGTACAECCLARDPYCAWDGASCTHYRPSLGKRRFRRQDIRHGNPALQCLGQSQEEEA
NOVlb, ETYGTACAECCLARDPYCAWDGASCTHYRPSLGKRRFRRQDIRHGNPALQCLGQSQEEEA
NOVla VGLVAATMVYGTEHNSTFLECLPKSPQAAVRWLLQRPGDEGPDQVKTDERVLHTERGLLF
NOVlb VGLVAATMWGTEHNSTFLECLPKSPQAAVRWLLQRPGDEGPDQVKTDERVLHTERGLLF
NOVla RRLSRFDAGTYTCTTLEHGFSQTWRLALWIVASQLDNLFPPEPKPEEPPARGGLASTP
NOVlb RRLSRFDAGTYTCTTLEHGFSQTVVRLALWIVASQLDNLFPPEPKPEEPPARGGLASTP
NOVla PKAWYKDILQLIGFANLPRVDEYCERVWCRGTTECSGCFRSRSRGKQARGKSWAGLELGK
NOVlb PKAWKDILQLIGFANLPRWEYCERVWCRGTTECSGCFRSRSRGKQARGKSWAGLELGK
NOVla KMKSRVHAEHNRTPREVEAT
NOVlb KMKSRVHAEHNRTPREVEAT
NOVla (SEQ ID NO: 2) NOVlb (SEQ ID NO: 4) Further analysis of the NOV1 a protein yielded the following properties shown in Table 1 C.

Table 1C. Protein Sequence Properties NOVla SignalP analysis: Cleavage site between residues 23 and 24 PSORT II analysis: ' PSG: a new signal peptide prediction method N-region: length 0; pos.chg 0; neg.chg 0 H-region: length 31; peak value 9.35 PSG score: 4.95 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 1.50 possible cleavage site: between 22 and 23 » > Seems to have a cleavable signal peptide (1 to 22) ALOM: Klein et al's method for TM region allocation Init position for calculation: 23 Tentative number of TMS(s) for the threshold 0.5: 3 Number of TMS(s) for threshold 0.5: 1 INTEGRAL Likelihood = -2.02 Transmembrane 345 - 361 PERIPHERAL Likelihood = 2.86 (at 150) ALOM score: -2.02 (number of TMSs: 1) 'MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 11 Charge difference: 0.5 C( 1.5) - N( 1.0) C > N: C-terminal side will be inside » >Caution: Inconsistent mtop result with signal peptide » > membrane topology: type 1a (cytoplasmic tail 362 to 800) MITDISC: discrimination of mitochondrial targeting seq R content: 4 Hyd Moment(75): 2.13 Hyd Moment(95): 2.46 G content: 7 D/E content: 1 S/T content: 9 Score: -2.94 Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 73 NRS~AI
NUCDISC: discrimination of nuclear localization signals pat4: none pat7: PSLGKRR (3) at 570 bipartite: none content of basic residues: 11.4&
NLS Score: -0.22 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: none SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif.
type 1: none type 2: none NMYR: N-myristoylation pattern : none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: too long tail 'Dileucine motif in the tail: found LL at 633 ~i LL at 658 checking 63 PROSITE DNA binding motifs: none checking 71 PR05ITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: cytoplasmic Reliability: 89 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues Final Results (k = 9/23):
44.4 %: endoplasmic reticulum 22.2 %: Golgi 22_2 %: extracellular, including cell wall 11_1 %: plasma membrane » prediction for CG103945-02 is end (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.

Table 1D.
Geneseq Results for NOVla NOVla Identities/

Geneseq Protein/Organism/Length Residues/SimilaritiesExpect [Patent #, for IdentifierDate] Match the MatchedValue ResiduesRegion AAG65620Novel human protein (NHP)1..800 781/800 0.0 sequence (97%) - Homo Sapiens, 782 aa. 1..782 781/800 (97%) [W0200170806-A2, 27-SEP-2001]

AAG65619Novel human protein (NHP)1..800 781/800 0.0 sequence (97%) - Homo Sapiens, 875 aa. 94..875 781/800 (97%) [W0200170806-A2, 27-SEP-2001]

AAB23609Human secreted protein L.800 781/800 0.0 SEQ ID NO: (97%) 18 - Homo sapiens, 782 1..782 781/800 aa. (97%) [W0200049134-Al, 24-AUG-2000]

AAB23636Human secreted protein 1..800 7811803 0.0 SEQ >D NO: (97%) 92 - Homo Sapiens, 785 2..785 781/803 aa. (97%) [WO200049134-Al, 24-AUG-2000]

AAG78481Human ZSMF-16 - Homo 1..800 778/800 0.0 sapiens, (97%) 779 aa. [US2001049432-Al,1..779 779/800 (97!0) 06-DEC-2001]

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

Table lE. Public BLASTP
Results for NOVla NOVla Identities/

Protein Residues/SimilaritiesExpect for AccessionProtein/Organism/Length Match the Matched Value Number ResiduesPortion Q9NS98 Semaphorin sem2 (FLJ000141..800 781/800 (97%)0.0 protein) - Homo Sapiens 1..782 781/800 (97%) (Human), 782 aa.

CAC42673 Sequence 1 from Patent 1..800 778/800 (97%)0.0 ~ 1..779 779/800 (97%) - Homo Sapiens (human), 779 aa.

Q9QX23 Semaphorin M-SemaK - 1..795 399/805 (49%)0.0 Mus musculus (Mouse), 775 1..770 525/805 (64%) aa.

P70275 Semaphorin 3E precursor 1..795 398/805 (49%)0.0 (Semaphorin H) (Sema 1..?70 524/805 (64%) H) - Mus musculus (Mouse), 775 aa.

042237 Semaphorin 3E precursor 1..797 398/806 (49%)0.0 (Collapsin-5) (COLL-5) 5..782 519/806 (64%) - Gallus gallus (Chicken), 785 aa.

PFam analysis predicts that the NOV 1 a protein contains the domains shown in the Table 1F.
Table 1F. Domain Analysis of NOVla Identities/

Pfam Domain NOVla Match RegionSimilarities Expect Value for the Matched Region Sema 76..521 217/497 (44%) 1.3e-176 360/497 (72%) PSI 539..622 14/101 (14%) 0.76 56/101 (SS%) ig 614..675 15/66 (23%) 0.0061 45/66 (68%) Example 2.
The NOV2 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 2A.

2A. NOV2 CG106951-O1 ~SEO mNO: S
Sequence ORF Start: ATG at 1400 ORF Stop: TGA at 5456 TGGTCCAGAGCTCATTGTCCTTGTTGATAAAATGATAGATTTGGACTCAATATCC
CCTTTCTTTTCTTCTCTGCATCTCTGCCTTTGTGTCCAGAGCGTGTTTTCCCTTTGCAAGTTTCTCTCCATTC
TGCACATTATGAGTTTCAGCATTTCTGTTGCCCTAGAAAGTCTATCTTTGAGATCTTGCACTGTTTCTCTTTT
TACAGTGTCTCATAAACTCCCTTCTTGGATTCAGAACCACCCTTTCTTTCCCATTATCCTGTCAAACTGCTTC
TTGCCATGGTCCAGGGGTAGGAGGATGGCAGGCAGGAGGTGCTTCTCTGGGGCTCTTAGTGTCTCAATTCTTC
ATTTTTATTTCATAGT
CTTAGGTCACCTTTTTTTACATTTTCAAATATATTTTTTGTTCAGCAGAGGGCTCCCTTCCCATCCCTCTTGC
AGCCCGGGCAGCTAGGATTTGAAGCTTGCCCCTTGAATCTTTCTCTCCCGCCTTCTAGCCATCAGAAACACTA
GATCACTTAAACTTGTAAACAATTCGGCCTCGCTCCTTGTGATTGCGCTAAACCTTCCGTCCTCAGCTGAGAA
CGCTCCACCACCTCCCCGGATCGCTCATCTCTTGGCTGCCCTCCCACTGTTCCTGATGTTATTTTACTCCCCG
TATCCCCTACTCGTTCTTCACAATTCTGTAGGGTGCGTATTACTAACCCCAGTTTACAGCTGAGGAAACTGAG
GCTTGGAGAGGTTCGCTCGGTATCGTACAGTTTGCAAGGTTAACCCTAATCCGGCCAGTTCTGGCTTTCCAGC
CCAGCCCAGCAGCCTAGCCTCCCTCTCTGCCGCTGCAGGTTATAACGGCTCTCCCCCGTTTTACACGAGGTCC
CTTCCCCTTCAAATCCACAGGCAGGAAGATCGTTCCGAACTGACGGGGCTGGGGAATGTGGGAGTCCGGAGTG
~CATTCGAGATGGGGTGACCGAGAACGGCAAGGCGGGATGTGGCAAACGGCGGCAAGTGCTCGGAGTCCTAGGT
CTTGCCGCCGGAATGCCGGCCGGGGAAGGGGCTTCGGCCCACCGGGCTGGTCACCACACTCGGCAGGCCCGGG
CAGCGCATCCAGCGGCTGCTGGGAGCCCGAGCGCAGCGGGCGCGGGCCCGGGTGGGGACTGCACCGGAGCG
GAGAGCTGGAGGCCGTTCCTGCGCGGCCGCCCCATTCCCAGACCGGCCGCCAGCCCATCTGGTTAGCTCCC
CGCTCCGCGCCGCCCGGGAGTCGGGAGCCGCGGGGAACCGGGCACCTGCACCCGCCTCTGGGAGTGAGTGG
TGGACAGTAGGGGGCTGGCTTCTCTCAC
CAGAGGGGCCTATCATGGTGCTTGCAGG
TGTC
ACCTCTTCAGACTCAGCCTTGCCAATGTCTCTCTTCTTCAGGCCACA
TCGTCGCCGGCCGGAAGGTGTTCATGTGTGGAACCAATGCCTTTTCCCCCATGTGCAC
GTGGGCCACCGCTTCGCACTGCCCAATATAACTCCAAGTGGCT
TATTGGGCTGTTTGCATACTTCTTCCTGCGGGAGAACGCAGTG
CGCGTGGCCCGCGTGTGCAAGAATGACGTGGGGGGCCGATTCC
ATAACGAGCTGCAGAGTGCCTTCCACTTGCCAGAGCAGGACCTCATCTATGGAGTTTTCACAACCAACGTA
CAGCATCGCGGCTTCTGCTGTCTGCGCCTTCAACCTCAGTGCTATCTCCCAGGCTTTCAATGGCCCATTTC
GCCTGAGACCGGTCCCAACGAGAACCTGACGGAGCGCAGCCTGCAGGACGCGCAGCGCCTCTTCCTGATGAGC
CTGGGACGGGAAGCAGCAACGTTGCAGCACACTCGAGGACAGCTCCAACATGAGCCTCTGGACCCAGAACATC

TCGGCTTCCAGGTCCGCCAGCGAAGTTGCAGCAACCCTGCTC
TCTGTCTCGGGCTGCACACGGAGGAGGCACTATGTGCCACACAGGCC
TTCCCGTCATCCTGCCAGCCTCCAGCATGGAGGAGGCCACCGGCTGTGCAGGGTTCAATCTCATCCAC
GGCCACGGGCATCTCCTGCTTCTTGGGCTCTGGGCTCCTGACCCTAGCAGTGTACCTGTCTTGCCAGC
CAGCGTCAGTCCCAGGAGTCCACACTGGTCCATCCTGCCACCCCCAACCATTTGCACTACAAGGGCGG
CCCCGAAGAATGAAAAGTACACACCCATGGAATTCAAGACCCTGAACAAGAATAACTTGATCCCTGAT
AGCCAACTTCTACCCATTGCAGCAGACCAAZ'GTGTACACGACTACTTACTACCCAAGCCCCCTGAACA
CCCT
~TTATCTTCCAACCCACTGTCACGCTGACACTATGCTCCCATGCCTGGGCTGTGGACCTACTGGGCATTTC,AC,r CACA
AGCTGAGTCTGGCCTTCTCCAGCTGGCCCCAAAAAAGGCCTTTGCTACATCCTGATTATCTCTGAAAGTAATC
AATCAAGTGGCTCCAGTAGCTCTGGATTTTCTGCCAGGGCTGGGCCATTGTGGTGCTGCCCCAGTATGACATG
~GGACCAAGGCCAGCGCAGGTTATCCACCTCTGCCTGGAAGTCTATACTCTACCCAGGGCATCCCTCTGGTCAG
GCCTTGCCCTCAATGCACGAAAGGTGGCCCAGGAGAGAGGATCAATGCCACAGGAGGCAGAAGTCTGGCCTCT
~GTGCCTCTATGGAGACTATCTTCCAGTTGCTGCTCAACAGAGTTGTTGGCTGAGACCTGCTTGGGAGTCTCTG
AATTAAAGATGATATCCAGTCTCC
2a, CG106951-O1 SEQ ID NO: 6 1352 as MW at 145674.1kD
MPAGEGASAHRAGHHTRQARGGSRPSSRGMQGAPSRSSARLEAGGCSARRGRSAPAPSSFSLPLPSFSPFACN
SSPTAPSLLLLPRSPPPCSLRAPGRELVGARGLVPEPSSAEPGGSAAHPAAAGSPSAAGAGPGGDCTGALRAG
GRSCAAAPFPDRPPAHLVSSRRSAPPGSREPRGTGHLHPPLGVSGSSWCLACVSWMPCGFSPSPVAHHLVPGP
PDTPAQQLRCGWTVGGWLLSLVRGLLPCLPPGARTAEGPTMVLAGPLAVSLLLPSLTLLVSHLSSSQDVSSEP
SSEQQLCALSKHPTVAFEDLQPWVSNFTYPGARDFSQLALDPSGNQLIVGARNYLFRLSLANVSLLQATEWAS
SEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAFSPMCTSRQVGNLSRTTEKINGVARCPYDPRHNS
TAVISSQGELYAATVIDFSGRDPAIYRSLGSGPPLRTAQYNSKWLNEPNFVAAYDIGLFAYFFLRENAVEHDC
GRTVYSRVARVCKNDVGGRFLLEDTWTTFMKARLNCSRPGEVPFYYNELQSAFHLPEQDLIYGVFTTNVNSIA
ASAVCAFNLSAISQAFNGPFRYQENPRAAWLPIANPIPNFQCGTLPETGPNENLTERSLQDAQRLFLMSEAVQ
PVTPEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESGTILKALSTASRSLHGCYLEELHVLPPGRREPLR
SLRILHSARALFVGLRDGVLRVPLERCAAYRSQGACLGARDPYCGWDGKQQRCSTLEDSSNMSLWTQNITACP
VRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWAL
CSTSCGIGFQVRQRSCSNPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRA
CDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRTCTNPEPRNGGLPCVG
QACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEEALCATQACPEG
TDDGAQSRSRHCEELLPGSSACAGNSSQSRPCPYSEIPVILPASSMEEATGCAGFNLIHLVAT
LTLAVYLSCQHCQRQSQESTLVHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLTPDDRAN
TTTYYPSPLNKHSFRPEASPGQRCFPNS

?b, CG1069S1-04 SEQ ID NO: 7 3631 by Sequence__ ORF Start: ATG at 1S4 ORF Stop: TGA at 3544 TGGACAGTAGGGGGCTGGCTTCTCTCACTGGTCAGGGGTCTTCTCCCCTGTC
CAGAGGGGCCTATCATGGTGCTTGCAGGCCCCCTGGCTGTCTCGCTGTTGCT
AGCAAGCACCCCACCGTGGCCTTTGAAGACCTGCAGCCGTGGGTCTCTAACTTCACCT
GGTGTTCATGTGTGGAACCAATGCCTTTTCCCCCATGTGCACCAGCAGACAGGTGGGGAACCTCAG
ACTGAGAAGATCAATGGTGTGGCCCGCTGCCCCTATGACCCACGCCACAACTCCACAGCTGTCATC
AGGGGGAGCTCTATGCAGCCACGGTCATCGACTTCTCAGGTCGGGACCCTGCCATCTACCGCAGCC
ATAACGAGCTGCAGAGTGCCTT
'rCr~u't'UC'1'A'1'C'sCCCAGGCTTTCAATGGCCCATTTCGCTACCAGGAGAACCCCAGGGCTG
AGCCAACCCCATCCCCAATTTCCAGTGTGGCACCCTGCCTGAGACCGGTCCCAACGAGAA
ACTCTACATTGGCACCGAGTCGGGCACCATCCTGAAGGCGCTGTCCACGGCGAGCCGCAGCCT
CCTACCGCAGCCAGGGGGCATGCCTGGGGGCCCGGGACCCGTACTGTGGCTGGGACGGGAAGCAGCAACGTTG
CAGCACACTCGAGGACAGCTCCAACATGAGCCTCTGGACCCAGAACATCACCGCCTGTCCTGTGCGGAATGTG
ACACGGGATGGGGGCTTCGGCCCATGGTCACCATGGCAACCATGTGAGCACTTGGATGGGGACAACTCAGGCT
TCCACATCGCCAACTGCTCCAGGAATGGGGCGTGGACCCCGTGGTCATCGTGGGCGCTGTGCAGCACGTCC
CCGC
CTCCCAGGGTCCAGCGCCTGTGCTGGAAACAGCAGCCAGAGCCGCCCCTGCCCCTACAGCGAGATTCCCGTCA
CATCTCCTGCTTCTTGGGCTCTGGGCTCCTGACCCTAGCAGTGTACCTGTCTTGCCAGCACTGCCAGCGTCAG
TCCCAGGAGTCCACACTGGTCCATCCTGCCACCCCCAACCATTTGCACTACAAGGGCGGAGGCACCCCGAAGA
ATGAAAAGTACACACCCATGGAATTCAAGACCCTGAACAAGAATAACTTGATCCCTGATGACAGAGCCAACTT
AAGGCACAGAGCAGATGGAGATGGGACAGTGGAGCCAGTTTGGTTTCT

CG10695I-04 SEQ m NO: 8 1130 as ~MW at 123700.9kD
FSPSPVAHHLVPGPPDTPAQQLRCGWTVGGWLLSLVRGLLPCLPPGARTAEGPIMVLAGPLAVSLLLPS
RLSLANVSLLQATEWASSEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAFSPMCTSRQVGNLSRT
EKINGVARCPYDPRHNSTAVISSQGELYAATVIDFSGRDPAIYRSLGSGPPLRTAQYNSKWLNEPNFVAAYD
GLFAYFFLRENAVEHDCGRTVYSRVARVCKNDVGGRFLLEDTWTTFMKARLNCSRPGEVPFYYNELQSAFHL
EQDLIYGVFTTNVNSIAASAVCAFNLSAISQAFNGPFRYQENPRAAWLPIANPIPNFQCGTLPETGPNENLT
RSLQDAQRLFLMSEAVQPVTPEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESGTILKALSTASRSLHG
YLEELHVLPPGRREPLRSLRILHSARALFVGLRDGVLRVPLERCAAYRSQGACLGARDPYCGWDGKQQRCST
EDSSNMSLWTQNITACPVRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAIH
ANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGS
SKCSSNCGGGMQSRRRACENGNSCLGCGVEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCR
PLADPHGLQFGRRRTETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVR
RTCTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTSPAPSPEGWSP
SEWSKCTDDGAQSRSRHCEELLPGSSACAGNSSQSRPCPYSEIPVILPASSMEEATGCAGFNLIHLVATGIS
FLGSGLLTLAVYLSCQHCQRQSQESTLVHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLIPDDRANFYP
~OOTNVYTTTYYPSPLNKHSFRPEASPGQRCFPNS
209829549 SEQ ID NO: 9 X1203 Sequence ~ORF Start: at I ~ORF Stop: end of GAGCTCGATCCTGTGATTCCCCTCGACCCCGCTGTGGGGGCCTTGACTGCCTGGGGCCAGCCATCCACATCGC
CAACTGCTCCAGGAATGGGGCGTGGACCCCGTGGTCATCGTGGGCGCTGTGCAGCACGTCCTGTGGCATCGGC
mmrraccmrrr_rrnrr~an Att~rTC~CACTCAACCCTGCTCCCCGCCACGGGGGCCGCATCTGCGTGGGCAAGAGCC
CAAGTGCAGCAGCAACTGTGGAGGGGGCATGCGGTCGCGGCGTCGGGCCTGCGAGAACGGCAACTCCTGCCTG
GGCTGCGGCGTGGAGTTCAAGACGTGCAACCCCGAGGGCTGCCCCGAAGTGCGGCGCAACACCCCCTGGACGC
CCCGCGGACGGCTCC
ATGTGCCACACAGGCCTGCCCAGAAGGCTGGTCGCCCTGGTCTGAGTGGAGT
CTGGAAACAGCAGCCAGAGCCGCCCCTGCGTCGAC

209829549 SEQ ID NO: 10 401 as ~MW at 43284.SkD
PWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIG
SCSNPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMRSRRRACENGNSCL
KTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRTETRTCPADGS
ALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRTCTNPEPRNG~GLPCVGDAAEYQDC
VRGAWSCWTSWSPCSASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWS
AOSRSRHCEELLPGSSACAGNSSQSRPCVD
>.d, 209829553 SEQ ID NO: 11 03 by _ Sequence ORF Start: at 1 ~ ORF Stop: end of CCCATGGTCACCATGGCAACCATGTGAGCACTTGGATGGGGACAACTCAGGCTCTTGCCTGTGTC
TCCTGTGATTCCCCTCGACCCCGCTGTGGGGGCCTTGACTGCCTGGGGCCAGCCATCCACATCGC
AAGTTGCAGCAACCCTGCTCCCCGCCACGGGGGCCGCATCTGCGTGGGCAAGAGCC
AATGAGAACACGCCTTGCCCGGTGCCCATCTTCTGGGCTTCCTGGGGCTCCTGGAG
CTGGTGGAGGACCTCCTGCGCAGCGGGAGCACCTCCCCGCACACGGTGAGCG
CTAACCCGGAGTCCCGCAACGGGGGCCTGCCCTGCGTGGGCGATGCTGCCGAGTACCAGGACTGC
GGCTTGCCCAGTTCGGGGTGCTTGGTCCTGCTGGACCTCATGGTCTCCATGCTCAGCTTCCTGTG
CACTATCAACGCACCCGTTCCTGCACCAGCCCCGCACCCTCCCCAGGTGAGGACATCTGTCTCGG
CGGAGGAGGCACTATGTGCCACACAGGCCTGCCCAGAAGGCTGGTCGCCCTGGTCTGAGTGGAGT
CTGGAAACAGCAGCCAGAGCCGCCCCTGCGTCGAC
209829553 ~SEQ ID NO: 12 X401 as ]MW at 43246.4kD
PCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIG
NPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCL
NPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRTETRTCPADGS
EDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRTCTNPESRNGGLPCVGDAAEYQDC
AWSCWTSWSPCSASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWS
RSRHCEELLPGSSACAGNSSQSRPCVD

!e, 209829642 SEQ ID NO: 13 1203 by Sequence ORF Start: at 1 ORF Stop: end of TGGCAACCATGTGAGCACTTGGATGGGGACAACTCAGGCTCTTGCCTGTGTC
CTCGACCCCGCTGTGGGGGCCTTGACTGCCTGGGGCCAGCCATCCACATCGC
TGCCCAGTTCGGGGTGCTTGGTCCTGCTGGACCTCATGGTCTCCATGCTCAGCTTCCTGTG
ATCAACGCACCCGTTCCTGCACCAGCCCCGCACCCTCCCCAGGTGAGGACATCTGTCTCGG
GGAGGCACTATGTGCCACACAGGCCTGCCCAGAAGGCTGGTCGCCCTGGTCTGAGTGGAGT
209829642 ~SEQ ID NO: 14 X401 as BMW at 432$6.4kD
SPWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIG
RSCSNPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCL
FKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRTETRTCPADGS
DALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRTCTNPEPRNGGLPCVGDAAEYQDC
PVRGAWSCWTSWSPCSASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWS
GAQSRSRHCEELLPGSSACAGNSSQSRPCVD
>.f, 209829670 SEQ ID NO:1$ ~ 1203 by _ Sequence ORh Start: at 1 ~~ORF Stop: end of CAACTGCTCCAGGAATGGGGCGTGGACCCCGTGGTCATCGTGGGCGCTGTGCAGCACGTCCTGTGGCATCGGC
CCGTTCCTGCACCAGCCCCGCACCCTCCCCAGGTGAGGACATCTGTCTCGG
TG,TGCCACACAGGCCTGCCCAGAAGGCTGGTCGCCCTGGTCTGAGTGGAGT

209829670 ~SEQ ID NO: 16 ~40I as BMW at 43240.SkD
PWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPTIHIANCSRNGAWTPWSSWALCSTSCGIG
SCSNPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGSWSKCGSNCGGGMQSRRRACENGNSCL
~VEVLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRTCTNPEPRNGGLPCVGDAAEY'QDC
.GAWSCWTSWSPCSASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWS
~SRSRHCEELLPGSSACAGNSSQSRPCVD
?g, CGI0695I-02 SEQ ID NO: 17 _ 4_23_3 by , Sequence ORF Start: ATG at 2 ORF Stop: TGA at 3281 ACCCTGGAGCCCGGGATTTCTCCCAGCTGGCTTT
AGGAGTGTCAGAACTACGTGCGAGTCCTGATCGTCGCCGGCCGGAAGGTGTTCATGTGTGGAACCAATGCCTT
TTCCCCCATGTGCACCAGCAGACAGGTGGGGAACCTCAGCCGGACTACTGAGAAGATCAATGGTGTGGCCCGC
TGCCCCTATGACCCACGCCACAACTCCACAGCTGTCATCTCCTCCCAGGGGGAGCTCTATGCAGCCACGGTCA
TCGACTTCTCAGGTCGGGACCCTGCCATCTACCGCAGCCTGGGCAGTGGGCCACCGCTTCGCACTGCCCAATA
TAACTCCAAGTGGCTTAATGAGCCAAACTTCGTGGCAGCCTATGATATTGGGCTGTTTGCATACTTCTTCCTG
CGGGAGAACGCAGTGGAGCACGACTGTGGACGCACCGTGTACTCTCGCGTGGCCCGCGTGTGCAAGAATGACG
CGAGGTCCCCTTCTACTATAACGAGCTGCAGAGTGCCTTCCACTTGCCAGAGCAGGACCTCATCTATGGAGTT
TTCACAACCAACGTAAACAGCATCGCGGCTTCTGCTGTCTGCGCCTTCAACCTCAGTGCTATCTCCCAGGCTT
ACTCTACATTGGCACCGAGTCGGG
CTGCGCAGCCTGCGCATCCTGCACAGCGCCCGCGCGCTCTTCGTGGGGCTGA
CACTGGAGAGGTGCGCCGCCTACCGCAGCCAGGGGGCATGCCTGGGGGCCCG
CGGGAAGCAGCAACGTTGCAGCACACTCGAGGACAGCTCCAACATGAGCCTC
TGGGGACAACTCAGGCTCTTGCCTGTGTCGAGCTCGATCCTGTGATTCCCC
ACAGCGAGATTCCCGTCATCCTGCCAGCCTCCAGCATGGAGGAGGCCACCGGCTGTGCAGGG
ACCCATTGCAGCAGACCAATGTGTACACGACTACTTACTACC

