WO2002006465A9

WO2002006465A9 - 7677, a novel human atpase family member and uses therefor

Info

Publication number: WO2002006465A9
Application number: PCT/US2001/022165
Authority: WO
Inventors: Rachel Meyers; Mark Williamson
Original assignee: Millennium Pharm Inc; Rachel Meyers; Mark Williamson
Priority date: 2000-07-13
Filing date: 2001-07-12
Publication date: 2002-09-06
Also published as: WO2002006465A2; EP1299551A2; WO2002006465A3; AU2001275917A1

Abstract

The invention provides isolated nucleic acids molecules, designated 7677 nucleic acid molecules, which encode novel ATPase family members. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 7677 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a 7677 gene has been introduced or disrupted. The invention still further provides isolated 7677 proteins, fusion proteins, antigenic peptides and anti-7677 antibodies. Diagnostic methods utilizing compositions of the invention are also provided.

Description

7677, A NOVEL HUMAN ATPASE FAMI Y MEMBER AND

USES THEREFOR

Background of the Invention

Enzymes that bind to and hydrolyze ATP play a pivotal role in translating chemically stored energy into biological activity. Proteins that bind and hydrolyze ATP are frequently involved in the early steps of DNA replication. ATPases can function in a variety of cellular processes including, selective ion transport events, actin-based motility, membrane traffic and numerous biosynthetic pathways. Multiple ATPase families exist, including ion pumps, DEAD box-helicases, ABC transporters, and AAA (ATPases Associated to a variety of cellular Activities).

The AAA family of ATPases is characterized by a highly conserved AAA motif. This motif forms a 230 AA domain that imparts ATPase activity. Members of this class have 1 or 2 domains.

AAA proteins play essential roles in cellular housekeeping, cell division and differentiation and have been identified in prokaryotes and eukaryotes. All members of the AAA family are Mg²⁺ dependent ATPases and comprise a conserved region that binds ATP. Cytosolic, transmembrane, as well as, membrane-associated AAA family members have been identified in various cellular locations and multimeric states.

The biological role of the AAA^' family members in the cell is diverse. Currently, members of this ATPase family are known to be involved in organelle biogenesis, cell- cycle regulation, vesicle-mediated transport and biogenesis, assembly of proteins through membranes, peroxisome biogenesis, gene expression in yeast and in human, and 26S proteasome function. For a review, see, Confalonieri et al. (1995) BioEssays 7:639-650. AAA-family members also include metalloproteases. See also Patel, S. et al. (1998) Trends Cell Biol 8(2)65-71.

Several members of the AAA family are involved in the biogenesis of peroxisomes. These organelles contain enzymes responsible for fatty acid oxidation and the elimination of peroxides. AAA family members, such as the PAS genes of S. cerevisiae, appear to be required for peroxisome growth, and proliferation (Subramani et al. (1993) Annu. Rev. Cell Biol. P:445-478). Furthermore, mutations in the AAA proteins Pexlp or P^"ex6p accumulate abnormal peroxisomal vesicles, suggesting a defect in vesicle fusion during peroxisome assembly (Song et al. (1993) J. Cell Biol. 723:535-548 and Heyman et al. (1994) J. Cell Biol. 727:1269-1273).

AAA family members are also known to regulate transcription. Nelbock et al. described the TBP1 protein that binds human HTv^* TAT transactivator, thus impairing its activity in cotransfection experiments (Nelbock et al. (1990) Science 248: 1650-1653). TBP1 has since been identified as an AAA family member which acts as a transcriptional activator for various promoters (Ohana et al. (1993) Proc. Natl. Acad. Scie. P0:138-142). Various ATP-dependent proteases, such as the regulatory components Lon, Clp, and RuvB are also members of the AAA ATPase family. Evidence suggests the Lon and Clp proteases are involved in DNA replication, recombination and restriction. For instance, human Lon binds specifically to single-stranded DNA in a region of the mitochondrial genome involved in regulation of DNA replication and transcription. It has been suggested that Lon may target and remodel specific DNA binding proteins either for selective degredation or for assembly (Fu et al. (1998) Biochemistry 37:1905-1909). Dubiel et al. discovered that subunit 4 of the human proteasome was in fact a member of the AAA family (Dubiel et al. (1992) J. Biol. Chem. 267:22699-22702). Subsequently, at least 5 of the 26S-proteasome subunits already described as transcription factors or cell cycle proteins have now been identified as representatives of the AAA family. Therefore, members of the family are likely to play an essential role in ATP- dependent and ubiquitin-dependent degradation of abnormal proteins and short-lived regulatory proteins and in antigen processing.

Macromolecular machines (protein complexes) carry out nearly every major process in a cell with highly coordinated moving parts driven by energy dependent conformational changes. Examples of such structures include the proteasomes, spliceosomes, ribosomes, peroxisomes and chromosomal replicases. The intricacy of these machines require additional devices to assist in their assembly. The AAA family of ATPase is thought of as a class of molecular chaperones that assist in the noncovalent assembly of other proteins or protein complexes. Thus, the AAA family members play critical regulatory roles in the assembly or regulation of various molecular machines associated with diverse cellular activities. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize novel ATPases. The present invention advances the state of the art by providing a novel human ATPase-like nucleic acid and polypeptide.

Summary of the Invention The present invention is based, in part, on the discovery of a novel human ATPase, referred to herein as "7677". The nucleotide sequence of a cDNA encoding 7677 is shown in SEQ ID NO:l, and the amino acid sequence of a 7677 polypeptide is shown in SEQ ID NO:2. In addition, the nucleotide sequence of the coding region is depicted in SEQ ID NO:3. Accordingly, in one aspect, the invention features a nucleic acid molecule which encodes a 7677 protein or polypeptide, e.g., a biologically active portion of the 7677 protein. In a preferred embodiment, the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2. In other embodiments, the invention provides an isolated 7677 nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number . In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number . In other embodiments, the invention provides a nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, SEQ 3D NO:3, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number , wherein the nucleic acid encodes a full length 7677 protein or an active fragment thereof. In a related aspect, the invention further provides nucleic acid constructs which include a 7677 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 7677 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 7677 nucleic acid molecules and polypeptides, and methods of producing polypeptides using such constructs. In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 7677-encoding nucleic acids.

In still another related aspect, isolated nucleic acid molecules that are antisense to a 7677 encoding nucleic acid molecule are provided. In another aspect, the invention features, 7677 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 7677-mediated or -related disorders. In another embodiment, the invention provides 7677 polypeptides having a 7677 activity. Preferred polypeptides are 7677 proteins including at least one ATPase domain, and, preferably, having a 7677 activity, e.g., a 7677 activity as described herein.

In other embodiments, the invention provides 7677 polypeptides, e.g., a 7677 polypeptide having the amino acid sequence shown in SEQ ID NO:2; the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession

Number ; an amino acid sequence mat is substantially identical to the arnino acid sequence shown in SEQ ID NO:2; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:3, or the sequence of the DNA insert of the plasmid deposited with ATCC

Accession Number , wherein the nucleic acid encodes a full length 7677 protein or an active fragment thereof.

In a related aspect, the invention further provides nucleic acid constructs which include a 7677 nucleic acid molecule described herein.

In a related aspect, the invention provides 7677 polypeptides or fragments operatively linked to non-7677 polypeptides to form fusion proteins. In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 7677 polypeptides.

In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 7677 polypeptides or nucleic acids.

In still another aspect, the invention provides a process for modulating 7677 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression of the 7677 polypeptides or nucleic acids, such as conditions involving aberrant or deficient cellular proliferation or differentiation such as lung cancer and colon cancer or in subjects at risk of developing cancer using for example a 7677 modulator.

The invention also provides assays such as methods or kits with instructions for use for deternήning the activity of or the presence or absence of 7677 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis, such as contacting the sample with a compound such as an antibody or a probe or primer that selectively binds, hybridizes, or modulates the 7677 molecules of the invention particularly for lung, colon or an other cancer. In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 7677 polypeptide or nucleic acid molecule, including for disease diagnosis.

In another aspect, the invention provides a method of identifying a nucleic acid or polypeptide associated with a disease such as cancer using PCR or bmding partners. In a further aspect, the invention provides a method of identifying a subject having or at risk for developing a disease such as cancer using a hybridization probe or a primer comprising the 7677 nucleic acids of the invention or using a binding partner such as an antibody of the 7677 polypeptides of the invention.

In still a further aspect, the invention provides a method for evaluating the efficacy of a treatment of a disease such as cancer or aberrant cellular proliferation and/or differentiation, by assessing the change in expression level of a 7677 nucleic acid or 7677 polypetide.

In a further aspect, the invention provides a method of diagnosing a disease such as cancer or aberrant cellular proliferation and/or differentiation in a subject by evaluating the difference in the expression or activity of a 7677 nucleic acid or a 7677 polypeptide, relative to a normal subject or a cohort of normal subjects. Brief Description of the Drawings Figures 1A-C depicts a cDNA sequence (SEQ ID NO:l) and predicted amino acid sequence (SEQ ID NO:2) of human 7677. The melMonine-initiated open reading frame of human 7677 (without the 5' and 3' untranslated regions) extends from nucleotide position 1 to position 1995 of SEQ ID NO:3, not including the terminal codon.

Figures 2A-L represent data generated by the 7677 protein. Figure 2A, in part, depicts a hydropathy plot of human 7677. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) and N glycosylation site (Ngly) are indicated by short vertical lines just below the hydropathy trace. The location of the transmembrane domains is also indicated. The numbers corresponding to the amino acid sequence of human 7677 are indicated. Polypeptides of the invention include fragments which include: all or a part of a hydrophobic sequence (a sequence above the dashed line); or all or part of a hydrophiUc fragment (a sequence below the dashed line). Other fragments include a cysteine residue or an N-glycosylation site. Figures 2A-B include the prediction of protein subcellular localization sites using PSORT software. Signal peptide predictions and MEMSAT transmembrane predictions are also shown. Figures 2C-F, in part, include results from the Prosite database of protein families and domains, which identifies biologically significant sites. Figures 2F-H depict the ahgnment of the ATPase domains of human 7677 with a consensus amino acid sequence.derived from a hidden Markov model. The upper sequence is the consensus amino acid sequence, while the lower amino acid sequence corresponds to, for example, amino acids 263 to 459 or 260 to380 of SEQ ID NO:2. Finally, Figures 2K-L include results from the ProDom protein domain database which identify homologous ATPase domains. The lower the sequence is the consensus arnino acid sequence, while the upper arnino acid sequence corresponds to a portion of SEQ ID NO:2.

Figure 3 depicts variable expression of 7677 RNA in all xenograph friendly cell lines tested.

Figure 4 is a Phase I panel bar graph depicting the relative expression of 7677 RNA relative to a no template control in a panel of human tissues or cells, including but not limited to heart, brain, breast, ovary, pancreas, prostate, colon, kidney, liver, fetal liver, lung, spleen, tonsil, lymph node, thymus, epithelial, endothelial, skeletal, fibroblasts, skin, adipose, bone cells (e.g., osteoclasts and osteoblasts), among others, detected using realtime quantitative RT-PCR Taq Man analysis. The graph indicates significant expression in human fetal heart, brain cortex, fetal liver, epithelial cells, and human umbilical vein cells (HUVEC). Figure 5 is a graph of a Taq Man array depicting 7677 RNA expression relative to the progression of cells through the cell cycle and shows increased expression of 7677 RNA in Go/Gi phase (1=0) of synchronized cells of the human breast epithelial cell line, MCF-10A.

Figure 6 is an oncology panel bar graph depicting the expression of 7677 RNA relative to a no template control showing an increased expression in 4/6 clinical colon tumors in comparison to 3/4 clinical normal colon tissues and 4/4 colon tumors metastases in comparison to 3/4 clinical normal colon tissues, which expression was detected using Taq Man analysis.

Figure 7 is an oncology panel bar graph depicting the expression of 7677 RNA relative to a no template control showing in part an increased expression in 3/6 lung tumors in comparison to normal lung tissues, which expression was detected using Taq Man analysis. Also, the relative expression of breast normal and tumor, lung normal and tumor, colon normal, tumor and metastases, and brain normal and tumor are depicted.

Figure 8 is a graph of an HPMG array depicting 7677 RNA expression relative to the progression of cells through the cell cycle and shows increased expression of 7677 RNA in Go/Gi phase (t=3, 12 and 24) of synchronized cells of a breast cancer cell line, MCF-7 .

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

Detailed Description Human 7677

The human 7677 sequence (Figures 1A-C; SEQ ID NO:l), which is approximately 2745 nucleotides long including untranslated regions, contains a predicted methionine- initiated coding sequence of about 1995 nucleotides (nucleotides 270-2367 of SEQ ID

NO:l; nucleotides 1-1995 of SEQ ID NO:3), not including the terminal codon. The coding sequence encodes a 665 arnino acid protein (SEQ ID NO:2).

This mature protein form is approximately 665 amino acid residues in length (from about arnino acid 1 to amino acid 665 of SEQ ID NO:2). Human 7677 contains a predicted transmembrane domain which extends from about arnino acid residue 385-401 of SEQ ID NO:2.

The 7677 protein contains a significant number of structural characteristics in common with members of the ATPase family. The term "family" when referring to the protein and nucleic acid molecules of the invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins.

The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as "ATPase" or "7677" nucleic acid and polypeptide molecules, which play a role in or function in the conversion of chemical energy into biological energy. In one embodiment, the 7677 molecules modulate the activity of one or more proteins involved in cellular housekeeping processes, such as cell division and differentiation. In another embodiment, the 7677 molecules of the present invention are capable of modulating actin-based motility mechanisms. In yet another embodiment, the 7677 molecules of the present invention are involved in the functioning of selective ion transportation through membranes. ^' Since the 7677 nucleic acid was found to be expressed in cells of the fetal heart, brain, breast, colon, fetal Uver, as well as epithelial cells, aortic smooth muscle cells (SMC) and HUVEC cells as shown in Figure 4 in particular, the encoded protein ATPase is at least expected to catalyze cell type specific ATPase-related reactions in those cells.

Comparison of the 7677 nucleic acids of SEQ ID NO:l to various databases reveal that 7677 partially aligns with several other human expressed sequence tag (EST) cDNA sequences. The aligned EST-cDNA sequences recently reported in DBEST (DBEST accession nos. AW241815, AA621580, AI040917, AW957342, and BE019356) do not have annotations that associate the ESTs with the area of oncology. However, the EST- cDNA sequence accession no. AW241815 has been reported as similar to hypothetical H. influenzae hypothetical protein P45262, which in turn was annotated as an ATP-binding protein.

Another EST-cDNA sequence (GenBank accession no. U56249) aligned with approximately 20% of the C-teiminus of 7677 and was reported as human HELA mRNA isolated as a false positive during two-hybrid screening. Further, another EST-cDNA sequence (Patent DB accession no. T25215) aligned with approximately 60 nucleotides of the 3 ' end and parts of the non-translated region with an 87% identity and was reported as identifying gene signatures in 3'-directed human cDNA-library, e.g. for diagnosis of abnormal cell function.

As illustrated above, the 7677 molecules of the invention have upregulated expression in various tumors versus normal tissues. In addition the 7677 molecules of the invention have been found to be cell cycle regulated in several different tumor cell lines as show, for example, in Figure 3. Important pathways involved in tuinorgenesis include protein degradation via the proteasome, DNA damage repair and cell cycle regulation. As such, without being bound by theory, the 7677 molecules of the invention may play a role in regulating aspects of one or more of these pathways. The 7677 protein of the invention has homology to RuvB DNA helicase. A partial protein, a putative helicase, that is identical in part to the 7677 protein has been reported as being similar to E. coli RuvB helicase. (Adamson et al, (2000) locus AF218313 accession AAF80563). Moreover, a rat RuvB-like protein, TTP49a, also shows significant homology to bacterial RuvB helicase (Makino et al, (1999) Journal of Biol Chem 274(22): 15329-35). It has been reported that TIP49a may play a role in nuclear processes such as recombination and transcription. As such, the 7677 protein of the invention may also play a role in such in nuclear processes. Stimulation of 7677 activity is desirable in situations in which 7677 is abnormally downregulated and/or in which increased 7677 activity is likely to have a beneficial effect.

Likewise, inhibition of 7677 activity is desirable in situations in which 7677 is abnormally upregulated and/or in which decreased 7677 activity is likely to have a beneficial effect. Inhibition or over stimulation of the activity of ATPases can lead to cellular growth related disorders. As used herein, a "cellular growth related disorder" includes a disorder, disease, or condition characterized by a deregulation, e.g., an upregulation or a downregulation, of cellular growth. Cellular growth deregulation may be due to a deregulation of cellular proUferation, cell cycle progression, cellular differentiation and/or ceUular hypertrophy. Examples of cellular growth related disorders include cardiovascular disorders such as heart failure, hypertension, atrial fibrillation, dilated cardiomyopathy, idiopathic cardiomyopathy, or angina; proUferative disorders or differentiative disorders such as cancer, e.g., melanoma, prostate cancer, cervical cancer, breast cancer, colon cancer, or sarcoma. As used herein, the term "ATPase" includes a protein or polypeptide which is capable of translating chemical energy into biological energy.