CCAGGTCTCTCATGGTTATCTTCCAACCCACTGTCACGCTGACACTATGCTGCCATGCCTGGGCTGTGGACCT
ACTGGGCATTTGAGGAACTGGAGAATGGAGATGGCAAGAGGGCAGGCTTTTAAGTTTGGGTTGGAGACAACTT
CCTGTGGCCCCCACAAGCTGAGTCTGGCCTTCTCCAGCTGGCCCCAAAAAAGGCCTTTGCTACATCCTGATTA
ACCCAGGG
AATCTGGG
~AGAAGTCTGGCCTCTGTGCCTCTATGGAGACTATCTTCCAGTTGCTGCTCAACAGAGTTGTTGGCTGAGACCT
GCTTGGGAGTCTCTGCTGGCCCTTCATCTGTTCAGGAACACACACACACACACACTCACACACGCACACACAA
TCACAATTTGCTACAGCAACAAAAAAGACATTGGGCTGTGGCATTATTAATTAAAGATGATATCCAGTCTCC
CG1069$1-02 ~SEQ m N0:18 X1093 as BMW at 119865.3kD
MVLAGPLAVSLLLPSLTLLVSHLSSSQDVSSEPSSEQQLCALSKHPTVAFEDLQPWVSNFTYPGARDFSQLAL
DPSGNQLIVGARNYLFRLSLANVSLLQATEWASSEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAF
SPMCTSRQVGNLSRTTEKINGVARCPYDPRHNSTAVISSQGELYAATVIDFSGRDPAIYRSLGSGPPLRTAQY

EVPFYYNELQSAFHLPEQDLIYGVFTTNVNSIAASAVCAFNLSAISQAFNGPFRYQENPRAAWLPIANPIPNF
QCGTLPETGPNENLTERSLQDAQRLFLMSEAVQPVTPEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESG
TILKALSTASRSLHGCYLEELHVLPPGRREPLRSLRILHSARALFVGLRDGVLRVPLERCAAYRSQGACLGAR
DPYCGWDGKQQRCSTLEDSSNMSLWTQNITACPVRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLCRARSCDSP
RPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHGGRICVGKSREERFCNE
NTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCLGCGVEFKTCNPEGCPEVRRNTPWTPWLPVNVT
QGGARQEQRFRFTCRAPLADPHGLQFGRRRTETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGP
WSSCSRDCELGFRVRKRTCTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRT
RSCTSPAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSACAGNSSQSR
PCPYSEIPVILPASSMEEATGCAGFNLIHLVATGISCFLGSGLLTLAVYLSCQHCQRQSQESTLVHPATPNHL
HYKGGGTPKNEKYTPMEFKTLNKNNLIPDDRANFYPLQQTNVYTTTYYPSPLNKHSFRPEASFGQRCFPNS
?h, CG1069$1-03 SEQ mNO:19 1203 by Sequence ORF Start: at 7 ORF Ston: at 1198 GGATCCGGCCCATGGTCACCATGGCAACCATGTGAGCACTTGGATGGGGACAACTCAGGCTCTTGCCTGTGTC
GAGCTCGATCCTGTGATTCCCCTCGACCCCGCTGTGGGGGCCTTGACTGCCTGGGGCCAACCATCCACATCGC
CAACTGCTCCAGGAATGGGGCGTGGACCCCGTGGTCATCGTGGGCGCTGTGCAGCACGTCCTGTGGCATCGGC
TCTTCTGGGCTTCCTGGGGCTCCTGGAG
ATGTGCCACACAGGCCTGCCCAGAAGGCTGGTCGCCCTGGTCTGAGTGGAGT

CG106951-03 ~SEQ ID NO: 20 397 as MW at 42882.1kD
PWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPTIHIANCSRNGAWTPWSSWALCSTSCGIGFQ
SCSNPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGSWSKCGSNCGGGMQSRRRACENGNSCLGC
umrr'rpFt~rpF~mR1 TpwTPwLPVNVTOGGAROEORFRFTCRAPLADPHGLOFGRRRTETRTCPADGSGS
SWSPCSASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKC
ELLPGSSACAGNSSQSRPC
V2i, SNP13382456 of SEQ ID NO: 21 _ 6408 bp~
106951-Ol, DNA Sequence ORF Start: ATG at 1400 Stop: TGA at 5456 Pos: 5770 SNP Change: C to T
pCATGCTGCCTCTTCCAACTTGATTTTTACCCCAGACTGGGCTACCAGACTGGTATGCC:CACAC:A'1'GCCCG'1.d egree.1"1' TGCACATTATGAGTTTCAGCATTTCTGTTGCCCTAGAAAGTCTATCTTTGAGATCTTGCACTGTTTCTCTTTT
TACAGTGTCTCATAAACTCCCTTCTTGGATTCAGAACCACCCTTTCTTTCCCATTATCCTGTCAAACTGCTTC
TGCTTTATCTGGGTTTTCCTTTACCCAGAATTTTATTATGTAAAATGCTTCACTCAGACTTTGTTCTAATTAT
CCAATTTTTGGCATACTCTAGAAAGTCTTTTGATATTTTCCTTCCTCCAACTTATCTATTTTTATTTCATAGT
TCTCTTTGGTTATCTCTTAGAATCACACTTTCCTGGTTTTAATTTTTCAAATCCTTTGTCTTTCTCACTCGTT
CTTAGGTCACCTTTTTTTACATTTTCAAATATATTTTTTGTTCAGCAGAGGGCTCCCTTCCCATCCCTCTTGC
GATCACTTAAACTTGTAAACAATTCGGCCTCGCTCCTTGTGATTGCGCTAAACCTTCCGTCCTCAGCTGAGAA
CGCTCCACCACCTCCCCGGATCGCTCATCTCTTGGCTGCCCTCCCACTGTTCCTGATGTTATTTTACTCCCCG
TATCCCCTACTCGTTCTTCACAATTCTGTAGGGTGCGTATTACTAACCCCAGTTTACAGCTGAGGAAACTGAG
GCTTGGAGAGGTTCGCTCGGTATCGTACAGTTTGCAAGGTTAACCCTAATCCGGCCAGTTCTGGCTTTCCAGC
CCAGCCCAGCAGCCTAGCCTCCCTCTCTGCCGCTGCAGGTTATAACGGCTCTCCCCCGTTTTACACGAGGTCC
CTTCCCCTTCAAATCCACAGGCAGGAAGATCGTTCCGAACTGACGGGGCTGGGGAATGTGGGAGTCCGGAGTG
CATTCGAGATGGGGTGACCGAGAACGGCAAGGCGGGATGTGGCAAACGGCGGCAAGTGCTCGGAGTCCTAGGT
CTTGCCGCCGGAATGCCGGCCGGGGAAGGGGCTTCGGCCCACCGGGCTGGTCACCACACTCGGCAGGCCCGGG
TCCAGCGGCTGCTGGGAGCCCGAGCGCAGCGGGCGCGGGCCCGGGTGGGGACTGCACCGGAGCG
AAGGTGTGGATGGACAGTAGGGGGCTGGCTTCTCTCAC
GCTAGGACTGCAGAGGGGCCTATCATGGTGCTTGCAGG
AGCAAGCACCCCACCGTGGCCTTTGAAGACCTGC
CGTGGGAGCCAGGAACTACCTCTTCAGACTCAGCCTTGCCAATGTCTCTCTTCTTCAGGCCACA
GTGGGCCACCGCTTCGCACTGCCCAATATAACTCCAAGTGGCT
TATTGGGCTGTTTGCATACTTCTTCCTGCGGGAGAACGCAGTG
CGCGTGGCCCGCGTGTGCAAGAATGACGTGGGGGGCCGATTCC
AGGCCCGGCTCAACTGCTCCCGCCCGGGCGAGGTCCCCTTCTA
(~CCA(iA(tCAC~GACCTCATCTATGGAGTTTTCACAACCAACGTA

TCGAGGACAGCTCCAACATGAGCCTCTGGACCCAGAACATC
TGGGGGCTTCGGCCCATGGTCACCATGGCAACCATGTGAGC
TGTCGAGCTCGATCCTGTGATTCCCCTCGACCCCGCTGTGG
TCGCCAACTGCTCCAGGAATGGGGCGTGGACCCCGTGGTCA
AATGAGAACACGCCTTGCCC
TGCGCAGCGGGAGCACCTCCCCGCACACGGTGAGCGGGGGCTGGGCCGCCTGGGGCCCGTGGTCGTCCTGCTC
CCGGGACTGCGAGCTGGGCTTCCGCGTCCGCAAGAGAACGTGCACTAACCCGGAGCCCCGCAACGGGGGCCTG
CCCTGCGTGGGCGATGCTGCCGAGTACCAGGACTGCAACCCCCAGGCTTGCCCAGTTCGGGGTGCTTGGTCCT
GCTGGACCTCATGGTCTCCATGCTCAGCTTCCTGTGGTGGGGGTCACTATCAACGCACCCGTTCCTGCACCAG
AAGTGCACTGACGACGGAGCCCAGAGCCGAAGCCGGC
TGCCAGCCTCCAGCATGGAGGAGGCCACCGGCTGTGCAGGGTTCAATCTCATCCAC
CTCCTGCTTCTTGGGCTCTGGGCTCCTGACCCTAGCAGTGTACCTGTCTTGCCAGC
CAGGAGTCCACACTGGTCCATCCTGCCACCCCCAACCATTTGCACTACAAGGGCGG
nnaar_mnrnrnrrrAmrrAA~r'TC~AAGACCCTGAACAAGAATAACTTGATCCCTGAT
AACACAGCTTCCGGCCCGAGGCCTCACCTGGACAACGGTGCTTCCCCAACAGCTGATACCGCCGTCCTGGGGA
CTTGGGCTTCTTGCCTTCATAAGGCACAGAGCAGATGGAGATGGGACAGTGGAGCCAGTTTGGTTTTCTCCCT
CTGCACTAGGCCAAGAACTTGCTGCCTTGCCTGTGGGGGGTCCCATCCGGCTTCAGAGAGCTCTGGCTGGCAT
TGACCATGGGGGAAAGGGCTGGTTTCAGGCTGACATATGGCCGCAGGTCCAGTTCAGCCCAGGTCTCTCATGG
TTATCTTCCAACCCACTGTCACGCTGACACTATGCTGCCATGCCTGGGCTGTGGACCTACTGGGCATTTGAGG
AATTGGAGAATGGAGATGGCAAGAGGGCAGGCTTTTAAGTTTGGGTTGGAGACAACTTCCTGTGGCCCCCACA
AGCTGAGTCTGGCCTTCTCCAGCTGGCCCCAAAAAAGGCCTTTGCTACATCCTGATTATCTCTGAAAGTAATC
AATCAAGTGGCTCCAGTAGCTCTGGATTTTCTGCCAGGGCTGGGCCATTGTGGTGCTGCCCCAGTATGACATG
GGACCAAGGCCAGCGCAGGTTATCCACCTCTGCCTGGAAGTCTATACTCTACCCAGGGCATCCCTCTGGTCAG
ATCCAGTCTCC

V2i, SNP133S2456 of SEQ ID NO: 22 .1352 as MW at 145674.11cD
106951-Ol, Protein Sequence SNP Change: no cha KPAGEGASAHRAGHHTRQARGGSRPSSRGMQGAPSRSSARLEAGGCSARRGRSAPAPSSFSLPLPSFSPFACN
SSPTAPSLLLLPRSPPPCSLRAPGRELVGARGLVPEPSSAEPGGSAAHPAAAGSPSAAGAGPGGDCTGALRAG

PDTPAQQLRCGWTVGGWLLSLVRGLLPCLPPGARTAEGPIMVLAGPLAVSLLLPSLTLLVSHLSSSQDVSSEP
SSEQQLCALSKHPTVAFEDLQPWVSNFTYPGARDFSQLALDPSGNQLIVGARNYLFRLSLANVSLLQATEWAS
SEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAFSPMCTSRQVGNLSRTTEKINGVARCPYDPRHNS
TAVISSQGELYAATVIDFSGRDPAIYRSLGSGPPLRTAQYNSKWLNEPNFVAAYDIGLFAYFFLRENAVEHDC
GRTVYSRVARVCKNDVGGRFLLEDTWTTFMKARLNCSRPGEVPFYYNELQSAFHLPEQDLIYGVFTTNVNSIA
ASAVCAFNLSAISQAFNGPFRYQENPRAAWLPIANPIPNFQCGTLPETGPNENLTERSLQDAQRLFLMSEAVQ
PVTPEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESGTILKALSTASRSLHGCYLEELHVLPPGRREPLR
SLRILHSAR.ALFVGLRDGVLRVPLERCAAYRSQGACLGARDPYCGWDGKQQRCSTLEDSSNMSLWTQNITACP
VRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWAL
CSTSCGIGFQVRQRSCSNPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRA
CGGGHYQRTRSCTSPAPSPGEDICLGLHTEEALCATQACPEG
CAGNSSQSRPCPYSEIPVILPASSMEEATGCAGFNLIHLVAT
VHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLIPDDRAN
PLNKHSFRPEASPGQRCFPNS
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 MPAGEGASAHRAGHHTRQARGGSRPSSRGMQGAPSRSSARLEAGGCSARRGRSAPAPSSF
NOV2b ____________________________________________________________ NOV2c ____________________________________________________________ NOV2d ____________________________________________________________ NOV2e ____________________________________________________________ NOV2f ____________________________________________________________ NOV2g ____________________________________________________________ NOV2h -___________________________________________________________ NOV2a SLPLPSFSPFACNSSPTAPSLLLLPRSPPPCSLRAPGRELVGARGLVPEPSSAEPGGSAA
NOV2b ____________________________________________________________ NOV2C ____________________________________________________________ NOV2d ____________________________________________________________ NOV2e ____________________________________________________________ NOV2f ____________________________________________________________ NOV2g ____________________________________________________________ NOV2h ____________________________________________________________ NOV2a HPAAAGSPSAAGAGPGGDCTGALRAGGRSCAAAPFPDRPPAHLVSSRRSAPPGSREPRGT
NOV2b ____________________________________________________________ NOV2C ____________________________________________________________ NOV2d ____________________________________._______________________ NOV2e ____________________________________________________________ NOV2f ____________________________________________________________ NOV2g ____________________________________________________________ NOV2h ____________________________________________________________ NOV2a GHLHPPLGVSGSSWCLACVSWMPCGFSPSPVAHHLVPGPPDTPAQQLRCGWTVGGWLLSL
NOV2b ---------------------MPCGFSPSPVAHHLVPGPPDTPAQQLRCGWTVGGWLLSL
____________________________________________________________ 'NOV2c 'NOV2d ____________________________________________________________ INOV2e ____________________________________________________________ NOV2f ____________________________________________________________ NOV2g ___________________________________-________________________ NOV2h ____________________________________________________________ NOV2a VRGLLPCLPPGARTAEGPIMVLAGPLAVSLLLPSLTLLVSHLSSSQDVSSEPSSEQQLCA
NOV2b VRGLLPCLPPGARTAEGPIMVLAGPLAVSLLLPSLTLLVSHLSSSQDVSSEPSSEQQLCA
NOV2c ____________________________________________________________ NOV2d _____________________________________-______________________ NOV2e ____________________________________________________________ NOV2f ____________________________________________________________ NOV2g -------------------~GPLAVSLLLPSLTLLVSHLSSSQDVSSEPSSEQQLCA
NOV2h _____________________________________________.______________ NOV2a LSKHPTVAFEDLQPWVSNFTYPGARDFSQLALDPSGNQLIVGARNYLFRLSLANVSLLQA
NOV2b LSKHPTVAFEDLQPWVSNFTYPGARDFSQLALDPSGNQLIVGARNYLFRLSLANVSLLQA
NOV2c ______________________________________________-_____________ NOV2d ____________________________________________________________ NOV2e ___-________________________________________________________ NOV2f ____________________________________________________________ NOV2g LSKHPTVAFEDLQPWVSNFTYPGARDFSQLALDPSGNQLIVGARNYLFRLSLANVSLLQA
NOV2h ____________________________________________________________ NOV2a TEWASSEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAFSPMCTSRQVGNLSRT
NOV2b TEWASSEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAFSPMCTSRQVGNLSRT
____________________________________________________________ NOV2c NOV2d ____________________________________________________________ NOV2e ____________________________________________________________ NOV2f ____________________________________________________________ NOV2g TEWASSEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAFSPMCTSRQVGNLSRT
NOV2h ____________________________________________________________ NOV2a TEKINGVARCPYDPRHNSTAVISSQGELYAATVIDFSGRDPAIYRSLGSGPPLRTAQYNS
NOV2b TEKINGVARCPYDPRHNSTAVISSQGELYAATVIDFSGRDPAIYRSLGSGPPLRTAQYNS
NOV2c ____________________________________________________________ NOV2d ____________________________________________________________ NOV2e ____________________________________________________________ NOV2f ____________________________________________________________ NOV2g TEKINGVARCPYDPRHNSTAVISSQGELYAATVIDFSGRDPAIYRSLGSGPPLRTAQYNS
NOV2h ____________________________________________________________ NOV2a KWLNEPNFVAAYDIGLFAYFFLRENAVEHDCGRTVYSRVARVCKNDVGGRFLLEDTWTTF
NOV2b KWLNEPNFVAAYDIGLFAYFFLRENAVEHDCGRTVYSRVARVCKNDVGGRFLLEDTWTTF
NOV2c ____________________________________________________________ NOV2d ____________________________________________________________ NOV2e ____________________________________________________________ NOV2f ____________________________________________________________ NOV2g KWLNEPNFVAAYDIGLFAYFFLRENAVEHDCGRTVYSRVARVCKNDVGGRFLLEDTWTTF
NOV2h _______-____________________________________________________ NOV2a MKARLNCSRPGEVPFYYNELQSAFHLPEQDLIYGVFTTNVNSIAASAVCAFNLSAISQAF
NOV2b MKARLNCSRPGEVPFYYNELQSAFHLPEQDLIYGVFTTNVNSIAASAVCAFNLSAISQAF
NOV2c ____________________________________________________________ NOV2d ____________________________________________________________ NOV2e ____________________________________________________________ NOV2f _____________________________!______________________________ NOV2g MKARLNCSRPGEVPFYYNELQSAFHLPEQDLIYGVFTTNVNSIAASAVCAFNLSAISQAF
NOV2h _____________________________-___________________________ NOV2a NGPFRYQENPRAAWLPIANPIPNFQCGTLPETGPNENLTERSLQDAQRLFLMSEAVQPVT
NOV2b NGPFRYQENPRAAWLPIANPIPNFQCGTLPETGPNENLTERSLQDAQRLFLMSEAVQPVT
NOV2C ______________________________________-____________-________ NOV2d ____________________________________________________________ NOV2e _____________-______________________________________________ NOV2f ____________________________________________________-_______ NOV2g NGPFRYQENPRAAWLPIANPIPNFQCGTLPETGPNENLTERSLQDAQRLFLMSEAVQPVT
NOV2h ____________________________________________________________ NOV2a PEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESGTILKALSTASRSLHGCYLEELHV
NOV2b PEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESGTILKALSTASRSLHGCYLEELHV
NOV2c ____________________________________________________________ NOV2d ____________________________________________-_______________ NOV2e ________________________________-____________-___-__________ NOV2f ____________________________________________________________ NOV2g PEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESGTILKALSTASRSLHGCYLEELHV
NOV2h _____________________________________-______________________ NOV2a LPPGRREPLRSLRILHSARALFVGLRDGVLRVPLERCAAYRSQGACLGARDPYCGWDGKQ
NOV2b LPPGRREPLRSLRILHSARALFVGLRDGVLRVPLERCAAYRSQGACLGARDPYCGWDGKQ
NOV2c _______-____________________________________________________ NOV2d _____________________________________-_-____________________ NOV2e ____________________________________________________________ NOV2f _________________________________-__________________________ NOV2g LPPGRREPLRSLRILHSARALFVGLRDGVLRVPLERCAAYRSQGACLGARDPYCGWDGKQ
NOV2h ________________________________-_______________________-___ NOV2a QRCSTLEDSSNMSLWTQNITACPVRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLCRARSC
NOV2b QRCSTLEDSSNMSLWTQNITACPVRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLCRARSC
NOV2c --------------------___________GSGPWSPWQPCEHLDGDNSGSCLCRARSC
NOV2d ------------------_____________GSGPWSPWQPCEHLDGDNSGSCLCRARSC
NOV2e -------------------____________GSGPWSPWQPCEHLDGDNSGSCLCRARSC
NOV2f ------------------__-__________GSGPWSPWQPCEHLDGDNSGSCLCRARSC
NOV2g QRCSTLEDSSNMSLWTQNITACPVRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLCRARSC
NOV2h ------------------_______-_______GpWSPWQPCEHLDGDNSGSCLCRARSC
NOV2a DSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHG
NOV2b DSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGTGFQVRQRSCSNPAPRHG
NOV2c DSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHG
NOV2d DSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHG
NOV2e DSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHG
NOV2f DSPRPRCGGLDCLGPTIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHG
NOV2g DSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHG
NOV2h DSPRPRCGGLDCLGPTIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHG
NOV2a GRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCLGCG
NOV2b GRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCLGCG
NOV2c GRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMRSRRRACENGNSCLGCG
NOV2d GRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCLGCG
NOV2e GRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCLGCG
NOV2f GRICVGKSREERFCNENTPCPVPIFWASWGSWSKCGSNCGGGMQSRRRACENGNSCLGCG
NOV2g GRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCLGCG
NOV2h GRICVGKSREERFCNENTPCPVPIFWASWGSWSKCGSNCGGGMQSRRRACENGNSCLGCG
'NOV2a VEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRT

NOV2b VEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRT
NOV2c VEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRT
NOV2d VEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRT
NOV2e VEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRT
NOV2f VEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRT
'NOV2g VEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRT
NOV2h VEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRT
INOV2a ETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRT
NOV2b ETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRT
NOV2c ETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRT
NOV2d ETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRT
NOV2e ETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRT
NOV2f ETRTCPADGSGSCDTDALVEVLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRT
NOV2g ETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRT
NOV2h ETRTCPADGSGSCDTDALVEVLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRT
NOV2a CTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTS
NOV2b CTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTS
NOV2c CTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTS
NOV2d CTNPESRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTS
NOV2e CTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTS
NOV2f CTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTS
NOV2g CTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTS
NOV2h CTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTS
NOV2a PAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSAC
NOV2b PAPSP---------------------EGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSAC

NOV2d PAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSAC
NOV2e PAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSAC
NOV2f PAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSAC
NOV2g PAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSAC
NOV2h PAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSAC
NOV2a AGNSSQSRPCPYSEIPVILPASSMEEATGCAGFNLIHLVATGISCFLGSGLLTLAVYLSC
NOV2b AGNSSQSRPCPYSEIPVILPASSMEEATGCAGFNLIHLVATGISCFLGSGLLTLAVYLSC
NOV2c AGNSSQSRPCVD-_______________________________________________ NOV2d AGNSSQSRPCVD-_______________________________________________ NOV2e AGNSSQSRPCVD-_______________________________________________ NOV2f AGNSSQSRPCVD-_______________________________________________ NOV2g AGNSSQSRPCPYSEIPVILPASSMEEATGCAGFNLIHLVATGISCFLGSGLLTLAVYLSC
NOV2h AGNSSQSRPC-_________________________________________________ NOV2a QHCQRQSQESTLVHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLIPDDRANFYPLQQ
NOV2b QHCQRQSQESTLVHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLIPDDRANFYPLQQ
NOV2c ____________________________________________________________ NOV2d ____________________________________________________________ NOV2e ____________________________________________________________ NOV2f ____________________________________________________________ NOV2g QHCQRQSQESTLVHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLIPDDRANFYPLQQ
NOV2h ____________________________________________________________ NOV2a TNVYTTTYYPSPLNKHSFRPEASPGQRCFPNS
NOV2b TNVYTTTYYPSPLNKHSFRPEASPGQRCFPNS
NOV2c ________________________________ NOV2d ________________________________ NOV2e ________________________________ NOV2f ________________________________ NOV2g TNVYTTTYYPSPLNKHSFRPEASPGQRCFPNS
NOV2h ________________________________ NOV2a (SEQ ID NO: 6) NOV2b (SEQ ID NO: 8) NOV2c (SEQ ID NO: 10) NOV2d (SEQ ID NO: 12) NOV2e (SEQ ID NO: 14) NOV2f (SEQ ID NO: 16) NOV2g (SEQ ID NO: 18) NOV2h (SEQ ID NO: 20) Further analysis of the NOV2a protein yielded the following properties shown in Table 2C.
Table 2C. Protein Sequence Properties NOV2a SignalP analysis: ' No Known Signal Sequence Predicted PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length 11; pos.chg 1; neg.chg 1 H-region: length 5; peak value -8.91 PSG score: -13.31 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -7.65 possible cleavage site: between 53 and 54 »> Seems to have no N-terminal signal peptide ALOM: Klein et al's method for TM region allocation Init position for calculation: 1 Tentative number of TMS(s) for the threshold 0.5: 3 Number of TMS(s) for threshold 0.5: 1 INTEGRAL Likelihood = -4.83 Transmembrane 259 - 275 PERIPHERAL Likelihood = 1.54 (at 232) ALOM score: -4.83 (number of TMSs: 1) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 266 Charge difference: -3.5 C(-2.5) - N( 1.0) N >= C: N-terminal side will be inside » > membrane topology: type 2 (cytoplasmic tail 1 to 259) MITDISC: discrimination of mitochondrial targeting seq R content. 7 Hyd Moment(75): 4.89 Hyd Moment(95): 4.42 G content: 7 D/E content: 2 S/T content: 8 Score. -1.94 Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 104 LRAIPG
NUCDISC: discrimination of nuclear localization signals pat4: none pat7: none bipartite. none content of basic residues: 9.3%
NLS Score: -0_47 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals: none SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif:
type 1: none type 2: none NMYR: N-myristoylation pattern : none Prenylation motif: none memY~RL: transport motif from cell surface to Golgi: none Tyrosines in the tail: too long tail Dileucine motif in the tail: found LL at 81 LL at 82 LL at 83 LL at 237 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 1 total: 0 residues Final Results (k = 9/23):
47.8 %: nuclear 26.1 %: mitochondrial 8.7 %: cytoplasmic 4.3 %: Golgi 4.3 %: plasma membrane 4.3 %: extracellular, including cell wall 4.3 %: peroxisomal » prediction for CG106951-O1 is nuc (k=23) A search of the NOV2a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table ZD.

Table 2D.
Geneseq Results for NOV2a NOV2a Identities/

Geneseq Protein/Organism/LengthResidues/similarities Expect [Patent for the Identifier#, Date] Match Value Matched Region Residues AAE18212Human MOL4 protein - 1..1352 1352/1352 0.0 Homo (100%) sapiens,1352 aa. 1..1352 1352/1352 (100%) [W0200206339-A2, 24-JAN-2002]

AAG68293Human semaphorin G-like202..13521150/1151 0.0 NHP (99%) protein SEQ ID NO:l 1..1151 1150/1151 0 - Homo (99%) Sapiens, 1151 aa.

[W0200188133-A2, 22 NOV-2001]

AAG68294Human semaphorin G-like202..13521135/1151 0.0 NHP (98%) protein SEQ ID N0:12 1..1136 1135/1151 - Homo (98%) sapiens,1136 aa.

[W0200188133-A2, 22 NOV-2001]

AAG68290Human semaphorin G-like~ 260..13521092/1093 0.0 NHP (99%) protein SEQ ID N0:4 1..1093 1092/1093 - Homo (99%) Sapiens, 1093 aa.

[W0200188133-A2, 22 NOV-2001]

AAG68292Human semaphorin G-like260..13521077/1093 0.0 NHP (98%) protein SEQ ID N0:8 1..1078 1077/1093 - Homo (98%) sapiens, 1078 aa.

[W0200188133-A2, 22-NOV-2001]

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 NOV2a Protein Identities!

AccessionProtein/Organism/LengthResidues/Similarities E~Pect for the Number Matched PortionValue R sidues Q9P283 Hypothetical protein 151..13521202/1202 (100%)0.0 Homo sapiens (Human),12021..1202 120211202 (100%) as (fragment).

Q60519 Semaphorin SB precursor260..13521021/1093 (93%)0.0 (Semaphorin G) (Sema 1..1093 1053/1093 (95%) G) - Mus musculus (Mouse), 1093 aa.

Q13591 Semaphorin SA precursor299..1336616/1043 (59%)0.0 (Semaphorin F) (Sema 30..1071781/1043 (74%) F) - Homo Sapiens (Human),1074 aa.

Q62217 Semaphorin SA precursor299..1336617/1046 (58%)0.0 (Semaphorin F) (Sema 30..1074776/1046 (73%) F) - Mus musculus (Mouse), 1077 aa.

Q8BXLT8 Sema domain - Mus musculus299..1109507/811 (62%) 0.0 (Mouse), 844 aa. 30..839 632/811 (77%) 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 Sema 327..738 217/491 (44%) 7e-202 372/491 (76%) PSI 756..803 18/67 (27%) 2.Se-14 40/67 (60%) tsp_1 869..920 23/54 (43%) 3.Se-12 3 8/54 (70%) tsp_I 927..971 ; 17/53 (32%) 4 3e-06 31/53 (58%) tsp 1 1058..1108 24/53 (45%) 9.1e-11 ( 64%) tsp 1 1115..1165 23/53 (43%) 5.9e-08 ( 35/53 66%) tsp_1 1170..1210 17/53 (32%) 0.0034 ( 51 %) Example 3.
The NOV3 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 3A.
3A. NOV3 Sa, CG121295-O1 SEQ ID N_O: _23 ~~750 by Sequence ORF Start: ATG at 41 ORF Ston: TGA at 701 _GAATGGATTATTTGCTCATGATTTTCTCTCTGCTG
AGGCGCTGAGCTCAGCGCGGTGGGTGAGAACGGCG
TGTGCTAGCCAAAAAGACAAGAAGTGCTGGAATTTTTGCCAAGC
AGTGAGAGGAAGAAAAATCAGAAGAAGTTCAGAGGAACACC
TTGGTGACAGACCTTCGGGGCCTGTCTGAAGCCA

CG121295-Ol ~SEp ID NO: 24 X220 as BMW at 25403.9kD
FSLLFVACQGAPETAVLGAELSAVGENGGEKPTPSPPWRLRRSKRCSCSSLMDKECWFCHLDIIW
LSLDNRHWPYGLGSPRSKRALENLLPTKATDRENRCQCASQKDKKCWNFCQAGKELRAEDIMEKD
KDCSKLGKKCIYQQLVRGRKIRRSSEEHLRQTRSETMRNSVKSSFHDPKLKGKPSRERYVTHNRAH
Further analysis of the NOV3a protein yielded the following properties shown in Table 3B.
Table 3B. Protein Sequence Properties NOV3a SignalP analysis: Cleavage site between residues 1 ~ and 19 PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length 2; pos.chg 0; neg.chg 1 H-region: length 17; peak value 0.00 PSG score: -4.40 GvH: von Heijne's method for signal seq_ recognition GvH score (threshold: -2.1): 1.68 possible cleavage site: between 17 and 18 »> Seems to have no N-terminal signal peptide ALOM: Klein et al's method for TM region allocation Init position for calculation: 1 Tentative number of TMS(s) for the threshold 0.5: 1 Number of TMS(s) for threshold 0.5: 0 PERIPHERAL Likelihood = 6.31 (at 67) ALOM score: -1.59 (number of TMSs: 0) MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment(75): 4.56 Hyd Moment(95): 7.21 G content: 1 D/E content: 2 S/T content: 1 Score: -7.31 Gavel: prediction of cleavage sites for mitochondrial preseq cleavage site motif not found NUCDISC: discrimination of nuclear localization signals pat4: none pat7: PPWRLRR (3) at 43 pat7: PWRLRRS (4) at 44 pat7: PRSKRAL (5) at 96 bipartite: KKCIYQQLVRGRKIRRS at 161 content of basic residues: 18.6 NLS Score: 1.05 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):
69.6 %. nuclear 13.0 %. mitochondrial 8.7 %: extracellular, including cell wall 8.7 %. cytoplasmic » prediction for CG121295-O1 is nuc (k=23) A search of the NOV3a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 3C.

Table 3C.
Geneseq Results for NOV3a NOV3a Identities/

Geneseq Protein/Organism/Length Residues/SimilaritiesExpect [Patent #, for IdentifierDate] Match the Matched Value ResiduesRegion ABU03518Angiogenesis-associated 1..220 211/220 (95%)e-125 human protein sequence #63 - Homo Sapiens,1..212 212/220 (95%) 212 aa.

[W0200279492-A2,10-OCT-2002]

ABP65215Hypoxia-regulated protein1..220 211/220 (95%)e-125 #89 - Homo Sapiens, 212 aa. [WO200246465-A2,1..212 212/220 (95%) 13-JUN-2002]

AAG64862Heart muscle cell differentiation1..220 211/220 (95%)e-125 related protein SEQ 1D NO: 65 1..212 212/220 (95%) - Homo Sapiens, 212 aa. [W0200148151-Al, 05-JUL-2001 ]

AAB99933Human ETl protein sequence1..220 ~ 211/220 e-125 SEQ ID (95%) N0:65 - Homo Sapiens, 1..212 212/220 (95%) 212 aa.

[W0200148150-Al, 05-JLJL-2001]

AAB00197Preproendothelin-1 - Homo1..220 211/220 (95%)e-125 Sapiens, 212 aa. [W0200055314-A2, 1..212 212/220 (95%) 21-SEP-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 3D.
Table 3D.
Public BLASTP
Results for NOV3a NOV3a Protein Identities/

AccessionProtein/Organism/LengthResidues/Similarities Expect for the Number Matched PortionValue Residues P05305 Endothelin-1 precursor 1..220 211/220 (95%)e-124 (ET-1) -Homo Sapiens (Human), 1..212 212/220 (95%) 212 aa.

P17322 Endothelin-1 precursor 1_.219 148/220 (67%)3e-80 (ET-1) -Bos taurus (Bovine}, 1..202 167/220 (75%) 202 aa.

P09558 Endothelin-1 precursor 1..219 145/221 (65%)7e-78 (ET-1) - Sus scrofa (Pig), 203 aa. 1..203 168/221 (75%) P22387 Endothelin-1 precursor 1..219 147/220 (66%}le-77 (ET-1) -Mus musculus (Mouse), 1..202 1651220 (74%) 202 aa.

Q9BG76 Preproendothelin-1 - 1..219 142/220 (64%)9e-76 Ovis aries (Sheep), 202 aa. 1..202 164/220 (74%) PFam analysis predicts that the NOV3a protein contains the domains shown in the Table 3E.
Table 3E. Domain Analysis of NOV3a Identities/
Pfam Domain NOV3a Match Region Similarities Expect Value for the Matched Region endothelin 48..78 26/31 (84%) 8.6e-20 31/31 (100%) Example 4.
The NOV4 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 4A.
4A. NOV4 '4a, CG124756-Ol S~EQ ID NO: 25 ! 1076 by _ . Sequence ORF Start: ATG at 75 ' ORF Stop: TGA at 834 TGATGATGAAGATCCCATGGGGCAGCATCCCAGTACTGATGTTGCTCCTGCTCCTGGGCCTAATCGATATC
CCAGGCCCAGCTCAGCTGCACCGGGCCCCCAGCCATCCCTGGCATCCCGGGTATCCCTGGGACACCTGGCC
AAAGGTGGCCCAGGGGCCCCTGGAGCCCCAGGCCCCAAAGGTGAATCGGGAGACTACAAGGCCACCCAGAAAA
TCGCCTTCTCTGCCACAAGAACCATCAACGTCCCCCTGCGCCGGGACCAGACCATCCGCTTCGACCACGTGAT
CACCAACATGAACAACAATTATGAGCCCCGCAGTGGCAAGTTCACCTGCAAGGTGCCCGGTCTCTACTACTTC
ATGCCTACAACACCTTCCAGGTCACCACCGGTGGCATGGTCCTCAAGCTGGAGCAGGG
GCAGGCCACCGACAAGAACTCACTACTGGGCATGGAGGGTGCCAACAGCATCTTTTCC
(GCAACGCTCACTCTACCCCCAACACCACCCCTTGCCCAGCCAATGCACACAGTAGGGCTTGGTGAATGCTGCT
GAGTGAATGAGTAAATAAACTCTTCAAGGCCAAGGG
IO
CG124756-O1 SEQ ID NO: 26 253 as ~MW at 26721.S1cD
GSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLAGDHGE
GIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQKIAFSATRTINVPLRRDQTIRFDHVI
EPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQG
TDKNSLLGMEGANSIFSGFLLFPDMEA

V4b, CG124756-02 SEQ ID NO: 27 816 by _ A Sequence _ __ __ ~ ORF Start: ATG at 48 _ O__RF_Sto_p: T_GA at 807 _ ~GTAACCTTCACATTGTCTTCTCCACAGGAGGCGTCTGACACAGTATGATGATGAAGATCCCATGGGGCAG
.'CCCAGTACTGATGTTGCTCCTGCTCCTGGGCCTAATCGATATCTCCCAGGCCCAGCTCAGCTGCACCGGG
:CCAGCCATCCCTGGCATCCCGGGTATCCCTGGGACACCTGGCCCCGATGGCCAACCTGGGACCCCAGGGA
CCTGGAGCC
CGGGTTCCTGCTCTTTCCAGATATGGAG
4b, CG124756-OZ ~SEQ ID NO: 28 X253 as BMW at 26721.SkD
PWGSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLAGDHGE
DPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQKIAFSATRTINVPLRRDQTIRFDHVI
IFSGFLLFPDMEA
V4c, SNP13382475 of SEQ ID NO: 29 1076 by _ 124756-Ol, DNA Sequence O~' Start: ATG at 75 ORF Stop: TGA at 834 :. . _ .__ .
SNP Pos: 302 SNP Chance. G to T
TCCCAGTACTGATGTTGCTCCTGCTCCTGGGCCTAATCGATATC
CCCAGCCATCCCTGGCATCCCGGGTATCCCTGGGACACCTGGCC
TAAGGGAGACCCAGGGATTCCTGGGAATCCAGGAAAAGTCGGCCCCAAGGGCCCCATGGGCCCT
ACAACAATTATGAGCCCCGCAGTGGCAAGTTCACCTGCAAGGTGCCCGGTCTCTACTACTTC
CAGCTCTCGAGGGAACCTGTGCGTGAACCTCATGCGTGGCCGGGAGCGTGCACAGAAGGTGG
GACTATGCCTACAACACCTTCCAGGTCACCACCGGTGGCATGGTCCTCAAGCTGGAGCAGGG
CTTTCCAGATATGGAGGCCTGACCTGTGGGCTGCTTCACATCCACCCCGGCTCCCCCTGCCA
TCTACCCCCAACACCACCCCTTGCCCAGCCAATGCACACAGTAGGGCTTGGTGAATGCTGCT

V4c, SNP13382475 of SEQ ID NO: 30 253 as ~MW at 26707.SkD
124756-Ol, Protein Sequence ~SNp pos: 76 SNP Chance: Glu to MKIPWGSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLAGDHGE
_DKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQKIAFSATRTINVPLRRDQTIRFDHVI
iMNNNYEPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQG
iVFLQATDKNSLLGMEGANSIFSGFLLFPDMEA
V4d, SNP13382476 of SEQ ID NO: 31 1076.,bp.,...
124756-01, DNA Sequence ORF Start: ATG at 75 O_R_F _S_top:_YTGA_at 8_34 SNP Pos: 433 SNP~~Chan~e: A to~G
TGATGATGAAGATCCCATGGGGCAGCATCCCAGTACTGATGTTGCTCCTGCTCCTGGGCCTAATCGATATC
CCAGGCCCAGCTCAGCTGCACCGGGCCCCCAGCCATCCCTGGCATCCCGGGTATCCCTGGGACACCTGGCC
TCAACGTCCCCCTGCGCCGGGACCAGACCATCCGCTTCGACCACGTGAT
TCACCTTCTGTGACTATGCCTACAACACCTTCCAGGTCACCACCGGTGGCATGGTCCTCAAGCTGGAGCAGGG
GGAGAACGTCTTCCTGCAGGCCACCGACAAGAACTCACTACTGGGCATGGAGGGTGCCAACAGCATCTTTTCC
GGGTTCCTGCTCTTTCCAGATATGGAGGCCTGACCTGTGGGCTGCTTCACATCCACCCCGGCTCCCCCTGCCA
GCAACGCTCACTCTACCCCCAACACCACCCCTTGCCCAGCCAATGCACACAGTAGGGCTTGGTGAATGCTGCT
[GAGTGAATGAGTAAATAAACTCTTCAAGGCCAAGGG
NOV4d, SNP13382476 of SEQ ID NO: 32 ~ 2_53 as ~MW at 26749.6kD _ CG124756-Ol, Protein Sequence SNP Pos: 120 Y ~ SNP Change:~Gln to Arg MMMKIPWGSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLAGDHGE
FGEKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKAT_RKIAFSATRTINVPLRRDQTIRFDHVI
TNMNNNYEPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVZ'FCDYAYNTFQVTTGGMVLKLEQG
ENVFLQATDKNSLLGMEGANSIFSGFLLFPDMEA
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 MMMKIPWGSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKG
NOV4b MMMKIPWGSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKG
NOV4a EKGLPGLAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQ
NOV4b EKGLPGLAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQ
NOV4a KIAFSATRTINVPLRRDQTIRFDHVITNMNNNYEPRSGKFTCKVPGLYYFTYHASSRGNL
NOV4b KIAFSATRTINVPLRRDQTIRFDHVITNr4rINNYEPRSGKFTCKVPGLYYFTYHASSRGNL
NOV4a CVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQGENVFLQATDKNSLLGMEGANS
NOV4b CVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQGENVFLQATDKNSLLGMEGANS
NOV4a IFSGFLLFPDMEA
NOV4b IFSGFLLFPDMEA
NOV4a (SEQ ID NO: 26) NOV4b (SEQ ID NO: 28) Further analysis of the NOV4a protein yielded the following properties shown in Table 4C.
Table 4C. Protein Sequence Properties NOV4a SignaIP analysis: Cleavage site between residues 28 and 29 PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length 4; pos.chg 1; neg.chg 0 H-region: length 18; peak value 11.91 PSG score: 7.51 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 4.21 possible cleavage site: between 27 and 28 » > Seems to have a cleavable signal peptide (1 to 27) ALOM: Klein et al's method for TM region allocation Init position for calculation: 28 Tentative number of TMS(s) for the threshold 0.5: 0 number of TMS(s) .. fixed PERIPHERAL Likelihood = 2.60 (at 232) ALOM score: 2.60 (number of TMSs: 0) MTOP: Prediction of membrane topology (Hartmann et al_) Center position for calculation: l3 Charge difference: -3.0 C(-1.0) - N( 2.0) N >= C: N-terminal side will be inside MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment(75): 6.93 Hyd Moment(95): 5.45 G content: 2 D/E content: 1 S/T content: 1 Score. -5.61 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: 9.5%
NLS Score: -0.47 'KDEL: ER retention motif in the C-terminus: none iER Membrane Retention Signals: none iSKL: peroxisomal targeting signal in the C-terminus: none IIPTS2: 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 PROSTTE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: cytoplasmic _ Reliability: 76.7 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues Final Results (k = 9/23).
22.2 %: extracellular, including cell wall 22.2 %. vacuolar 22.2 %: mitochondrial 22.2 %: endoplasmic reticulum ll.l %: Golgi » prediction for CG124756-01 is exc (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 Protein/Organism/Length Residues/SimilaritiesExpect [Patent #, for Ident'erDate] Match the Matched Value ResiduesRegion AAM40607Human polypeptide SEQ 1..253 253/253 (100%)e-151 - Homo Sapiens, 255 aa. 3..255 253/253 (100%) [W0200153312 Al, 26-JLJL-2001]

AAM38821Human polypeptide SEQ I ..253 253/253 (100%)e-151 - Homo Sapiens, 253 aa. 1..253 253/253 (100%) [W0200153312-Al, 26-JUL-2001]

ABB57231Mouse ischaemic condition3..253 201/253 (79%)e-117 related protein sequence SEQ 1..253 218/253 (85%) ID N0:599 -Mus musculus, 253 aa.

[W0200188188-A2, 22 NOV-2001]

AAU32411Novel human secreted 1..248 203/267 (76%)e-103 protein #2902 -Homo Sapiens, 309 aa. 3..269 212/267 (79%) [WO200179449-A2, 25-OCT-2001]

AAU30709Novel human secreted 23..248 195/243 (80%)e-102 protein #1200 -Homo Sapiens, 287 aa. 2..244 199/243 (81%) [W0200179449-A2, 25-OCT-2001]

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

Table 4E.
Public BLASTP
Results for NOV4a Protein NOV4a Identities) AccessionProtein/Organism/LengthResidues/Similarities Expect for the Number Matched PortionValue Residues C1HUQB complement subcomponent1..253 253/253 (100%)e-151 Clq chain B precursor [validated]1..253 253/253 (100%) -human, 253 aa.

P02746 Complement Clq subcomponent,3..253 251/251 (100%)e-150 B

chain precursor - Homo 1..251 251/251 (100%) sapiens (Human), 251 aa.

P14106 Complement Clq subcomponent,3..253 2011253 (79%)e-117 B

chain precursor - Mus 1..253 219/253 (86%) musculus (Mouse), 253 aa.

I49560 complement Clq B chain 3..253 201/253 (79%)e-117 precursor -mouse, 253 aa. 1..253 ' 218/253 (85%) P31721 Complement Clq subcomponent,3..252 197/252 (78%)e-115 B

chain precursor - Rattus1..252 217/252 (85%) norvegicus (Rat), 253 aa.

PFam analysis predicts that the NOV4a protein contains the domains shown in the Table 4F.
Table 4F. Domain Analysis of NOV4a Identities/
Pfam Domain NOV4a Match Region Similarities Expect Value for the Matched Region Collagen 51..110 35/60 (58%) 8.7e-09 45/60 (75%) Clq 123..247 69/138 (50%) 2.4e-72 124/138 (90%) Example 5.
The NOVS clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table SA.

able SA. NOVS Sequence Analysis fOVSa, CG50353-Ol SEQ 1D NO: 33 ~ ' 1628 by ANA Sequence OItF Start: ATG at 1 ORF Stop: TGA at 1048 TGAACCGGAAAGCGCGGCGCTGCCTGGGCCACCTCTTTCTCAGCCTGGGCATGGTCTGTCTCCTAGCATGTG
CTTCTCCTCAGTGGTAGCTCTGGGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCG
GCGATCTGCCAGAGCCGGCCCGACGCCATCATCGTCATAGGAGAAGGCTCACAAATGGGCCTGGACGAGTGT
CGGG
GCGCACGGAGATGTACACGTGCAAGTGAGCCCCGTGTGCACACCACCCTCCCGCTGCAAGTCAGATTGCTGGG
T PTT P~ml~l~T /'~I~P~TTTI~I~T T l~P~ml~l~l~P~Tl~m/'Il"~!'tml'fP~I~T l1/~T
mlYlYm~ ~n ~~mm~mr~mmmm~m~nm~ ~n iv~-w .~r~nm,. .r CCCAATGCTGCTCCACCCTCCCCCAGACACAGCCCAGGTCCCTCCGCGGCTGGAGCGAAGCCTTCTGCAGCAG
GAACTCTGGACCCCTGGGCCTCATCACAGCAATATTTAACAATTTATTCTGATAAAAATAATATTAATTTATT
(AGGATGATTTTGTTGCTAGGACAAGGAGCCGTGTAGAAGTGTACATAACTATTCTTTATGCAGATATTTCTAC
TAGCTGATTTTGCAGGTACCCACCTTGCAGCACTAGATGTTTAAGTACAAGAGGAGACATCTTTTATGCATAT
ATAGATATACACACACGAAAAA
a, CG50353-O1 SEQ ID NO: 34 349 as MW at 38980.7kD
LFLSLGMVCLLACGFSSVVALGATVICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDEC
SALGERTVFGKELKVGSRDGAFTYAIIAAGVAHAITAACTHGNLSDCGCDKEKQGQYHRDEG
YGIGFAKVFVDAREIMKNARRLMNLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCWTTLP
KYNAAVQVEVVRASRLRQPTFLRIKQLRSYRKPMKTDLVYIEKSPNYCEEDPVTGSVGTQGR
5b, 228753443 SEQ ID NO: 35 966 by Sequence ORF Start: at 1 ORF Stop: end of sequence ~~
CTCTGGGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCGGGCGATCTGCCAGA
TCATTGCCGCCGGCGTGGCCCACGCCATCACAGCTGCCTGTACCCAGGGCA
CACAGTTTCGGGAGCT
TCAAGAAGCCACTGTCGTACCGCAAGCCCATGGACACGGACCTGGTGTACATCGAGA
TGT

228753443 ~SEQ ID NO: 36 322 as MW at 36054.9kD
VICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSR
AIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQN
LHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKR
KKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARV
>c, 169475673 SEQ ID NO:_37 J966 by Sequence ~pRF Start: at 1 J' ORF Ston: end of TCGTCATAGGAGAAGGCTCACAAATGGGCCTGGACGAGTGTCAGTTTCAGTTCCG
CTCATGAACTTGCACAACAACGAGGCAGGCCGAAAGATCCTGGAGGAGAACATGAAGCTGGAAT
169475673 ~SEQ ID NO: 38 X322 as BMW at 36054.9kD
VICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSR
AIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQN
VYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARV
VSd, 228753459 SEQ ID NO: 39 f 966 by __. __ .. _ _ ~ "~",~~,.~, ..~_____.._ _ _.__~._.. ~ ~"
~1 Sequence ORF Start: _at 1 _ ~ ORF Stop: end of sequence TCTCTGGGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCGGGCGATCTGCCAGA
TTCACCTACGCCATCATTGCCGCCGGCGTGGCCCACGCCATCACAGCTGCCTGTACCCAGGGCA
CTGCTCCGCCGACATCCGCTACGGCATCGGCTTCGCCAAGGTCTTTGTGGATGCCCGGGAGATCAAGCAGAAT
GCCCGGACTCTCATGAACTTGCACAACAACGAGGCAGGCCGAAAGATCCTGGAGGAGAACATGAAGCTGGAAT
GTAAGTGCCACGGCGTGTCAGGCTCGTGCACCACCAAGACGTGCTGGACCACACTGCCACAGTTTCGGGAGCT
CCCACCTTCCTGAAGATCAAGAAGCCACTGTCGTACCGCAAGCCCATGGACACGGACCTGGTGTACATCGAGA

Sd, 228753459 SEQ m NO: 40 322 as MW at 36054.9kD
CQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSR
KKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARV
VSe, 228753462 SEQ ID NO: 41 966 by Sequence ORF Start: at 1 ~y ORF Stop: end of sequence TCTCTGGGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCGGGCGATCTGCCAGA
.TGGCCGCTGGAACTGCTCTGCACTGGGAGAGCGCACCGTCTTCGGGAAGGAGCTCAAAGTGGGGAGCCGG
228753462 SEQ m NO: 42 322 as MW at 36083.OkD
TVICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSR
YAIIAAGVVHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQN
NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKR
IKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARV
if, 228753446 SEQ ID NO: 43 985 by ___ __ Sequence ~ORF Start: at 2 ~ ~~: ORF Stop: end of AGGAGAAGGCTCACAAATGGGCCTGGACG
TCCTGGAG
CCAGCCGCAACAAGCGGCCCACCTTCCTGAAGATCAAGAAGCCACTGTCGTACCGCAAGCCCATGGACACG
CCTGGTGTACATCGAGAAGTCGCCCAACTACTGCGAGGAGGACCCGGTGACCGGCAGTGTGGGCACCCAGG
CGCGCCTGCAACAAGACGGCTCCCCAGGCCAGCGGCTGTGACCTCATGTGCTGTGGGCGTGGCTACAACAC
ACCAGTACGCCCGCGTGTGGCAGTGCAACTGTAAGTTCCACTGGTGCTACTATGTCAAGTGCAACACGTGC