Human 7677 contains the following regions or other structural features (for general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://www.psc.edu/general/sofrware/packages fanVpfam.html): an APTase domain (AAA_5) derived from modular architecture analysis (SMART); one transmembrane domain (predicted by MEMSAT, Jones et al. (1994)

Biochemistry 33:3038-3049) at about amino acids 385 to 401 of SEQ ID NO:2; three N-glycosylation sites (PS00001) located at about amino acids 334-337, 415- 418 and 516-519 of SEQ ID NO:2; one glycosaminoglycan attachment site (PS00002) located at about amino acids

203-206; four cAMP- and cGMP-dependent protein kinase phosphorylation sites (PS00004) located at about arnino acids 72-75, 136-139, 286-289 and 320-323 of SEQ ID NO:2; nine predicted protein kinase C phosphorylation sites (PS00005) located at about amino acids 54-56, 139-141, 153-155, 230-232, 285-287, 289-291, 403-405, 456-458 and

509-511 of SEQ ID NO:2; eleven predicted casein kinase II phosphorylation sites (PS00006) located at about amino 4-7, 34-37, 87-90, 92-95, 156-159, 235-238, 254-257, 336-339, 416-419, 436-439 and 477-480 of SEQ ID NO:2; three predicted tyrosine kinase phosphorylation sites (PS00007) located at about arnino acids 427-434, 494-500 and 624-631 of SEQ JJD NO:2; eight predicted N-myristoylation sites (PS00008) located at about amino acids 53- 58, 201-206, 241-246, 271-276, 446-451, 484-489, 512-517 and 569-574 of SEQ ID NO:2; two predicted amidation sites (PS00009) located at about amino acids 134-137 and 139-142 of SEQ ID NO:2; one ATP/GTP-binding site motif A (PS00017) located at about amino acids 268-

275 ofSEQ ID NO:2; and one leucine zipper pattern (PS00029) located at about amino acids 604-625 of SEQ ID NO:2.

The ATPase-Uke protein of the invention possesses a NB-ARC domain, which is a signaling motif of ceU death regulators, from aa 266-278, AAA domains from aa 263-459 and 260-380, an adenylate kinase domain from aa 266-274, and an ultradead 3 domain from aa 206-383, as predicted by HMMer, Version 2. The NB-ARC domain is a novel signaling motif shared by plant resistant gene products and regulators of cell death in animals. See for example, Van der Biezen et al. (1998) Curr Biol 5:229-227. Adenylate kinase is a small monomeric enzyme that catalyzes the reversible transfer of MgATP to AMP. In mammals there are three different isozymes: AK1 (or myokinase), which is cytosohc; AK2, which is located in the outer compartment of mitochondria; and, AK3 (or GTP:AMP phosphotransferase), which is located in the mitochondrial matrix and which uses MgGTP instead of MgATP. The RNA heUces domain is found in a family of RNA heUces thought to be involved in duplex unwinding during viral RNA repUcation.

Members of this family are found in a variety of single stranded RNA viruses. See for example, Gorbalenya et al. (1989) NAR 17:4713-4730. The AAA domain (ATPase Associated with various cellular Activities) is found in a family of proteins that often perform chaperone-like functions that assist in the assembly, operation, or disassembly of protein complexes. See for example, Confalonieri et al. (1995) Bioessays 77:639-650 and Neuwald et al. (1999) Genome Research 9:27 A3. For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http//www.psc.edu/genera software/packages/pfarnypfam.h1n l.

A plasmid containing the nucleotide sequence encoding human 7677 was deposited with American Type Culture CoUection (ATCC), 10801 University Boulevard, Manassas,

VA 20110-2209, on and assigned Accession Number . This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

The 7677 protein contains a significant number of structural characteristics in common with members of the ATPase family. The term "family'' when referring to the protein and nucleic acid molecules of the invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as weU as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins. Members of a family can also have common functional characteristics.

A novel human ATPase-like gene sequence, referred to as 7677, is provided. This gene sequence and variants and fragments thereof are encompassed by the term "ATPase- like" molecules or sequences as used herein. The ATPase-like sequences find use in modulating a ATPase function. By "modulating" is intended the upregulating or downregulating of a response. The sequences of the invention find use in modulating organelle biogenesis, ceU-cycle regulation, protein degredation, vesicle-mediated transport, assembly of proteins through membranes, peroxisome biogenesis, gene expression, and 26S proteasome function response. That is, the compositions of the invention, affect the targeted activity in either a positive or negative fashion. Proteins and or antibodies of the invention are also useful in modulating the above mentioned cellular process. The present invention provides isolated nucleic acid molecules comprising nucleotide sequences encoding the ATPase-like polypeptides whose amino acid sequences are given in SEQ ID NO:2, or a variant or fragment of the polypeptides. Nucleotide sequences encoding the ATPase-like polypeptides of the invention are set forth in SEQ ID NO:l.

As used herein, the term "ATPase domain" includes an amino acid sequence of about 50-300 arnino acid residues in length and having a bit score for the aUgnment of the sequence to the ATPase domain (HMM) of at least 50. Preferably, an ATPase domain includes at least about 75-200 amino acids, more preferably about 100-200 amino acid residues, or about 150-200 amino acids and has a bit score for the aUgnment of the sequence to the ATPase domain (HMM) of at least 60 or greater. The ATPase domain, AAA (ATPases associated with various cellular activities) (HMM) has been assigned the PFAM Accession PF00004 (http;//pfam.wustl.edu/). An aUgnment of the ATPase domain (amino acids 260-380 of SEQ ID NO:2) of human 7677 with a consensus arnino acid sequence derived from a hidden Markov model is depicted in Figure 2.

In a preferred embodiment 7677 polypeptide or protein has a "ATPase domain" or a region which includes at least about 75-150 more preferably about 100-140 or 125-135 amino acid residues and has at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100% homology with an "ATPase domain," e.g., the ATPase domain of human 7677 (e.g., amino acid residues 260-380 of SEQ ID NO:2).

To identify the presence of an "ATPase" domain in a 7677 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for deterniining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al., (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al., (1990) Meth. Enzymol. 183:146-159; Gribskov et al., (1987)

Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al., (1994) J. Mol. Biol. 235:1501- 1531; and Stultz et al., (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference.

In one embodiment, a 7677 protein includes at least one transmembrane domain. As used herein, the term "transmembrane domain" includes an amino acid sequence of about 15 amino acid residues in length that spans a phosphoUpid membrane. More preferably, a transmembrane domain includes about at least 18, 20, 22, 24, 25, 30, 35 or 40 arnino acid residues and spans a phosphoUpid membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an α-heUcal structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, http:/ pfam.wustl.edu/cgi-bin/getdesc7name— 7tm-l, and Zagotta W.N. et al., (1996) Annual Rev. Neuronsci. 19: 235-63, the contents of which are incorporated herein by reference. In a preferred embodiment, a 7677 polypeptide or protein has at least one transmembrane domain or a region which includes at least 18, 20, 22, 24, 25, 30, 35 or 40 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a "transmembrane domain," e.g., at least one transmembrane domain of human 7677 (e.g., amino acid residues 385-401 of SEQ ID NO:2).

In another embodiment, a 7677 protein includes at least one "non-transmembrane domain." As used herein, "non-transmembrane domains" are domains that reside outside of the membrane. When referring to plasma membranes, non-transmembrane domains include extracellular domains (i.e., outside of the cell) and intracellular domains (i.e., within the cell). When referring to membrane-bound proteins found in intracellular organelles (e.g., mitochondria, endoplasmic reticulum, peroxisomes and microsomes), non- transmembrane domains include those domains of the protein that reside in the cytosol (i.e., the cytoplasm), the lumen of the organelle, or the matrix or the intermembrane space (the latter two relate specifically to mitochondria organelles). The C-terminal arnino acid residue of a non-transmembrane domain is adjacent to an N-terminal amino acid residue of a transmembrane domain in a naturaUy-occurring 7677, or 7677-like protein. In a preferred embodiment, a 7677 polypeptide or protein has a "non- transmembrane domain" or a region which includes at least about 1-500, preferably about

200-450, more preferably about 225-400, and even more preferably about 250-350 arnino acid residues, and has at least about 60%, 70% 80% 90% 95%, 99% or 100% homology with a "non-transmembrane domain", e.g., a non-transmembrane domain of human 7677 (e.g., residues 1-384 and 402-665 of SEQ ID NO:2). Preferably, a non-transmembrane domain is capable of catalytic activity (e.g., catalyzing an acylation reaction). A non-transmembrane domain located at the N-terminus of a 7677 protein or polypeptide is referred to herein as an "N-terminal non-transmembrane domain." As used herein, an ''N-terminal non-transmembrane domain" includes an amino acid sequence having about 1-450, preferably about 30-425, more preferably about 50-400, or even more preferably about 80-375 arnino acid residues in length and is located outside the boundaries of a membrane. For example, an N-terminal non-transmembrane domai is located at about amino acid residues 1-384 of SEQ ID NO:2.

Similarly, a non-transmembrane domain located at the C-terminus of a 7677 protein or polypeptide is referred to herein as a "C-terminal non-transmembrane domain." As used herein, an "C-terminal non-transmembrane domain" includes an amino acid sequence having about 1-450, preferably about 15-400, preferably about 20-350, more preferably about 25-300 amino acid residues in length and is located outside the boundaries of a membrane. For example, an C-terminal non-transmembrane domain is located at about amino acid residues 402-665 of SEQ ID NO:2.

As the 7677 polypeptides of the invention may modulate 7677-mediated activities, they may be useful for developing novel diagnostic and therapeutic agents for 7677- mediated or related disorders, as described below.

As used herein, a "7677 activity", "biological activity of 7677" or "functional activity of 7677", refers to an activity exerted by a 7677 protein, polypeptide or nucleic acid molecule on e.g., a 7677-responsive cell or on a 7677 substrate, e.g., a Upid or protein substrate, as determined in vivo or in vitro. In one embodiment, a 7677 activity is a direct activity, such as an association with a 7677 target molecule. A "target molecule" or "binding partner" is a molecule with which a 7677 protein binds or interacts in nature, e.g. A 7677 activity can be a direct activity, such as an association with or an enzymatic activity on a second protein, or an indirect activity, such as a ceUular signaling activity mediated by interaction of the ATPase-like protein with a second protein. In a preferred embodiment, an

ATPase-like activity includes at least one or more of the following activities: (1) modulating (stimulating and/or enhancing or inhibiting) ceuular division; (2) modulating organelle biogenesis; (3) modulating protein sorting; (4) modulating gene expression; (5) modulating protein degredation; and (6) modulating the function of the 26S proteosome.

Accordingly, 7677 protein may mediate various disorders, including cellular prohferative and/or differentiative disorders, brain disorders, heart disorders, blood vessel disorders, and platelet disorders.

Examples of ceUular prohferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, mcluding but not limited to those of prostate, colon, lung, breast and Uver origin. As used herein, the terms "cancer", "hyperproUferative" and "neoplastic" refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proUferating ceU growth. HyperproUferative and neoplastic disease states maybe categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. "Pathologic hyperproUferative" ceUs occur in disease states characterized by maUgnant tumor growth. Examples of non-pathologic hyperproUferative cells include proUferation of cells associated with wound repair.

The terms "cancer" or "neoplasms" include maUgnancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genitourinary tract, as well as adenocarcinomas which include maUgnancies such as most colon cancers, renal-ceU carcinoma, prostate cancer and/or testicular tumors, non-small ceU carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

The term "carcinoma" is art recognized and refers to maUgnancies of epitheUal or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include maUgnant tumors composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor ceUs form recognizable glandular structures.

The term "sarcoma" is art recognized and refers to maUgnant tumors of mesenchymal derivation. The 7677 nucleic acid and protein of the invention can be used to treat and/or diagnose a variety of proliferative disorders. E.g., such disorders include hematopoietic neoplastic disorders. As used herein, the term "hematopoietic neoplastic disorders" includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor ceUs thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L., (1991) Crit. Rev. in Oncol/Hemotol. 11:267-97); lymphoid malignancies include, but are not Umited to acute lymphoblastic leukemia (ALL) which includes B- lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobuUnemia (WM). Additional forms of maUgnant lymphomas include, but are not Umited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-ceU lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

Disorders involving the brain include, but are not Umited to, disorders involving neurons, and disorders involving gUa, such as astrocytes, oUgodendrocytes, ependymal cells, and microgUa; cerebral edema, raised intracranial pressure and herniation, and hydrocephalus; malformations and developmental diseases, such as neural tube defects, forebrain anomaUes, posterior fossa anomaUes, and syringomyeUa and hydromyelia; perinatal brain injury; cerebrovascular diseases, such as those related to hypoxia, ischemia, and infarction, mcluding hypotension, hypoperfusion, and low-flow states—global cerebral ischemia and focal cerebral ischerm^--infarction from obstruction of local blood supply, intracranial hemorrhage, including intracerebral (intraparenchymal) hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, and vascular malformations, hypertensive cerebrovascular disease, including lacunar infarcts, sUt hemorrhages, and hypertensive encephalopathy; infections, such as acute meningitis, including acute pyogenic (bacterial) meningitis and acute aseptic (viral) meningitis, acute focal suppurative infections, including brain abscess, subdural empyema, and extradural abscess, chronic bacterial meningoencephaUtis, including tuberculosis and mycobacterioses, neurosyphiUs, and neuroborreUosis (Lyme disease), viral meningoencephaUtis, including arthropod-borne (Arbo) viral encephaUtis, Herpes simplex virus Type 1, Herpes simplex virus Type 2, Varicella-zoster virus (Herpes zoster), cytomegalovirus, poUomyeUtis, rabies, and human immunodeficiency virus 1, including HTV- 1 meningoencephaUtis (subacute encephaUtis), vacuolar myelopathy, ATDS-associated myopathy, peripheral neuropathy, and AIDS in children, progressive multifocal leukoencephalopathy, subacute sclerosing panencephaUtis, fungal meningoencephaUtis, other infectious diseases of the nervous system; transmissible spongiform encephalopathies (prion diseases); demyeUnating diseases, including multiple sclerosis, multiple sclerosis variants, acute disseminated encephalomyeUtis and acute necrotizing hemorrhagic encephalomyeUtis, and other diseases with demyelination; degenerative diseases, such as degenerative diseases affecting the cerebral cortex, including Alzheimer disease and Pick disease, degenerative diseases of basal gangha and brain stem, including Parkinsonism, idiopathic Parkinson disease (paralysis agitans), progressive supranuclear palsy, corticobasal degenration, multiple system atrophy, mcluding striatonigral degenration, Shy-Drager syndrome, and oUvopontocerebellar atrophy, and Huntington disease; spinocerebeUar degenerations, including spinocerebeUar ataxias, including Friedreich ataxia, and ataxia-telanglectasia, degenerative diseases affecting motor neurons, including amyotrophic lateral sclerosis (motor neuron disease), bulbospinal atrophy (Kennedy syndrome), and spinal muscular atrophy; inborn errors of metabolism, such as leukodystrophies, mcluding Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy, PeUzaeus-Merzbacher disease, and Canavan disease, mitochondrial encephalomyopathies, including Leigh disease and other mitochondrial encephalomyopathies; toxic and acquired metaboUc diseases, including vitamin deficiencies such as tmamine (vitamin B_t) deficiency and vitamin B₁₂ deficiency, neurologic sequelae of metaboUc disturbances, including hypoglycemia, hyperglycemia, and hepatic encephatopathy, toxic disorders, including carbon monoxide, methanol, ethanol, and radiation, including combined methotrexate and radiation-induced injury, tumors, such as gUomas, including astrocytoma, including fibriUary (diffuse) astrocytoma and glioblastoma multiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain stem ghoma, oUgodendrogUoma, and ependymoma and related paraventricular mass lesions, neuronal tumors, poorly differentiated neoplasms, including meduUoblastoma, other parenchymal tumors, mcluding primary brain lymphoma, germ cell tumors, and pineal parenchymal tumors, meningiomas, metastatic tumors, paraneoplastic syndromes, peripheral nerve sheath tumors, mcluding schwannoma, neurofibroma, and maUgnant peripheral nerve sheath tumor (maUgnant schwannoma), and neurocutaneous syndromes (phakomatoses), including neurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lihdau disease. Disorders involving the heart, include but are not Umited to, heart failure, including but not Umited to, cardiac hypertrophy, left-sided heart failure, and right-sided heart failure; ischemic heart disease, including but not Umited to angina pectoris, myocardial infarction, chronic ischemic heart disease, and sudden cardiac death; hypertensive heart disease, including but not Umited to, systemic (left-sided) hypertensive heart disease and pulmonary (right-sided) hypertensive heart disease; valvular heart disease, including but not Umited to, valvular degeneration caused by calcification, such as calcific aortic stenosis, calcification of a congenitally bicuspid aortic valve, and mitral annular calcification, and myxomatous degeneration of the mitral valve (mitral valve prolapse), rheumatic fever and rheumatic heart disease, infective endocarditis, and noninfected vegetations, such as nonbacterial thrombotic endocarditis and endocarditis of systemic lupus erythematosus (Libman-Sacks disease), carcinoid heart disease, and compUcations of artificial valves; myocardial disease, including but not limited to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and myocarditis; pericardial disease, including but not limited to, pericardial effusion and hemopericardium and pericarditis, including acute pericarditis and healed pericarditis, and rheumatoid heart disease; neoplastic heart disease, including but not limited to, primary cardiac tumors, such as myxoma, Upoma, papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac effects of noncardiac neoplasms; congenital heart disease, mcluding but not Umited to, left-to-right shunts—late cyanosis, such as atrial septal defect, ventricular septal defect, patent ductus arteriosus, and atrioventricular septal defect, right-to-left shunts-early cyanosis, such as tetralogy of faUot, transposition of great arteries, truncus arteriosus, tricuspid atresia, and total anomalous pulmonary venous connection, obstructive congenital anomaUes, such as coarctation of aorta, pulmonary stenosis and atresia, and aortic stenosis and atresia, and disorders involving cardiac transplantation.