228753446 1SE0 ID NO: 44 X328 as BMW at 36733.6kD
EFALRSLGATVICNKIPGLAPRQR.AICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKE
VGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA
TKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVR
RNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNT
ig, 228753465 SEQ ID NO: 45 966 by Sequence ~gp' Start: at 1 ~ ORF Stop: end of sequence ~TCTGGGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCGGGCGATCTGCCAGA
T
CAAGGACAAGTACAACGAGGCCGTTCACGTGGAGCCTGTGCGTGCCAGCCGCAACAAGCGG
AAGATCAAGAAGCCACTGTCGTACCGCAAGCCCATGGACACGGACCTGGTGTACATCGAGA
ACTGCGAGGAGGACCCGGTGACCGGCAGTGTGGGCACCCAGGGCCGCGCCTGCAACAAGAC
TGTCAAGTGCAACACGTGCAGCGAGCGCACGGAGATGT
g, 228753465 SEQ ID NO: 46 322 as ~MW at 36173.OkD
VICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSR
AIIAAGVAHAITAACTQGNLSDCDCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQN
LHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKR
KKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARV
FHWCCYVKCNTCSERTEMYTCKLE
Sh q 28753438 O~ ~Ovy47 ~~~~966 b "...~ p ..d.~...~...._~..._.._...~._.~~____...._._ Se,~.uence,.
.._......__...____._._....._..___.__.._...__..at...l..._.....~..___._._._~~_Sto .._~.._end of sequence,.w., .._..._.....____..__ CTCTGGGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCGGGCGATCTGCCAGA
TGGCCGCTGGAACTGCTCTGCACTGGGAGAGCGCACCGTCTTCGGGAAGGAGCTCAAAGTGGGGAGCCGG
GCTGCGTTCACCTACGCCATCATTGCCGCCGGCGTGGCCCACGCCATCACAGCTGCCTGTACCCAGGGCA
TCCGCTACGGCATCGGCTTCGCCAAGGTCTTTGTGGATGCCCGGGAGATCAAGCAGAAT
TGTCAAGTGCAACACGTGCAGCGAGCGCACGGAGATGT
145 ' 228753438 SEQ 117 NO: 48 322 as ~MW at 35998.9kD
PRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSR
ITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQN
LEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKR
DTDLVYIEKSTNCCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARV
TCSERTEMYTCKLE
228753449 ~SEO ID NO: 49 Sequence ~ORF Start: at 1 ORF Stop: end of AGATCTCTGGGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCGGGCGATCTGCCAGA
GCCGGCCCGACGCCATCATCGTCATAGGAGAAGGCTCACAAATGGGCCTGGACGAGTGTCAGTTTCAGTTCCG
CAATGGCCGCTGGAACTGCTCTGCACTGGGAGAGCGCACCGTCTTCGGGAAGGAGCTCAAAGTGGGGAGCCGG
GAGGCTGCGTTCACCTACGCCATCATTGCCGCCGGCGTGGCCCACGCCATCACAGCTGCCTGTACCCAGGGCA
CTGCTCTGCCGACATCCGCTACGGCATCGGCTTCGCCAAGGTCTTTGTGGATGCCCGGGAGATCAAGCAGAAT
GCCCGGACTCTCATGAACTTGCACAACAACGAGGCAGGCCGAAAGATCCTGGAGGAGAACATGAAGCTGGGAT
ACCGCAAGCCCATGGACACGGACCTGGTGTACATCGAGA
ACGCCCGCGTG
228753449 ~SEQ ID NO: 50 X322 as BMW at 35926.8kD
VICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSR
AIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQN
LHNNEAGRKILEENMKLGCKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKR
KKPLSYRKPMDTDLVYIEKSTNCCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARV
VSj, CG503S3-02 SEQ ID NO:_5_l 966 by A Sequence ~ORF Start: at 7 ORF Stop: at 961 GGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCGGGCGATCTGCCAGA
ACGCCATCATCGTCATAGGAGAAGGCTCACAAATGGGCCTGGACGAGTGTCAGTTTCAGTTCCG
CTGGAACTGCTCTGCACTGGGAGAGCGCACCGTCTTCGGGAAGGAGCTCAAAGTGGGGAGCCGG
CTGCTCTGCCGACATCCGCTACGGCATCGGCTTCGCCAAGGTCTTTGTGGATGCCCGGGAGATCAAGCAGAAT
GCCCGGACTCTCATGAACTTGCACAACAACGAGGCAGGCCGAAAGATCCTGGAGGAGAACATGAAGCTGGAAT
CCCACCTTCCTGAAGATCAAGAAGCCACTGTCGTACCGCAAGCCCATGGACACGGACCTGGTGTACATCGAGA
TGTCAAGTGCAACACGTGCAGCGAGCGCACGGAGATGT

Sj, CG50353-02 ~SEQ ID NO: 52 318 as MW at 35569.4kD
CNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREA
IAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNAR
nnaFanRxrr.FFnmrtKr.FCKCHGVSGSCTTKTCWTTLPOFRELGYVLKDKYNEAVHVEPVRASRNKRPT
TEMYTCK
CG50353-03 SEQIDN0:53 X1057 ANA Sequence ORF Start: ATG at 1 ORF Stop: TGA at 1048 TGAACCGGAAAGCGCGGCGCTGCCTGGGCCACCTCTTTCTCAGCCTGGGCATGGTCTGTCTCCTAGCATGTG
CTTCTCCTCAGTGGTAGCTCTGGGCGCAACGGTCATCTGTAACAAGATCCCAGGCCTGGCTCCCAGACAGCG
GCGATCTGCCAGAGCCGGCCCGACGCCATCATCGTCATAGGAGAAGGCTCACAAATGGGCCTGGACGAGTGT
AAGTGGGGAGCCGGGACGGTGCGTTCACCTACGCCATCATTGCCGCCGGCGTGGCCCACGCCATCACAGCTGC
CTGTACCCATGGCAACCTGAGCGACTGTGGCTGCGACAAAGAGAAGCAAGGCCAGTACCACCGGGACGAGGGC
TGGAAGTGGGGTGGCTGCTCTGCCGACATCCGCTACGGCATCGGCTTCGCCAAGGTCTTCGTGGACGCTCGGG
AGATCATGAAGAACGCGCGGCGCCTCATGAACCTGCATAACAATGAGGCCGGCAGGAAGGTTCTAGAGGACCG
rama~ArrmarAGTGC_AAGTGCCACGGCGTGTCTGGCTCCTGCACCACCAAAACCTGCTGGACCACGCTGCCC
ATCGCAAGCCCATGAAGACGGACCT
TGCAACAAGACGGCTCCCCAGGCCAGCGGCTGTGACCTCATGTGCTGTGGGCGTGGCTACAACACCCACC
ACGCCCGCGTGTGGCAGTGCAACTGTAAGTTCCACTGGTGCTGCTATGTCAAGTGCAACACGTGCAGCGA
CACGGAGATGTACACGTGCAAGTGAGCCCCGT
CG50353-03 SEQ ID NO: 54 349 as ~MW at 38980.7kD
LSLGMVCLLACGFSSVVALGATVICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDEC
LGERTVFGKELKVGSRDGAFTYAIIAAGVAHAITAACTHGNLSDCGCDKEKQGQYHRDEG
IGFAKVFVDAREIMKNARRLMNLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCWTTLP
NAAVQVEVVRASRLRQPTFLRIKQLRSYRKPMKTDLVYIEKSPNYCEEDPVTGSVGTQGR
LMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK

BSI, SNPI3382474 of SEQ ID NO: 55 1628 bp_ ___ 0353-O1, DNA Sequence ORF Start: ATG at I OR_F Stop: T_GA at1048 SNP Pos: 951 SNP Chance: G to T
CGGCCCGACGCCATCATCGTCATAGGAGAAGGCTCACAAATGGGCCTGGACGAGTGT
CCATGGCAACCTGAGCGACTGTGGCTGCGACAAAGAGAAGCAAGGCCAGTACCACCGGGACGAGGGC
TGGGGTGGCTGCTCTGCCGACATCCGCTACGGCATCGGCTTCGCCAAGGTCTTCGTGGACGCTCGGG
TGAAGAACGCGCGGCGCCTCATGAACCTGCATAACAATGAGGCCGGCAGGAAGGTTCTAGAGGACCG
TGAAGACGGACCT
GAACTCTGGACCCCTGGGCCTCATCACAGCAATATTTAACAATTTATTCTGATAAAAATAATATTAATTTATT
TAATTAAAAAGAATTCTTCCACCTCGTCGGGATCCGTTTTCTGCAATCAAAGTGGACTGCTTGCTTTCCTAGC
AGGATGATTTTGTTGCTAGGACAAGGAGCCGTGTAGAAGTGTACATAACTATTCTTTATGCAGATATTTCTAC
TAGCTGATTTTGCAGGTACCCACCTTGCAGCACTAGATGTTTAAGTACAAGAGGAGACATCTTTTATGCATAT
SNP13382474 of ~ SEQ ID NO: 56 349 as MW at 38989.7kD
301, Protein Sequence SNP Pos: 317 ~ SNP Change: Gln to His ~RWNCSALGERTVFGKELKVGSRDGAFTYAIIAAGVAHAITAACTHGNLSDCGCDKEKQGQYHRDEG
iADIRYGIGFAKVFVDAREIMKNARRLMNLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCWTTLP
iLLKEKYNAAVQVEVVRASRLRQPTFLRIKQLRSYRKPMKTDLVYTEKSPNYCEEDPVTGSVGTQGR
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 MNRKARRCLGHLFLSLGMVCLLACGFSSWALGATVICNKIPGLAPRQRAICQSRPDAII
NOVSb ___-_________-_______________RSLGATVICNKIPGLAPRQRAICQSRPDAII
NOV5c -----------------------------RSLGATVICNKIPGLAPRQRAICQSRPDAII
NOVSd --------------------------__-RSLGATVICNKIPGLAPRQRAICQSRPDAII
NOVSe --------------------------___RSLGATVICNKIPGLAPRQRAICQSRPDAII
NOV5f -- .-----------------__S~FALRSLGATVICNKIPGLAPRQRAICQSRPDAII
NOVSg ----------------------------_RSLGATVICNKTPGLAPRQRAICQSRPDAII
NOV5h ----------------------------_RSI,GATVICNKTPGLAPRQRAICQSRPDAII
NOVSi ---------------------------__RSLGATVICNKIPGLAPRQRAICQSRPDAII
NOVSj --------------------------__-__I,GATVICNKIPGLAPRQRAICQSRPDAII
NOVSk MNRKARRCLGHLFLSLGMVCLLACGFSSWALGATVICNKTPGLAPRQRAICQSRPDAII
NOVSa VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSRDGAFTYAIIAAGVAHAIT
NOVSb VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAIT
NOV5c VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAIT
NOVSd VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAIT
NOVSe VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVVHAIT
NOVSf VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAIT
NOVSg VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAIT
NOVSh VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAIT
NOVSi VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAIT
NOVSj VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAIT
NOVSk VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSRDGAFTYAIIAAGVAHAIT
NOVSa AACTHGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIMKNARRLM
NOV5b AACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
NOVSc AACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
'NOVSd AACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
'NOVSe AACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
NOVSf AACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
NOVSg AACTQGNLSDCDCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
NOVSh AACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
NOVSi AACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
NOVSj AACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM
NOVSk AACTHGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIMKNARRLM
NOVSa NLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCWTTLPKFREVGHLLKEKYNAAVQVEV
NOVSb NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOVSc NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOV5d NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOVSe NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOV5f NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOV5g NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOV5h NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOVSi NLHNNEAGRKILEENMKLGCKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOVSj NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEP
NOVSk NLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCWTTLPKFREVGHLLKEKYNAAVQVEV
NOVSa VRASRLRQPTFLRIKQLRSYRKPMKTDLWIEKSPNYCEEDPVTGSVGTQGRACNKTAPQ
NOVSb VRASRNKRPTFLKIKKPLSYRKPMDTDLWIEKSPNYCEEDpVTGSVGTQGRACNKTAPQ
NOVSc VRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQ
NOVSd VRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDpVTGSVGTQGRACNKTAPQ
NOVSe VRASRNKRPTFLKIKKPLSYRKPMDTDLWIEKSPNYCEEDpWGSVGTQGRACNKTAPQ
NOVSf VRASRNKRPTFLKIKKPLSYRKPMDTDLWIEKSPNYCEEDPVTGSVGTQGRACNKTAPQ
NOVSg VRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNXCEEDPVTGSVGTQGRACNKTAPQ
NOVSh VRASRNKRPTFLKIKKPLSYRKPMDTDLWIEKSTNCCEEDPVTGSVGTQGRACNKTAPQ

NOVSi VRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSTNCCEEDPVTGSVGTQGRACNKTAPQ
NOVSj VR.ASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQ
NOVSk VRASRLRQPTFLRIKQLRSYRKPMKTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQ
'NOVSa ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK--'NOVSb ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKLE
INOVSc ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKLE
NOVSd ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKLE
NOVSe ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKLE
NOVSf ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCYYVKCNTCSERTEMYTCKLE
NOVSg ASGCDLMCCGRGYNTHQYARVWQYNCKFHWCCYVKCNTCSERTEMYTCKLE
NOVSh ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKLE
NOVSi ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKLE
NOVSj ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK--NOVSk ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK--NOVSa(SEQID 34) NO:

NOVSb(SEQID 36) NO:

NOVSc(SEQID 38) NO:

NOV5d(SEQID 40) NO:

NOVSe(SEQID 42) NO:

NOVSf(SEQID 44) NO:

NOV5g(SEQID 46) NO:

NOVSh(SEQID 48) NO:

NOVSi(SEQID 50) NO:

NOVSj(SEQID 52) NO:

NOVSk(SEQID 54) NO:

Further analysis of the NOVSa protein yielded the following properties shown in Table SC.
Table SC. Protein Sequence Properties NOVSa SignalP analysis: Cleavage site between residues 32 and 33 PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length 7; pos.chg 4; neg_chg 0 H-region: length 32; peak value 10.30 PSG score: 5.90 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -0.60 possible cleavage site: between 27 and 28 » > Seems to have a cleavable signal peptide (1 to 27) ALOM: Klein et al's method for TM region allocation Init position for calculation: 28 Tentative number of TMS(s) for the threshold 0_5: 2 Number of TMS(s) for threshold 0.5: 0 PERIPHERAL Likelihood = 6.89 (at 151) ALOM score: 0.05 (number of TMSs: 0) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 13 Charge difference: -2.5 C( 3.0) - N( 5.5) N >= C: N-terminal side will be inside MITDISC: discrimination of mitochondrial targeting seq R content: 6 Hyd Moment(75): 11.39 Hyd Moment(95): 26.83 G content: 5 D/E content: 1 S/T content: 5 Score: 1.59 Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at G5 SRP~DA
NUCDISC: discrimination of nuclear localization signals pat4: none pat7: none bipartite: none content of basic residues: 14.6 NLS Score. -0.47 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals:
XXRR-like motif in the N-terminus: NRKA
none SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC. possible vacuolar targeting motif: found TLPK at 217 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: 55.5 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues Final Results (k = 9/23):
65.2 &: mitochondrial I3.0 %: Golgi j 8.7 %: extracellular, including cell wall 8.7 %: endoplasmic reticulum 4.3 %: cytoplasmic I
» prediction for CG50353-O1 is mit (Ic=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/Length . Residues/SimilaritiesExpect [Patent #, for IdentifierDate] Match the Matched Value ResiduesRegion ABJ10594Human novel protein NOVSa1..349 349/349 (100%)0.0 SEQ ID

NO: 16 - Homo Sapiens, ~ 1..349~ 349/349 349 aa. (100%) [W0200259315-A2, Ol-AUG-2002]

AAY57598Human Wnt-7a protein 1..349 321/349 (91%)0.0 - Homo Sapiens, 349 aa. [W09957248-Al,1..349 335/349 (95%) 11 NOV-1999]

AAY70737' Human Wnt-7a protein ' 1..349321/349 (91 0.0 - Homo %) Sapiens, 349 aa. [W0200021555-Al,1..349 3351349 (95%) 20-APR-2000]

AAB19789Human Wnt-7a protein 1..349 321/349 (91%)0.0 involved in kidney tubulogenesis 1..349 335/349 (95%) - Homo sapiens, 349 aa. [W0200061630-Al, 19-OCT-2000]

AAE34043WNT-7A protein - Unidentified,1..349 317/349 (90%)0.0 aa. [W0200290992-A2, 1..349 333/349 (94%) 14 NOV-2002]

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 5E. Public BLASTP
Results for NOVSa NOVSa Identities/

Protein ~ Residues/SimilaritiesExpect for AccessionProtein/Organism/LengthMatch the Matched Value Number ResiduesPortion 000755 Wnt-7a protein precursor1..349 321/349 (91 0.0 - Homo %) Sapiens (Human), 349 1..349 335/349 (95%) aa.

Q96H90 Hypothetical protein 1..349 317/349 (90%)0.0 - Homo Sapiens (Human), 349 aa. 1..349 333/349 (94%) AAH49093 Hypothetical protein 1..349 315/349 (90%)0.0 - Mus musculus (Mouse), 433 85..433 332/349 (94%) as (fragment).

Q9DBY3 Wingless-related MMTV 1..349 315/349 (90%)0.0 integration site 7A - Mus musculus 1..349 332/349 (94%) (Mouse), 349 aa.

P24383 Wnt-7a protein precursor1..349 313/349 (89%)0.0 - Mus musculus (Mouse), 349 1..349 330/349 (93%) aa.

PFam analysis predicts that the NOVSa protein contains the domains shown in the Table SF.
Table~~SF. Domain Analysis of NOVSa ~~' Identities!
Pfam Domain NOVSa Match Region Similarities Expect Value for the Matched Region wnt 37..349 180/352 (51%) 3.2e-212 298/352 (85%) Example 6.
The NOV6 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 6A.

6A. NOV6 ia, CG50709-03 SEQ ID NO: 57 ~93 by __ _ Sequence ORF Start: at 1 '~ORF ~Ston: end of TTGGGCACTGCGGCAGCCCCGGCACAGGGCGGGGCCC
CTGCCGCCCTCACCCACACCCTGGCCCGGGCCTGCAGCGCTGGGCGCATGGAGCGCTGCACCTGTGATGACTC
CTGAGCAACTTCCTGGGGTCCAAGAGAGGAAACAAGGACCTGCGGGCACGGGCAGACGCCCACAATACCCACG
TGGGCATCAAGGCTGTGAAGAGTGGCCTCAGGACCACGTGTAAGTGCCATGGCGTATCAGGCTCCTGTGCCGT
GCGCACCTGCTGGAAGCAGCTCTCCCCGTTCCGTGAGACGGGCCAGGTGCTGAAACTGCGCTATGACTCGGCT
GTCAAGGTGTCCAGTGCCACCAATGAGGCCTTGGGCCGCCTAGAGCTGTGGGCCCCTGCCAGGCAGGGCAGCC
6a, CG50709-03 SEQ ID NO: 58 331 as MW at 36432.2kD
VLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWN
RTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKF
GSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSA
ATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGR
SRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH
ib, 282997951 SEQ ID NO: 59 928 by Sequence ORF Start: at 2 ~ ORF Stop: end of CACCGGATCCCAGTGTGACCTGCTGAAGCTGTCCCGGCGGCAGAAGCAGCTCTGCCGGAGGGAGCCCGGCCTG
CTCTGCCGCCCTCACCCACACCCTGGCCCGGGCCTGCAGCGCTGGGCGCATGGAGCGCTGCACCTGTGATGAC
TCAGGCTCCTGTGCC
TACTCACCTGGCACAGCAGGTAGGGTGTGCTCCCGGGAGGCCAGCTGCAGCAGCCTGTGCTGCGGGC
ATGACACCCAGAGCCGCCTGGTGGCCTTCTCCTGCCACTGCCAGGTGCAGTGGTGCTGCTACGTGGA
GCAATGTGTGCAGGAGGAGCTTGTGTACACCTGCAAGCTCGAGGGC

282997951 ~SEQ ID NO: 60 X309 as BMW at 34226.6kD
QCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKETAFLYAVS
LTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNFLGSKRGNKDLRARADAHNTH
KAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSAVKVSSATNEALGRLELWAPARQGS
GLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVE
CG50709-OS ~SEQ ID NO: 61 ~ 1464 Sequence ~ORF Start: ATG at 38 ORF Stop: TAG at 1109 TTGGGCACTGCGGCAGCCCCGGCACAGGGCGGGGCCCACCTGAAGCAGTGTGACCTGCTGAAGCTGTC
GCCAGTTTCAGTTCCGGCATGAGCGCTGGAACTGTAGCCTGGAGGGCAGGACGGGCCTGCTCA
CAAAGAGACAGCTTTCCTGTACGCGGTGTCCTCTGCCGCCCTCACCCACACCCTGGCCCGGGC
GGGCGCATGGAGCGCTGCACCTGTGATGACTCTCCGGGGCTGGAGAGCCGGCAGGCCTGGCAG
GCGGTGACAACCTCAAGTACAGCACCAAGTTTCTGAGCAACTTCCTGGGGTCCAAGAGAGGAA
GCGGGCACGGGCAGACGCCCACAATACCCACGTGGGCATCAAGGCTGTGAAGAGTGGCCTCAG
AAGTGCCATGGCGTATCAGGCTCCTGTGCCGTGCGCACCTGCTGGAAGCAGCTCTCCCCGTTC
GCCAGGTGCTGAAACTGCGCTATGACTCGGCTGTCAAGGTGTCCAGTGCCACCAATGAGGCCT
AGAGCTGTGGGCCCCTGCCAGGCAGGGCAGCCTCACCAAAGGCCTGGCCCCAAGGTCTGGGGA
ATGGAGGACTCACCCAGCTTCTGCCGGCCCAGCAAGTACTCACCTGGCACAGCAGGTAGGGTG
AGGCCAGCTGCAGCAGCCTGTGCTGCGGGCGGGGCTATGACACCCAGAGCCGCCTGGTGGCCT
CTGCCAGGTGCAGTGGTGCTGCTACGTGGAGTGCCAGCAATGTGTGCAGGAGGAGCTTGTGTA
GGGCAGACTGTCATCACATGCATGCATAAACCGGCATGTGTGCCAATGCACACG
TTCCTTGGCCAGCCTTTTGCCTCCCTCGATACTCAACAAAGAGAAGCAAAGCCT
ATTCCATCTTGCTTC
CG50709-OS ~SEQ ID NO: 62 X357 as BMW at 38970.2kD
RPPPALALAGLCLLALPAAAASYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPG
AETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCD
~SPGLESRQAWQWGVCGDNLKYSTKFLSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSC
..VRTCWKQLSPFRETGQVLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCR
SKYSPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH

277582109 ~SEp ID NO: 64 X364 as BMW at 39615.9kD
TGSTMRPPPALALAGLCLLALPAAAASYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCR
REPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMER
CTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGV
SGSCAVRTCWKQLSPFRETGQVLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSP
SFCRPSKYSPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKHLEG
277582117 SEQ ID NO: 65 X1024 Sequence ORF Start: at 2 ORF Stop: end of TGCTGCGCACCTCGGCCTGCTTGAGTGCCAGTTTCA
TGG
TCAGGCTCCTGTGCCGTGCGCACCTGCTGGAAGCAGCTCTCCCCGTTCCGTGAGACGGGCCAGGTGCT
TGCGCTATGACTCGGCTGTCAAGGTGTCCAGTGCCACCAATGAGGCCTTGGGCCGCCTAGAGCTGTGG

277582117 ~SEQ ID NO: 66 341 as MW at 37431.2kD
Y H'CiL'1'CiKr:
VL'1'Yr'YCiLCi'1'AAAYAQCiGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQ
ERWNCSLEGRTGLLKRGFKETAFLYAVSSAALTHTLAR.ACSAGRMERCTCDDSPGLESRQAWQWGVCGDN
STKFLSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVL
YDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASC
CCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKHLEG
NOV6f, CG50709-O1 SEQ ID NO: 67 ' _1021 by _ DNA Sequence ORF Start: at 3~~ORF Stop: TAG at 996-- J.._",~, T_CCTGACCGGGCGGGAAGTCCTGACGCCCTTCCCAGGATTGGGCACTGCGGCAGCCCCGGCACAGGGCGGGGC
CCACCTGAAGCAGTGTGACCTGCTGAAGCTGTCCCGGCGGCAGAAGCAGCTCTGCCGGAGGGAGCCCGGCCTG
TGGGCCTGCTCAAGAGAGGCTTCAAAGAGACAGCTTTCCTGTACGCGGTGTC
CCTGGCCCGGGCCTGCAGCGCTGGGCGCATGGAGCGCTGCACCTGTGATGAC
CG50709-O1 SEQ ID NO: 68 X331 as BMW at 36462.3kD
REVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWN
EGRMGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKF
FLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSA
SSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGR
TQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH

ig, CG50709-02 SEQ ~ NO: 69 933 by Sequence ORF Start: ATG at 274- ~~ ~ ORF Stop: TAG at 928 GCCTGGCTGAGACCCTGAGGGATGCTGCGCACCTCGGCCTGCTTGAGTGCCAGTTTCAGTTCCGGCATGAGCG
~CTGGAACTGTAGCCTGGAGGGCAGGACGGGCCTGCTCAAGAGAGGCTTCAAAGAGACAGCTTTCCTGTACGCG
GTGTCCTCTGCCGCCCTCACCCACACCCTGGCCCGGGCCTGCAGCGCTGGGCGCATGGAGCGCTGCACCTGTG
TGA

CG50709-02 SEQ ID NO: 70 218 as MW at 24076.1kD
PGLESRQAWQWGVCGDNLKYSTKFLSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKC
RTCWKQLSPFRETGQVLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYME
KYSPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH
ih, CG50709-04 SEQ ID NO: 71 _,_849 by Se uence _...__.._._ ___~__..__........._._._.. _- p: end of q ~ Start. at 1 'Sto -_ .__.___..__.._ CGGCAGGCCTGGCAGTGGGGCGTGTGCGGTGACAACCTCAAGTACAGCACCAAGTTTCTGAG
CCTGCTGGAAGCAGCTCTCCCCGTTCCGTGAGACGGGCCAGGTGCTGAAACTGCGCTATGACTCGGCTGTCAA
GGTGTCCAGTGCCACCAATGAGGCCTTGGGCCGCCTAGAGCTGTGGGCCCCTGCCAGGCAGGGCAGCCTCACC
anarrr~mar;~ce~CAAGC~TCTGGGGACCTGGTGTACATGGAGGACTCACCCAGCTTCTGCCGGCCCAGCAAGT
I~10V6h, CG50709-04 ~SEQ ID NO: 72 ~~283 as ~MW at 31272.41eD
Protein Sequence KQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRMGLLKRGFKETAFLYAVSSA
ALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNFLGSKRGNKDLRARADAHNTHVG
IKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSAVKVSSATNEALGRLELWAPARQGSLT
KGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQV ___-ii, CG50709-06 SEQ ID NO:_73 1093 by Sequence ORF Start:~ATG at 14 ORF Stop: end of CGGCACAGGGCGGGGCCCACCTGAAGCAGTGTGACCTGCTGAAGCTGTCCCGGCGGCAGAAGCAGCTCTGCCG
ACCCACGTGGGCATCAAGGCTGTGAAGAGTGGCCTCAGGACCACGTGTAAGTGCCATGGCGT
CAGT

CG50709-06 ~SEQ ID NO: 74 360 as MW at 39269.6kD
RPPPALALAGLCLLALPAAAASYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPG
AETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCD
SPGLESRQAWQWGVCGDNLKYSTKFLSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSC
VRTCWKQLSPFRETGQVLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCR
SKYSPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKHLEG
CG50709-07 ~SEQIDN0:75 1024 Sequence ~ORF Start: at 2 ~ORF Stop: end of TGAGCGCTGGAACTGTAGCCTGGAGGGCAGGACGGGCCTGCTCAAGAGAGGCTTCAAAGAGACA
TACGCGGTGTCCTCTGCCGCCCTCACCCACACCCTGGCCCGGGCCTGCAGCGCTGGGCGCATGG
TACCCACGTGGGCATCAAGGCTGTGAAGAGTGGCCTCAGGACCACGTGTAAGTGCCATG
CAGTGCCACCAATGAGGCCTTGGGCCGCCTAGAGCTGTGG
CACCCAGCTTCTGCCGGCCCAGCAAGTACTCACCTGGCACAGCAGGTAGGGTGTGCTCCCGGGAGGCCAGCTG
CAGCAGCCTGTGCTGCGGGCGGGGCTATGACACCCAGAGCCGCCTGGTGGCCTTCTCCTGCCACTGCCAGGTG
CAGTGGTGCTGCTACGTGGAGTGCCAGCAATGTGTGCAGGAGGAGCTTGTGTACACCTGCAAGCACCTCGAGG
6j, CG50709-07 ~SEQ ID NO: 76 341 as MW at 37431.2kD
SYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQ
ERWNCSLEGRTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDN
STKFLSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVL
YDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASC
CCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKHLEG

61c, SNP13381605 of SEQ ID NO: 77 993 by )709-03, DNA Sequence ORF Start: a_t_1 ORF Stop: end of see SNP Pos: 653 ~~ - SNP Change: C to T
CT
CAAGTACTCACCTGGCACAGCAGGTAGGGTGTGCTCCCGGGAGGCCAGCTGCAGCAGCCTGTGCTGCGGGCGG
TGTGTGCAGGAGGAGCTTGTGTACACCTGCAAGCAC
SNP13381605 of SEQ m NO: 78 _ 331 as ~MW at 36458.3kD
-03, Protein Sequence ~Np pos: 218 ~ ~~SNP Change: Ser to Leu LTGREVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWN
CSLEGRTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKF
LSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYD_LA
VKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGR
GYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH _ _~,.,_~m~-~~~~
S
I~OV61, SNP13381606 of SEQ ID NO: 79 993 by __ _ _ CG50709-03, DNA Sequence ORF Start: at 1 ORF Stop: endYof sequence SNP Pos: 743 SNP Change: T to C
CTGACCGGGCGGGAAGTCCTGACGCCCTTCCCAGGATTGGGCACTGCGGCAGCCCCGGCACAGGGCGGGGCCC
GACCCTGAGGGATGCTGCGCACCTCGGCCTGCTTGAGTGCCAGTTTCAGTTCCGGCATGAGCGCTGGAAC
AGCCTGGAGGGCAGGACGGGCCTGCTCAAGAGAGGCTTCAAAGAGACAGCTTTCCTGTACGCGGTGTCCT
nr~ar~r~mrnr~~rnr~nr~t~r~rt~~f'CCC~C~C~CCTGCAGCGCTGGGCGCATGGAGCGCTGCACCTGTGATGA
CTC
CACG
CAAGTACTCACCTGGCACAGCAGGTAGGGTGTGCTCCCGGGAGGCCAGCTGCAGCAGCCTGTGCTGCGGGCGG
GGCTATGACACCCAGAGCCGCCTGGTGGCCTTCTCCTGCCACTGCCAGGTGCAGTGGTGCTGCTACGTGGAGT

NOV6I, SNP13381606 of SEQ ID NO_: 80 331 as ,~MW_at 36416._2kD_ CG50709-03, Protein Sequence SNP Pos: 248 ~ SNP Change: Leu to Pro PFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWN
LLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKF
RGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSA
EALGRLELWAPARQGSLTKG_PAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGR
VAFSCHCQVQWCCYVECQQCVQEELVYTCKH
~6m, SNP13378337 of SEQ ID NO_ : 81 _ 993 by 0709-03, DNA Sequence ORF Start: at l ORF Ston: end of Pos: 764 SNP Change: T to C
ACAGCACCAAGTTT
ATCAGGCTCCTGTGCCGT
CGGTGTACATGGAGGACTCACCCAGCTTCTGCCGGCCCAG
TGACACCCAGAGCCGCCTGGTGGCCTTCTCCTGCCACTGCCAGGTGCAGTGGTGCTGCTACGTGGAGT
CAATGTGTGCAGGAGGAGCTTGTGTACACCTGCAAGCAC
SNP13378337 of SEQ ID NO: 82 331 as MW at 36416.2kD
-03, Protein Sequence SNP Pos: 255 ~ SNP Change: Leu to Pro rPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWN

VEALGRLELWAPARQGSLTKGLAPRSGD_PVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGR
LVAFSCHCQVQWCCYVECQQCVQEELVYTCKH

SNP13381607 of SEQ ID NO: 83 X993 by _ -03, DNA Sequence ORF Start: at 1 ~ORF Stop: end of Pos: 799 SNP Change: C to T
AAGTGCCATGGCGTATCAGGCTCCTGTGCCGT
AGAGCTGTGGGCCCCTGCCAGGCAGGGCAGCC
ATGACACCCAGAGCCGCCTGGTGGCCTTCTCCTGCCACTGCCAGGTGCAGTGGTGCTGCTACGTGGAGT
6n, SNP13381607 of SEQ ID NO: 84 331 as ~MWat 36422.2kD_ 1709-03, Protein Sequence SNP Pos: 267 SNP Change: Pro to Ser EVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWN
GRTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKF
LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSA
SATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCR_SSKYSPGTAGRVCSREASCSSLCCGR
QSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH

SNP13378336 of SEQ )D NO: 86 331 as M_W at 36372.2kD
-03, Protein Sequence SNP Pos: 294 SNP Change: Tyr to LTGREVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWN
CSLEGRTGLLKRGFKETAFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKF
LSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSA
VKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGR
GCDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH
6p, SNP13378335 of SEQ m NO: 87 =993 by )709-03, DNA Sequence ORF Start: at 1 ORF Stop: end of SNP Pos: 977 ISNP Change: T to C
CTGACCGGGCGGGAAGTCCTGACGCCCTTCCCAGGATTGGGCACTGCGGCAGCCCCGGCACAGGGCGGGGCCC
CTGCCGCCCTCACCCACACCCTGGCCCGGGCCTGCAGCGCTGGGCGCATGGAGCGCTGCACCTGTGATGACTC
TCCGGGGCTGGAGAGCCGACAGGCCTGGCAATGGGGCGTGTGCGGTGACAACCTCAAGTACAGCACCAAGTTT
ATGACTCGGCT
AAAGGCCTGGCCCCAAGGTCTGGGGACCTGGTGTACATGGAGGACTCACCCAGCTTCTGCCGGCCCAG
ACTCACCTGGCACAGCAGGTAGGGTGTGCTCCCGGGAGGCCAGCTGCAGCAGCCTGTGCTGCGGGCGG
TGACACCCAGAGCCGCCTGGTGGCCTTCTCCTGCCACTGCCAGGTGCAGTGGTGCTGCTACGTGGAGT
CAATGTGTGCAGGAGGAGCTTGCGTACACCTGCAAGCAC
T6p, SNP13378335 of SEQ ID NO:. 88 331_ as .MW at 36404.1kD _ 0709-03, Protein Sequence ' SNP Pos. 326 SNP Change: Val to Ala PGLAETLRDAAHLGLLECQFQFRHERWN
LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKLRYDSA
SATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSLCCGR
QSRLVAFSCHCQVQWCCYVECQQCVQEELAYTCKH
A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table 6B.
Table 6B. Comparison of the NOV6 protein sequences.
NOV6a ------------------------------LTGREVLTPFPGLGTAAAPAQGGAHLKQCD
NOV6b ______________________________________________________TGSQCD
NOV6c ----MRPPPALALAGLCLLALPAAAASYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCD
NOV6d TGSTMRPPPALALAGLCLLALPAAAASYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCD
NOV6e -----------------------TGSSYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCD
NOV6f ------------------------------LTGREVLTPFPGLGTAAAPAQGGAHLKQCD
NOV6g ____________________________________________________________ NOV6h ________________________________________________________KQCD
NOV6i ----MRPPPALALAGLCLLALPAAAASYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCD

NOV6j -----------------------TGSSYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCD
'NOV6a LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKET
'NOV6b LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKET
'NOV6c LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKET
NOV6d LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKET
NOV6e LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKET
NOV6f LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRMGLLKRGFKET
NOV6g _____________________________________________-______________ NOV6h LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRMGLLKRGFKET
NOV6i LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKET
NOV6j LLKLSRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKET
NOV6a AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6b AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6c AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6d AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6e AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6f AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6g -----------------------MERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6h AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6i AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6j AFLYAVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNF
NOV6a LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6b LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6c LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6d LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6e LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6f LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6g LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6h LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6i LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6j LGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQ
NOV6a VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6b VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6c VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6d VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6e VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6f VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6g VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6h VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6i VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6j VLKLRYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKY
NOV6a SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6b SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6c SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6d SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6e SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6f SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6g SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6h SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQV---------------------NOV6i SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6j SPGTAGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCK
NOV6a H---~NOV6b LEG-NOV6cH---NOV6dHLEG

NOV6eHLEG

NOV6fH---NOV6gH---NOV6h----NOV6iHLEG

NOV6jHLEG

NOV6a(SEQ ID 58) NO:

NOV6b(SEQ ID 60) NO:

NOV6c(SEQ ID 62) NO:

NOV6d(SEQ ID 64) NO:

NOV6e(SEQ ID 66) NO:

NOV6f(SEQ ID 68) NO:

NOV6g(SEQ ID 70) NO:

NOV6h(SEQ ID 72) NO:

NOV6i(SEQ ID 74) NO:

~NOV6j(SEQ ID ....7.6Y), NO:...

Further analysis of the NOV6a protein yielded the following properties shown in Table 6C.
Table 6C. Protein Sequence Properties NOV6a SignalP analysis: No Known Signal Sequence Predicted PSORT II analysis:
PSG: a new signal peptide prediction method N-region. length 5; pos.chg 1; neg.chg 1 H-region: length 21; peak value 7.18 PSG score: 2.78 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -5.38 possible cleavage site: between 22 and 23 » > 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 = 4.08 (at 90) ALOM score: 4.08 (number of TMSs: 0) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 6 Charge difference: 3.5 C( 4.5) - N( 1.0) C > N: C-terminal side will be inside » >Caution: Inconsistent mtop result with signal peptide MITDISC: discrimination of mitochondrial targeting seq R content: 1 Hyd Moment(75): 11.91 Hyd Moment(95): 8.21 G content: 5 D/E content: 2 S/T content. 3 Score: -6.56 Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 53 CRR~EP
',NUCDISC: discrimination of nuclear localization signals ' pat4: none pat7: none bipartite: none content of basic residues: 14.2%
NLS Score: -0.47 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals:
XXRR-like motif in the N-terminus: TGRE
KKXX-like motif in the C-terminus: YTCK
SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: found KLSRRQKQL at 33 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: 76.7 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues Final Results (k = 9/23).
78.3 %: nuclear 13.0 %: mitochondrial 8.7 %: cytoplasmic » prediction for CG50709-03 is nuc (k=23) A search of the NOV6a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 6D.
Table 6D.
Geneseq Results for NOV6a NOV6a Identities/

Geneseq Protein/Organism/Length Residues/SimilaritiesExpect [Patent #, for IdentifierDate] Match the Matched Value ResiduesRegion AAE17306Human WNT15 protein, 1..331 330/331 (99%)0.0 sbg389686WNT15a #2 - 31..361 330/331 (99%) Homo Sapiens, 361 aa. [WO200198342-Al, 27-DEC-2001]

AAE17305Human WNT15 protein, 1..331 330/331 (99%)0.0 sbg389686WNT15a #1 - 17..347 330/331 (99%) Homo sapiens, 704 aa. [W0200198342-Al, 27-DEC-2001 ]

ABB77769Amino acid sequence of 1..331 330/331 (99%)0.0 human Wnt (ZwntS) polypeptide variant4..334 330/331 (99%) - Homo Sapiens, 334 aa. [W0200231148-A2, 18-APR-2002]

ABB77768Amino acid sequence of 1..331 330/331 (99%)0.0 human Wnt (ZwntS) polypeptide - 31..361 330/331 (99%) Homo Sapiens, 361 aa. [W020023I148 A2, 18-APR-2002]

ABB83080Wnt family related protein1..331 330/331 (99%)0.0 2 - Homo sapiens, 363 aa. [W0200250278-A2,33..363 330/331 (99%) 27-JUN-2oo2]

s 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 6E.

Table -6E.
Public BLASTP
Results for NOV6a NOV6a Protein Identities/

AccessionProteinJOrganism/Length Residues/Similarities Expect for the Number Matched PortionValue Residues 014905 Wnt-9b protein precursor1..331 331/331 (100%)0.0 (Wnt-15) (Wnt-14b) - Homo sapiens27..357331/331 (100%) (Human), 357 aa.

Q8C718 WNT14B - Mus musculus 1..330 310/330 (93%) 3 0:0 (Mouse), 359 aa. 29..358319/330 (95%) 035468 Wnt-9b protein precursor1..330 310/330 (93%) 0.0 (Wnt-15) (Wnt-14b) - Mus musculus29..358319/330 (95%) (Mouse), 359 aa.

014904 Wnt-9a protein precursor1..330 209/335 (62%) e-124 (Wnt-14) -Homo Sapiens (Human), 33..364255/335 (75%) 365 aa.

Q8RSM2 Wnt-9a protein precursor1..330 208/335 (62%) e-123 (Wnt-14) -Mus musculus (Mouse), 33..364255/335 (76%) 365 aa.

PFam analysis predicts that the N0V6a protein contains the domains shown in the Table 6F.
Table 6F. Domain Analysis of NOV6a Identities/
Pfam Domain NOV6a Match Region Similarities Expect Value for the Matched Region wnt ~ 28..330 132/354 (37%) 2.1e-104 234/354 (66%) Example 7.
The N0V7 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 7A.

7A. NOV7 Sequence Analysis Via, CG53054-02 SEQ ID NO: 89 1128 by Sequence ORF Start: ATG at 31 ORF Stop: TGA at 1102 ACCCGATCTGGTGGCTGACGGGCAGCGAGCCCCTGACCATCCTCCCGCTGACCCTGGA
CGAGTGCCAGTTCC
CCACAACAACCTCGTGGGTGTGAAGGTGATCAAGGCTGGGGTGGAGACCACCTGCAAGTGC
rrcTCATGCACGGTGCGGACCTGCTGGCGGCAGTTGGCGCCTTTCCATGAGGTGGGCAAGC
CATCTCCCCACCACGGGGCCGTGCCTCGGGGGCAGGTGGCAGCGACCCGCTGCCCCGCACTCCAGAGCTGGTG
CACCTGGATGACTCGCCTAGCTTCTGCCTGGCTGGCCGCTTCTCCCCGGGCACCGCTGGCCGTAGGTGCCACC
GTGAGAAGAACTGCGAGAGCATCTGCTGTGGCCGCGGCCATAACACACAGAGCCGGGTGGTGACAAGGCCCTG
CG53054-02 SEQ 1D NO: 90 357 as ~MW at 39756.1kD
APLGYFLLLCSLKQALGSYPIWWLTGSEPLTILPLTLEPEAAAQAHYKACDRLKLERKQRRMCRRDPGVAET
VEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFKETAFLYAISSAGLTHALAKACSAGRMERCTCDEA
DLENREAWQWGGCGDNLKYSSKFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVR
CWRQLAPFHEVGKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFCL
GRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREEVYTCKG
lb,170251039 SEQ ID NO: 91 1029 by Sequence OIZF Start: at 1 ~~RF Stop: end of ATGCCATCTCCTCGGCTGGCCTGACGCACGCACTGGCCAAGGCGTGCAGCGCGGGCCGCAT
AAGTACAGCAGCAAGTTCGTCAAGGAATTCCTGGGCAGACGGTCAAGCAAGGATCTGCGAGCCCGTG
TGCCTCGGGGGCAGGTGGCAGCGACCCGCTGCCCCGCACTCCAGAGCTGGTGCACCT
TTCTGCCTGGCTGGCCGCTTCTCCCCGGGCACCGCTGGCCGTAGGTGCCACCGTGAG
TCTGCTGTGGCCGCGGCCATAACACACAGAGCCGGGTGGTGACAAGGCCCTGCCAGT
rTGCTATGTGCtAGTGCAGGCAGTGCACGCAGCGTGAGGAGGTCTACACCTGCAAGGG

170251039 ~SEQ ID NO: 92 X343 as BMW at 38208.1kD
IWWLTGSEPLTILPLTLEPEAGAQAHYKACDRLKLERKQRRMCRRDPGVVETLVEAVSMSALECQFQF
DTCTLEGRYRASLLKRGFKETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGD
SKFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEVGKHL
TALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFCLAGRFSPGTAGRRCHRE
ICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREEVYTCKGVD
~c,170251076 SEQ ID NO: 93 ~~~.~w1029 by .~.e~
Sequence ORF Start: at 1 ORF Stou: end of TGTGCCG
CGCTTTGAGCGCTGGAACTGCACGCTGGAGGGCCGCTACCGGGCCAGCCTGCTCAAGCGAGGCTTCAAGGAGA
CTGTACCTGCGATGAGGCACCCGACCTGGAGAACCGTGAGGCCTGGCAGTGGGGGGGCTGCGGAGAC
AAGTACAGCAGCAAGTTCGTCAAGGAATTCCTGGGCAGACGGTCAAGCAAGGATCTGCGAGCCCGTG
TGAGACGGCACTCAAGGTGGGCAGCACCACCAATGAAGCTGCCGGCGAGGCAGGTGCCATCT
TGTGGCCGCGGCCATAACACACAGAGCCGGGTGGTGACAAGGCCCTGCCAGT
ATGTGGAGTGCAGGCAGTGCACGCAGCGTGAGGAGGTCTACACCTGCAAGGG
170251076 ~SEQ ID NO: 94 X343 as BMW at 38194.1kD
PIWWLTGSEPLTILPLTLEPEAAAQAHYKACDRLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQF
WNCTLEGRYRASLLKRGFKETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGD
SSKFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEVGKHL
FTALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFCLAGRFSPGTAGRRCHRE

7d, CG53054-Ol SEQ ID NO: 95~ ~c-1085 by ~__ Sequence ORF Start: ATG at 13 ORF Ston: TGA at 1078 CAAGGCCTGCGACCGGCTGAAGCTGGAGCGGAAGCAGCGGCGCATGTGCCGCCGGGACCCGGGCGTGGCAGAG
ACGCTGGTGGAGGCCGTGAGCATGAGTGCGCTCGAGTGCCAGTTCCAGTTCCGCTTTGAGCGCTGGAACTGCA
CTCGGCTGGCCTGACGCACGCACTGGCCAAGGCGTGCAGCGCGGGCCGCATGGAGCGCTGTACCTGCGATGAG
TGCCCGCTCAGCCATGAACCGCCACAACAACGA
GTGCGGACCTGCTGGCGGCAGTTGGCGCCTTTCCATGAGGTGGGCAAGCATCTGAAGCACAAGTATGAGTCGG
CACTCAAGGTGGGCAGCACCACCAATGAAGCTGCCGGCGAGGCAGGTGCCATCTCCCCACCACGGGGCCGTGC
CCCCGGGCACCGCTGGCCGTAGGTGCCACCGTGAGAAGAACTGCGAGAGCATCT
CG53054-O1 ~SEQ m NO: 96 X355 as BMW at 39194.1kD
ALLYSSLGVWCTCSPSYFGLTGSEPLTILPLTLEPEAAAQAHYKACDRLKLERKQRRMCRRDPGVAETLVE
VSMSALECQFQFRFERWNCTLEGRYRASLLKRGFKETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDL
NREGWKWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNNEAGRQVIKAGVETTCKCHGVSGSCTVRTC
RQLAPFHEVGKHLKHKYESALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFCLAG
FSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREEVYTCKG
~e, CG53054-03 SEQ ~ NO: 97 1029 by Sequence ORF Start: at 7 OR_F Stop: _at 1024 _ _CAGCTACCCGATCTGGTGGCTGACGGGCAGCGAGCCCCTGACCATCCTCCCGCTGACCCTGGAGCCAG

TCTCCTCGGCTGGCCTGACGCACGCACTGGCCAAGGCGTGCAGCGCGGGCCGCAT
TTCCTGGGCAGACGGTCAAGCAAGGATCTGCGAGCCCGTG
TGCACGGTGCGGACCTGCTGGCGGCAGTTGGCGCCTTTCCATGAGGTGGGCAAGCATCTG
CTCCCCGGGCACCGCTGGCCGTAGGTGCCACCGTGAG
AACACACAGAGCCGGGTGGTGACAAGGCCCTGCCAGT

CG53054-03 ~SEQ ID NO: 98 1339 as 1MW at 37835.8kD
WWLTGSEPLTILPLTLEPEAAAQAHYKACDRLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRF
CTLEGRYRASLLKRGFKETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGDNL
KFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEVGKHLKH
CESICCGRGHNTQSRVUZ'RPCQCQVRWCCYVECRQCTQREEVYTCKG
NOV7f, CG53054-04 SEQ ID NO_: 99 _ 1631 by _ _ DNA Sequence ORF S_tar_t:~ATGaat 12 y ~,pRF Stop:mTGA at_~1107 GGCGCGGCAAGATGCTGGATGGGTCCCCGCTGGCGCGCTGGCTGGCCGCGGCCTTCGGGCTGACGCTGCTGCT
CGCCGCGCTGCGCCCTTCGGCCGCCTACTTCGGGCTGACGGGCAGCGAGCCCCTGACCATCCTCCCGCTGACC
TGAGTGCGCTCGAGTGCCA
AAGTACAGCAGCAAGTTCGTCAAGGAATTCCTGGGCAGACGGTCAAGCAAGGATCTG
TGAAGCTGCCGGCGAGGCA
TAACACACAGAGCCGGGTGGTGACAAGG
CAGTGCACGCAGCGTGAGGAGGTCTACA
ACACCTGCACAGGCTGAGTTCCTGGGCTCGACCAGCCCAGCTGCGTGGGGTACAGGCATTGCACACAGT
AGTCCTAGCTGCATGGGGTGCAGGCATTGCACAGAGCATGAATGGGCCTACACCTGCCAAGGCTGAATCCCTG
GGCCCAGCCAGCCCTGCTGCACATGGCACAGGCATTGCACACGGTGTGAGGAGTGTACACCTGCAAGGGCTGA
CG53054-04 ~SEO ID NO: 100 X365 as IMW at 40319.7kD
GSPLARWLAAAFGLTLLLAALRPSAAYFGLTGSEPLTILPLTLEPEAAAQAHYKACDRLKLERKQRRMCR
GVAETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFKETAFLYAISSAGLTHALAKACSAGRM
TCDEAPDLENREAWQWGGCGDNLKYSSKFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHG
SCTVRTCWRQLAPFHEVGKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHL
PSFCLAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREEVYTCKG
A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table 7B.

Table 7B. Comparison of the NOV7 protein sequences.
NOV7a --------MAPLGYFLLLCSLKQALGSYPIWWLTGSEPLTILPLTLEPEAAAQAHYKACD
NOV7b ------------------------GSSYPIWWLTGSEPLTILPLTLEPEAGAQAHYKACD
NOV?c ------------------------GSSYPIWWLTGSEPLTILPLTLEPEAAAQAHYKACD
NOV7d -----------MALLYSSLGVWCTCSPSYFGLTGSEPLTILPLTLEPEAAAQAHYKACD
NOV7e --------------------------SYPIWWLTGSEPLTILPLTLEPEAAAQAHYKACD
NOV7f MLDGSPLARWLAAAFGLTLLLAALRPSAAYFGLTGSEPLTILPLTLEPEAAAQAHYKACD
NOV7a RLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFK
NOV7b RLKLERKQRRMCRRDPGVVETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFK
NOV7c RLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFK
NOV7d RLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFK
NOV7e RLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFK
NOV7f RLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFK
NOV7a ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGDNLKYSSKFVK
NOV'7b ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGDNLKYSSKFVK
NOV7c ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGDNLKYSSKFVK
NOV7d ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREGWKWGGCSEDIEFGGMVSR
NOV7e ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGDNLKYSSKFVK
NOV7f ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGDNLKYSSKFVK
NOV7a EFLGRRSSK-DLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV
NOV7b EFLGRRSSK-DLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV
NOV7c EFLGRRSSK-DLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV
NOV7d EFADARENRPDARSAMNRHNNEAGRQVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV
NOV7e EFLGRRSSK-DLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV
NOV7f EFLGRRSSK-DLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEV
NOV7a GKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV7b GKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV7c GKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV7d GKHLKHKYESALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV7e GKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV7f GKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFC
NOV7a LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV7b LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV7c LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV7d LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV7e LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV7f LAGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREE
NOV7a VYTCKG--NOV7b VYTCKGVD
NOV7c VYTCKGVD
NOV7d VYTCKG--NOV7e VYTCKG--NOV7f VYTCKG--NOV7a (SEQ ID NO: 90) NOV7b (SEQ ID NO: 92) NOV7c (SEQ ID NO: 94) NOV7d (SEQ ID NO: 96) NOV7e (SEQ ID NO: 98) NOV7f (SEQ ID NO: 100) Further analysis of the NOV7a protein yielded the following properties shown in Table 7C.
Table 7C. Protein Sequence Properties NOV7a SignaIP 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 13; peak value 9.00 PSG score: 4.60 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 0.73 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 = 3.76 (at 114) ALOM score: 3.76 (number of TMSs: 0) 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 MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment(75): 1.56 Hyd Moment(95): 3.50 G content: 3 D/E content: 1 S/T content: 4 Score: -6.15 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: 14.8&
NLS Score: -0.47 ~KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals:
KICXX-like motif in the C-terminus: YTCK
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 memY~RL: 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: 70_6 ~ICOIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues Final Results (k = 9/23):
55.6 %: extracellular, including cell wall 22.2 %: mitochondrial 11.1 %: vacuolar 11.1 %: nuclear » prediction for CG53054-02 is exc (k=9) A search of the NOV7a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 7D.

Table 7D.
Geneseq Results for NOV7a NOV7a Identities/

Geneseq Protein/Organism/Length Residues/SimilaritiesExpect [Patent #, for IdentifierDate] Match the MatchedValue ResiduesRegion AAE34048WNT-14 protein - Unidentified,2..357 339/356 0.0 365 (95%) aa. [W0200290992 A2, 13..365 343/356 (96%) 14 NOV-2002]

ABU55894Human WNT-14 protein 2..357 339/356 0.0 - Homo (95%) sapiens, 365 aa. [W0200277204-A2,13..365 343/356 (96%) 03-OCT-2002]

ABG69638Human secreted protein 2..357 311/357 0.0 SCEP-18 - (87%}

Homo Sapiens, 366 aa. 13..366 327/357 (91 %) [W0200248337-A2, 20-JUN-2002]

AA018744Human NOVB protein - 25..357 302/334 0.0 Homo Sapiens, (90%) 355 aa. [W0200257450-A2,22..355 316/334 (94%) 25-JUL=2002]

AAE17305Human WNT15 protein, 22..356 210/338 e-124 (62%) sbg389686WNT15a #1 - 14..346 257/338 Homo (75%) sapiens, 704 aa. [W0200198342 Al, 27-DEC-200 / ]
n. ..

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

NOV7a Identities/

Protein Residues/SimilaritiesExpect for AccessionProtein/Organism/LengthMatch the Matched Value Number ResiduesPortion 014904 Wnt-9a protein precursor2..357 339/356 (95%)0.0 (Wnt-14) -Homo Sapiens (Human), 13..365 343/356 (96%) 365 aa.