Disorders involving blood vessels include, but are not limited to, responses of vascular cell walls to injury, such as endotheUal dysfunction and endotheUal activation and intimal thickening; vascular diseases mcluding, but not limited to, congenital anomaUes, such as arteriovenous fistula, atherosclerosis, and hypertensive vascular disease, such as hypertension; inflammatory disease-the vascuUtides, such as giant ceU (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneόus lymph node syndrome), microscopic polyangUtis (microscopic polyarteritis, hypersensitivity or leukocytoclastic angUtis), Wegener granulomatosis, thromboangUtis obUterans (Buerger disease), vascuUtis associated with other disorders, and infectious arteritis; Raynaud disease; aneurysms and dissection, such as abdominal aortic aneurysms, syphiUtic (luetic) aneurysms, and aortic dissection (dissecting hematoma); disorders of veins and lymphatics, such as varicose veins, thrombophlebitis and phlebotbrombosis, obstruction of superior vena cava (superior vena cava syndrome), obstruction of inferior vena cava (inferior vena cava syndrome), and lymphangitis and lymphedema; tumors, including benign tumors and tumor-like conditions, such as hemangioma, lymphangioma, glomus tumor (glomangioma), vascular ectasias, and baciUary angiomatosis, and intermediate-grade (borderline low-grade maUgnant) tumors, such as Kaposi sarcoma and hemangloendotheUoma, and maUgnant tumors, such as angiosarcoma and hemangiopericytoma; and pathology of therapeutic interventions in vascular disease, such as baUoon angioplasty and related techniques and vascular replacement, such as coronary artery bypass graft surgery.

The 7677 protein, fragments thereof, and derivatives and other variants of the sequence in SEQ ID NO:2 are collectively referred to as "polypeptides or proteins of the invention" or "7677 polypeptides or proteins". Nucleic acid molecules encoding such polypeptides or proteins are coUectively referred to as "nucleic acids of the invention" or "7677 nucleic acids." 7677 molecules refer to 7677 nucleic acids, polypeptides, and antibodies. As used herein, the term "nucleic acid molecule" includes DNA molecules (e.g., a cDNA or genomic DNA) and RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

The term "isolated or purified nucleic acid molecule" includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term "isolated" includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and or 3' ends 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 nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of 5' and/or 3' nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the ceU from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other ceUular material, or culture medium when produced by recombinant techniques, or substantiaUy free of chemical precursors or other chemicals when chemicaUy synthesized.

As used herein, the term "hybridizes under stringent conditions" describes conditions for hybridization and washing. Stringent conditions are known to those skuled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 -6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used. A preferred, example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, foUowed by one or more washes in 0.2X SSC, 0.1% SDS at 50°C. Another example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1 % SDS at 55°C. A further example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C. Preferably, stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, foUowed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C. Particularly preferred stringency conditions (and the conditions that should be used if the practitioner is uncertain about what conditions should be appUed to determine if a molecule is within a hybridization limitation of the invention) are 0.5M Sodium Phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:l, or SEQ ID NO:3, corresponds to a naturaUy-occurring nucleic acid molecule.

As used herein, a "naturaUy-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules which include an open reading frame encoding a 7677 protein, preferably a mammaUan 7677 protein, and can further include non-coding regulatory sequences, and introns.

An "isolated" or "purified" polypeptide or protein is substantiaUy free of cellular material or other contaminating proteins from the ceU or tissue source from which the protein is derived, or substantiaUy free from chemical precursors or other chemicals when chemicaUy synthesized. In one embodiment, the language "substantiaUy free" means preparation of 7677 protein having less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-7677 protein (also referred to herein as a "contaminating protein"), or of chemical precursors or non-7677 chemicals. When the 7677 protein or biologicaUy 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 protein preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 miUigrams in dry weight. A "non-essential" amino acid residue is a residue that can be altered from the wild- type sequence of 7677 (e.g., the sequence of SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession

Number ) without aboUshing or more preferably, without substantiaUy altering a biological activity, whereas an "essential" arnino acid residue results in such a change. For example, amino acid residues that are conserved among the polypeptides of the present invention, e.g., those present in the ATPase domain, are predicted to be particularly unamenable to alteration. A "conservative arnino acid substitution" is one in which the arnino acid residue is replaced with an amino acid residue having a similar side chain. FamiUes of amino acid residues having similar side chains have been defined in the art. These famiUes 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, phenylalariine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential arnino acid residue in a 7677 protein is preferably replaced with another arnino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along aU or part of a 7677 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 7677 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

As used herein, a "biologically active portion" of a 7677 protein includes a fragment of a 7677 protein which participates in an interaction between a 7677 molecule and a non- 7677 molecule. BiologicaUy active portions of a 7677 protein include peptides comprising a ino acid sequences sufficiently homologous to or derived from the amino acid sequence of the 7677 protein, e.g., the amino acid sequence shown in SEQ JD NO:2, which include less amino acids than the fuU length 7677 proteins, and exhibit at least one activity of a 7677 protein. TypicaUy, biologically active portions comprise a domain or motif with at least one activity of the 7677 protein, e.g., ATPase activity. A biologicaUy active portion of a 7677 protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or more amino acids in length. Biologically active portions of a 7677 protein can be used as targets for developing agents which modulate a 7677 mediated activity, e.g., ATPase activity. Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aUgned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second arnino acid or nucleic acid sequence for optimal aUgnment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%,

100% of the length of the reference sequence (e.g., when aUgning a second sequence to the 7677 amino acid sequence of SEQ ID NO:2 having 665_amino acid residues, at least 200, preferably at least , more preferably at least 333, even more preferably at least 400, and even more preferably at least 466, 533, 599 or 665 amino acid residues are aUgned). 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 identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal aUgnment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accompUshed using a mathematical algorithm. In a preferred embodiment, the percent identity between two arnino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be appUed to determine if a molecule is within a sequence identity or homology limitation of the invention) is using a Blossum 62 scoring matrix with a gap open penalty of

12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The percent identity between two arnino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a^'search against pubUc databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al., (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to 7677 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to 7677 protein molecules of the invention. To obtain gapped ahgnments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

"Misexpression or aberrant expression", as used herein, refers to a non-wild type pattem of gene expression, at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined ceU type or tissue type; a pattern of expression that differs from wild type in terms of the spUcing size, arnino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extraceUular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus. "Subject", as used herein, can refer to a mammal, e.g., a human, or to an experimental or animal or disease model. The subject can also be a non-human animal, e.g., a horse, cow, goat, or other domestic animal.

A "purified preparation of cells", as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured ceUs or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject ceUs.

Various aspects of the invention are described in further detail below.

Isolated Nucleic Acid Molecules

In one aspect, the invention provides, an isolated or purified, nucleic acid molecule that encodes a 7677 polypeptide described herein, e.g., a fuU length 7677 protein or a fragment thereof, e.g., a biologicaUy active portion of 7677 protein. Also included is a nucleic acid fragment suitable for use as a hybridization probe, which can be used, e.g., to a identify nucleic acid molecule encoding a polypeptide of the invention, 7677 mRNA, and fragments suitable for use as primers, e.g., PCR primers for the amplification or mutation of nucleic acid molecules.

In one embodiment, an isolated nucleic acid molecule of the invention includes the nucleotide sequence shown in SEQ ID NO:l, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , or a portion of any of these nucleotide sequences. In one embodiment, the nucleic acid molecule includes sequences encoding the human 7677 protein (i.e., "the coding region", from nucleotides 270-2367 of SEQ ID NO:l, not including the terminal codon), as weU as 5' untranslated sequences (nucleotides 1-269 of SEQ ID NO:l). Alternatively, the nucleic acid molecule can include only the coding region of SEQ ID NO: 1 (e.g., nucleotides 270-2367 of SEQ ID NO: 1, coiresponding to SEQ ID NO:3) and, e.g., no flanking sequences which normaUy accompany the subject sequence. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to the mature protein of SEQ ID NO:2.

In another embodiment, an isolated nucleic acid molecule of the invention includes a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ

ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , or a portion of any of these nucleotide sequences. In other embodiments, the nucleic acid molecule of the invention is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1 , SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number such that it can hybridize to the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , thereby forming a stable duplex.

In one embodiment, an isolated nucleic acid molecule of the present invention includes a nucleotide sequence which is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number . In the case of an isolated nucleic acid molecule which is longer than or equivalent in length to the reference sequence, e.g., SEQ ID NO:l, or SEQ ID NO:3, the comparison is made with the fuU length of the reference sequence. Where the isolated nucleic acid molecule is shorter than the reference sequence, e.g., shorter than SEQ ID NO:l, or SEQ ID NO:3, the comparison is made to a segment of the reference sequence of the same length (excluding any loop required by the homology calculation).

7677 Nucleic Acid Fragments A nucleic acid molecule of the invention can include only a portion of the nucleic acid sequence of SEQ JD NO:l, SEQ JD NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number . For example, such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of a 7677 protein, e.g., an immunogenic or biologicaUy active portion of a 7677 protein. A fragment can comprise: nucleotides 1056- 1644 of SEQ ID NO:l (787-1375 of SEQ ID NO:3) or 1047-1407 of SEQ ID NO:l (778- 1138 of SEQ ID NO:3), which encodes an ATPase domain of human 7677. The nucleotide sequence deterrnined from the cloning of the 7677 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 7677 family members, or fragments thereof, as well as 7677 homologues, or fragments thereof, from other species. a another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, of the coding region and extends into either (or both) the 5' or 3' noncoding region. Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof particularly fragments thereof which are at least 150 arnino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.

A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domain, region, or functional site described herein. Thus, for example, the nucleic acid fragment can include an ATPase domain. In a preferred embodiment the fragment is at least, 50, 100, 200, 300, 400, 500, 600, 700, or 900 base pairs in length.

7677 probes and primers are provided. TypicaUy a probe/primer is an isolated or purified oUgonucleotide. The oUgonucleotide typically includes a region of nucleotide sequence that hybridizes under stringent conditions to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ JD NO:l, SEQ JD NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , or of a naturally occurring aUeUc variant or mutant of SEQ ID NO: 1 , SEQ JD NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with

ATCC as Accession Number .

In a preferred embodiment the nucleic acid is a probe which is at least 5 or 10, and less than 200, more preferably less than 100, or less than 50, base pairs in length. It should be identical, or differ by 1, or less than in 5 or 10 bases, from a sequence disclosed herein. If aUgnment is needed for this comparison the sequences should be aUgned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.

A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes an ATPase domain (e.g., about amino acid residues 263-459 or 260- 380 ofSEQ ID NO:2).

In another embodiment a set of primers is provided, e.g., primers suitable for use in a PCR, which can be used to amplify a selected region of a 7677 sequence, e.g., a region described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturaUy occurring variant. E.g., primers suitable for ampUfying all or a portion of any of the foUowing regions are provided: an ATPase domain (e.g., about amino acid residues 263-459 or 260-380 of SEQ ID NO:2).

A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.

A nucleic acid fragment encoding a "biologicaUy active portion of a 7677 polypeptide" can be prepared by isolating a portion of the nucleotide sequence of SEQ JD NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number , which encodes a polypeptide having a

7677 biological activity (e.g., the biological activities of the 7677 proteins as described herein), expressing the encoded portion of the 7677 protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the 7677 protein. For example, a nucleic acid fragment encoding a biologicaUy active portion of 7677 includes an ATPase domain (e.g., about arnino acid residues 263-459 or 260-380 of SEQ JD NO:2). A nucleic acid fragment encoding a biologicaUy active portion of a 7677 polypeptide, may comprise a nucleotide sequence which is greater than 300-1200 or more nucleotides in length.

In preferred embodiments, nucleic acids include a nucleotide sequence which is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, and 1900 nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO:l, or SEQ JD NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number

7677 Nucleic Acid Variants

The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 1 , SEQ TD NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number . Such differences can be due to degeneracy of the genetic code (and result in a nucleic acid which encodes the same 7677 proteins as those encoded by the nucleotide sequence disclosed herein. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence which differs, by at least 1, but less than 5, 10, 20, 50, or 100 arnino acid residues that shown in SEQ ID NO:2. If aUgnment is needed for this comparison the sequences should be aUgned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.

Nucleic acids of the invention can be chosen for having codons, which are preferred, or not preferred, for a particular expression system. E.g., the nucleic acid can be one in which at least one colon, at preferably at least 10%, or 20% of the codons has been altered such that the sequence is optimized for expression in E. coU, yeast, human, insect, or CHO ceUs.

Nucleic acid variants can be naturaUy occurring, such as aUeUc variants (same locus), homologs (different locus), and orhologs (different organism) or can be non-naturaUy occurring. Non-naturaUy occurring variants can be made by mutagenesis techniques, including those apphed to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product). In apreferred embodiment, the nucleic acid differs from that of SEQ TD NO:l, SEQ

TD NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with

ATCC as Accession Number , e.g., as foUows: by at least one but less than 10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20% of the in the subject nucleic acid. If necessary for this analysis the sequences should be aUgned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.

Orthologs, homologs, and aUeUc variants can be identified using methods known in the art. These variants comprise a nucleotide sequence encoding a polypeptide that is 50%, at least about 55%, typicaUy at least about 70-75%, more typicaUy at least about 80-85%, and most typicaUy at least about 90-95% or more identical to the amino acid sequence shown in

SEQ ID NO:2 or a fragment of this sequence. Such nucleic acid molecules can readily be obtained as being able to hybridize under stringent conditions, to the nucleotide sequence shown in SEQ ID NO:3 or a fragment of this sequence. Nucleic acid molecules corresponding to orthologs, homologs, and aUeUc variants of the 7677 cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the 7677 gene. Preferred variants include those that are correlated with ATPase activity. AUeUc variants of 7677, e.g., human 7677, include both functional and nonfunctional proteins. Functional allehc variants are naturaUy occurring arnino acid sequence variants of the 7677 protein within a population that maintain the abiUty to modulate the phosphorylation state of itself or another protein or polypeptide. Functional alleUc variants will typicaUy contain only conservative substitution of one or more amino acids of SEQ ID NO:2, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein. Non-functional aUelic variants are naturaUy-occurring amino acid sequence variants of the 7677, e.g., human 7677 protein within a population that do not have the abiUty to modulate the translation of chemical to biological energy. Non-functional allehc variants wUl typicaUy contain a non-conservative substitution, a deletion, or insertion, or premature truncation of the amino acid sequence of SEQ ID NO:2, or a substitution, insertion, or deletion in critical residues or critical regions of the protein.

Moreover, nucleic acid molecules encoding other 7677 family members and, thus, which have a nucleotide sequence which differs from the 7677 sequences of SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number are intended to be within the scope of the invention.

Antisense Nucleic Acid Molecules, Ribozvmes and Modified 7677 Nucleic Acid

Molecules

In another aspect, the invention features, an isolated nucleic acid molecule which is antisense to 7677. An "antisense" nucleic acid can include a nucleotide sequence which 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. The antisense nucleic acid can be complementary to an entire 7677 coding strand, or to only a portion thereof (e.g., the coding region of human 7677 rørre onding to SEQ JD NO:3). In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding 7677 (e.g., the

5' and 3' untranslated regions). An antisense nucleic acid can be designed such that it is complementary to the entire coding region of 7677 mRNA, but more preferably is an oUgonucleotide which is antisense to only a portion of the coding or noncoding region of 7677 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of 7677 mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oUgonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50; 55, 60, 65, 70, 75, 80, or more nucleotides in length.

An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic Ugation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oUgonucleotide) can be chemicaUy synthesized using naturaUy occurring nucleotides or variously modified nucleotides designed to increase the biological stabiUty of the molecules or to increase the physical stabiUty of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologicaUy 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 wiU be of an antisense orientation to a target nucleic acid of interest, described further in the foUowing subsection). The antisense nucleic acid molecules of the invention are typicaUy adniinistered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to ceUular mRNA and/or genomic DNA encoding a 7677 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemicaUy. For systemic administration, antisense molecules can be modified such that they specificaUy bind to receptors or antigens expressed on a selected ceU surface, e.g., by Unking the antisense nucleic acid molecules to peptides or antibodies which bind to ceU surface receptors or antigens. The antisense nucleic acid molecules can also be deUvered to ceUs using the vectors described herein. To achieve sufficient intraceUular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol IQ promoter are preferred. In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run paraUel to each other (Gaultier et al., (1987) Nucleic Acids. Res. 15:6625- 6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al., (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. A ribozyme having specificity for a 7677-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of a 7677 cDNA disclosed herein (i.e., SEQ JD NO:l, or SEQ JD NO:3), and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach, (1988) Nature 334:585-591). For example, a derivative of a Tetrahymena L-19 TVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a 7677-encoding mRNA. See, e.g., Cech et al. U.S. Patent No.4,987,071; and Cech et al. U.S. Patent No. 5,116,742. Alternatively, 7677 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418. 7677 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the 7677 (e.g., the 7677 promoter and/or enhancers) to form triple heUcal structures that prevent transcription of the 7677 gene in target ceUs. See generaUy, Helene, C, (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al., (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L.J., (1992) Bioassays 14(12):807-15. The potential sequences that can be targeted for triple helix formation can be increased by creating a so-caUed "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3 -5' manner, such that they base pair with first one strand of a duplex and then the other, eUrninating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex. The invention also provides detectably labeled oUgonucleotide primer and probe molecules. Typically, such labels are chermluminescent, fluorescent, radioactive, or colorimetric. A 7677 nucleic acid molecule can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stabiUty, hybridization, or solubihty of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al., (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms "peptide nucleic acid" or "PNA" refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oUgomers can be performed using standard sohd phase peptide synthesis protocols as described in Hyrup B. et al., (1996) supra; Perry-OKeefe et al., Proc. Nail. Acad. Sci. 93: 14670-675.

PNAs of 7677 nucleic acid molecules can be used in therapeutic and diagnostic appUcations. For example, PNAs can be used as antisense or antigene agents for sequence- specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting repUcation. PNAs of 7677 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B., (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al., (1996) supra; Perry-CKeefe supra).

In other embodiments, the oUgonucleotide may include other appended groups such as peptides (e.g., for targeting host ceU receptors in vivo), or agents facilitating transport across the ceU membrane (see, e.g., Letsinger et al., (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al., (1987) Proc. Natl. Acad. Sci. USA 84.'648-652; PCT PubUcation No. W088/09810) or the blood-brain barrier (see, e.g., PCT Pubhcation No.. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al., (1988) Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon, (1988) Pharm. Res. 5:539-549). To this end, the oUgonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross- linking agent, transport agent, or hybridization-triggered cleavage agent).

The invention also includes molecular beacon oUgonucleotide primer and probe molecules having at least one region which is complementary to a 7677 nucleic acid of the invention, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantitating the presence of the 7677 nucleic acid of the invention in a sample. Molecular beacon nucleic acids are described, for example, in Lizardi et al., U.S. Patent No. 5,854,033; Nazarenko et al., U.S. Patent No. 5,866,336, and Livak et al., U.S. Patent 5,876,930.

Isolated 7677 Polypeptides

In another aspect, the invention features, an isolated 7677 protein, or fragment, e.g., a biologicaUy active portion, for use as immunogens or antigens to raise or test (or more generaUy to bind) anti-7677 antibodies. 7677 protein can be isolated from cells or tissue sources using standard protein purification techniques. 7677 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemicaUy.

Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA spUcing events, and alternative translational and postranslational events. The polypeptide can be expressed in systems, e.g., cultured ceUs, which result in substantiaUy the same postranslational modifications present when expressed the polypeptide is expressed in a native ceU, or in systems which result in the alteration or omission of postranslational modifications, e.g., gylcosylation or cleavage, present when expressed in a native ceU. In a preferred embodiment, a 7677 polypeptide has one or more of the following characteristics:

(i) it binds to and hydrolyzes ATP, playing a pivotal role in translating chemically stored energy into biological energy;

(n) it has a molecular weight, e.g., a deduced molecular weight, amino acid composition or other physical characteristic of the polypeptide of SEQ ID NO:2;

(in) it has an overall sequence similarity of at least 50%, preferably at least 60%, more preferably at least 70, 80, 90, or 95%, with a polypeptide of SEQ ID NO:2;

(iv) it has an ATPase domain which preferably has an overaU sequence similarity of about 70%, 80%, 90% or 95% with amino acid residues 263-459 or 260-380 of SEQ ID NO:2;

(v) it has at least 70%, preferably 80%, and most preferably 95% of the cysteines found in the amino acid sequence of the native protein. In a preferred embodiment the 7677 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID NO:2. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ JD NO:2 by at least one residue but less than 20%, 15%, 10% or 5% of the residues in it differ from the corresponding sequence in SEQ ID NO:2. (If this comparison requires aUgnment the sequences should be aUgned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non-essential residue or a conservative substitution. In a preferred embodiment the differences are not in the ATPase domain. In another preferred embodiment one or more differences are in non- active site residues, e.g. outside of the ATPase domain.

Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g., a change in an amino acid residue which is not essential for activity. Such 7677 proteins differ in amino acid sequence from SEQ ID NO:2, yet retain biological activity.

In one embodiment, a biologicaUy active portion of a 7677 protein includes an ATPase domain. In another embodiment, a biologically active portion of a 7677 protein includes a protein kinase C phosphorylation domain. Moreover, other biologicaUy 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 7677 protein.

In a preferred embodiment, the 7677 protein has an amino acid sequence shown in SEQ 3D NO:2. In other embodiments, the 7677 protein is substantially identical to SEQ ID NO:2. In yet another embodiment, the 7677 protein is substantiaUy identical to SEQ 3D NO:2 and retains the functional activity of the protein of SEQ 3D NO:2, as described in detail above. Accordingly, in another embodiment, the 7677 protein is a protein which includes an arnino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to SEQ 3D NO:2.

7677 Chimeric or Fusion Proteins

In another aspect, the invention provides 7677 chimeric or fusion proteins. As used herein, a 7677 "chimeric protein" or "fusion protein" includes a 7677 polypeptide linked to a non-7677 polypeptide. A "non-7677 polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the 7677 protein, e.g., a protein which is different from the 7677 protein and which is derived from the same or a different organism. The 7677 polypeptide of the fusion protein can correspond to aU or a portion e.g., a fragment described herein of a 7677 amino acid sequence. In a preferred embodiment, a 7677 fusion protein includes at least one (or two) biologicaUy active portion of a 7677 protein. The non-7677 polypeptide can be fused to the N-teπninus or C-terminus of the 7677 polypeptide.

The fusion protein can include a moiety which has a high affinity for a Ugand. For example, the fusion protein can be a GST-7677 fusion protein in which the 7677 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can faciUtate the purification of recombinant 7677. Alternatively, the fusion protein can be a 7677 protein containing a heterologous signal sequence at its N-terrninus. In certain host ceUs (e.g., mammalian host cells), expression and or secretion of 7677 can be increased through use of a heterologous signal sequence.

Fusion proteins can include aU or a part of a serum protein, e.g., an IgG constant region, or human serum albumin.

The 7677 fusion proteins of the invention can be incorporated into pharmaceutical compositions and adniinistered to a subject in vivo. The 7677 fusion proteins can be used to affect the bioavailabiUty of a 7677 substrate. 7677 fusion proteins may be useful therapeuticaUy for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a 7677 protein; (n) mis-regulation of the 7677 gene; and (Hi) aberrant post-translational modification of a 7677 protein.

Moreover, the 7677-fusion proteins of the invention can be used as immunogens to produce anti-7677 antibodies in a subject, to purify 7677 ligands and in screening assays to identify molecules which inhibit the interaction of 7677 with a 7677 substrate.

Expression vectors are commerciaUy available that already encode a fusion moiety (e.g., a GST polypeptide). A 7677-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the 7677 protein.

Variants of 7677 Proteins In another aspect, the invention also features a variant of a 7677 polypeptide, e.g., which functions as an agonist (mimetics) or as an antagonist. Variants of the 7677 proteins can be generated by mutagenesis, e.g., discrete point mutation, the insertion or deletion of sequences or the truncation of a 7677 protein. An agonist of the 7677 proteins can retain substantiaUy the same, or a subset, of the biological activities of the naturaUy occurring . form of a 7677 protein. An antagonist of a 7677 protein can inhibit one or more of the activities of the naturally occurring form of the 7677 protein by, for example, competitively modulating a 7677-mediated activity of a 7677 protein. Thus, specific biological effects can be ehcited by treatment with a variant of Umited function. Preferably, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturaUy occurring form of the 7677 protein.

Variants of a 7677 protein can be identified by screening combinatorial Ubraries of mutants, e.g., truncation mutants, of a 7677 protein for agonist or antagonist activity. Libraries of fragments e.g., N terminal, C terminal, or internal fragments, of a 7677 protein coding sequence can be used to generate a variegated population of fragments for screening and subsequent selection of variants of a 7677 protein.

Variants in which a cysteine residues is added or deleted or in which a residue which is glycosylated is added or deleted are particularly preferred. Methods for screening gene products of combinatorial Ubraries made by point mutations or truncation, and for screening cDNA Ubraries for gene products having a selected property. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the Ubraries, can be used in combination with the screening assays to identify 7677 variants (Arkin and Yourvan, (1992) Proc. Natl. Acad. Sci. USA 59:7811-7815; Delgrave et al., (1993) Protein Engineering 6(3):327-331).

CeU based assays can be exploited to analyze a variegated 7677 Ubrary. For example, a Ubrary of expression vectors can be transfected into a ceU line, e.g., a ceU line, which ordinarily responds to 7677 in a substrate-dependent manner. The transfected cells are then contacted with 7677 and the effect of the expression of the mutant on signaling by the 7677 substrate can be detected, e.g., by momtoring the activity of an intraceUular factor or by measuring ATPase activity. An intraceUular factor is any substance having an activity that is affected by the activity of 7677 such as an intracellular second messenger (e.g., calcium, cAMP, IP₃ or diacylgycerol) Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the 7677 substrate, and the individual clones further characterized.

In another aspect, the invention features a method of making a 7677 polypeptide, e.g., a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring 7677 polypeptide, e.g., a naturaUy occurring 7677 polypeptide. The method includes: altering the sequence of a 7677 polypeptide, e.g., altering the sequence, e.g., by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.

In another aspect, the invention features a method of making a fragment or analog of a 7677 polypeptide a biological activity of a naturally occurring 7677 polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of a 7677 polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.

Anti-7677 Antibodies

In another aspect, the invention provides an anti-7677 antibody. The term "antibody" as used herein refers to an immunoglobulin molecule or immunologicaUy active portion thereof, i.e., an antigen-binding portion. Examples of immunologicaUy active portions of immunoglobuUn molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.

The antibody can be a polyclonal, monoclonal, recombinant, e.g., a chimeric or humanized, fuUy human, non-human, e.g., murine, or single chain antibody. In a preferred embodiment it has effector function and can fix complement. The antibody can be coupled to a toxin or imaging agent.

A fuU-length 7677 protein or, antigenic peptide fragment of 7677 can be used as an immunogen or can be used to identify anti-7677 antibodies made with other immunogens, e.g., ceUs, membrane preparations, and the like. The antigenic peptide of 7677 should include at least 8 amino acid residues of the amino acid sequence shown in SEQ 3D NO:2 and encompasses an epitope of 7677. Preferably, the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 arnino acid residues.

Fragments of 7677 which include, e.g., residues 151-191 of SEQ 3D NO:2 can be used to make, e.g., used as immunogens, or used to characterize the specificity of an antibody or antibodies against what are beUeved to be hydrophiUc regions of the 7677 protein. Similarly, a fragment of 7677 which includes, e.g., residues 381-401 of SEQ ID

NO:2 can be used to make an antibody against what is beUeved to be a hydrophobic region of the 7677 protein; a fragment of 7677 which includes residues 263-459 or 260-380 of SEQ ID NO:2 can be used to make an antibody against the ATPase region of the 7677 protein.

Antibodies reactive with, or specific for, any of these regions, or other regions or domains described herein are provided.

In a preferred embodiment the antibody fails to bind an Fc receptor, e.g. it is a type which does not support Fc receptor binding or has been modified, e.g., by deletion or other mutation, such that is does not have a functional Fc receptor binding region.

Preferred epitopes encompassed by the antigenic peptide are regions of 7677 are located on the surface of the protein, e.g., hydrophiUc regions, as weU as regions with high antigenicity. For example, an Emini surface probabihty analysis of the human 7677 protein sequence can be used to indicate the regions that have a particularly high probabihty of being localized to the surface of the 7677 protein and are thus likely to constitute surface residues useful for targeting antibody production.

In a preferred embodiment the antibody binds an epitope on any domain or region on 7677 proteins described herein. Chimeric, humanized, but most preferably, completely human antibodies are desirable for appUcations which include repeated administration, e.g., therapeutic treatment

(and some diagnostic appUcations) of human patients.

The anti-7677 antibody can be a single chain antibody. A single-chain antibody

(scJFV) maybe engineered (see, for example, Colcher, D. et al., Ann. NY Acad. Sci. 1999 Jun 30;880:263-80; and Reiter, Y., Clin. Cancer Res. 1996 Feb;2(2):245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target 7677 protein. An anti-7677 antibody (e.g., monoclonal antibody) can be used to isolate 7677 by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an anti-7677 antibody can be used to detect 7677 protein (e.g., in a cellular lysate or ceU supernatant) in order to evaluate the abundance and pattern of expression of the protein. Anti-7677 antibodies can be used diagnosticaUy 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 faciUtated by coupling (i.e., physically Unking) the antibody to a detectable substance (i.e., antibody labeling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, biolurninescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylchoUnesterase; examples of suitable prosthetic group complexes include sfreptøvidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbeUiferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazmylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of biolurninescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵1, ¹³¹1, ³⁵S or ³H.

Recombinant Expression Vectors. Host Cells and Genetically Engineered Cells In another aspect, the invention includes, vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g., repUcation defective retroviruses, adenoviruses and adeno-associated viruses. A vector can include a 7677 nucleic acid in a form suitable for expression of the nucleic acid in a host ceU. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as weU as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host ceU to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., 7677 proteins, mutant forms of 7677 proteins, fusion proteins, and the like).

The recombinant expression vectors of the invention can be designed for expression of 7677 proteins in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in E. coU, insect ceUs (e.g., using baculovirus expression vectors), yeast ceUs or mammalian cells. Suitable host cells are discussed further in

Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. Expression of proteins in prokaryotes is most often carried out in E. coU 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, usuaUy to the amino terminus of the recombinant protein. Such fusion vectors typicaUy serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a Ugand in affinity purification. Often, 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, D.B. and Johnson, K.S., (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRTT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Purified fusion proteins can be used in 7677 activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for 7677 proteins. In a preferred embodiment, a fusion protein expressed in a retroviral expression vector of the present invention can be used to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks).

To maximize recombinant protein expression in E. coli is to express the protein in host bacteria with an impaired capacity to proteolyticaUy cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Cahfornia (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 preferentiaUy utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111 -2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

The 7677 expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector or a vector suitable for expression in mammaUan cells. When used in mammaUan ceUs, 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.

In another embodiment, the recombinant mammaUan expression vector is capable of directing expression of the nucleic acid preferentially in a particular ceU type (e.g., tissue- specific regulatory elements are used to express the nucleic acid). Non-Umiting examples of suitable tissue-specific promoters include the albumin promoter (Uver-specific; Pinkert et al., (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton, (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore, (1989) EMBO J. 8:729-733) and immunoglobuUns (Banerji et al., (1983) Cell 33 :729-740; Queen and Baltimore, (1983) Cell 33 :741 -748), neuron-specific promoters (e.g., the neurofilament promoter, Byrne and Ruddle, (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al., (1985) Science 230:912-916). and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European AppUcation Publication No. 264,166). Developmentally- regulated promoters are also encompassed, for example, the murine hox promoters (Kessel and Grass, (1990) Science 249:374-379) and the α-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. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of ceU types. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986. Another aspect the invention provides a host ceU which includes a nucleic acid molecule described herein, e.g., a 7677 nucleic acid molecule within a recombinant expression vector or a 7677 nucleic acid molecule containing sequences which aUow it to homologously recombine into a specific site of the host cell's genome. The terms "host ceU" and "recombinant host ceU" are used interchangeably herein. Such terms refer not only to the particular subj ect ceU but rather also to the progeny or potential progeny of such a ceU. 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 ceU, but are still included within the scope of the term as used herein.

A host ceU can be any prokaryotic or eukaryotic ceU. For example, a 7677 protein can be expressed in bacterial cells such as E. coli, insect ceUs, yeast or mammaUan cells (such as Chinese hamster ovary cells (CHO) or COS ceUs). Other suitable host ceUs are lcnown to those sldlled in the art.

Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, Upofection, or electroporation

A host ceU of the invention can be used to produce (i.e., express) a 7677 protein. Accordingly, the invention further provides methods for producing a 7677 protein using the host ceUs of the invention. In one embodiment, the method includes culturing the host ceU of the invention (into which a recombinant expression vector encoding a 7677 protein has been introduced) in a suitable medium such that a 7677 protein is produced. In another embodiment, the method further includes isolating a 7677 protein from the medium or the host ceU.

In another aspect, the invention features, a ceU or purified preparation of cells which include a 7677 transgene, or which otherwise misexpress 7677. The ceU preparation can consist of human or non-human ceUs, e.g., rodent cells, e.g., mouse or rat cells, rabbit ceUs, or pig ceUs. In preferred embodiments, the ceU or ceUs include a 7677 transgene, e.g., a heterologous form of a 7677, e.g., a gene derived from humans (in the case of a non-human ceU). The 7677 transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the ceU or cells include a gene which misexpress an endogenous 7677, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders which are related to mutated or mis- expressed 7677 aUeles or for use in drug screening.

In another aspect, the invention features, a human ceU, e.g., a hematopoietic stem ceU, transformed with nucleic acid which encodes a subject 7677 polypeptide. Also provided are cells or a purified preparation thereof, e.g., human ceUs, in which an endogenous 7677 is under the control of a regulatory sequence that does not normaUy control the expression of the endogenous 7677 gene. The expression characteristics of an endogenous gene within a cell, e.g., a ceU line or microorganism, can be modified by inserting a heterologous DNA regulatory element into the genome of the ceU such that the inserted regulatory element is operably linked to the endogenous 7677 gene. For example, an endogenous 7677 gene, e.g., a gene which is "transcriptionaUy silent," e.g., not normaUy expressed, or expressed only at very low levels, may be activated by inserting a regulatory element which is capable of promoting the expression of a normaUy expressed gene product in that ceU. Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, US 5,272,071; WO 91/06667, pubUshed on May 16, 1991.