Q8RSM2 Wnt-9a protein precursor2..357 333/356 (93%)0.0 (Wnt-14) -Mus musculus (Mouse), 13..365 340/356 (94%) 365 aa.

042280 Wnt-9a protein precursor25..356 283/333 (84%)e-173 (Wnt-14) -Gallus gallus (Chicken},24..353 310/333 (92%) 354 aa.

Q8C718 WNT14B - Mus musculus 8..356 216/354 (61%)e-125 (Mouse), 359 aa. 12..358 ~ 264/354 (74%) 035468 Wnt-9b protein precursor8..356 216/354 (61%)e-125 (Wnt-15}

(Wnt-14b) - Mus musculus12..358 264/354 (74%) (Mouse), 359 aa.

PFam analysis predicts that the NOV7a protein contains the domains shown in the Table 7F.
Table 7F. Domain Analysis of NOV7a Identities/
Pfam Domain NOV7a Match Region Similarities Expect Value for the Matched Region wnt 50..356 1291359 (36%) 4.6e-103 234/359 (65%) Example 8.
The NOV8 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 8A.
Table 8A. NOV8 Sequence Analysis _ I~10V8a, CG53473-02 SEQ ID NO:101 514 by DNA Sequence OItF Start: ATG at 37 , O1ZF Stop: TGA at 400 CGCGCGCCCGAACGAAGCCGCGGCCCGGGCACAGCCATGGCCCGGCGGGCGGGGGGCGCTCGGATGTTCGGCA
CCCCATTGGGGACAGCTCCCCACACCTCCCTGAGGGACCAGCGACTGCAGCTGAGTC
TACAGGAGGCTGCTGGTACAAATACTGCAGAAATGACACCAATAATGGGGCAGACACAACAGCGTGGCTTAG
TTGTGCCCACCCAGGGAAGGTGCTGAATGGGACCCTGTTGATGGCCATCAACAGGGTCCCATTCAGCACAGG
LVOVBb, CG53473-O1 SEQ ID N0:103 , 646 by DNA Sequence ORF Start: ATG at 62 ORF Stop: TGA at 398 AGCGCGCCCGAACGAAGCCGCGGCCCGGGCACAGCATGGCCCGCGGCGGGAGGGCGCTCGGATGTTCGGCAGC
CTCCTGCACTTCGCCCTGCTCGCTGCCGGCGTCGTCCCGCTCAGCTGGGATCTCCCGGAGCCCCGCAGCCGAG
CCAGCAAGATCCGAGTGCACTCGCGAGGCAAGCTCTGGGCCATCGGTCACTTCATGGGCAAGAAGAGTCTGGA
GCCTTCCAGCCCATCCCCATTGGGGACAGCTCCCCACACCTCCCTGAGGGACCAGCGACTGCAGCTGAGTCAT
ACTGCAGAAATGACACCAATAATGGGGCAGACACAACAGCGTGGCTTAGAT
....~.~....-............ .,.n.a..~~wn mivmniv ~n mnmW w T ml~/~m!'nT TI~TI~T T
T
(TCTCTGTTACTCCATTACTGTGATTTCTGGCTGGGTCACCAGAAATATCGCTGATGCAGACACAGATTATGTT'--CCTGCTGTATTTCCTGCTTCCCTGTTGAATTGGTGAATAAAACCTTGCTCTATACATACAAA

CG53473-Ol ~SEQ ID NO: 104 112 as ~MW at 12402.SkD
RASKIRVHSRGKLWAIGHFMGKKSLEPSSPSPLGTAPHTSLRDQRL
QYRRLLVQILQK
SNP13376396 of SEQ ID NO: 107_x(514 by _ f-02, DNA Sequence ORF Start: ATG at 37 ~ ORF Stop: TGA at 400 SNP Pos:190 SNP Change: A to G
AGCCATGGCCCGGCGGGCGGGGGGCGCTCGGATGTTCGGCA
TCCCCATTGGGGACAGCTCCCCACACCTCCCTGAGGGACCAGCGACTGCAGCTGAGTC
TCCTCCTGCTAAAGAAGGCTCTGGGCGTGAGCCTCAGCCGCCCCGCACCCCAAATCCA
GGTACAAATACTGCAGAAATGACACCAATAATGGGGCAGACACAACAGCGTGGCTTAG
GGAAGGTGCTGAATGGGACCCTGTTGATGGCCATCAACAGGGTCCCATTCAGCACAGG

SNP1337639$ of SEQ m NO: 109 $14 by 3-02, DNA Sequence ORF Start: ATG at 37 ORF Stop: _TGA_at 400 SNP Pos: 2$3 SNP Change: C to A
TCCGAGTGCACTCGCGAGGCAACCTCTGGGCCACCGGTCACTTCATGGGCAAGAAGAGTCTG
TCTGCTCGGAATCCTCCTGCTAAAGAAGGCTCTGGGCGTGAGCCTCAGCCGCCCCGCACCCCAAATCCA
AGGAGGCTGCTGGTACAAATACTGCAGAAATGACACCAATAATGGGGCAGACACAACAGCGTGGCTTAG
8f, SNP13376394 of SEQ ID NO: 111 514 by .473-02, DNA Sequence ORF Start: ATG at 37 ORF Stop: T_AA at 4_00 SNP Pos: 401 SNP-Change: G to A
CGCCCGAACGAAGCCGCGGCCCGGGCACAGCCATGGCCCGGCGGGCGGGGGGCGCTCGGATGTTCGGCA
CCTGCTCTTCGCCCTGCTCGCTGCCGGCGTCGCCCCGCTCAGCTGGGATCTCCCGGAGCCCCGCAGCCG
AGCAAGATCCGAGTGCACTCGCGAGGCAACCTCTGGGCCACCGGTCACTTCATGGGCAAGAAGAGTCTG
CTTCCAGCCCATCCCCATTGGGGACAGCTCCCCACACCTCCCTGAGGGACCAGCGACTGCAGCTGAGTC
TCTGCTCGGAATCCTCCTGCTAAAGAAGGCTCTGGGCGTGAGCCTCAGCCGCCCCGCACCCCAAATCCA
CAACAGGGTCCCATTCAGCACAGG
A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table 8B.

Table 8B. Comparison of the NOV8 protein sequences.
NOVBa MARRAGGARMFGSLLLFALLAAGVAPLSWDLPEPRSRASKIRVHSRGNLWATGHFMGKKS
NOVBb ---------MFGSLLHFALLAAGWPLSWDLPEPRSRASKIRVHSRGKLWA-IGHFMGKKS
NOVSc ______________________________________________GHI,WAIGHFM-___ NOVBa LEPSSPSPLGTAPHTSLRDQRLQLSHDLLGILLLKKALGVSLSRPAPQIQYRRLLVQILQ
NOVBb LEPSSPSPLGTAPHTSLRDQRLQLSHDLLGILLLKKALGVSLSRPAPQIQYRRLLVQILQ
NOVBc ____________________________________________________________ NOVBa K
NOV8b K
NOVBc -NOVBa (SEQ ID NO: 102) NOVBb (SEQ ID NO: 104) NOVBc (SEQ ID NO: 106) Further analysis of the NOVBa protein yielded the following properties shown in Table 8C.
Table 8C. Protein Sequence Properties NOVBa SignalP analysis: Cleavage site between residues 27 and 28 PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length 9; pos.chg 3; neg.chg 0 H-region: length 20; peak value 10.93 PSG score: 6.53 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2_1): 2.23 possible cleavage site: between 26 and 27 » > Seems to have a cleavable signal peptide (1 to 26) ALOM: Klein et al's method for TM region allocation Init position for calculation: 27 Tentative number of TMS(s) for the threshold 0.5: 0 number of TMS(s) _. fixed PERIPHERAL Likelihood = 1.85 (at 87) ALOM score: 1.85 (number of TMSs: 0) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 13 Charge difference: -1.5 C( 2.5) - N( 4.0) N >= C: N-terminal side will be inside MITDISC: discrimination of mitochondrial targeting seq R content: 3 Hyd Moment(75): 12.45 Hyd Moment(95): 10.60 G content: 4 D/E content: 1 5/T content: 2 Score: -2.16 Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 19 ARM~FG

NUCDISC: discrimination of nuclear localization signals pat4: none pat7: none bipartite: none content of basic residues: 14_9 NLS Score: -0.47 IKDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals:
XXRR-like motif in the N-terminus: ARRA
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: 89 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 &: mitochondrial 22.2 ~s: nuclear prediction for CG53473-02 is exc (k=9) A search of the NOV 8a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 8D.

Table SD.
Geneseq Results for NOVBa .

NOVBa Identities/

Geneseq Protein/Organism/Length ResidueslSimilaritiesExpect [Patent #, for IdentifierDate] Match the Matched Value ResiduesRegion AAE17605Humanextracellularmessenger1..121 121/121 (100%)le-63 (XMES)-7 protein - Homo 1..121 121/121 (100%) Sapiens, 121 aa. [W0200194587 A2, 13-DEC-2001]

ABP51992NOVNEURhomologous amino 1..121 114/121 (94%)3e-58 acid sequence SEQ ID N0:29 1..121 114/121 (94%) - Homo Sapiens, 121 aa. [US2002068279-Al, 06-JUN-2002]

ABP51987NOVNEUR homologous amino4..121 1121118 (94%)7e-58 acid sequence SEQ ID N0:24 1..118 112h 18 (94%) - Homo Sapiens, 118 aa. [US2002068279-A1, 06-JUN-2002]

ABP51989NOVNEURhomologous amino 4..121 111/11'8 le-56 acid (94%) sequence SEQ ID N0:26 1..118 111/118 (94%) - Homo Sapiens, 118 aa. [US2002068279-A1, 06-JUN-2002]

ABI'S1990NOVNEUR homologous amino10..121 108/112 (96%)7e-56 acid sequence SEQ ID N0:27 1..112 108/112 (96%) - Homo Sapiens, 112 aa. [US2002068279-Al, 06-JUN-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 8E.

Table 8E.
Public BLASTP
Results for NOVBa NOVBa Identities/

Protein Residues!SimilaritiesExpect for AccessionProtein/Organism/Length Match the MatchedValue Number ResiduesPortion P08949 Neuromedin B-32 precursor1..121 1201121 2e-62 (99%) [Contains: Neuromedin 1..121 120/121 B] - Homo (99%) Sapiens (Human), 121 .
aa.

Q9CR53 Neuromedin B-32 precursor1..121 89/121 (73%)2e-43 [Contains: Neuromedin 1..121 991121 (81%) B] - Mus musculus (Mouse), 121 aa.

A37178 neuromedin B precursor 1..115 84/115 (73%)2e-4.1 - rat, 117 aa.

1..115 94/115 (81 %) A28945 neuromedin B precursor 1..73 69/73 (94%)Se-33 - human, 76 1..73 69/73 (94%) P01297 Neuromedin B-32 [Contains:25..56 30/32 (93%)2e-11 Neuromedin B] - Sus scrofa1..32 30/32 (93%) (Pig), 32 aa.

PFam analysis predicts that the NOVBa protein contains the domains shown in the Table 8F.
Table 8F. Domain Analysis of NOVBa Identities/
Pfam Domain NOVBa Match Region Similarities Expect Value for the Matched Region Bombesin 47..56 8/10 (80%) 0.26 10/10 (100%) Example 9.
The NOV9 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 9A.

9A. NOV9 Sequence Analysis >a, CG55184-03 SEQ ID NO: I 13 614 by Sequence ORF Start: ATG at 4 ORF Stop: TAG at 607 ACGGACTCCAAGGGCTCCTCTTCCTCCCCGCTGGGGATATCGGTCCGGGCGGCCAACTCCAAGGTCGCC
CGGCGGTGCGGAGCACCAACCACGAGCCATCCGAGATGAGCAACAAGACGCGCATCATTTACTTCGATC
CCTGGTGAATGTGGGTAATTTTTTCACATTGGAGTCTGTCTTTGTAGCACCAAGAAAAGGAATTTACAG
AGTTTTCACGTGATTAAAGTCTACCAGAGCCAAACTATCCAGGTTAACTTGATGTTAAATGGAAAACCA
TATCTGCCTTTGCGGGGGACAAAGATGTTACTCGTGAAGCTGCCACGAATGGTGTCCTGCTCTACCTAG
AAA(zc,ATAAGGTTTACCTAAAACTGGAGAAAGGTAATTTGGTTGGAGGCTGGCAGTATTCCACGTTTTC
CG55184-03 ~SEQ ID N0:114 X201 as BMW at 21807.9kD
MGSGRRALSAVPAVLLVLTLPGLPVWAQNDTEPIVLEGKCLVVCDSNPATDSKGSSSSPLGISVRAANSKVAF
SAVRSTNHEPSEMSNKTRIIYFDQILVNVGNFFTLESVFVAPRKGIYSFSFHVIKVYQSQTIQVNLMLNGKPV
ISAFAGDKDVTREAATNGVLLYLDKEDKVYLKLEKGNLVGGWQYSTFSGFLVFPL_,v_,.~ . - ~"
fib, CGSSI84-O1 SEQ ID_NO: 115 _ .614 by Sequence ORF Stazt:~ATG at 4 ORF Stop:YTAG at 607 AGACGCGCATCATTTACTTCGATC
AGCACCAAGAAAAGGAATTTACAG
TAAGGTTTACCTAAAACTGGAGAAAGGTAATTTGGTTGGAGGCTGGCAGTATTCCACGTTTTC
GTGTTCCCCCTATAGGATTC
CG55184-OI ~SEQ ID NO: I16 201 as MW at 21807.9kD
MGSGRRALSAVPAVLLVLTLPGLPVWAQNDTEPIVLEGKCLVVCDSNPATDSKGSSSSPLGISVRAANSKVAF
SAVRSTNHEPSEMSNKTRIIYFDQILVNVGNFFTLESVFVAPRKGIYSFSFHVIKVYQSQTIQVNLMLNGKPV
ISAFAGDKDVTREAATNGVLLYLDKEDKVYLKLEKGNLVGGWQYSTFSGFLVFPL _-NOV9c, CGSS I84-02 SEQ ID NO: 117 522 bp- _ __~
DNA Sequence ORF Start: at 1 ~ OR_F Stop: end of sequence CAGAACGACACGGAGCCCATTGTGCTGGAGGGCAAGTGTCTGGTGGTGTGCGACTCGAACCCGGCCACGGACT
CCAAGGGCTCCTCTTCCTCCCCGCTGGGGATATCGGTCCGGGCGGCCAACTCCAAGGTCGCCTTCTCGGCGGT
GCGGAGCACCAACCACGAGCCATCCGAGATGAGCAACAAGACGCGCATCATTTACTTCGATCAGATCCTGGTG
TTAAAGTCTACCAGAGCCAAACTATCCAGGTTAACTTGATGTTAAATGGAAAACCAGTAATATCTGC
GGGGGACAAAGATGTTACTCGTGAAGCTGCCACGAATGGTGTCCTGCTCTACCTAGATAAAGAGGAT
CG55184-02 ~SEQ ID NO: 118 174 as ~MW at 19080.6kD
KCLWCDSNPATDSKGSSSSPLGISVRAANSKVAFSAVRSTNHEPSEMSNKTRIIYFDQILV
FVAPRKGIYSFSFFiVIKVYQSQTIQVNLMLNGKPVISAFAGDKDVTREAATNGVLLYLDKED
VGGWQYSTFSGFLVFPL '~
Vie, CG55184-OS SEQ m NO: 121 45 b -'_..~ ~_._._Jr.__ Sequence ~ORF Start: at 1 RF Ston: end of . q NOV9e, CG55I84-OS SEQ ID NO: 122 15 as MW at I588.71eD
Protein Se uence . ~,__~___~~;
_r.._._.___~._~.~ ___. __ ~ .._.____..._._~~ -:~
ANSKVAFSAVRSTNfi A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table 9B.

Table 9B. Comparison of the NOV9 protein sequences.
NOV9a MGSGRRALSAVPAVLLVLTLPGLPVWAQNDTEPIVLEGKCLVVCDSNPATDSKGSSSSPL
NOV9b MGSGRRALSAVPAVLLVLTLPGLPVWAQNDTEPIVLEGKCLWCDSNPATDSKGSSSSPL
NOV9c ---------------------------QNDTEPIVLEGKCLVVCDSNPATDSKGSSSSPL
NOV9d ____________________________________________________________ NOV9e ____________________________________________________________ NOV9a GISVRAANSKVAFSAVRSTNHEPSEMSNKTRIIYFDQILVNVGNFFTLESVFVAPRKGIY
NOV9b GISVRAANSKVAFSAVRSTNHEPSEMSNKTRIIYFDQILVNVGNFFTLESVFVAPRKGIY
NOV9c GISVRAANSKVAFSAVRSTNHEPSEMSNKTRTIYFDQILVNVGNFFTLESVFVAPRKGIY
NOV9d _____p,~7S~AFSAVRSTNH-______________________________________ NOV9e ______~JS~AFSAVRSTNH-______________________________________ NOV9a SFSFHVIKVYQSQTIQVNLMLNGKPVISAFAGDKDVTREAATNGVLLYLDKEDKVYLKLE
NOV9b SFSFHVIKVYQSQTIQVNLMLNGKPVISAFAGDKDVTREAATNGVLLYLDKEDKVYLKLE
NOV9c SFSFHVIKVYQSQTIQVNLMLNGKPVISAFAGDKDVTREAATNGVLLYLDKEDKVYLKL,E
NOV9d _______________________________-____________________________ NOV9e __-___________-_________________________________-___________ NOV9a KGNLVGGWQYSTFSGFLVFPL
NOV9b KGNLVGGWQYSTFSGFLVFPL
NOV9c KGNLVGGWQYSTFSGFLVFPL
NOV9d _____________________ NOV9e ___________________._ NOV9a (SEQ ID NO: 114) NOV9b (SEQ ID NO: 116) NOV9c (SEQ ID NO: 118) NOV9d (SEQ ID NO: 120) NOV9e (SEQ ID N0: 122) Further analysis of the NOV9a protein yielded the following properties shown in Table 9C.
Table 9C. Protein Sequence Properties NOV9a SignalP analysis: Cleavage site between residues 28 and 29 PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length 6; pos.chg 2; ~neg.chg 0 Ii-region: length 23; peak value 10.04 PSG score: 5.64 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 0.95 possible cleavage site. between 27 and 28 » > Seems to have a cleavable signal peptide (1 to 27) ALOM: Klein et al's method for TM region allocation Tnit position for calculation: 28 Tentative number of TMS(s) for the threshold 0.5: 1 Number of TMS(s) for threshold 0.5: 0 PERIPHERAL Likelihood = 5.67 (at 60) ALOM score: 0.10 {number of TMSs: 0) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 23 Charge difference: -5.0 C(-2.0) - N( 3.0) N >= C: N-terminal side will be inside MITDISC: discrimination of mitochondrial targeting seq R content: 2 Hyd Moment(75): 11.01 Hyd Moment(95): 9.83 G content: 3 D/E content: l S/T content: 3 Score: -2.58 Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 16 RRA~LS
NUCDISC: discrimination of nuclear localization signals pat4: none pat7: none bipartite: none content of basic residues: 9.5%
NLS Score: -0.47 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals:
XXRR-like motif in the N-terminus: GSGR
none SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none ACtinin-type actin-binding motif:
type 1: none type 2: none NMYR: N-myristoylation pattern : none Prenylation motif. none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: none Dileucine motif in the tail: none checking 63 PROSITE DNA binding motifs: . none (checking 71 PROSITE ribosomal protein motifs: none 'checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: cytoplasmic Reliability: 94.1 COIL: Lupas's algorithm to detect coiled-coil regions total: 0 residues Final Results (k = 9/23):
33.3 ~: extracellular, including cell wall 33.3 ~: mitochondrial 22.2 ~: endoplasmic reticulum 11.1 ~: Golgi » prediction for CG55184-03 is exc (k=9) A search of the NOV9a protein against the Geneseq database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 9D.
Table 9D.
Geneseq Results for NOV9a NOV9a ~ Identities/

Geneseq Protein/Organism/Length Residues/SimilaritiesExpect [Patent #, for IdentifierDate] Match the Matched Value ResiduesRegion AAE16346Human cerebellin-like 1..201 201/201 (100%)e-11 protein, I

POLY10 -Homo Sapiens, 1..201 201/201 (100%) 201 aa.

[W0200185767-A2, 15 NOV-2001]

ABB84924Human PR01382 protein 1..201 ~ 201/201 e-111 sequence (100%) SEQ ID N0:216 - Homo Sapiens,1..201 201/201 (100%) aa. [W0200200690-A2, 03-JAN-2002]

ABB95530Human angiogenesis related1..201 201/201 (100%)e-111 protein PRO1382 SEQ 1D NO: 216 1..201 201/201 (100%) - Homo Sapiens, 201 aa. [W0200208284-A2, 31-JAN-2002]

AA015422Human genset metabolic 1..201 201/201 (100%)e-111 gene (GMG-8) protein - Homo 1..201 2011201 (100%) Sapiens, 201 aa. [W0200255694-A2, 18-JUL-2002]
.

_ AAB66151Protein of the invention 1..201 201/201 (100%)e-111 #63 -Unidentified, 201 aa. 1..201 201/201 (100%) [W0200078961-A1, 28-DEG-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 9E.

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

AccessionProtein/Organism/Length Residues!Similarities Expect for the Number Matched PortionValue Residues Q9NTU7 Cerebellin-like glycoprotein1..201 201/201 (100%)e-111 precursor - Homo sapiens1..201 201/201 (100%) (Human), 201 aa.

Q8BME9 3 CEREBELLIN-like glycoprotein1..201 193/201 (96%) e-105 precursor - Mus musculus1..198 195/201 (96%) (Mouse), 198 aa.

Q8BMF0 CEREBELLIN-like gIycoprotein1..201 192/201 (95%) e-104 precursor - Mus musculus1..198 1941201 (95%) (Mouse), 198 aa.

Q8BGU2 Cerebellin 2 precursor 7..201 145/196 (73%) 2e-76 protein - Mus musculus (Mouse), 224 31..224170/196 (85%) aa.

P98087 Cerebellin-like glycoprotein7..201 144/196 (73%) 6e-76 Rattus norvegicus (Rat),31..224169/196 (85%) 224 aa.

PFam analysis predicts that the NOV9a protein contains the domains shown in the Table 9F.
Table 9F. Domain Analysis of NOV9a Identities/
Pfam Domain NOV9a Match Region ~ Similarities Expect Value for the Matched Region Clq 72..198 48/137 (35%) 1.4e-48 ' 113/137 (82%) Example 10.
The NOV 10 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 10A.

Oa, CG55274-OS SEQ ID NO: 124 ~86 as ~MW at 9590.OkD
i Sequence , ~EFDKAAEDVRKLPTRPADNKELKKLDGLYKQAIIGDINIEYLGMLDLKGKAKCAAWTLQKRLSKEDAT
ISKAKEPIEK
V l Ob, CG55274-O1 SEQ m N0:125 _ 280 by _ A Sequence ORF Start: ATG at 7 --~ Stop: TAG at 265 :ACCATGGCACTGCAGGCTGAATTCGACAAGGCTGCAGAAGACGTGAGGAAGCTGCCAACAAGACCAGCAG
~ATAAAGAACTGAAAAAACTCGATGGACTTTACAAACAAGCTATAATTGGAGACATTAATATTGAGTATCT

SAC'GAGTGTCTCTATTTCTAAGGCAAAAGAGCCGATAGAAAAATAGGACATTTAGAATA
Ob, CG55274-Ol ~SEQ ID NO: I26 ~86 as MW at 9624.1kD
iSequence __ EFDKAAEDVRKLPTRPADNKELKKLDGLYKQAIIGDINIEYLGMLDFKGKAKCAAWTLQKRLSKEDAT
SVSISKAKEPIEK
.Oc, CG55274-02 SEQ ID NO: 127 289 by __ ._.. ._.___.__:__.__ .__._ __...__.._..__~~-._.._........ _ ._. ...___ ....
Sequence ~ORF Start. ATG at 17 ORF Sto : TAG at 272 AGATGAGAAAGAACTGAAAAAACTCGATGGACTTTACAAACAAGCTATAATTGGAGACATTAATATTG
CCTGGGAATGCTGGATTTAAAGGGCAAGGCCAAATGCGCAGCATGGACCCTCCAAAAAAGGTTGTCAAA
iATGCAACGAGTGTCTCTATTTCTAAGGCAAAAGAGCCGATAGAAAAATAGGACATTTAGAATACA
Oc, CG55274-02 ~SEQ ID NO: 128 a ~85 as -__~MW at 9528.9kD
i_.Sequence.....__. ..________. __._.._.... _..._.___ _ .. __. ..._____. .
_.._._ .. _....._.._.._....._.__ _ ... ._.__.___..._..._........_ nRnKAAFnVRKLPTRPDEKELKKLDGLYKOAIIGDINIEYLGMLDLKGKAKCAAWTLQKRLSKEDATS
ISKAKEPIEK
IS

NOV 10e, CG55274-04 ~ ~ SEQ ID N0: 132 a 18 as MW at 2053.4kD
Protein Sequence QAIIGDINIEYLGMLDFK
A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table l OB.
Table lOB. Comparison of the NOV10 protein sequences.
NOVlOa MALQAEFDKAAEDVRKLPTRPADNKELKKLDGLYKQAIIGDINIEYLGMLDLKGKAKCAA
NOVlOb MALQAEFDKAAEDVRKLPTRPADNKELKKLDGLYKQAIIGDINIEYLGMLDFKGKAKCAA
NOVlOc MALQADRDKAAEDVRKLPTRPDE-KELKKLDGLYKQAIIGDINIEYLGMLDLKGKAKCAA
NOVlOd -----------------------------------QAIIGDINIEYLGMLDLKGK-----NOVlOe ___________________________________QAIIGDINIEYLGMLDFK-______ NOVlOa WTLQKRLSKEDATSVSISKAKEPIEK
NOVlOb WTLQKRLSKEDATSVSISKAKEPIEK
NOVlOc WTLQKRLSKEDATSVSISKAKEPIEK
NOVlOd __________________________ NOVlOe __________________________ NOVlOa (SEQ ID NO: 124) NOVlOb (SEQ ID NO: 126) NOVlOc (SEQ ID NO: 128) NOVlOd (SEQ ID NO: 130) NOVlOe (SEQ ID NO: 132) Further analysis of the NOVlOa protein yielded the following properties shown in Table lOC.
Table lOC. Protein Sequence Properties NOVlOa SignalP analysis: No Known Signal Sequence Predicted PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length 9; pos.chg 1; neg.chg 2 H-region: length 2; peak value 0.00 PSG score: -4.40 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): -10.68 possible cleavage site: between 58 and 59 »> Seems to have no N-terminal signal peptide ALOM: Klein et al's method for TM region allocation Init position for calculation: 1 Tentative number of TMS(s) for the threshold 0.5: 0 number of TMS(s) _. fixed PERIPHERAL Likelihood = 7.11 (at 36) ALOM score: 7.11 (number of TMSs: 0) MITDISC: discrimination of mitochondrial targeting seq R content: 0 Hyd Moment(75): 3.71 Hyd Moment(95): 2.95 G content: 0 D/E content: 2 S/T content: 0 Score: -7.75 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: 19.8 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 yAC: 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 I!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):
82.6 %. nuclear 4.3 %: cytoskeletal 4.3 %: mitochondria!
4.3 %: cytoplasmic 4.3 %. peroacisomal » prediction for CG55274-05 is nuc (k=23) 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 lOD.
Table ~
10D.
Geneseq Results for NOVlOa NOVlOa Identities/

Geneseq Protein/Organism/Length Residues/SimilaritiesExpect [Patent #, for IdentifierDate] Match the MatchedValue ResiduesRegion AAB81814 Human endozepine-like 1..86 85/86 (98%)le-42 ENDOS SEQ

ID NO: 10 - Homo Sapiens,1..86 85/86 (98%) 86 aa.