Transgenic Animals

The invention provides non-human transgenic animals. Such animals are useful for studying the function and/or activity of a 7677 protein and for identifying and/or evaluating modulators of 7677 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 ceUs of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA or a rearrangement, e.g., a deletion of endogenous chromosomal DNA, which preferably is integrated into or occurs in the genome of the ceUs of a transgenic animal. A transgene can direct the expression of an encoded gene product in one or more ceU types or tissues of the transgenic animal, other transgenes, e.g., a knockout, reduce expression. Thus, a transgenic animal can be one in which an endogenous 7677 gene has been altered by, e.g., by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a ceU of the animal, e.g., an embryonic ceU of the animal, prior to development of the animal.

Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a transgene of the invention to direct expression of a 7677 protein to particular cells. A transgenic founder animal can be identified based upon the presence of a 7677 transgene in its genome and/or expression of 7677 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 a 7677 protein can further be bred to other transgenic animals carrying other transgenes. 7677 proteins or polypeptides can be expressed in transgenic animals or plants, e.g., a nucleic acid encoding the protein or polypeptide can be introduced into the genome of an animal, preferred embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Suitable animals are mice, pigs, cows, goats, and sheep. The invention also includes a population of cells from a transgenic animal, as discussed herein.

Uses

The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, momtoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic). The isolated nucleic acid molecules of the invention can be used, for example, to express a 7677 protein (e.g., via a recombinant expression vector in a host ceU in gene therapy appUcations), to detect a 7677 mRNA (e.g., in a biological sample) or a genetic alteration in a 7677 gene, and to modulate 7677 activity, as described further below. The 7677 proteins can be used to treat disorders characterized by insufficient or excessive production of a 7677 substrate or production of 7677 inhibitors. In addition, the 7677 proteins can be used to screen for naturaUy occurring 7677 substrates, to screen for drugs or compounds which modulate 7677 activity, as well as to treat disorders characterized by insufficient or excessive production of 7677 protein or production of 7677 protein forms which have decreased, aberrant or unwanted activity compared to 7677 wild-type protein. Such disorders include those characterized by aberrant signaling or aberrant, e.g., hyperproUferative, cell growth. Moreover, the anti-7677 antibodies of the invention can be used to detect and isolate 7677 proteins, regulate the bioavailabiUty of 7677 proteins, and modulate 7677 activity. A method of evaluating a compound for the abiUty to interact with, e.g., bind, a subject 7677 polypeptide is provided. The method includes: contacting the compound with the subject 7677 polypeptide; and evaluating abiUty of the compound to interact with, e.g., to bind or form a complex with the subject 7677 polypeptide. This method can be performed in vitro, e.g., in a ceU free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturaUy occurring molecules which interact with subject 7677 polypeptide. It can also be used to find natural or synthetic inhibitors of subject 7677 polypeptide. Screening methods are discussed in more detail below.

Screening Assays:

The invention provides methods (also referred to herein as "screening assays") for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, smaU molecules or other drugs) which bind to 7677 proteins, have a stimulatory or inhibitory effect on, for example, 7677 expression or 7677 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a 7677 substrate. Compounds thus identified can be used to modulate the activity of target gene products (e.g., 7677 genes) in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions.

In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a 7677 protein or polypeptide or a biologicaUy active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a 7677 protein or polypeptide or a biologicaUy active portion thereof.

The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial Ubrary methods known in the art, including: biological Ubraries; peptoid Ubraries [Ubraries of molecules having the functionaUties of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive] (see, e.g., Zuckermann, R.N. et al., J.

Med. Chem. 1994, 37: 2678-85); spatiaUy addressable paraUel soUd phase or solution phase

Ubraries; synthetic Ubrary methods requiring deconvolution; the 'one-bead one-compound' Ubrary method; and synthetic library methods using affinity chromatography selection.

The biological Ubrary and peptoid Ubrary approaches are Umited to peptide Ubraries, while the other four approaches are appUcable to peptide, non-peptide oUgomer or small molecule Ubraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular Ubraries 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. USA 91 :11422; Zuckermann et al., (1994). J. Med. Chem.

37:2678; Cho et al., (1993) Science 261:1303; CarreU et al., (1994) Angew. Chem. Int. Ed.

Engl. 33:2059; CareU et al., (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al., (1994) J. Med. Chem. 37:1233. Libraries of compounds may be presented in solution (e.g., Houghten, ( 1992)

Biotechniques 13:412-421), or on beads (Lam, (1991) Nature 354:82-84), chips (Fodor,

(1993) Nature 364:555-556), bacteria or spores (Ladner, United States Patent No.

5,223,409), plasmids (CuU 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. 87:6378-6382); (FeUci, (1991) J. Mol.

Biol. 222:301-310); (Ladner supra.). In one embodiment, an assay is a cell-based assay in which a ceU which expresses a 7677 protein or biologically active portion thereof is contacted with a test compound, and the abiUty of the test compound to modulate 7677 activity is determined. Determining the abiUty of the test compound to modulate 7677 activity can be accompUshed by monitoring, for example, ATPase activity. The ceU, for example, can be of mammaUan origin, e.g., human. CeU homogenates, or fractions, preferably membrane containing fractions, can also be tested.

The abUity of the test compound to modulate 7677 binding to a compound, e.g., a 7677 substrate, or to bind to 7677 can also be evaluated. This can be accompUshed, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to 7677 can be determined by detecting the labeled compound, e.g., substrate, in a complex. Altematively, 7677 could be coupled with a radioisotope or enzymatic label to monitor the abiUty of a test compound to modulate 7677 binding to a 7677 substrate in a complex. For example, compounds (e.g., 7677 substrates) can be labeled with ¹²⁵1, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintiUation counting. Alternatively, compounds can be enzymaticaUy labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by deterrnination of conversion of an appropriate substrate to product. The abiUty of a compound (e.g., a 7677 substrate) to interact with 7677 with or without the labeling of any of the interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with 7677 without the labeling of either the compound or the 7677. McConneU, H. M. et al., (1992) Science 257:1906-1912. As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that measures the rate at which a ceU acidifies its environment using a Ught- addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and 7677.

In yet another embodiment, a ceU-free assay is provided in which a 7677 protein or biologically active portion thereof is contacted with a test compound and the abiUty of the test compound to bind to the 7677 protein or biologicaUy active portion thereof is evaluated. Preferred biologicaUy active portions of the 7677 proteins to be used in assays of the present invention include fragments which participate in interactions with non-7677 molecules, e.g., fragments with high surface probabihty scores.

Soluble and/or membrane-bound forms of isolated proteins (e.g., 7677 proteins or biologically active portions thereof) can be used in the cell-free assays of the invention. When membrane-bound forms of the protein are used, it may be desirable to utiUze a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N- methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_n, 3-[(3-cholanήdopropyl)dimethylanmιinio]-l- propane sulfonate (CHAPS), 3-[(3-cholarmdopropyl)ά^ethylarnπώuo]-2-hydroxy-l- propane sulfonate (CHAPSO), or N-dodecyl-N^-dimethyl-3-ammonio-l-propane sulfonate.

CeU-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

In one embodiment, assays are performed where the abiUty of an agent to block ATPase activity within a ceU is evaluated.

The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FET) (see, for example, Lakowicz et ah, U.S. Patent No. 5,631,169; Stavrianopoulos, et al., U.S. Patent No.4,868,103). A fluorophore label on the first, 'donor' molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, 'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of Ught, such that the 'acceptor' molecule label may be differentiated from that of the 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be i assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means weU known in the art (e.g., using a fluorimeter). In another embodiment, determining the abiUty of the 7677 protein to bind to a target molecule can be accompUshed using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C, (1991) Anal. Chem. 63:2338-2345 and Szabo et al., (1995) Curr. Opin. Struct. Biol. 5:699-705). "Surface plasmon resonance" or "BIA" detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of Ught near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules. In one embodiment, the target gene product or the test substance is anchored onto a sohd phase. The target gene product/test compound complexes anchored on the sohd phase can be detected at the end of the reaction, Preferably, the target gene product can be anchored onto a soUd surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein. It may be desirable to immobiUze either 7677, an anti-7677 antibody or its target molecule to faciUtate 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 a 7677 protein, or interaction of a 7677 protein with a target molecule in the presence and absence of a candidate compound, can be accompUshed 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 which adds a domain that aUows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/7677 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or 7677 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). FoUowing incubation, the beads or microtiter plate weUs 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 above. Altematively, the complexes can be dissociated from the matrix, and the level of

7677 binding or activity determined using standard techniques. Other techniques for immobiUzing either a 7677 protein or a target molecule on matrices include using conjugation of biotin and sfreptavidin. Biotinylated 7677 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-sUccinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the weUs of streptavidin-coated 96 well plates (Pierce Chemical).

In order to conduct the assay, the non-immobiUzed component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed wiU remain immobUized on the sohd surface. The detection of complexes anchored on the sohd surface can be accompUshed in a number of ways. Where the previously non-immobiUzed component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the in-mobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactive with 7677 protein or target molecules but which do not interfere with binding of the 7677 protein to its target molecule. Such antibodies can be derivatized to the weUs of the plate, and unbound target or 7677 protein trapped in the weUs by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include irnmunodetection of complexes using antibodies reactive with the 7677 protein or target molecule, as weU as enzyme-linked assays which rely on detecting an enzymatic activity associated with the 7677 protein or target molecule. Alternatively, ceU free assays can be conducted in a Uquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minton, A.P., Trends Biochem Sci 1993 Aug;18(8):284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel, F. et al., eds. Current Protocols in Molecular Biology

1999, J. WUey: New York.); and immunoprecipitation (see, for example, Ausubel, F. et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, N.H., J Mol. Recognit. 1998 Winter;ll(l-6):141-8; Hage, D.S., and Tweed, S.A., J. Chromatogr. B Biomed. Sci. Appl. 1997 Oct 10;699(l-2):499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

In a preferred embodiment, the assay includes contacting the 7677 protein or biologically active portion thereof with a known compound which binds 7677 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 7677 protein, wherein deterrnining the abiUty of the test compound to interact with a 7677 protein includes determining the abiUty of the test compound to preferentiaUy bind to 7677 or biologicaUy active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.

The target gene products of the invention can, in vivo, interact with one or more ceUular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such ceUular and extraceUular macromolecules are referred to herein as

"binding partners." Compounds that disrupt such interactions can be useful in regulating the activity of the target gene product. Such compounds can include, but are not Umited to molecules such as antibodies, peptides, and small molecules. The preferred target genes/products for use in this embodiment are the 7677 genes herein identified. In an alternative embodiment, the invention provides methods for determining the abiUty of the test compound to modulate the activity of a 7677 protein through modulation of the activity of a downstream effector of a 7677 target molecule. For example, the activity of the effector molecule on an appropriate target can be determined, or the binding of the effector to an appropriate target can be determined, as previously described. To identify compounds that interfere with the interaction between the target gene product and its cellular or extraceUular binding partner(s), e.g., a substrate, a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to aUow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence of the test compound. The test compound can be initiaUy included in the reaction mixture, or can be added at a time subsequent to the addition of the target gene and its ceUular or extraceUular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene product and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.

These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a soUd phase, and detecting complexes anchored on the sohd phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a Uquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.

In a heterogeneous assay system, either the target gene product or the interactive ceUular or extraceUular binding partner, is anchored onto a sohd surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored species can be immobiUzed by non-covalent or covalent attachments. Alternatively, an immobiUzed antibody specific for the species to be anchored can be used to anchor the species to the soUd surface.

In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed wiU remain immobiUzed on the sohd surface. Where the non-immobiUzed species is pre- labeled, the detection of label immobiUzed on the surface indicates that complexes were formed. Where the non-immobihzed species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobiUzed species (the antibody, in turn, can be directly labeled or mdirectly labeled with, e.g., a labeled anti-Ig antibody). Dφending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a Uquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobiUzed antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the Uquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified. In an alternate embodiment of the invention, a homogeneous assay can be used. For example, a preformed complex of the target gene product and the interactive ceUular or extraceUular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Patent No.4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrapt target gene product-bmding partner interaction can be identified.

In yet another aspect, the 7677 proteins can be used as "bait proteins" in a two- hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al.,

(1993) Cell 72:223-232; Madura et al., (1993) J. Biol. Chem. 268:12046-12054; Bartel et al., (1993) Biotechniques 14:920-924; Iwabucbi et al., (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with 7677 ("7677- binding proteins" or "7677-bp") and are involved in 7677 activity. Such 7677-bps can be activators or inhibitors of signals by the 7677 proteins or 7677 targets as, for example, downstream elements of a 7677-mediated signaling 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 a 7677 protein is fused to a gene encoding the DNA bmding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a Ubrary 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. (Alternatively the: 7677 protein can be the fused to the activator domain.) If the "bait" and the "prey" proteins are able to interact, in vivo, forming a 7677-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity aUows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and ceU colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the 7677 protein.

In another embodiment, modulators of 7677 expression are identified. For example, a ceU or ceU free mixture is contacted with a candidate compound and the expression of 7677 mRNA or protein evaluated relative to the level of expression of 7677 mRNA or protein in the absence of the candidate compound. When expression of 7677 mRNA or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of 7677 mRNA or protein expression. Alternatively, when expression of 7677 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of 7677 mRNA or protein expression. The level of 7677 mRNA or protein expression can be determined by methods described herein for detecting 7677 mRNA or protein.

In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a ceU- based or a ceU free assay, and the abiUty of the agent to modulate the activity of a 7677 protein can be confirmed in vivo, e.g., in an animal.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a 7677 modulating agent, an antisense 7677 nucleic acid molecule, a 7677-specific antibody, or a 7677-binding partner) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above- described screening assays can be used for treatments as described herein.

Detection Assays

Portions or fragments of the nucleic acid sequences identified herein can be used as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome e.g., to locate gene regions associated with genetic disease or to associate 7677 with a disease; (ii) identify an individual from a minute biological sample (tissue typing); and (in) aid in forensic identification of a biological sample. These appUcations are described in the subsections below.

Chromosome Mapping

The 7677 nucleotide sequences or portions thereof can be used to map the location of the 7677 genes on a chromosome. This process is called chromosome mapping. Chromosome mapping is useful in correlating the 7677 sequences with genes associated with disease. Briefly, 7677 genes can be mapped to chromosomes by preparing PCR primers

(preferably 15-25 bp in length) from the 7677 nucleotide sequences. These primers can then be used for PCR screening of somatic ceU hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the 7677 sequences wiU yield an amplified fragment. A panel of somatic cell hybrids in which each ceU line contains either a single human chromosome or a small number of human chromosomes, and a fuU set of mouse chromosomes, can allow easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al., (1983) Science 220:919-924).

Other mapping strategies e.g., in situ hybridization (described in Fan, Y. et al., (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA Ubraries can be used to map 7677 to a chromosomal location. 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. 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 wiU 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 individuaUy 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 actuaUy are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene famiUes, 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, for example, in V. McKusick, MendeUan Inheritance in Man, avaUable on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through Unkage analysis (co-inheritance of physicaUy adjacent genes), described in, for example, Egeland, J. et al., (1987) Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the 7677 gene, can be deterrnined. If a mutation is observed in some or aU 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 corifirm the presence of a mutation and to distinguish mutations from polymorphisms.

Tissue Typing 7677 sequences can be used to identify individuals from biological samples using, e.g., restriction fragment length polymorphism (RFLP). In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, the fragments separated, e.g., in a Southern blot, and probed to yield bands for identification. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).

Furthermore, the sequences of the present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the 7677 nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to ampUfy 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 aUeUc differences. AUeUc variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. 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 of SEQ 3D NO: 1 can provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding ampUfied sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO: 3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000. If a panel of reagents from 7677 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely smaU tissue samples.

Use of Partial 7677 Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensic biology. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saUva, or semen found at a crime scene. The ampUfied sequence can then be compared to a standard, thereby aUowing identification of the origin of the biological sample.

The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reUabiUty of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO: 1 (e.g., fragments derived from the noncoding regions of SEQ 3D NO:l having a length of at least 20 bases, preferably at least 30 bases) are particularly appropriate for this use.

The 7677 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., a tissue containing ATPase activity. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such 7677 probes can be used to identify tissue by species and/or by organ type.

In a similar fashion, these reagents, e.g., 7677 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a rnixture of different types of cells in a culture).

Predictive Medicine

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

GeneraUy, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes 7677.

Such disorders include, e.g., a disorder associated with the misexpression of 7677, or Upid metabolism related disorder.

The method includes one or more of the foUowing: detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the 7677 gene, or detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5' control region; detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the 7677 gene; detecting, in a tissue of the subject, the misexpression of the 7677 gene, at the mRNA level, e.g., detecting a non-wild type level of a mRNA ; detecting, in a tissue of the subject, the misexpression of the gene, at the protein level, e.g., detecting a non-wild type level of a 7677 polypeptide.

In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the 7677 gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.

For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oUgonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ 3D NO:l naturally occurring mutants thereof or 5' or 3' flanking sequences naturaUy associated with the 7677 gene; (n) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.

In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the 7677 gene; the presence of a non-wild type spUcing pattern of a messenger RNA transcript of the gene; or anon-wild type level of 7677.

Methods of the invention can be used prenataUy or to determine if a subject's offspring wiU be at risk for a disorder.

In preferred embodiments the method includes determining the structure of a 7677 gene, an abnormal structure being indicative of risk for the disorder. In preferred embodiments the method includes contacting a sample form the subject with an antibody to the 7677 protein or a nucleic acid, which hybridizes specifically with the gene. These and other, embodiments are discussed below.

Diagnostic and Prognostic Assays

The presence, level, or absence of 7677 protein or nucleic acid in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting 7677 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes 7677 protein such that the presence of 7677 protein or nucleic acid is detected in the biological sample. The term "biological sample" includes tissues, ceUs and biological fluids isolated from a subject, as weU as tissues, ceUs and fluids present within a subject. A preferred biological sample is serum. The level of expression of the 7677 gene can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the 7677 genes; measuring the amount of protein encoded by the 7677 genes; or measuring the activity of the protein encoded by the 7677 genes.

The level of mRNA corresponding to the 7677 gene in a ceU can be determined both by in situ and by in vitro formats.

The isolated mRNA can be used in hybridization or amplification assays that include, but are not Umited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a fuU-length 7677 nucleic acid, such as the nucleic acid of SEQ JD NO:l, or the DNA insert of the plasmid deposited with ATCC as Accession Number , or a portion thereof, such as an oUgonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specificaUy hybridize under stringent conditions to 7677 mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays are described herein. In one format, mRNA (or cDNA) is immobiUzed on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and fransferring the mRNA from the gel to a membrane, such as nifroceUulose. In an alternative format, the probes are immobiUzed on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array. A skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the 7677 genes. The level of mRNA in a sample that is encoded by one of 7677 can be evaluated with nucleic acid amplification, e.g., by rtPCR (MuUis, 1987, U.S. Patent No.4,683,202), Ugase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence repUcation (GuateUi et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional ampUfication system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta RepUcase (Lizardi et al., 1988, Bio/Technology 6:1197), rolling circle repUcation (Lizardi et al., U.S. Patent No. 5,854,033) or any other nucleic acid ampUfication method, foUowed by the detection of the ampUfied molecules using techniques known in the art. As used herein, ampUfication primers are defined as being a pair of nucleic acid molecules that can anneal to 5 ' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, ampUfication primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the ampUfication of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers. For in situ methods, a ceU or tissue sample can be prepared/processed and immobiUzed on a support, typically a glass sUde, and then contacted with a probe that can hybridize to mRNA that encodes the 7677 gene being analyzed. ha another embodiment, the methods further contacting a control sample with a compound or agent capable of detecting 7677 mRNA, or genomic DNA, and comparing the presence of 7677 mRNA or genomic DNA in the control sample with the presence of 7677 mRNA or genomic DNA in the test sample.

A variety of methods can be used to determine the level of protein encoded by 7677. hi general, these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample. In a preferred embodiment, the antibody bears 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 Unking) a detectable substance to the probe or antibody, as weU as indirect labeling of the probe or antibody by reactivity with a detectable substance. Examples of detectable substances are . provided herein.

The detection methods can be used to detect 7677 protein in a biological sample in vitro as well as in vivo. In vitro techniques for detection of 7677 protein include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. In vivo techniques for detection of 7677 protein include introducing into a subject a labeled anti- 7677 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 another embodiment, the methods further include contacting the control sample with a compound or agent capable of detecting 7677 protein, and comparing the presence of 7677 protein in the control sample with the presence of 7677 protein in the test sample. The invention also includes kits for detecting the presence of 7677 in a biological sample. For example, the kit can include a compound or agent capable of detecting 7677 protein or mRNA in a biological sample; and 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 7677 protein or nucleic acid.

For antibody-based kits, the kit can include: (1) a first antibody (e.g., attached to a sohd support) which binds to a polypeptide corresponding to a marker of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent. For oUgonucleotide-based kits, the kit can include: (1) an oUgonucleotide, e.g., a detectably labeled oUgonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention. The kit can also includes a buffering agent, a preservative, or a protein-stabiUzing agent. The kit can also includes components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit can be enclosed within an individual container and aU of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. The diagnostic methods described herein can identify subjects having, or at risk of developing, a disease or disorder associated with misexpressed or aberrant or unwanted 7677 expression or activity. As used herein, the term "unwanted" includes an unwanted phenomenon involved in a biological response such as pain or deregulated ceU proUferation. In one embodiment, a disease or disorder associated with aberrant or unwanted 7677 expression or activity is identified. A test sample is obtained from a subject and 7677 protein or nucleic acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level, e.g., the presence or absence, of 7677 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted 7677 expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest, including a biological fluid (e.g., serum), ceU sample, or tissue.

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, smaU molecule, or other drag candidate) to treat a disease or disorder associated with aberrant or unwanted 7677 expression or activity. For example, such methods can be used to deteπnine whether a subject can be effectively treated with an agent for a ceUular growth related disorder.

The methods of the invention can also be used to detect genetic alterations in a 7677 gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in 7677 protein activity or nucleic acid expression, such as a cellular growth related disorder. In preferred embodiments, the methods include detecting, in a sample from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a 7677-protein, or the mis-expression of the 7677 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a 7677 gene; 2) an addition of one or more nucleotides to a 7677 gene; 3) a substitution of one or more nucleotides of a 7677 gene, 4) a chromosomal rearrangement of a 7677 gene; 5) an alteration in the level of a messenger RNA transcript of a 7677 gene, 6) aberrant modification of a 7677 gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type spUcing pattern of a messenger RNA transcript of a 7677 gene, 8) a non-wild type level of a 7677-protein, 9) allelic loss of a 7677 gene, and 10) inappropriate post-translational modification of a 7677-protein.

An alteration can be detected without a probe/primer in a polymerase chain reaction, such as anchor PCR or RACE PCR, or, alternatively, in a Ugation chain reaction (LCR), the latter of which can be particularly useful for detecting point mutations in the 7677-gene. This method can include the steps of coUecting a sample of ceUs from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the sample, contacting the nucleic acid sample with one or more primers which specificaUy hybridize to a 7677 gene under conditions such that hybridization and ampUfication of the 7677-gene (if present) occurs, and detecting the presence or absence of an ampUfication product, or detecting the size of the ampUfication product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preUminary ampUfication step in conjunction with any of the techniques used for detecting mutations described herein. Alternative ampUfication methods include: self sustained sequence repUcation (GuateUi, J.C. et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional ampUfication system (Kwoh, D.Y. et al., (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta RepUcase (Lizardi, P.M. et al., (1988) Bio-Technology 6:1197), or other nucleic acid ampUfication methods, foUowed by the detection of the ampUfied molecules using techniques known to those of skill in the art.

In another embodiment, mutations in a 7677 gene from a sample cell can be identified by detecting alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, ampUfied (optionaUy), digested with one or more restriction endonucleases, and fragment length sizes are determined, e.g., 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, for example, U.S. Patent No. 5,498,531) 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 7677 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, two-dimensional arrays, e.g., chip based arrays. Such arrays include a pluraUty of addresses, each of which is positionaUy distinguishable from the other. A different probe is located at each address of the pluraUty. The arrays can have a high density of addresses, e.g., can contain hundreds or thousands of oUgonucleotides probes (Cronin, M.T. et al., (1996) Human Mutation 7: 244-255; Kozal, M.J. et al., (1996) Nature Medicine 2:753-759). For example, genetic mutations in 7677 can be identified in two dimensional arrays containing Ught-generated DNA probes as described in Cronin, M.T. 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 step is followed by a second hybridization array that aUows the characterization of specific mutations by using smaUer, 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 7677 gene and detect mutations by comparing the sequence of the sample 7677 with the corresponding wild-type (control) sequence. Automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry.

Other methods for detecting mutations in the 7677 gene include methods in which protection from cleavage agents is used to detect mismatched bases in 3JNA/RNA or RNA/DNA heteroduplexes (Myers et al., (1985) Science 230:1242; Cotton et al., (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al., (1992) Methods Enzymol. 217:286- 295).

In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so caUed 'DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in 7677 cDNAs obtained from samples of ceUs. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the ymidine DNA glycosylase from HeLa ceUs cleaves T at G/T mismatches (Hsu et al., (1994) Carcinogenesis 15:1657-1662; U.S. Patent No. 5,459,039).

In other embodiments, alterations in electrophoretic mobiUty will be used to identify mutations in 7677 genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobiUty between mutant and wUd type nucleic acids (Orita et al., (1989) Proc. Natl. Acad. Sci. USA: 86:2766, see also Cotton, (l993)Mutat. Res. 285:125-144; and Hayashi, (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control 7677 nucleic acids wiU be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration¹ in electrophoretic mobiUty 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 a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobiUty (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) (Myers et al., (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high- melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobiUty of control and sample DNA (Rosenbaum andReissner, (1987) Biophys. Chem. 265:12753). Examples of other techniques for detecting point mutations include, but are not

Umited to, selective oUgonucleotide hybridization, selective ampUfication, or selective primer extension (Saiki et al., (1986) Nature 324:163); Saiki et al., (1989) Proc. Natl. Acad. Sci. USA 86:6230).

Alternatively, aUele specific ampUfication technology which depends on selective PCR amplification may be used in conjunction with the instant invention. OUgonucleotides used as primers for specific ampUfication may carry the mutation of interest in the center of the molecule (so that ampUfication depends on differential hybridization) (Gibbs et al., (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner, (1993) Tibtech 1_1 :238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al., (1992) Mol. Cell Probes 6: 1). It is anticipated that in certain embodiments amplification may also be performed using Taq Ugase for ampUfication (Barany, (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, Ugation wiU occur only if there is a perfect match at the 3' end 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 ampUfication. The methods described herein may be performed, for example, by utilizing prepackaged 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 7677 gene.

Use of 7677 _'Molecules as Surrogate Markers

The 7677 molecules of the invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drag activity, or as markers of the pharmacogenomic profile of a subject. Using the methods described herein, the presence, absence and/or quantity of the 7677 molecules of the invention may be detected, and may be correlated with one or more biological states in vivo. For example, the 7677 molecules of the invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states. As used herein, a "surrogate marker" is an objective biochemical marker which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such markers is independent of the disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder. Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentiaUy dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker, and an analysis of HJN infection may be made using HIV RΝA levels as a surrogate marker, weU

< in advance of the undesirable clinical outcomes of myocardial infarction or fully-developed

AIDS). Examples of the use of surrogate markers in the art include: Koomen et al. (2000) J. Mass. Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

The 7677 molecules of the invention are also useful as pharmacodynamic markers. As used herein, a "pharmacodynamic marker" is an objective biochemical marker which correlates specifically with drag effects. The presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity of the marker is indicative of the presence or activity of the drag in a subject. For example, a pharmacodynamic marker may be , indicative of the concentration of the drag in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level of the drag, in this fashion, the distribution or uptake of the drug may be monitored by the pharmacodynamic marker. Similarly, the presence or quantity of the pharmacodynamic marker may be related to the presence or quantity of the metaboUc product of a drug, such that the presence or quantity of the marker is indicative of the relative breakdown rate of the drug in vivo. Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drag effects, particularly when the drag is administered in low doses.. Since even a smaU amount of a drug may be sufficient to activate multiple rounds of marker (e.g., a 7677 marker) transcription or expression, the ampUfied marker may be in a quantity which is more readily detectable than the drag itself. Also, the marker may be more easily detected due to the nature of the marker itself; for example, using the methods described herein, anti-7677 antibodies may be employed in an immune-based detection system for a 7677 protein marker, or 7677-specific radiolabeled probes may be used to detect a 7677 mRΝA marker. Furthermore, the use of a pharmacodynamic marker may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples of the use of pharmacodynamic markers in the art include: Matsuda et al. US 6,033,862; Hattis et al. ^' (1991) Env. Health Perspect. 90: 229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; andΝicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl.3: S16-S20. The 7677 molecules of the invention are also useful as pharmacogenomic markers. As used herein, a "pharmacogenomic marker" is an objective biochemical marker which correlates with a specific clinical drug response or susceptibUity in a subject (see, e.g., McLeod et al (1999) Eur. J. Cancer 35(12): 1650-1652). The presence or quantity of the pharmacogenomic marker is related to the predicted response of the subject to a specific drug or class of drugs prior to administration of the drug. By assessing the presence or quantity of one or more pharmacogenomic markers in a subject, a drag therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA, or protein (e.g., 7677 protein or RNA) for specific tumor markers in a subject, a drag or course of treatment may be selected that is optimized for the treatment of the specific tumor likely to be present in the subject. Similarly, the presence or absence of a specific sequence mutation in 7677 DNA may correlate 7677 drug response. The use of pharmacogenomic markers therefore permits the apphcation of the most appropriate treatment for each subject without having to adrriinister the therapy.

Pharmaceutical Compositions

The nucleic acid and polypeptides, fragments thereof, as weU as anti-7677 antibodies (also referred to herein as "active compounds") of the invention can be incorporated into pharmaceutical compositions. Such compositions typicaUy include the nucleic acid molecule, protein, or antibody and a pharmaceuticaUy acceptable carrier. As used herein the language "pharmaceuticaUy acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteraL e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal adm istration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous appUcation can include the foUowing 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; buffers such as acetates, citrates or phosphates and agents for the adjustment of tomcity such as sodium chloride or dextrose. 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 aclministration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™

(BASF, Parsippany, NJ) or phosphate buffered saline (PBS), hi aU cases, the composition must be sterile and should be fluid to the extent that easy syringabmty exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contan inating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and Uquid polyetheylene 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 wiU be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitoL, sorbitol, sodium chloride in the composition. 3?rolonged 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 in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, foUowed by filtered sterilization. GeneraUy, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.

In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which 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. 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, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, piUs, capsules, troches and the like can contain any of the foUowing ingredients, or compounds of a similar nature: a binder such as microcrystaUine cellulose, gum fragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a gtidant such as coUoidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl saUcylate, or orange flavoring. For ac iinistration by inhalation, the compounds are dehvered 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 adrninistration, penefrants appropriate to the barrier to be permeated are used in the formulation. Such penefrants are generaUy known in the art, and include, for example, for transmucosal adrninistration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accompUshed through the use of nasal sprays or suppositories. For transdermal aciministration, 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 deUvery.

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

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of aclrrήnistration and uniformity of dosage. Dosage unit form as used herein refers to physicaUy discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in ceU cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeuticaUy effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. WhUe compounds that exhibit toxic side effects may be used, care should be taken to design a deUvery system that targets such compounds to the site of affected tissue in order to rninimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the ceU culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds Ues preferably within a range of circulating concentrations that include the ED₅₀ with Uttle or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of admmistration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initiaUy from ceU culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in ceU culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance Uquid chromatography. As defined herein, a therapeuticaUy effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg kg, 3 to 8 mg kg, 4 to 7 mg/kg, or 5 to 6 mg kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not Umited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeuticaUy effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

For antibodies, the preferred dosage is 0.1 mg/kg of body weight (generaUy 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mgkg to 100 mg/kg is usually appropriate. Generally, partiaUy human antibodies and fuUy human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent adrninistration is often possible. Modifications such as lipidation can be used to stabiUze antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for Upidation of antibodies is described by Cruikshank et al., ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

The present invention encompasses agents which modulate expression or activity. An agent may, for example, be a smaU molecule. For example, such smaU molecules include, but are not Umited to, peptides, peptidonώnetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e,. including heteroorganic and organometalhc compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

Exemplary doses include miUigram or microgram amounts of the smaU molecule per kilogram of subject or sample weight (e.g., about lmicrogram per kilogram to about 500 rniUigrams per kilogram, about 100 micrograms per kuogram to about 5 milligrams per kilogram, or about lmicrogram per kuogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the smaU molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drag combination, and the degree of expression or activity to be modulated.

An antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to ceUs. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colcbicin, doxorubicin, daunorabicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, Udocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetaboUtes (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., medύoretharriine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracycUnes (e.g., daunorabicin (formerly daunomycin) and doxorubicin), antibiotics

(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin

(AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be constraed as Umited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, .alpha. - interferon, .beta.-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ('TL-1"), interleukin-2 ("JL-2"), interleukin-6 ("IL-6"), granulocyte macrophase colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.

The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be dehvered to a subject by, for example, intravenous injection, local adrninistration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al., (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene deUvery vehicle is imbedded. Altematively, where the complete gene deUvery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene deUvery system.

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

Methods of Treatment:

The present 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 or unwanted 7677 expression or activity. With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specificaUy tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

"Treatment", as used herein, is defined as the apphcation or acϊrrώustration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or ceU tine from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, aUeviate, reUeve, alter, remedy, ameUorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not Umited to, smaU molecules, peptides, antibodies, ribozymes and antisense OUgonucleotides. "Pharmacogenomics", as used herein, refers to the apphcation of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specificaUy, the term refers the study of how a patient's genes determine his or her response to a drag (e.g., a patient's "drag response phenotype", or "drag response genotype".) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the 7677 molecules of the present invention or 7677 modulators according to that individual's drag response genotype. Pharmacogenomics aUows a clinician or physician to target prophylactic or therapeutic treatments to patients who wiU most benefit from the treatment and to avoid treatment of patients who wiU experience toxic drag-related side effects.