[W0200125436-A2, 12-APR-2001]

ABU11538 Human MDDT polypeptide 1..86 64/86 (74%)le-26 - Homo sapiens,100 aa. 13..97 70/86 (80%) [W0200279449-A2, 10-OCT-2002) AAB81811 Human endozepine-like 3..86 61/84 (72%)Se-25 ID NO: 8 - Homo Sapiens, 11..93 68/84 (80%) 96 aa.

[W0200125436-A2, 12-APR-2001]

ABJ05397 Frog acyl coenzyme A binding4..86 57/83 (68%)2e-23 protein (ACBP) - Rana sp, 86 aa. 2..83 66183 (78%) [W0200261096-Al, 08-AUG-2002]

ABJ05396 Duck acyl coenzyme A binding4..86 55/83 (66%)3e-23 protein (ACBP) 2 - Anas sp, 86 2..83 66/83 (79%) aa.

[W0200261096-Al, 08 AUG-2002) In a BLAST search of public sequence databases, the NOVlOa protein was found to have homology to the proteins shown in the BLASTP data in Table 10E.

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

Protein Residues/SimilaritiesExpect for AccessionProtein/Organism/Length Match the MatchedValue Number ResiduesPortion Q8N6N7 Similar to RII~EN cDNA 1..86 64/86 (74%)4e-26 gene - Homo Sapiens (Human),1..85 70/86 (80%) 88 aa.

Q9D258 9230116B18Rik protein - 1..86 60186 (69%)3e-25 Mus musculus (Mouse), 88 aa. 1..85 70/86 (80%) A57711 diazepam-binding inhibitor1..86 58/86 (67%)2e-23 - laughing frog, 88 aa. 1..85 68/86 (78%) P45883 Acyl-CoA-binding protein 4..86 57/83 (68%)4e-23 homolog (ACBP) (Diazepam binding 3..84 66/83 (78%) inhibitor homology (DBI) - Rana ridibunda (Laughing frog) (Marsh frog), 87 aa.

P45882 Acyl-CoA-binding protein 4..86 55/83 (66%)7e-23 (ACBP) (Diazepam binding inhibitor)19..10066/83 (79%) (DBI) (Endozepine) (EP) - Anas platyrhynchos (Domestic duck), 103 aa.

PFam analysis predicts that the NOV l0a protein contains the domains shown in the Table l OF.
Table l OF. Domain Analysis of NOVlOa Identities/
Pfam Domain NOVlOa Match Region Similarities Expect Value for the Matched Region ACBP 3..86 42/90 (47%) S.le-18 66/90 (73%) Example 11.
The NOV 11 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 11 A.

11A. NOVll ~la, GG55379-04 SEQ m,N0~133 6291 by _ Sequence ORF Start:~ATG at 1 ORF Stop: TAG at 3763 AAGGATGGGGACACCCTGCTGGAGCACGACCACTTACACCTGCTGCCCAATGGTTCCCTGTGGCTGTCCCAGC
CACTAGCACCCAATGGCAGTGACGAGTCAGTCCCTGAGGCTGTGGGGGTCATTGAAGGCAACTATTCGTGCCT
CTGCACCCGGAGTCTCAGACGGTGGAGGAGAACGGGACAGCTCGCTTTGAGTGCCACATTGAAGGGCTGCCAG
CTCCCATCATTACTTGGGAGAAGGACCAGGTGACATTGCCTGAGGAGCCTCGGCTCATCGTGCTTCCCAACGG
CGTCCTTCAGATCCTGGATGTTCAGGAGAGTGATGCAGGCCCCTACCGCTGCGTGGCCACCAACTCAGCTCGC
CCCCTTTTGTGTCCTGGGTCCGAGACGGGAAGCCCATCTCCACAGATGTCATCGTCCTGGGC
ACTAATTGCCAACGCGCAGCCCTGGCACTCCGGCGTCTATGTCTGCCGCGCCAACAAGCCCC
TTCGCCACTGCAGCCGCTGAGCTCCGTGTGCTGCTAGCGGCTCCCGCCATCACTCAGGCGCC
CGCTGGCCGTGGTGGTGCGCGAGGGGCTGCCCAGCGCCCCCACGCGGGTCACTGCTACG
CTCTCCACTACCAGAAGGCACGGGGTATGGACAATGTGGAATACCAGTTTGCAGTGAACAACGACACCACAGA
ACTACAGGTTCGGGACCTGGAACCCAACACAGATTATGAGTTCTACGTGGTGGCCTACTCCCAGCTGGGAGCC
AGCCGCACCTCCACCCCAGCACTGGTGCACACACTGGATGATGTCCCCAGTGCAGCACCCCAGCTCTCCCTGT
CCAGCCCCAACCCTTCGGACATCAGGGTGGCGTGGCTGCCCCTGCCCCCCAGCCTGAGCAATGGGCAGGTGGT
GAAGTACAAGATAGAATACGGTTTGGGAAAGGAAGGTGAGTGGGGGGATCAGATTTTCTCTACTGAGGTGCGA
CAGCAGCCGGCTTCGGGGCCCCCTCCCAGTGGATGCATCACAGGACGCCCAGTATGCACAACCAGAGCCATGT
CCCTTTTGCCCCTGCAGAGTTGAAGGTGCAGGCAAAGATGGAGTCCCTGGTCGTGTCATGGCAGCCACCCCCT
CACCCCACCCAGATCTCTGGCTACAAACTATATTGGCGGGAGGTGGGGGCTGAGGAGGAGGCCAATGGCGATC
GCCTGCCAGGGGGCCGTGGAGACCAGGCTTGGGATGTGGGGCCTGTCCGGCTCAAGAAGAAAGTGAAGCAGTA
TGTGGAAGGGCAAGACGGAGAAGGCGCCGGCACCAGACATGCCTATCCAGAGGGGACCACCCC
CCACGTCCATGCGGAATCAAACAGCTCCACATCCATCTGGCTTCGGTGGAAAAAGCCAGATTT
AAGATTGTCAACTACACTGTGCGCTTCAGCCCCTGGGGGCTCAGGAATGCCTCCCTGGTCACC
GTTCTGGAGAAGACATCCTCATTGGCGGCTTGAAGCCATTCACCAAATACGAGTTTGCAGTGC
CGTGGACATGGATGGGCCTTTCGGCTCTGTGGTGGAGCGCTCCACCCTGCCTGACCGGCCCTC
TCCGACCTGCGACTGAGCCCCCTGACACCGTCCACGGTTCGGCTGCACTGGTGCCCCCCCACA
ATGGGTGGCGGTGTTTCTGAAGGCCGGAGTCACTCCAAAAGAAAGATCTCCTGGGCTCAACCAAG
TCAAATCCCCCTGCCCTCTAGGAGCCAGCCCAGGCCTGCCCAGATCCCCGGTCTCCTCCTCTGCCTAGCTCTT
CCCAGAGGATGTGGTTTGGGGCAGGCAGGTATGGATCACATAGGATGCGATACCTGTGGCCGTGTATGTCCAC
ATGTGTGCCTGTAGATACATCATCAAGCCCTTTGGAGCTTCCTAAGTTGCTTTGGCTGAGGGGAGAGGAAAAC

TTCACTCCCCCCATACTCTTTGTGATACACATGTGACATGTGAAAGACATACGAGACATAGCTAC
CTGACAAAGACAGTCC
#CCCAGCTCCTTATTGCCCAAATAGAGAGGGTGGCCCTGGCTCCCCTCCGAGCAACTCTGCATTTAATTTTGTA
~TCCTCTCTCCTGTTTTCCTTCCTCTGCTATCTCTCACACCTCTCCCAGACTATGTCATCTTGTTCTCCTGCCT
#GGGTTCAAACTCTGCATCCTTCTCTAACAACGTGACTACCTCATGTCTGCTTCAAGGCCCCCGTGCCCTTCCT
#GTATCCGCGGCTGCCGCGCACTCGCCTGCCATCCTCCTGCCTCCTCTTCACTCAGTGCTTCTGCTTGCCCTGC
#TTGGGATCCAAGGGTTCTCCATGGATGGATCCAAGTCATAGAGGGGAATGTTTGAGACAGGGAAGGGGGCTGT
#GAGGCTTGACTTGATCTGGATTGGGGATGACAGGAATCTCACCCTCTGGGGTGCTGGCAAGGAGGTCTTTGCA
#CAGGAAAAGGGGTAGCTCATTTCAGTTTGTTTTTTCTTTAAATTGAATCCTCAAGTCATTTTCTGTTCACCTG
ECTCTGCCTTCTGGGCCAAATTCGAAATAACCAGTCCATTTTTCCTTTTTTTTTTTTTTTTTTTTTTTTAAATG
#GTGGAATGTCTCTCAGCACAGTTGCGGCTTCCTCAAACCCTGAAAGCATCTGTGTTTATTATACTCGGGTGTC
#ACTCACTGTTGATGTCTGCACCTACGTTTCCACCTCCTCCCCCTCCTTCAGCCAGCCTATGATAACACTAAAG
ATTATTAATGTTGGTTTTGTATCTCGTTAAAGACAGAATTGTCACTTGTAGTATTTCTGTAGCATTCAGCGCT
GCTGTGGCTAACACCACTGTGTATGTTTCATCATTGCTCTGAAGGTCAAAAGCCTCATTTTATTTTGCTGGTT
ATTATACCACATAGTCATTTTTCATGTTCTTGTTTAACAGGCACTGAGGTTCTGGTTTAAATTAAATAGCTGC
AAATGAGACAATTTATAACCCATTAGGTTGGGTGGAAAATTGTTTCTCAAAAGCAAATAAGTAATAAATCTGG
TATCTGCCTATAACTCACAGTTGATAAGAAAGTGGCCATTTCTCACTAGCACTATATATGATTTGGGCTCTGG
GTAATTTGGAAGTGTTAGGTTTGTGTCTTTGTAGCAGTATTTTTATTAGAAAAGAATCTATTGGCCTTTTACA
GGGTATTAATCCCTTTGTCACCTACCATTGATGCCTTAAGTTTTCTGAGTCTCAATTAAAAATCTTCCTTTTC
TTGATGCATGACAAGTGTAATCAGTACTTGCTCATTTATTTGTCTGTATTTAGTTTATGCTGTACTATTTAAT
#TATCCTTCCAGCGTTTTTTTTTTCTCCTTACAAATATGATACTCTTTAGTGTTAAGCTAAGGCATTGATTCAT
#GTATCTGTCCTTATAATGAATTAATAAACTATTTTCCAG #
la, CG55379-04 ~SEQ ID NO: 134 1254 as MW at 134608.7kD
VLNCSLGAAAAGPPTRVTWS
DTLLEHDHLHLLPNGSLWLSQPLAPNGSDESVPEAVGVIEGNYSCLAHGPLGVLASQTAWKLASLADFS
ESQTVEENGTARFECHIEGLPAPIITWEKDQVTLPEEPRLIVLPNGVLQILDVQESDAGPYRCVATNSAR
SQEALLSVAHRGSLASTRGQDVVIVAAPENTTVVSGQSVVMECVASADPTPFVSWVRDGKPISTDVIVLG
TRDFATAAAELRVLLAAPAITQAPEALSRTRASTARFVCRASGEPRPA
VITQIGLQDAGYYQCVAENSAGMACAAASLAVVVREGLPSAPTRVTAT
~SSAVLVAWERPEMHSEQIIGFSLHYQKARGMDNVEYQFAVNNDTTELQVRDLEPNTDYEFYWAYSQLGA
'STPALVHTLDDVPSAAPQLSLSSPNPSDIRVAWLPLPPSLSNGQVVKYKIEYGLGKEGEWGDQIFSTEVR
TQLMLNSLQPNKVYRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQAKMESLVVSWQPPP
'QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVPGRLYEVKLVAFNKHEDG
VNTKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFTTVKIVNYTVRFSPWGLRNASLVT
'SSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGSVVERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPT
fGEIVEYLILYSSNHTQPEHQWTLLTTQGNIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQE
~DSLDMHSVTGIIVGVCLGLLCLLACMCAGLRRSPHRESLPGLSSTATPGNPALYSRARLGPPSPPAAHEL
~VHPHPQDWSPPPSDVEDRAEVHSLMGGGVSEGRSHSKRKISWAQPSGLSWAGSWAGCELPQAGPRPALTR
~PPAGTGQTLLLQALVYDAIKGNGRKKSPPACRNQVEAEVIVHSDFSASNGNPDLHLQDLEPEDPLPPEAP
SGVGDPGQGAAWLDRELGGCELAAPGPDRLTCLPEAASASCSYPDLQPGEVLEETPGDSCQLKSPCPLGA
aLPRSPVSSSA

.lb, CG55379-Ol SEQ m NO:1_35 ~~3741 by Sequence ORF Start: ATG at 1 ORF Stop: end of AGCACCCAATGGCAGTGACGAGTCAGTCCCTGAGGCTGTGGGGGTCATTGAAGGCAACTATTCGTGCCT
rarrcmrrrrrmrnnrm~rmarceAC~ccAGACTGCTGTCGTCAAGCTTGCCAGTCTCGCAGACTTCTCT
TTACTTGGGAGAAGGACCAGGTGACATTGCCTGAGGAGCCTCGGCTCATCGTGCTTCCCAACGG
GATCCTGGATGTTCAGGAGAGTGATGCAGGCCCCTACCGCTGCGTGGCCACCAACTCAGCTCGC
CCCCTTTTGTGTCCTGGGTCCGAGACGGGAAGCCCATCTCCACAGATGTCATCGTCCTGGGC
ACTAATTGCCAACGCGCAGCCCTGGCACTCCGGCGTCTATGTCTGCCGCGCCAACAAGCCCC
ACTACCAGTGCGTGGCTGAGAACAGCGCGGGAATGGC
ACG
ATGAGTTCTACGTGGTGGCCTACTCCCAGCTGGGAGCC
CAAGATAGAATACGGTTTGGGAAAGGAAGATCAGATTTTCTCTACTGAGGTGCGAGGAAATGAGACA
ATGCTGAACTCGCTTCAGCCAAACAAGGTGTATCGAGTACGGATTTCGGCTGGTACAGCAGCCGGCT
CCCCCTCCCAGTGGATGCATCACAGGACGCCCAGTATGCACAACCAGAGCCATGTCCCTTTTGCCCC
TCTCTGGCTACAAACTATATTGGCGGGAGGTGGGGGCTGAGGAGGAGGCCAATGGCGATCGCCTGCCAGGGG
CCGTGGAGACCAGGCTTGGGATGTGGGGCCTGTCCGGCTCAAGAAGAAAGTGAAGCAGTATGAGCTGACCCA
CTAGTCCCTGGCCGGCTGTACGAGGTGAAGCTCGTGGCTTTCAACAAACATGAGGATGGCTATGCAGCAGTG
GGAAGGGCAAGACGGAGAAGGCGCCGGCACCAGACATGCCTATCCAGAGGGGACCACCCCTGCCTCCAGCCC
CGTCCATGCGGAATCAAACAGCTCCACATCCATCTGGCTTCGGTGGAAAAAGCCAGATTTCACCACAGTCAA
ATTGTCAACTACACTGTGCGCTTCAGCCCCTGGGGGCTCAGGAATGCCTCCCTGGTCACCTATTACAGTTCT
GAGAAGACATCCTCATTGGCGGCTTGAAGCCATTCACCAAATACGAGTTTGCAGTGCAGTCTCACGGCGTGG
CCTGCGACTGAGCCCCCTGACACCGTCCACGGTTCGGCTGCACTGGTGCCCCCCCACAGAGCCCAACGGGGAG
TGTGATCACGCTCCAGGAGAAGCTGTCA
TGAATTGGAGTCCCTT
AAGTGCTCAACCAAGCGGGCTGAGCTG
CATCTAACGGGAACCCTGACCTCCATCTCCAAGACCTGGAGCCTGAGGACCCCCTGCCTCCAG
TCTCATCTCGGGTGTTGGGGATCCAGGGCAGGGGGCAGCCTGGCTGGACAGGGAGTTGGGAGG
CCGGACCTCCAGCCAGGCGAGGTGCTAGAGGAGACCCCTGGAGATAGCTGCCAGCTCAAATCCCCCTGCCCTC
TAGGAGCCAGCCCAGGCCTGCCCAGATCCCCGGTCTCCTCCTCT

:I lb, CG55379-01 ~SEQ ID N0:136 1247 as ~MW at 133821.8kD
~iARGDAGRGRGLILALTFCLLAARGELLLPQETTVELSCGVGPLQVILGPEQAAVLNCSLGAAAAGPPTRVTWS
KDGDTLLEHDHLHLLANGSLWLSQPLAPNGSDESVPEAVGVIEGNYSCLAHGPPRVLASQTAWKLASLADFS
LHPESQTVEENGTARFECHIEGLPAPIITWEKDQVTLPEEPRLIVLPNGVLQILDVQESDAGPYRCVATNSAR
QHFSQEALLSVAHRGSLASTRGQDWIVAAPENTTWSGQSVVMECVASADPTPFVSWVRDGKPISTDVIVLG
RTNLLIANAQPWHSGVYVCRANKPRTRDFATAAAELRVLLAAPAITQAPEALSRTRASTARFVCRASGEPRPA
LRWLHNGAPLRPNGRVKVQGGGGSLVITQIGLQDAGWQCVAENSAGMACAAASLAVVVREGLPSAPTRVTAT
PLSSSAVLVAWERPEMHSEQIIGFSLHYQKARGMDNVEYQFAVNNDTTELQVRDLEPNTDYEFYWAYSQLGA
SRTSTPALVHTLDDVPSAAPQLSLSSPNPSDIRVAWLPLPPSLSNGQVVKYKIEYGLGKEDQIFSTEVRGNET
QLMLNSLQPNKWRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQAKMESLWSWQPPPHPTQ
ISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVPGRLYEVKLVAFNKHEDGYAAV
WKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFTTVKIVNYTVRFSPWGLRNASLVTYYSS
GEDILIGGLKPFTKYEFAVQSHGVDMDGPFGSWERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGE
IVEYLILYSSNHTQPEHQWTLLTTQGEGNIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLS
DSLDMHSVTGIIVGVCLGLLCLLACMCAGLRRSPHRESLPGLSSTATPGNPALYSRARLGPPSP.PAAHELESL
VHPHPQDWSPPPSDVEDRAEVHSLMGGGVSEGRSHSKRKVSAQPSGLSWAGSWAGCELPQAGPRPALTRALLP
PAGTGQTLLLQVLCSDQGNGRKKSPPACRNQVEAEVIVHSDFSASNGNPDLHLQDLEPEDPLPPEAPDLISGV
GDPGOGAAWLDRELGGCELAAPGPDRLTCLPEAASASCSYPDLqPGEVLEETPGDSCQLKSPCPLGASPGLPR
SPVSSS
',1 c, 258065951 SEQ ID NO: 137 ~ 1609 by Sequence ~ ORF Start: at l ORF Stop: at 1609 ACGCCAC
ATGAGTTCTACGTGGTGGCCTACTCCCAGCTGGGAGCCAGCC
ACAAGATAGAATACGGTTTGGGAAAGGAAGATCAGATTTTCTCTACTGAGGTGCGAGGAAATGAGACACAGC
TATGCTGAACTCGCTTCAGCCAAACAAGGTGTATCGAGTACGGATTTCGGCTGGTACAGCAGCCGGCTTCGG
AGGCAAAGATGGAGTCCCTGGTCGTGTCATGGCAGCCACCCCCTCACCCCACCCAGATCT
ATATTGGCGGGAGGTGGGGGCTGAGGAGGAGGCCAATGGCGATCGCCTGCCAGGGGGCCG
TGGGATGTGGGGCCTGTCCGGCTCAAGAAGAAAGTGAAGCAGTATGAGCTGACCCAGCTA
TCTGGCTTCGGTGGAAAAAGCCAGATTTCACCACAGTCAAGATT
GAGAAGACATCCTCATTGGCGGCTTGAAGCCATTCACCAAATACGAGTTTGCAGTGCAGTCTCACGGCGTGGA
CATGGATGGGCCTTTCGGCTCTGTGGTGGAGCGCTCCACCCTGCCTGACCGGCCCTCCACACCCCCATCCGAC
CTGCGACTGAGCCCCCTGACACCGTCCACGGTTCGGCTGCACTGGTGCCCCCCCACAGAGCCCAACGGGGAGA
TCGTGGAGTATCTGATCCTGTACAGCAGCAACCACACGCAGCCTGAGCACCAGTGGACCTTGCTCACCACGCA
ACTTCTTCAAGATGGGGGCGCGC
s :1 lc, 2$806$9$1 ~SEQ ID NO:138 $36 as MW at $9$32.7kD
LPSAPTRVTATPLSSSAVLVAWERPEMHSEQIIGFSLHYQKARGMDNVEYQFAVNNDTTEL
FYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNPSDIRVAWLPLPPSLSNGQWK
IFSTEVRGNETQLMLNSLQPNKWRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPA
VSWQPPPHPTQISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQL
FNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFTTVKI
RNASLWYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGSVVERSTLPDRPSTPPSD
LHWCPPTEPNGEIVEYLILYSSNHTQPEHQWTLLTTQGNIFSAEVHGLESDTRYFFKMGAR
Vlld, 209886264 SEQ ID NO: 139_ 1611 by A Se uence ___.~__..__.___.___.._.~.__-__._...___-_ q ORF Start: at-1~ ~ ~ ORF Ston: end of TGGACAATGTGGAATACCAGTTTGCAGTGAACAACGACACCACAGAACTA
CACAGATTATGAGTTCTACGTGGTGGCCTACTCCCAGCTGGGAGCCAGCC
CCCCAACCCTTCGGACATCAGGGTGGCGTGGCTGCCCCTGCCCCCCAGCCTGAGCAATGGGCAGGTGGTGAAG
TACAAGATAGAATACGGTTTGGGAAAGGAAGATCAGATTTTCTCTACTGAGGTGCGAGGAAATGAGACACAGC
TTATGTTGAACTCGCTTCAGCCAAACAAGGTGTATCGAGTACGGATTTCGGCTGGTACAGCAGCCGGCTTCGG
GGCAAAGATGGAGTCCCTGGTCGTATCATGGCAGCCACCCCCTCACCCCACCCAGATCT
TATTGGCGGGAGGTGGGGGCTGAGGAGGAGGCCAATGGCGATCGCCTGCCAGGGGGCCG
GGGATGTGGGGCCTGTCCGGCTCAAGAAGAAAGTGAAGCAGTATGAGCTGACCCAGCTA
AGGGCAAGACGGAGAAGGCGCCGGCACCAGACATGCCTATCCAGAGGGGACCACCCCTGCCTCCAGCCCACGT
CCATGCGGAATCAAACAGCTCCACATCCATCTGGCTTCGGTGGAAAAAGCCAGATTTCACCACAGTCAAGATT
GTCAACTACACTGTGCGCTTCAGCCCCTGGGGGCTCAGGAATGCCTCCCTGGTCACCTATTACACCAGTTCTG
GAGAAGACATCCTCATTGGCGGCTTGAAGCCATTCACCAAATACGAGTTTGCAGTGCAGTCTCACGGCGTGGA
CATGGATGGGCCTTTCGGCTCTGTGGTGGAGCGCTCCACCCTGCCTGACCGGCCCTCCACACCCCCATCCGAC
TCCTGTACAGCAGCAACCACACGCAGCCTGAGCACCAGTGGACCTTGCTCACCACGCA
TGCTGAGGTCCATGGCCTGGAGAGCGACACTCGGTACTTCTTCAAGATGGGGGCGCGC
ld, 209886264 ~SEQ ID NO: 140 $37 as ~MW at $9607.71eD
LAVWREGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFAVNNDTTEL
LEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNPSDIRVAWLPLPPSLSNGQWK
YGLGKEDQIFSTEVRGNETQLMLNSLQPNKWRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPA
QAKMESLWSWQPPPHPTQISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQL
LYEVKLVAFNKHEDGYAAWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFTTVKI
VRFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGSWERSTLPDRPSTPPSD
PLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQWTLLTTQGNIFSAEVHGLESDTRYFFKMGAR
PGPFSRLQDVITLQEKLSDSVD