In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted 7677 expression or activity, by administering to the subject a 7677 or an agent which modulates 7677 expression or at least one 7677 activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted 7677 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 7677 aberrance, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of 7677 aberrance, for example, a 7677, 7677 agonist or 7677 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

It is possible that some 7677 disorders can be caused, at least in part, by an abnormal level of gene product, or by the presence of a gene product exhibiting abnormal activity. As such, the reduction in the level and/or activity of such gene products would bring about the ameUoration of disorder symptoms. As discussed, successful treatment of 7677 disorders can be brought about by techniques that serve to inhibit the expression or activity of target gene products. For example, compounds, e.g., an agent identified using an assays described above, that proves to exhibit negative modulatory activity, can be used in accordance with the invention to prevent and/or ametiorate symptoms of 7677 disorders. Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (mcluding, for example, polyclonal monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab')₂ and FAb expression Ubrary fragments, scFV molecules, and epitope-bmding fragments thereof). Further, antisense and ribozyme molecules that inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. StiU further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above. It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene aUeles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into ceUs via gene therapy method. Alternatively, in instances in that the target gene encodes an extraceUular protein, it can be preferable to co-administer normal target gene protein into the ceU or tissue in order to maintain the requisite level of ceUular or tissue target gene activity. Another method by which nucleic acid molecules may be utilized in treating or preventing a disease characterized by 7677 expression is through the use of aptamer molecules specific for 7677 protein. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specificaUy bind to protein figands (see, e.g., Osbome, et al., Curr. Opin. Chem. Biol. 1997, 1(1): 5-9; and Patel, D.J., Curr. Opin. Chem. Biol. 1997 Jun;I(l):32-46). Since nucleic acid molecules may in many cases be more conveniently introduced into target ceUs than therapeutic protein molecules may be, aptamers offer a method by which 7677 protein activity may be specifically decreased without the introduction of drags or other molecules which may have pluripotent effects.

Antibodies can be generated that are both specific for target gene product and that reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of 7677 disorders. For a description of antibodies, see the Antibody section above.

In circumstances wherein injection of an animal or a human subject with a 7677 protein or epitope for stimulating antibody production is harmful to the subject, it is possible to generate an immune response against 7677 through the use of anti-idiotypic antibodies (see, for example, Herlyn, D., Ann. Med. 1999;31(l):66-78; and Bhattacharya- Chatterjee, M., and Foon, K.A., Cancer Treat. Res. 1998;94:51-68). If an anti-idiotypic antibody is introduced into a mammal or human subject, it should stimulate the production of anti-anu-idiotypic antibodies, which should be specific to the 7677 protein. Vaccines directed to a disease characterized by 7677 expression may also be generated in this fashion.

In instances where the target antigen is intracellular and whole antibodies are used, internahzing antibodies may be preferred. Lipofectin or Uposomes can be used to dehver the antibody or a fragment of the Fab region that binds to the target antigen into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target antigen is preferred. For example, peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intraceUular target antigens can also be administered. Such single chain antibodies can be admmistered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see e.g., Marasco et al., (1993, Proc. Nat Acad. Sci. USA 90:7889-7893).

The identified compounds that inhibit target gene expression, synthesis and/or activity can be adininistered to a patient at therapeuticaUy effective doses to prevent, treat or ameUorate 7677 disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in ameUoration of symptoms of the disorders. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in ceU cultures or experimental animals, e.g., for deterrnining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeuticaUy effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit large therapeutic indices are preferred. WhUe compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to rmnimize potential damage to uninfected ceUs and, thereby, reduce side effects.

The data obtained from the ceU culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds Ues preferably within a range of circulating concentrations that include the ED50 with tittle or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utiUzed. For any compound used in the method of the invention, the therapeuticaUy effective dose can be estimated initially from ceU culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as deterrnined in ceU culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

Another example of determination of effective dose for an individual is the abiUty to directly assay levels of "free" and "bound" compound in the serum of the test subject. Such assays may utilize antibody mimics and/or "biosensors" that have been created through molecular imprinting techniques. The compound which is able to modulate 7677 activity is used as a template, or "imprinting molecule", to spatiaUy organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated "negative image" of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in AnseU, R. J. et al., (1996) Current Opinion in Biotechnology 7:89-94 and in Shea, K.J., (1994) Trends in Polymer Science 2 : 166- 173. Such "imprinted" affinity matrixes are amenable to tigand- binding assays, whereby the immobiUzed monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrixes in this way can be seen in Vlatakis, G. et al., (1993) Nature 361 :645-647. Through the use of isotope- labeling, the "free" concentration of compound which modulates the expression or activity of 7677 can be readily monitored and used in calculations of IC₅0.

Such "imprinted" affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual ICso- A radimentary example of such a "biosensor" is discussed in Kriz, D. et al., (1995) Analytical Chemistry 67:2142-2144.

Another aspect of the invention pertains to methods of modulating 7677 expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell with a 7677 or agent that modulates one or more of the activities of 7677 protein activity associated with the cell: An agent that modulates 7677 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturaUy-occurring target molecule of a 7677 protein (e.g., a 7677 substrate or receptor), a 7677 antibody, a 7677 agonist or antagonist, a peptidomimetic of a 7677 agonist or antagonist, or other smaU molecule.

In one embodiment, the agent stimulates one or 7677 activities. Examples of such stimulatory agents include active 7677 protein and a nucleic acid molecule encoding 7677. In another embodiment, the agent inhibits one or more 7677 activities. Examples of such inhibitory agents include antisense 7677 nucleic acid molecules, anti-7677 antibodies, and 7677 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the ceU with the agent) or, alternatively, in vivo (e.g., by adrninistering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a 7677 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., upregulates or downregulates) 7677 expression or activity. In another embodiment, the method involves administering a 7677 protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted 7677 expression or activity.

Stimulation of 7677 activity is desirable in situations in which 7677 is abnormaUy downregulated and/or in which increased 7677 activity is likely to have a beneficial effect. For example, stimulation of 7677 activity is desirable in situations in which a 7677 is downregulated and/or in which increased 7677 activity is likely to have a beneficial effect. Likewise, inhibition of 7677 activity is desirable in situations in which 7677 is abnormaUy upregulated and/or in which decreased 7677 activity is likely to have a beneficial effect. The 7677 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of ceUular proUferative and/or differentiative disorders, cardiovascular disorders, as described above, as weU as disorders associated with bone metabolism, hematopoietic disorders, liver disorders, viral diseases, pain or metaboUc disorders. Aberrant expression and/or activity of 7677 molecules may mediate disorders associated with bone metaboUsm. "Bone metaboUsm" refers to direct or indirect effects in the formation or degeneration of bone structures, e.g., bone formation, bone resorption, etc., which may ultimately affect the concentrations in serum of calcium and phosphate. This term also includes activities mediated by 7677 molecules effects in bone cells, e.g. osteoclasts and osteoblasts, that may in turn result in bone formation and degeneration. For example, 7677 molecules may support different activities of bone resorbing osteoclasts such as the stimulation of differentiation of monocytes and mono-nuclear phagocytes into osteoclasts. Accordingly, 7677 molecules that modulate the production of bone ceUs can influence bone formation and degeneration, and thus may be used to treat bone disorders. Examples of such disorders include, but are not Umited to, osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis, anti- convulsant treatment, osteopenia, fibrogenesis-imperfecta ossium, secondary hyperparathyrodism, hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructive jaundice, drag induced metaboUsm, meduUary carcinoma, chronic renal disease, rickets,

keratoconjunctivitis, ulcerative colitis, asthma, aUergic asthma, cutaneous lupus erythematosus, scleroderma, vagimtis, proctitis, drag eraptions,leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyeUtis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red ceU anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens- Johnson syndrome, idiopathic sprue, Uchenplanus, Graves' disease, sarcoidosis, primary bitiary cirrhosis, uveitis posterior, and interstitial lung fibrosis), graft-versus-host disease, cases of transplantation, and allergy such as, atopic aUergy. Disorders which may be treated or diagnosed by methods described herein include, but are not Umited to, disorders associated with an accumulation in the liver of fibrous tissue, such as that resulting from an imbalance between production and degradation of the extraceUular matrix accompanied by the coUapse and condensation of preexisting fibers. The methods described herein can be used to diagnose or treat hepatoceUular necrosis or injury induced by a wide variety of agents including processes which disturb homeostasis, such as an inflammatory process, tissue damage resulting from toxic injury or altered hepatic blood flow, and infections (e.g., bacterial, viral and parasitic). For example, the methods can be used for the early detection of hepatic injury, such as portal hypertension or hepatic fibrosis. In addition, the methods can be employed to detect Uver fibrosis attributed to inborn errors of metabolsim, for example, fibrosis resulting from a storage disorder such as Gaucher's disease (Upid abnormaUties) or a glycogen storage disease, Al-antitrypsin ' deficiency; a disorder mediating the accumulation (e.g., storage) of an exogenous substance, for example, hemochromatosis (iron-overload syndrome) and copper storage diseases (Wilson's disease), disorders resulting in the accumulation of a toxic metaboUte (e.g., tyrosinemia, fructosemia and galactosemia) and peroxisomal disorders (e.g.,

ZeUweger syndrome). AdditionaUy, the methods described herein may be useful for the early detection and treatment of Uver injury associated with the administration of various chemicals or drugs, such as for example, methotrexate, isonizaid, oxyphenisatin, methyldopa, chlorpromazine, tolbutamide or alcohol, or which represents a hepatic manifestation of a vascular disorder such as obstruction of either the intrahepatic or extrahepatic bile flow or an alteration in hepatic circulation resulting, for example, from chronic heart failure, veno-occlusive disease, portal vein thrombosis or Budd-Chiari syndrome.

AdditionaUy, 7677 molecules may play an important role in the etiology of certain viral diseases, including but not limited to, Hepatitis B, Hepatitis C and Herpes Simplex Virus (HSV). Modulators of 7677 activity could be used to control viral diseases. The modulators can be used in the treatment and/or diagnosis of viral infected tissue or virus- associated tissue fibrosis, especially Uver and Uver fibrosis. Also, 7677 modulators can be used in the treatment and/or diagnosis of virus-associated carcinoma, especially hepatoceUular cancer. AdditionaUy, 7677 may play an important role in the regulation of metaboUsm or pain disorders. Diseases of metaboUc imbalance include, but are not Umited to, obesity, anorexia nervosa, cachexia, lipid disorders, and diabetes. Examples of pain disorders include, but are not limited to, pain response ehcited during various forms of tissue injury, e.g., inflammation, infection, and ischemia, usually referred to as hyperalgesia (described in, for example, Fields, H.L., (1987) Pain, New York:McGraw-Hill); pain associated with muscoloskeletal disorders, e.g., joint pain; tooth pain; headaches; pain associated with surgery; pain related to irritable bowel syndrome; or chest pain.

Pharmacogenomics The 7677 molecules of the present invention, as weU as agents, or modulators which have a stimulatory or inhibitory effect on 7677 activity (e.g., 7677 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylacticaUy or therapeuticaUy) 7677 associated disorders (e.g., ceUular growth related disorders) associated with aberrant or unwanted 7677 activity, hi conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metaboUsm of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologjcaUy active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a 7677 molecule or 7677 modulator as weU as tailoring the dosage and/or therapeutic regimen of treatment with a 7677 molecule or 7677 modulator. Pharmacogenomics deals with cUnicaUy significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11) :983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):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 drag action) or genetic conditions transmitted as single factors altering the way the body acts on drags (altered drag metaboUsm). These pharmacogenetic conditions can occur either as rare genetic defects or as naturaUy-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical compUcation is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drug response, known as "a genome-wide association", rehes primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a "bi-alleUc" gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase H/JJI drag trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high-resolution map can be generated from a combination of some ten mUlion known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tanored to groups of geneticaUy sinήlar individuals, taking into account traits that may be common among such geneticaUy similar individuals. Alternatively, a method termed the "candidate gene approach", can be utilized to identify genes that predict drag response. According to this method, if a gene that encodes a drug's target is known (e.g., a 7677 protein of the present invention), aU common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drag response.

Alternatively, a method termed the "gene expression profiling", can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a 7677 molecule or 7677 modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomics approaches can be used to determine' appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual. 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 7677 molecule or 7677 modulator, such as a modulator identified by one of the exemplary screening assays described herein.

The present invention further provides methods for identifying new agents, or combinations, that are based on identifying agents that modulate the activity of one or more of the gene products encoded by one or more of the 7677 genes of the present invention, wherein these products may be associated with resistance of the ceUs to a therapeutic agent. SpecificaUy, the activity of the proteins encoded by the 7677 genes of the present invention can be used as a basis for identifying agents for overcoming agent resistance. By blocking the activity of one or more of the resistance proteins, target ceUs, e.g., cancer cells, wiU become sensitive to treatment with an agent that the unmodified target ceUs were resistant to.

Monitoring the influence of agents (e.g., drags) on the expression or activity of a 7677 protein can be appUed in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase 7677 gene expression, protein levels, or upregulate 7677 activity, can be monitored in clinical trials of subjects exhibiting decreased 7677 gene expression, protein levels, or downregulated 7677 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease 7677 gene expression, protein levels, or downregulate 7677 activity, can be monitored in clinical trials of subjects exhibiting increased 7677 gene expression, protein levels, or upregulated 7677 activity. In such clinical trials, the expression or activity of a 7677 gene, and preferably, other genes that have been implicated in, for example, a 7677-associated disorder can be used as a "read out" or markers of the phenotype of a particular cell.

Other Embodiments In another aspect, the invention features, a method of analyzing a pluraUty of capture probes. The method can be used, e.g., to analyze gene expression. The method includes: providing a two dimensional array having a pluraUty of addresses, each address of the plurality being positionaUy distinguishable from each other address of the plurality, and each address of the pluraUty having a unique capture probe, e.g., a nucleic acid or peptide sequence; contacting the array with a 7677, preferably purified, nucleic acid, preferably purified, polypeptide, preferably purified, or antibody, and thereby evaluating the pluraUty of capture probes. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the pluraUty, is detected, e.g., by signal generated from a label attached to the 7677 nucleic acid, polypeptide, or antibody. The capture probes can be a set of nucleic acids from a selected sample, e.g., a sample of nucleic acids derived from a control or non-stimulated tissue or ceU.

The method can include contacting the 7677 nucleic acid, polypeptide, or antibody with a first array having a pluraUty of capture probes and a second array having a different pluraUty of capture probes. The results of each hybridization can be compared, e.g., to analyze differences in expression between a first and second sample. The first pluraUty of capture probes can be from a control sample, e.g., a wild type, normal, or non-diseased, non-stimulated, sample, e.g., a biological fluid, tissue, or ceU sample. The second pluraUty of capture probes can be from an experimental sample, e.g., a mutant type, at risk, disease- state or disorder-state, or stimulated, sample, e.g., a biological fluid, tissue, or ceU sample. The pluraUty of capture probes can be a pluraUty of nucleic acid probes each of which specifically hybridizes, with an aUele of 7677. Such methods can be used to diagnose a subject, e.g., to evaluate risk for a disease or disorder, to evaluate suitabiUty of a selected treatment for a subject, to evaluate whether a subject has a disease or disorder. 7677 is associated with ATPase activity, thus it is useful for disorders associated with abnormal Upid metaboUsm.

The method can be used to detect SNPs, as described above. In another aspect, the invention features, a method of analyzing a plurahty of probes. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a pluraUty of addresses, each address of the pluraUty being positionaUy distinguishable from each other address of the pluraUty having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which express or mis express 7677 or from a cell or subject in which a 7677 mediated response has been eUcited, e.g., by contact of the ceU with 7677 nucleic acid or protein, or adrninistration to the ceU or subject 7677 nucleic acid or protein; contacting the array with one or more inquiry probe, wherein an inquiry probe can be a nucleic acid, polypeptide, or antibody (which is preferably other than 7677 nucleic acid, polypeptide, or antibody); providing a two dimensional array having a pluraUty of addresses, each address of the pluraUty being positionaUy distinguishable from each other address of the pluraUty, and each address of the pluraUty having a unique capture probe, e.g., wherein the capture probes are from a ceU or subject which does not express 7677 (or does not express as highly as in the case of the 7677 positive pluraUty of capture probes) or from a ceU or subject which in which a 7677 mediated response has not been eUcited (or has been eUcited to a lesser extent than in the first sample); contacting the array with one or more inquiry probes (which is preferably other than a 7677 nucleic acid, polypeptide, or antibody), and thereby evaluating the plurahty of capture probes. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the pluraUty, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody. In another aspect, the invention features, a method of analyzing 7677, e.g., analyzing structure, function, or relatedness to other nucleic acid or amino acid sequences. The method includes: providing a 7677 nucleic acid or arnino acid sequence; comparing the 7677 sequence with one or more preferably a pluraUty of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database; to thereby analyze 7677.

Preferred databases include GenBank™. The method can include evaluating the sequence identity between a 7677 sequence and a database sequence. The method can be performed by accessing the database at a second site, e.g., over the internet. In another aspect, the invention features, a set of oligonucleotides, useful, e.g., for identifying SNP's, or identifying specific aUeles of 7677. The set includes a pluraUty of

OUgonucleotides, each of which has a different nucleotide at an interrogation position, e.g., an SNP or the site of a mutation. In a preferred embodiment, the OUgonucleotides of the plurahty identical in sequence with one another (except for differences in length). The OUgonucleotides can be provided with different labels, such that an OUgonucleotides which hybridizes to one allele provides a signal that is distinguishable from an OUgonucleotides which hybridizes to a second allele.

This invention is further iUustrated by the following examples which should not be constraed as limiting. The contents of aU references, patents and pubUshed patent appUcations cited throughout this apphcation are incorporated herein by reference.

EXAMPLES

Example 1: Identification and Characterization of Human 7677 cDNAs

The human 7677 sequence (Figure 1A-C; SEQ ID NO:l), which is approximately 2745 nucleotides long including untranslated regions, contains a predicted methionine- initiated coding sequence of about 1995 nucleotides (nucleotides 270-2369 of SEQ ID NO:l; nucleotides 1-1995 of SEQ ID NO:3). The coding sequence encodes a 665 amino acid protein (SEQ ID NO:2).