L 1 e, 209886345 SEQ ID NO: 141 ;1672 by _ Sequence ~ ORF Start: at 1 ORF Stop: at 1672 AGCTCCGCTGTGTTGGTGGCCTGGGAGCGGCCCGAGATGCCCAGCGAGCAGATCATCGGCTTCTCTCT
ACCAGAAGGCACGGGGCATGGACAATGTGGAATACCAGTTTGCAGTGAACAACGACACCACAGAACTA
TCGGGACCTGGAACCCAACACAGATTATGAGTTCTACGTGGTGGCCTACTCCCAGCTGGGAGCCAGCC
mccArrcCAGC~CTGGTGCACACACTGGATGATGTCCCCAGTGCAGCACCCCAGCTCTCCCTGTCCAG
TACAAGATAGAATACGGTTTGGGAAAGGAAGATCAGATTTTCTCTACTGAGGTGCGAGGAAATGAGACACAGC
TTATGTTGAACTCGCTTCAGCCAAACAAGGTGTATCGAGTACGGATTTCGGCTGGTACAGCAGCCGGCTTCGG
GGCCCCCTCCCAGTGGATGCATCACAGGACGCCCAGTATGCACAACCAGAGCCATGTCCCTTTTGCCCCTGCA
GAGTTGAAGGTGCAGGCAAGGATGGAGTCCCTGGTCGTGTCATGGCAGCCACCCCCTCACCCCACCCAGATCT
CTGGCTACAAACTATATTGGCGGGAGGTGGGGGCTGAGGAGGAGGCCAATGGCGATCGCCTGCCAGGGGGCCG
TGGAGACCAGGCTTGGGATGTGGGGCCTGTCCGGCTCAAGAAGAAAGTGAAGCAGTATGAGCTGACCCAGCTA
GTCCCTGGCCGGCTGTACGAGGTGAAGCTCGTGGCTTTCAACAAACATGAGGATGGCTATGCAGCAGTGTGGA
AGGGCAAGACGGAGAAGGCGCCGGCACCAGACATGCCTATCCAGAGGGGACCACCCCTGCCTCCAGCCCACGT
CCATGCGGAATCAAACAGCTCCACATCCATCTGGCTTCGGTGGAAAAAGCCAGATTTCACCACAGTCAAGATT
TTGGCGGCTTGAAGCCATTCACCAAATACGAGTTTGCAGTGCAGTCTCACGGCGTGGA
ACAGCAGCAACCACACGCAGCCTGAGCACCAGTGGACCTTGCTCACCACGCA
r~me~cAT(~GCCTGGAGAGCGACACTCGGTACTTCTTCAAGATGGGGGCGCGC
le, 209886345 ~SEQ ID NO: 142 557 as ~MW at 61878.3kD
REGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFAVNNDTTEL
DYEFYVVAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNPSDIRVAWLPLPPSLSNGQWK
EDQIFSTEVRGNETQLMLNSLQPNKVYRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPA
SLVVSWQPPPHPTQISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQL
LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFTTVKI
WGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGSVVERSTLPDRPSTPPSD
TVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQWTLLTTQGNIFSAEVHGLESDTRYFFKMGAR
RT.WDVTTT.GFKLSDSVDSFSWSVITAPRAPPRPATRY
l lf, 209886357 SEQ ID NO. 143 1611 by Sequence ORF Start: at 1 ORF Stop: end of ACCGCGTCGCTGGCCGTGGTGGTGCGCGAGGGGCTGCCCAGCGCCCCCACGCGGGTCACTGCTACGCCAC
~rnnrmrrrrmam~mmar~mc~CCCTGGGAGCGGCCCGAGATGCCCAGCGAGCAGATCATCGGCTTCTCTCT
TCACAGGACGCCCAGTATGCACAACCAGAGCCATGTCCCTTTTGCCCCTGCA
ATATTGGCGGGAGGTGGGGGCTGAGGAGGAGGCCAATGGCGATCGCCTGCCAGGGGGCCG
GCTTTCAACAAACATGAGGATGGCTATGCAGCAGTGTGGA
TGCCTATCCAGAGGGGACCACCCCTGCCTCCAGCCCACGT

TGTGATCACGCTCCAGGAGAAGCTGTCAGACTCGG
lf, 209886357 SEQ ID N0:144 537 as MW at 59607.7kD
VWREGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFAVNNDTTEL
PNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNPSDIRVAWLPLPPSLSNGQWK
LGKEDQIFSTEVRGNETQLMLNSLQPNKVYRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPA
YEVKLVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFTTVKI
RFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGSVVERSTLPDRPSTPPSD
LTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQWTLLTTQGNIFSAEVHGLESDTRYFFKMGAR
~lg, CG55379-02 SEQ.ID.NO: 145_............._........_............_1611 bp.
._....._...... ...
Sequence ORF Start: at 7 ORF Stop: at 1606 ACGCCAC
CCACTACCAGAAGGCACGGGGCATGGACAATGTGGAATACCAGTTTGCAGTGAACAACGACACCACAGAACTA
CAGGTTCGGGACCTGGAACCCAACACAGATTATGAGTTCTACGTGGTGGCCTACTCCCAGCTGGGAGCCAGCC
CCCCAACCCTTCGGACATCAGGGTGGCGTGGCTGCCCCTGCCCCCCAGCCTGAGCAATGGGCAGGTGGTGAAG
ATGTTGAACTCGCTTCAGCCAAACAAGGTGTATCGAGTACGGATTTCGGCTGGTACAGCAGCCGGCTTCGG
CCCCCTCCCAGTGGATGCATCACAGGACGCCCAGTATGCACAACCAGAGCCATGTCCCTTTTGCCCCTGCA
GTTGAAGGTGCAGGCAAAGATGGAGTCCCTGGTCGTATCATGGCAGCCACCCCCTCACCCCACCCAGATCT
GGCTACAAACTATATTGGCGGGAGGTGGGGGCTGAGGAGGAGGCCAATGGCGATCGCCTGCCAGGGGGCCG
TGGCTATGCAGCAGTGTGGA
CGGCACCAGACATGCCTATCCAGAGGGGACCACCCCTGCCTCCAGCCCACGT
CACATCCATCTGGCTTCGGTGGAAAAAGCCAGATTTCACCACAGTCAAGATT
AGCCCCTGGGGGCTCAGGAATGCCTCCCTGGTCACCTATTACACCAGTTCTG
ATCTGATCCTGTACAGCAGCAACCACACGCAGCCTGAGCACCAGTGGACCTTGCTCACCACGCA
CTTCAGTGCTGAGGTCCATGGCCTGGAGAGCGACACTCGGTACTTCTTCAAGATGGGGGCGCGC

lg, CG55379-02 SEQ m NO: 146 533 as MW at 59235.4kD
VWREGLPSAPTRWATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFAVNNDTTELQV
PNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNPSDIRVAWLPLPPSLSNGQWKYK
LGKEDQIFSTEVRGNETQLMLNSLQPNKVYRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAEL
.FQ~fESLWSWQPPPHPTQISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVP
EVKLVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFTTVKIVN
.FSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGSVVERSTLPDRPSTPPSDLR
~TPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQWTLLTTQGNIFSAEVHGLESDTRYFFKMGARTE
.lh, CG55379-03 SEQ ID NO: 147 .167_2 by ___ Sequence ORF Start: at 7 ORF Stop:-'at 1606 ATGAGTTCTACGTGGTGGCCTACTCCCAGCTGGGAGCCAGCC
CAAACAAGGTGTATCGAGTACGGATTTCGGCTGGTACAGCAGCCGGCTTCGG
TCACAGGACGCCCAGTATGCACAACCAGAGCCATGTCCCTTTTGCCCCTGCA
ATGGAGTCCCTGGTCGTGTCATGGCAGCCACCCCCTCACCCCACCCAGATCT
CCTGGCCGGCTGTACGAGGTGAAGCTCGTGGCTTTCAACAAACATGAGGATGGCTATGCAGCAGTGTGGA
GCAAGACGGAGAAGGCGCCGGCACCAGACATGCCTATCCAGAGGGGACCACCCCTGCCTCCAGCCCACGT
TGCGGAATCAAACAGCTCCACATCCATCTGGCTTCGGTGGAAAAAGCCAGATTTCACCACAGTCAAGATT
AACTACACTGTGCGCTTCAGCCCCTGGGGGCTCAGGAATGCCTCCCTGGTCACCTATTACACCAGTTCTG
TGGATGGGCCTTTCGGCTCTGTGGTGGAGCGCTCCACCCTGCCTGACCGGCCCTCCACACCCCCATCCGAC
TCTGATCCTGTACAGCAGCAACCACACGCAGCCTGAGCACCAGTGGACCTTGCTCACCACGCA
TGTGATCACGCTCCAGGAGAAGCTGTCAGACTCGG
ACC
lh, CG55379-03 SEQ m N0:148 533 as MW at 59263.4kD
SLAVVVREGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFAVNNDTTELQV
DLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNPSDIRVAWLPLPPSLSNGQWKYK
EYGLGKEDQIFSTEVRGNETQLMLNSLQPNKVYRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAEL
VQARMESLWSWQPPPHPTQISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVP
RLYEVKLVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFTTVKIVN
TVRFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGSWERSTLPDRPSTPPSDLR
SPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQWTLLTTQGNIFSAEVHGLESDTRYFFKMGARTE

A ClustalW comparison of the above protein sequences yields the following sequence alignment shown in Table 11B.
Table 11B. Comparison of the NOV11 protein sequences.
NOV2la MARGDAGRGRGLLALTFCLLAARGELLLPQETTVELSCGVGPLQVILGPEQAAVLNCSLG
NOVllb MARGDAGRGRGLLALTFCLLAARGELLLPQETTVELSCGVGPLQVILGPEQAAVLNCSLG
NOV11C ____________________________________________________________ NoVlld ____________________________________________________________ NOVlle ____________________________________________________________ NOVllf ____________________________________________________________ NOVllg ____________________________________________________________ NOVllh ____________________________________________________________ NOVlla AAAAGPPTRVTWSKDGDTLLEHDHLHLLPNGSLWLSQPLAPNGSDESVPEAVGVIEGNYS
NOVllb AAAAGPPTRVTWSKDGDTLLEHDHLHLLANGSLWLSQPLAPNGSDESVPEAVGVIEGNYS
NOVllc ____________________________________________________________ NOVlld ____________________________________________________________ NOVlle ____________________________________________________________ NOVl2f ____________________________________________________________ NOVllg ____________________________________________________________ NOVllh -___________________________________________________________ NOVlla CLAHGPLGVLASQTAWKLASLADFSLHPESQTVEENGTARFECHIEGLPAPIITWEKDQ
NOVllb CLAHGPPRVLASQTAWKLASLADFSLHPESQTVEENGTARFECHIEGLPAPIITWEKDQ
NOVllc _____________________________________________-______________ NOVlld ____________________________________________________________ NOVlle ____________________________________________________________ NOVllf ____________________________________________________________ NOVllg ____________________________________________________________ NOVllh ____________________________________________________________ NOVlla VTLPEEPRLIVLPNGVLQILDVQESDAGPYRCVATNSARQHFSQEALLSVAHRGSLASTR
NOVllb VTLPEEPRLIVLPNGVLQILDVQESDAGPYRCVATNSARQHFSQEALLSVAHRGSLASTR
NOVllc ____________________________________________________________ NOVlld ____________________________________________________________ NOVlle ____________________________________________________________ NOVllf ____________________________________________________________ g ____________________________________________________________ NOVllh ____________________________________________________________ NOVlla GQDWIVAAPENTTWSGQSVVMECVASADPTPFVSWVRDGKPISTDVIVLGRTNLLIAN
NOVllb GQDWIVAAPENTTWSGQSVVMECVASADPTPFVSWVRDGKPISTDVIVLGRTNLLIAN
NOVllc ____________________________________________________________ NOVlld ____________________________________________________________ NOVlle ____________________________________________________________ NOVllf ____________________________________________________________ NOVllg ____________________________________________________________ NOVlih ________________________________________~___________________ NOVlla AQPWHSGVYVCRANKPRTRDFATAAAELRVLLAAPAITQAPEALSRTRASTARFVCRASG
NOVllb AQPWHSGWVCRANKPRTRDFATAAAELRVLLAAPAITQAPEALSRTRASTARFVCRASG
NOVllc ____________________________________________________________ NOVlld ____________________________________________________________ NOVlle ____________________________________________________________ NOVl2f ____________________________________________________________ NOVllg ____________________________________________________________ NOVllh ____________________________________________________________ 'NOVlla EPRPALRWLHNGAPLRPNGRVKVQGGGGSLVITQIGLQDAGYYQCVAENSAGMACAAASL
',NOVIlb EPRPALRWLHNGAPLRPNGRVKVQGGGGSLVITQIGLQDAGYYQCVAENSAGMACAAASL
'NOVllc __________._____________________________________________GTASL
',NOVlld _______________________________________________________GTASL
NOVlIe _______________________________________________________GTASL
INOVllf _______________________________________________________GTASL
NOVllg _________________________________________________________p,SL
NOVllh _________________________________________________________p~SL
NOVlla AVVVREGLPSAPTRVTATPLSSSAVLVAWERPEMHSEQIIGFSLHYQKARGMDNVEYQFA
NOVllb AWVREGLPSAPTRWATPLSSSAVLVAWERPEMHSEQIIGFSLHYQKARGMDNVEYQFA
NOVllc AVWREGLPSAPTRVTATPLSSSAVLVAWERPEMHSEQIIGFSLHYQKARGMDNVEYQFA
NOVlld AVVVREGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQTIGFSLHYQKARGMDNVEYQFA
NOVlle AVVVREGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFA
NOVlIf AVVVREGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFA
NOVllg AVVVREGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFA
NOVllh AVVVREGLPSAPTRVTATPLSSSAVLVAWERPEMPSEQIIGFSLHYQKARGMDNVEYQFA
NOVlla VNNDTTELQVRDLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNP
NOVllb VNNDTTELQVRDLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNP

NOVlld VNNDTTELQVRDLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNP
NOVlle VNNDTTELQVRDLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNP
NOVllf VNNDTTELQVRDLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNP
NOVllg VNNDTTELQVRDLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNP
NOVllh VNNDTTELQVRDLEPNTDYEFYWAYSQLGASRTSTPALVHTLDDVPSAAPQLSLSSPNP
NOVlla SDIRVAWLPLPPSLSNGQWKYKIEYGLGKEGEWGDQIFSTEVRGNETQLMLNSLQPNKV
NOVllb SDIRVAWLPLPPSLSNGQWKYKIEYGLGKE----DQIFSTEVRGNETQLMLNSLQPNKV
NOVllc SDIRVAWLPLPPSLSNGQWKYKIEYGLGKE----DQIFSTEVRGNETQLMLNSLQPNKV
NOVlld SDIRVAWLPLPPSLSNGQWKYKIEYGLGKE----DQIFSTEVRGNETQLMLNSLQPNKV
NOVlle SDIRVAWLPLPPSLSNGQWKYKIEYGLGKE----DQIFSTEVRGNETQLMLNSLQPNKV
NOVllf SDIRVAWLPLPPSLSNGQVVKYKIEYGLGKE----DQIFSTEVRGNETQLMLNSLQPNKV
NOVllg SDIRVAWLPLPPSLSNGQWKYKIEYGLGKE----DQIFSTEVRGNETQLMLNSLQPNKV
NOVllh SDIRVAWLPLPPSLSNGQWKYKIEYGLGKE----DQIFSTEVRGNETQLMLNSLQPNKV
NOVlla YRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQAKMESLWSWQPPPHPT
NOVllb YRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQAKMESLWSWQPPPHPT
NOVllc YRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQAKMESLWSWQPPPHPT
NOVlld YRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQAKMESLWSWQPPPHPT
NOVlle YRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQARMESLWSWQPPPHPT
NOVllf YRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHWFAPAELKVQAKMESLWSWQPPPHPT
NOVllg YRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQAKMESLWSWQPPPHPT
NOVllh YRVRISAGTAAGFGAPSQWMHHRTPSMHNQSHVPFAPAELKVQARMESLWSWQPPPHPT
NOVlla QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVPGRLYEVK
NOVllb QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVPGRLYEVK
NOVllc QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLWGRLYEVK
NOVlld QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLWGRLYEVK
NOVlle QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVPGRLYEVK
NOVllf QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVPGRLYEVK
NOVllg QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVPGRLYEVK
NOVllh QISGYKLYWREVGAEEEANGDRLPGGRGDQAWDVGPVRLKKKVKQYELTQLVPGRLYEVK
NOVlla LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFT

NOVIlb LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFT
NOVllc LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFT
NOVlld LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFT
NOVlle LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFT
NOVllf LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFT
NOVllg LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFT
NOVllh LVAFNKHEDGYAAVWKGKTEKAPAPDMPIQRGPPLPPAHVHAESNSSTSIWLRWKKPDFT
NOVlla TVKIVNYTVRFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGS
NOVllb TVKIVNYTVRFSPWGLRNASLVTYYSS-GEDILIGGLKPFTKYEFAVQSHGVDMDGPFGS
NOVllc TVKIVNYTVRFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGS
NOVlld TVKIVNYTVRFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGS
NOVlle TVKIVNYTVRFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGS
NOVllf TVKIVNYTVRFSPWGLRNASLWYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGS
NOVllg TVKIVNYTVRFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGWMDGPFGS
NOVllh TVKIVNYTVRFSPWGLRNASLVTYYTSSGEDILIGGLKPFTKYEFAVQSHGVDMDGPFGS
NOVlla VVERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQW
NOVllb VVERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQW
NOVllc VVERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQW
NOVlld WERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQW
NOVlle VVERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQW
NOVllf WERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQW
NOVllg WERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQW
'NOVllh VVERSTLPDRPSTPPSDLRLSPLTPSTVRLHWCPPTEPNGEIVEYLILYSSNHTQPEHQW
NOVlla TLLTTQG--NIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLSDSLDMH
'NOVllb TLLTTQGEGNIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLSDSLDMH
',NOVllc TLLTTQG--NIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLSDSV---',NOVlld TLLTTQG--NIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLSDSVD--',NOVlle TLLTTQG--NIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLSDSVDSF
',NOVllf TLLTTQG--NIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLSDSVD--',NOVllg TLLTTQG--NIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLSDS----',NOVllh TLLTTQG--NIFSAEVHGLESDTRYFFKMGARTEVGPGPFSRLQDVITLQEKLSDS----INOVIIa SVTGIIVGVCLGLLCLLACMCAGLRRSPHRESLPGLSSTATPGNPALYSRARLGPPSPPA
INOVIIb SVTGIIVGVCLGLLCLLACMCAGLRRSPHRESLPGLSSTATPGNPALYSRARLGPPSPPA
~NOVllc ____________________________________________________________ _____________________ ~iNOVlld _______________________________________ ~~NOVlle SWSVITAPRAPPRPATRY-_________________________________________ I,NOVIIg ____________________________________________________________ INOVIIh '___________________________________________________________ i NOVlla AHELESLVHPHPQDWSPPPSDVEDRAEVHSLMGGGVSEGRSHSKRKISWAQPSGLSWAGS
NOVllb AHELESLVHPHPQDWSPPPSDVEDRAEVHSLMGGGVSEGRSHSKRK-VSAQPSGLSWAGS
NOVllc ____________________________________________________________ NOVlld ______-_____________________________________________________ NOVlle ____________________________________________________________ NOVllf ____________________________________________________________ NOVllg ______________________________-_____________________________ NOVllh ____________________________________________________________ NOVlla WAGCELPQAGPRPALTRALLPPAGTGQTLLLQALWDAIKGNGRKKSPPACRNQVEAEVI
NOVllb WAGCELPQAGPRPALTRALLPPAGTGQTLLLQVLCSD--QGNGRKKSPPACRNQVEAEVI
NOVllc ____________________________________________________________ NOVIId ____________________________________________________________ NOVlle ____________________________________________________________ NOVllf ____________________________________________________________ NOVllg ____________________________________________________________ NOVllh ____________________________________________________________ NOVlla VHSDFSASNGNPDLHLQDLEPEDPLPPEAPDLISGVGDPGQGAAWLDRELGGCELAAPGP
NOVllb VHSDFSASNGNPDLHLQDLEPEDPLPPEAPDLISGVGDPGQGAAWLDRELGGCELAAPGP
NOVllc ____________________________________________________________ NOVlld ____________________________________________________________ NOVlle ____________________________________________________________ NOVllf ____________________________________________________________ NOVllg ____________________________________________________________ 'NOVllh ____________________________________________________________ INOVIIa DRLTCLPEAASASCSYPDLQPGEVLEETPGDSCQLKSPCPLGASPGLPRSPVSSSA
NOVllb DRLTCLPEAASASCSYPDLQPGEVLEETPGDSCQLKSPCPLGASPGLPRSPVSSS-NOVllc ________________________________________________________ NOVlld ________________________________________________________ NOVlle ________________________________________________________ NOVllf ________________________________________________________ NOVllg ________________________________________________________ NOVllh ________________________________________________________ NOVlla (SEQ ID NO: 134) NOVllb (SEQ ID NO: 136) NOVllc (SEQ ID NO: 138) NOVlid (SEQ ID NO: 140) NOVlle (SEQ ID NO: 142) NOVllf (SEQ ID NO: l44) NOVllg (SEQ ID NO: 146) NOVllh (SEQ ID NO: 148) Further analysis of the NOV l 1 a protein yielded the following properties shown in Table 11C.
Table 11C. Protein Sequence Properties NOVlla SignalP analysis: Cleavage site between residues 25 and 26 PSORT II analysis:
PSG: a new signal peptide prediction method N-region: length 10; pos.chg 3; neg.chg 1 H-region: length 12; peak value 10.30 PSG score: 5.90 GvH: von Heijne's method for signal seq. recognition GvH score (threshold: -2.1): 1.11 possible cleavage site. between 24 and 25 » > Seems to have a cleavable signal peptide (1 to 24) ALOM: Klein et al's method for TM region allocation Init position for calculation: 25 Tentative number of TMS(s) for the threshold 0.5: 1 Number of TMS(s) for threshold 0.5: 1 INTEGRAL Likelihood =-11.89 Transmembrane 963 - 979 PERIPHERAL Likelihood = 0.90 (at 321) ALOM score. -11.89 (number of TMSs: 1) MTOP: Prediction of membrane topology (Hartmann et al.) Center position for calculation: 22 Charge difference. -5.0 C(-2.0) - N( 3.0) N >= C. N-terminal side will be inside » > membrane topology: type is (cytoplasmic tail 980 to 1254) MITDISC: discrimination of mitochondrial targeting seq R content. 4 Hyd Moment(75): 11.83 Hyd Moment(95): 5.63 G content: 5 D/E content: 2 S/T content: 1 Score: -4.74 Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 33 ARG~EL
NUCDISC: discrimination of nuclear localization signals pat4: none pat7: PVRLKKK (4) at 696 bipartite: none content of basic residues: 8.2g NLS Score: -0.13 KDEL: ER retention motif in the C-terminus: none ER Membrane Retention Signals:
XXRR-like motif in the N-terminus: ARGD
none SKL: peroxisomal targeting signal in the C-terminus: none PTS2: 2nd peroxisomal targeting signal: none VAC: possible vacuolar targeting motif: none RNA-binding motif: none Actinin-type actin-binding motif.
type 1: none type 2: none NMYR: N-myristoylation pattern : none Prenylation motif: none memYQRL: transport motif from cell surface to Golgi: none Tyrosines in the tail: too long tail Dileucine motif in the tail: found LL at 1097 LL at 1107 'checking 63 PROSITE DNA binding motifs: none checking 71 PROSITE ribosomal protein motifs: none checking 33 PROSITE prokaryotic DNA binding motifs: none NNCN: Reinhardt's method for Cytoplasmic/Nuclear discrimination Prediction: cytoplasmic Reliability: 55.5 COIL. Lupas's algorithm to detect coiled-coil regions total: 0 residues DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

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Claims (59)

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 141.

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 141.

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 141.

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 141.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

19. A method of treating a pathological state in a mammal, the method comprising administering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95% identical to a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:2n, wherein n is an integer between 1 and 141, 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 141.

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 141.

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 141.

24. A composition comprising an isolated nucleic acid molecule, said 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 141, and a carrier.

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 141, 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 141.

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 413~

acid sequence selected from the group consisting of SEQ ID NO:2n-1, wherein n is an integer between 1 and 141.

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

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

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

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

46. An isolated polypeptide comprising an amino acid sequence at least 95%
similar to SEQ ID NO: 2, wherein said amino acid sequence comprises at least one amino acid substitution, wherein said substitution is at amino acid position 43 when numbered in accordance with SEQ ID NO: 2.

47. An isolated nucleic acid molecule comprising an nucleic acid sequence at least 95% similar to SEQ ID NO: 1, wherein said nucleic acid sequence comprises at least one nucleic acid substitution, wherein said substitution is at nucleic acid position 135 when numbered in accordance with SEQ ID NO: 1.

48. An isolated polypeptide comprising an amino acid sequence at least 95%
similar to SEQ ID NO: 14, wherein said amino acid sequence comprises at least one amino acid substitution, wherein said substitution is at the amino acid position selected from the group consisting of 8, 54, 56, 92, 207, 240, 706, 891 and 923 when numbered in accordance with SEQ ID NO: 14.

49. An isolated nucleic acid molecule comprising an nucleic acid sequence at least 95% similar to SEQ ID NO: 13, wherein said nucleic acid sequence comprises at least one nucleic acid substitution, wherein said substitution is at the nucleic acid position selected from the group consisting of 272, 410, 416, 523, 869, 967, 2366, 2921 and 3018 when numbered in accordance with SEQ ID NO: 13.

50. An isolated polypeptide comprising an amino acid sequence at least 95%
similar to SEQ ID NO: 58, wherein said amino acid sequence comprises at least one amino acid substitution, wherein said substitution is at the amino acid position selected from the group consisting of 23, 56, 105, 125, 160, 183 and 215 when numbered in accordance with SEQ
ID NO: 58.

51. An isolated nucleic acid molecule comprising an nucleic acid sequence at least 95% similar to SEQ ID NO: 57, wherein said nucleic acid sequence comprises at least one nucleic acid substitution, wherein said substitution is at the nucleic acid position selected from the group consisting of 181, 278, 426, 485, 591, 661 and 756 when numbered in accordance with SEQ ID NO: 57.

52. An isolated polypeptide comprising an amino acid sequence at least 95%
similar to SEQ ID NO: 80, wherein said amino acid sequence comprises at least one amino acid substitution, wherein said substitution is at amino acid position 219 when numbered in accordance with SEQ ID NO: 80.

53. An isolated nucleic acid molecule comprising an nucleic acid sequence at least 95% similar to SEQ ID NO: 79, wherein said nucleic acid sequence comprises at least one nucleic acid substitution, wherein said substitution is at nucleic acid position 685 when numbered in accordance with SEQ ID NO: 79.

54. An isolated polypeptide comprising an amino acid sequence at least 95%
similar to SEQ ID NO: 92, wherein said amino acid sequence comprises at least one amino acid substitution, wherein said substitution is at amino acid position 470 when numbered in accordance with SEQ ID NO: 92.

55. An isolated nucleic acid molecule comprising an nucleic acid sequence at least 95% similar to SEQ ID NO: 91, wherein said nucleic acid sequence comprises at least one nucleic acid substitution, wherein said substitution is at nucleic acid position 1874 when numbered in accordance with SEQ ID NO: 91.

56. An isolated polypeptide comprising an amino acid sequence at least 95%
similar to SEQ ID NO: 100, wherein said amino acid sequence comprises at least one amino acid substitution, wherein said substitution is at the amino acid position selected from the group consisting of 11, 112 and 145 when numbered in accordance with SEQ ID NO: 100.

57. An isolated nucleic acid molecule comprising an nucleic acid sequence at least 95% similar to SEQ ID NO: 99, wherein said nucleic acid sequence comprises at least one nucleic acid substitution, wherein said substitution is at the nucleic acid position selected from the group consisting of 80, 383 and 482 when numbered in accordance with SEQ ID
NO: 99.

58. An isolated polypeptide comprising an amino acid sequence at least 95%
similar to SEQ ID NO: 122, wherein said amino acid sequence comprises at least one amino acid substitution, wherein said substitution is at the amino acid position selected from the group consisting of 12, 38, 54, 65, 66, 69, 80, 90, 91, 96, 100, 101, 102, 114, 122, 125, 126, 134, 135, 144, 148, 154, 155 and 156 when numbered in accordance with SEQ ID NO:
122.

59. An isolated nucleic acid molecule comprising an nucleic acid sequence at least 95% similar to SEQ ID NO: 121, wherein said nucleic acid sequence comprises at least one nucleic acid substitution, wherein said substitution is at the nucleic acid position selected from the group consisting of 35, 112, 160, 194, 197, 206, 240, 269, 273, 287, 298, 301, 305, 340, 365, 374, 376, 400, 404, 431, 442, 461, 463 and 468 when numbered in accordance with SEQ ID NO: 121.