Example 2: Expression and Tissue Distribution of 7677 mRNA Northern blot hybridizations with various RNA samples can be performed under standard conditions and washed under stringent conditions, i.e., 0.2xSSC at 65°C. A DNA probe corresponding to all or a portion of the 7677 cDNA (SEQ ID NO:l) can be used. The DNA was radioactively labeled with ³ P-dCTP using the Prime-It Kit (Stratagene, La JoUa, CA) according to the instructions of the supplier. Filters containing mRNA from mouse hematopoietic and endocrine tissues, and cancer ceU lines (Clontech, Palo Alto, CA) can be probed in ExpressHyb hybridization solution (Clontech) and washed at high stringency according to manufacturer's recommendations. TaqMan real-time quantitative RT-PCR was used to detect the presence of RNA transcript corresponding to human 7677 in several tissues. It was found that the corresponding orthologs of 7677 are expressed in a variety of tissues. The results of this screening are shown in Figures 3-8. Reverse Transcriptase PCR (RT-PCR) was used to detect the presence of RNA transcript corresponding to human 7677 in RNA prepared from tumor and normal tissues. Variable expression was found in aU xenograph friendly ceU lines tested as shown in Figure 3. Figure 4 illustrates the relative expression levels and tissue distribution of the 7677 gene in various tissues using Taq Man PCR. Both Taq Man as well as HMPGv2 array results show that the cell cycle was regulated in GO/Gl phase of synchronized ceUs of the human breast epitheUal ceU line, MCF-10A as shown in Figure 5.

Relative expression levels of the 7677 was assessed in colon and Uver cells using TaqMan PCR and increased expression was found in 4/6 colon tumor ceU lines in comparison to 3/4 normal colon tissue control; and 3/6 lung metastases in comparison to normal lung tissue control. The results of this comparison are shown in Figures 6-7.

Expression profiling results using in situ hybridization techniques have shown that 7677 mRNA has been detected in human colon, lung, and breast tumors. Moderate to strong expression (5/6) in lung adenocarcinomas and squamous ceU carcinomas in comparison to Uttle or no expression (1/3) found in normal lung tissue. AU colon tumors (4/4) demonstrated expression which ranged from low to high levels of intensity in comparison to Uttle or no expression (1/3) found in normal colon tissue. Both breast normal (2/2) and tumor (4/4) were weakly positive for expression of which one tumor exhibited very strong hybridization. As seen by these results, 7677 molecules have been found to be overexpressed in some tumor ceUs, where the molecules may be inappropriately propagating either ceU proUferation or ceU survival signals. As such, 7677 molecules may serve as specific and novel identifiers of such tumor cells. Further, inhibitors of the 7677 molecules are also useful for the treatment of cancer, preferably lung cancer, and useful as a diagnostic. Additional results have shown 7677 expression in an oncology plate π as foUows in

Table 1:

Table 1

Average Average Relative 7677 Beta 2 Expression

Colon N 31.8 22.5 1.6

Colon N 29.4 19.0 0.7 Colon N 26.4 18.4 3.7

Colon N 27.0 16.7 0.8

Colon T 24.0 16.1 4.2

Colon T 22.2 17.6 40.4

Colon T 24.1 16.0 3.6

Colon T 25.0 17.1 4.0

Colon T 27.0 17.4 1.3

Colon T 25.9 16.6 1.6

Colon T 25.0 17.9 7.3

Colon T 23.9 16.3 4.9

Liver Met 24.2 17.3 8.9

Liver Met 25.9 19.2 9.6

Liver Met 24.8 18.2 10.1

Liver Met 26.4 19.5 8.8

Liver Nor 25.8 16.4 1.5

Liver Nor 30.2 23.0 6.4

Brain N 25.4 20.0 22.4

Brain N 24.7 20.3 46.4

Astrocyt 26.1 20.4 19.2

Brain T 29.6 19.3 0.8

Brain T 24.8 16.1 2.3

Brain T 25.6 16.9 2.6

Brain T 29.1 , 19.0 0.9

HMVEC-Arr 23.0 16.1 8.5

HMVEC-Prol 22.8 16.8 16.0

Placenta 29.2 22.6 10.7

Fetal Adrenal 29.8 23.8 15.6

Fetal Adrenal 1 30.4 22.9 5.6

Fetal Liver 25.2 19.5 19.6

Fetal Liver 24.8 18.4 11.8

As seen by these results, 7677 molecules have been found to be overexpressed in some tumor cells (e.g. 2/8 colon tumor cells; 4/4/ Uver metastases), where the molecules may be inappropriately propagating either ceU proUferation or ceU survival signals. As such, 7677 molecules may serve as specific and novel identifiers of such tumor cells. Further, inhibitors of the 7677 molecules are also useful for the treatment of cancer, preferably colon cancer and Uver metastases, and useful as a diagnostic.

The 7677 molecules also have been found to be underexpressed in some tumor ceUs (e.g. 4/4 brain tumor ceUs), where the molecules may be inappropriately propagating either ceU proUferation or ceU survival signals. As such, 7677 molecules may likewise serve as specific and novel identifiers of such tumor ceUs. Further, activators of the 7677 molecules are also useful for the treatment of cancer, preferably brain cancer, and useful as a diagnostic.

In addition, the following data in Table 2 iUustrates expression of 7677 in a mouse angiogenic plate.

Table 2

Average Average Relative m7677.2 Beta 2 Expression

Islets Hyper/Norm 27.76 21.05 9.59

Islets Angiogenic 26.01 19.86 14.03

Islets Tumor 26.65 20.23 11.72

Xeno/tumor parental #1 27.54 18.29 1.64 Xeno/tumor parental #2 32.22 17.44 0.04

Xeno/tumor VEGF #1 32.18 18.03 0.05

Xeno/tumor VEGF #2 28.98 17.59 0.37

Spleen Normal 23.14 17.68 22.72

Liver Normal 24.25 17.12 7.16 Heart Normal 23.18 14.31 2.14

I idney Normal 24.91 16.06 2.17

Brain Normal 21.70 15.29 11.76

Colon Normal 25.29 17.69 5.14 Example 3: Recombinant Expression of 7677 in Bacterial CeUs

In this example, 7677 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. SpecificaUy, 7677 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression of the GST-7677 fusion protein in PEB199 is induced with 3P TG. The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.

Example 4: Expression of Recombinant 7677 Protein in COS CeUs

To express the 7677 gene in COS ceUs, the pcDNA/Amp vector by Invitrogen Corporation (San Diego, CA) is used. This vector contains an SV40 origin of replication, an ampicilUn resistance gene, an E. coli replication origin, a CMV promoter followed by a polyUnker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire 7677 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3' end of the fragment is cloned into the polyUnker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter. ■ To construct the plasmid, the 7677 DNA sequence is ampUfied by PCR using two primers. The 5' primer contains the restriction site of interest followed by approximately twenty nucleotides of the 7677 coding sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the 7677 coding sequence. The PCR ampUfied fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, MA). Preferably the two restriction sites chosen are different so that the 7677 gene is inserted in the correct orientation. The Ugation mixture is transformed into E. coli ceUs (strains HB101, DH5α, SURE, available from Stratagene Cloning Systems, La JoUa, CA, can be used), the transformed culture is plated on ampicilUn media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.

COS cells are subsequently transfected with the 7677-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran- mediated transfection, Upofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T.

Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. The expression of the 7677 polypeptide is detected by radiolabelUng (³⁵S-methionine or ³⁵S-cysteine available from NEN, Boston, MA, can be used) and immunoprecipitation (Harlow, E. and

Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold

Spring Harbor, NY, 1988) using an HA specific monoclonal antibody. Briefly, the ceUs are labeled for 8 hours with ³⁵S-methionine (or ³⁵S-cysteine). The culture media are then coUected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the ceU lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the 7677 coding sequence is cloned directly into the polyUnker of the pCDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS ceUs in the manner described above, and the expression of the 7677 polypeptide is detected by radiolabelUng and immunoprecipitation using a 7677 specific monoclonal antibody.

Equivalents Those skilled in the art wiU recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. An isolated 7677 nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60% identical to the nucleotide sequence of SEQ 3D NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number

b) a nucleic acid molecule comprising a fragment of at least 300 nucleotides of the nucleotide sequence of SEQ TD NO: 1 , SEQ ID NO:3, or the nucleotide sequence of the

DNA insert of the plasmid deposited with ATCC as Accession Number ; c) a nucleic acid molecule which encodes a polypeptide comprising the arnino acid sequence of SEQ ID NO:2, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number ; d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or the arnino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number , wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:2, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number ; e) a nucleic acid molecule which encodes a naturally occurring aUeUc variant of a polypeptide comprising the arnino acid sequence of SEQ 3D NO:2, or the arnino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number , wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:l, SEQ 3D NO:3, or a complement thereof, under stringent conditions; f) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ; and g) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number . ,

2. The isolated nucleic acid molecule of claim 1 , which is the nucleotide sequence SEQ 3D NO: 1.

3. A host ceU which contains the nucleic acid molecule of claim 1.

4. An isolated 7677 polypeptide selected from the group consisting of: a) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 60% identical to a nucleic acid comprising the nucleotide sequence of SEQ 3D NO:l, SEQ TD NO:3, or the nucleotide sequence of the

DNA insert of the plasmid deposited with ATCC as Accession Number , or a complement thereof; b) a naturally occurring aUeUc variant of a polypeptide comprising the amino acid sequence of SEQ 3D NO:2, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number , wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ 3D NO:l, SEQ 3D NO:3, or a complement thereof under stringent conditions; c) a fragment of a polypeptide comprising the amino acid sequence of SEQ 3D NO:2, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number , wherein the fragment comprises at least 15 contiguous amino acids of SEQ 3D NO:2; and d) the amino acid sequence of SEQ ID NO:2.

5. An antibody which selectively binds to a polypeptide of claim 4.

6. A method for producing a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ 3D NO:2, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number ; b) a polypeptide comprising a fragment of the amino acid sequence of SEQ ID

NO:2, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number , wherein the fragment comprises at least 15 contiguous amino acids of SEQ 3D NO:2, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number ; c) a naturaUy occurring aUetic variant of a polypeptide comprising the arnino acid sequence of SEQ 3D NO:2, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number , wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:l, SEQ ID NO:3; and d) the amino acid sequence of SEQ 3D NO:2; comprising culturing the host cell of claim 3 under conditions in which the nucleic acid molecule is expressed.

7. A method for detecting the presence of a nucleic acid molecule of claim 1 or a polypeptide encoded by the nucleic acid molecule in a sample, comprising: a) contacting the sample with a compound which selectively hybridizes to the nucleic acid molecule of claim 1 or binds to the polypeptide encoded by the nucleic acid molecule; and b) determining whether the compound hybridizes to the nucleic acid or binds to the polypeptide in the sample.

8. A kit comprising a compound which selectively hybindizes to a nucleic acid molecule of claim 1 or binds to a polypeptide encoded by the nucleic acid molecule and instructions for use.

9. A method for identifying a compound which binds to a polypeptide or modulates the activity of the polypeptide of claim 4 comprising the steps of: a) contacting a polypeptide, or a ceU expressing a polypeptide of claim 4 with a test compound; and b) determining whether the polypeptide binds to the test compound or determining the effect of the test compound on the activity of the polypeptide.

10. A method for modulating the activity of a polypeptide of claim 4 comprising contacting the polypeptide or a ceU expressing the polypeptide with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide.

11. A method of identifying a nucleic acid molecule associated with cancer comprising: a) contacting a sample from a subject with or at risk of developing cancer comprising nucleic acid molecules with a hybridization probe comprising at least 25 contiguous nucleotides of SEQ ID NO: 1 defined in claim 2; and

b) detecting the presence of a nucleic acid molecule in the sample that hybridizes to the probe, thereby identifying a nucleic acid molecule associated with cancer.

12. A method of identifying a nucleic acid associated with cancer comprising: a) contacting a sample from a subj ect having cancer or at risk of developing cancer comprising nucleic acid molecules with a first and a second ampUfication primer, the first primer comprising at least 25 contiguous nucleotides of SEQ ID NO:l defined in claim 2 and the second primer comprising at least 25 contiguous nucleotides from the complement ofSEQ TD NO:l; '

b) incubating the sample under conditions that aUow nucleic acid ampUfication; and

c) detecting the presence of a nucleic acid molecule in the sample that is ampUfied, thereby identifying the nucleic acid molecule associated with cancer.

13. A method of identifying a polypeptide associated with cancer comprising: a) contacting a sample comprising polypeptides with a 7677 binding partner of the

7677 polypeptide defined in claim 4; and

b) detecting the presence of a polypeptide in the sample that binds to the 7677 bmding partner, thereby identifying the polypeptide associated with cancer.

14. A method of identifying a subject having cancer or at risk for developing cancer comprising: a) contacting a sample obtained from the subject comprising nucleic acid molecules with a hybridization probe comprising at least 25 contiguous nucleotides of SEQ ID NO:l defined in claim 2; and

b) detecting the presence of a nucleic acid molecule in the sample that hybridizes to the probe, thereby identifying a subject having cancer or at risk for developing cancer.

15. A method of identifying a subj ect having cancer or at risk for developing cancer comprising: a) contacting a sample obtained from the subject comprising nucleic acid molecules with a first and a second ampUfication primer, the first primer comprising at least 25 contiguous nucleotides of SEQ ID NO:l defined in claim 2 and the second primer comprising at least 25 contiguous nucleotides from the complement of SEQ ID NO: 1 ;

c) detecting the presence of a nucleic acid molecule in the sample that is ampUfied, thereby identifying a subject having cancer or at risk for developing cancer.

16. A method of identifying a subj ect having cancer or at risk for developing cancer comprising: a) contacting a sample obtained from the subject comprising polypeptides with a 7677 binding partner of the 7677 polypeptide defined in claim 4; and

b) detecting the presence of a polypeptide in the sample that binds to the 7677 binding partner, thereby identifying a subject having cancer or at risk for developing cancer.

17. A method for identifying a compound capable of treating cancer or modulating ceUular proUferation and/or differentiation characterized by aberrant 7677 nucleic acid expression or 7677 polypeptide activity comprising assaying the abihty of the compound to modulate 7677 nucleic acid expression or 7677 polypeptide activity, thereby identifying a compound capable of treating cancer characterized by aberrant 7677 nucleic acid expression or 7677 polypeptide activity.

18. The method of claim 17, wherein the cancer or cellular proUferation and/or differentiation is lung, colon, Uver or brain cancer.

19. A method for treating a subject having a cancer or at risk of developing cancer or for modulating ceUular proUferation and or differentiation comprising administering to the subject a 7677 modulator of the nucleic acid molecule defined in claim 1 or the polypeptide encoded by the nucleic acid molecule or contacting a ceU with a 7677 modulator.

20. The method of claim 47, wherein the cancer or cellular proliferation and/or differentiation is lung, colon, Uver or brain cancer.

21. The method of claim 19, wherein the 7677 modulator is a) a smaU molecule; b) peptide; c) phosphopeptide; d) anti-7677 antibody; e) a 7677 polypeptide comprising the amino acid sequence of SEQ 3D NO:2, or a fragment thereof; f) a 7677 polypeptide comprising an amino acid sequence which is at least 90 percent identical to the amino acid sequence of SEQ ID NO:2, wherein the percent identity is calculated using the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4; or g) an isolated naturally occurring aUeUc variant of a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a complement of a nucleic acid molecule consisting of SEQ 3D NO:l at 6X SSC at 45°C, foUowed by one or more washes in 0.2X SSC, 0.1% SDS at 50-65°C.

5 22. The method of claim 19, wherein the 7677 modulator is a) an antisense 7677 nucleic acid molecule; b) is a ribozyme; c) the nucleotide sequence of SEQ 3D NO:l, or a fragment thereof; d) a nucleic acid molecule encoding a polypeptide comprising an amino acid o sequence which is at least 90 percent identical to the amino acid sequence of SEQ 3D

NO:2, wherein the percent identity is calculated using the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4; e) a nucleic acid molecule encoding a naturaUy occurring alleUc variant of a 5 polypeptide comprising the amino acid sequence of SEQ 3D NO:2, wherein the nucleic acid molecule which hybridizes to a complement of a nucleic acid molecule consisting of SEQ ID NO:l at 6X SSC at 45°C, foUowed by one or more washes in 0.2X SSC, 0.1% SDS at 50-65°C; or f) a gene therapy vector. 0

23. A method for evaluating the efficacy of a treatment of cancer or aberrant ceUular proUferation and/or differentiation, in a subject, comprising: ι treating a subject with a protocol under evaluation; assessing the expression level of a 7677 nucleic acid molecule defined in 5 claim 1 or 7677 polypeptide encoded by the 7677 nucleic acid molecule, wherein a change in the expression level of 7677 nucleic acid or 7677 polypeptide after the treatment, relative to the level before the treatment, is indicative of the efficacy of the treatment of cancer or aberrant cellular proUferation and/or differentiation.

0 24. The method of claim 23, wherein the cancer or abenant ceUular proliferation and/or differentiation is lung, Uver colon or brain cancer.

25. A method of diagnosing cancer or aberrant cellular proUferation and/or differentiation in a subject, comprising: evaluating the expression or activity of a 7677 nucleic acid molecule defined in claim 1 or a 7677 polypeptide encoded by the 7677 polypeptide, such that a difference in the level of 7677 nucleic acid or 7677 polypeptide relative to a normal subject or a cohort of normal subjects is indicative of cancer or aberrant ceUular proUferation and/or differentiation.

26. The method of claim 25, wherein the cancer or aberrant cellular proUferation and/or differentiation is lung, colon, liver or brain cancer.