US 20060053498A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0053498A1 Bejanin et al. (43) Pub. Date: Mar. 9, 2006

(54) FULL-LENGTH HUMAN CDNAS ENCODING Publication Classification POTENTIALLY SECRETED PROTEINS (51) Int. Cl. (76) Inventors: Stephane Bejanin, Rochechouart (FR); AOIK 67/00 (2006.01) Jean-Baptiste Dumas Milne Edwards, C07K I4/47 (2006.01) CI2O I/68 (2006.01) Paris (FR); Jean-Yves Giordano, Paris C07H 21/04 (2006.01) (FR); Severin Jobert, Paris (FR); CI2P 21/06 (2006.01) Hiroaki Tanaka, Antony (FR) (52) U.S. Cl...... 800/8; 435/6; 435/69.1; 435/320.1; Correspondence Address: 435/325; 530/350, 536/23.5 SALIWANCHIK LLOYD & SALWANCHIK (57) ABSTRACT A PROFESSIONAL ASSOCATION PO BOX 142950 The invention concerns GENSET polynucleotides and GAINESVILLE, FL 32614-2950 (US) polypeptides. Such GENSET products may be used as reagents in forensic analyses, as chromosome markers, as (21) Appl. No.: 10/475,075 tissue/cell/organelle-Specific markers, in the production of expression vectors. In addition, they may be used in Screen (22) PCT Filed: Apr. 18, 2001 ing and diagnosis assays for abnormal GENSET expression and/or biological activity and for Screening compounds that (86) PCT No.: PCT/IB01/00914 may be used in the treatment of GENSET-related disorders. Patent Application Publication Mar. 9, 2006 Sheet 1 of 4 US 2006/0053498A1

D 125A 125B 125C E."

1OO

COMPUTER SYSTEM

PROCESSOR

MEMORY

120 DATA RETRIEMNG DISPLAY DEVICE

Figure 1 Patent Application Publication Mar. 9, 2006 Sheet 2 of 4 US 2006/0053498A1

2O2 STORE NEW SEQUENCE TO A MEMORY

204 OPEN DATABASE OF SEQUENCES

2O6 READ FIRST SECUENCE IN DATABASE

21 O PERFORM COMPARISON OF NEW SEQUENCE AND 224 STORED SEQUENCE GO TO NEXT 212 SEQUENCE IN DATABASE

YES

MORE SEQUENCESN DATABASE?

Patent Application Publication Mar. 9, 2006 Sheet 3 of 4 US 2006/0053498A1

252 250 \N, START 254 STORE A FIRST SEQUENCE TO A MEMORY

256 STORE A SECOND SECUENCE TO A MEMORY

, 260 READ FIRST CHARACTER OF FIRST SEQUENCE

262 READ FIRST CHARACTER OF SECOND SEQUENCE

264

NO YES

MORE CHARACTERS TO READ?

NO 276

Figure 3 Patent Application Publication Mar. 9, 2006 Sheet 4 of 4 US 2006/0053498A1

30 3OO\N 304 STORE AFIRST SEQUENCE TO A MEMORY . 306

OPEN ADATABASE OF SEQUENCE FEATURES 308 READ FIRST FEATURE FROMDATABASE

r 310 COMPARE FEATURE AT TRIBUTES WITH THE FIRST SEQUENCE 326 a...... READ NEXT 316 FEATURE IN . . . . . DATABASE

YES 38

DISPLAY FOUND FEATURE TO THE USER

320

MORE FEATURES IN DATABASE US 2006/0053498A1 Mar. 9, 2006

FULL-LENGTH HUMAN CDNAS ENCODING matics Software may mischaracterize the genomic Sequences POTENTIALLY SECRETED PROTEINS obtained, i.e., labeling non-coding DNA as coding DNA and Vice versa. CROSS REFERENCE TO RELATED APPLICATIONS 0006 An alternative approach takes a more direct route to identifying and characterizing human genes. In this 0001. The present application is related to and claims priority from U.S. provisional Application Nos. 60/197.873, approach, complementary DNAS (cDNAS) are Synthesized filed Apr. 18, 2000, 60/224,009, filed Aug. 7, 2000, 60/260, from isolated messenger RNAs (mRNAs) which encode 328, filed Jan. 8, 2001, and 60/224,006, filed Aug. 4, 2000, human proteins. Using this approach, Sequencing is only the entire disclosures of each of which is herein incorporated performed on DNA which is derived from protein coding by reference. fragments of the genome. Often, only short Stretches of the cDNAS are Sequenced to obtain Sequences called expressed FIELD OF THE INVENTION sequence tags (ESTs). The ESTs may then be used to isolate or purify cDNAS which include Sequences adjacent to the 0002 The present invention is directed to GENSET polypeptides, fragments thereof, and the regulatory regions EST sequences. The cDNAS may contain all of the sequence located in the 5'- and 3'-ends of the genes encoding the of the EST which was used to obtain them or only a fragment polypeptides. The invention also concerns polypeptides of the sequence of the EST which was used to obtain them. encoded by GENSET genes and fragments thereof. The In addition, the cDNAS may contain the full coding present invention also relates to recombinant vectors includ sequence of the gene from which the EST was derived or, ing the polynucleotides of the present invention, particularly alternatively, the cDNAS may include fragments of the recombinant vectors comprising a GENSET gene regulatory coding Sequence of the gene from which the EST was region or a Sequence encoding a GENSET polypeptide, and derived. It will be appreciated that there may be several to host cells containing the polynucleotides of the invention, cDNAS which include the EST sequence as a result of as well as to methods of making Such vectors and host cells. alternate splicing or the activity of alternative promoters. The present invention further relates to the use of these recombinant vectors and host cells in the production of the 0007. In the past, these short EST sequences were often polypeptides of the invention. The invention further relates obtained from oligo-dT primed cDNA libraries. Accord to antibodies that specifically bind to the polypeptides of the ingly, they mainly corresponded to the 3' untranslated region invention and to methods for producing Such antibodies and of the mRNA. In part, the prevalence of EST sequences fragments thereof. The invention also provides methods of derived from the 3' end of the mRNA is a result of the fact detecting the presence of the polynucleotides and polypep that typical techniques for obtaining cDNAS are not well tides of the present invention in a Sample, methods of Suited for isolating cDNA sequences derived from the 5' diagnosis and Screening of abnormal GENSET polypeptide ends of mRNAs (Adams et al, Nature 377:3-174, 1996, expression and/or biological activity, methods of Screening Hillier et al., Genome Res. 6:807-828, 1996). In addition, in compounds for their ability to modulate the activity or those reported instances where longer cDNA sequences have expression of the GENSET polypeptides, and uses of Such been obtained, the reported Sequences typically correspond compounds. to coding Sequences and do not include the full 5' untrans lated region (5'UTR) of the mRNA from which the cDNA is derived. Indeed, 5'UTRS have been shown to affect either the BACKGROUND OF THE INVENTION stability or translation of mRNAS. Thus, regulation of gene 0003) The estimated 30,000-12,000 genes scattered along expression may be achieved through the use of alternative the human chromosomes offer tremendous promise for the 5'UTRS as shown, for instance, for the translation of the understanding, diagnosis, and treatment of human diseases. tissue inhibitor of metalloprotease mRNA in mitogenically In addition, probes capable of Specifically hybridizing to loci activated cells (Waterhouse et al., J Biol Chem. 265:5585-9. distributed throughout the human genome find application in 1990). Furthermore, modification of 5'UTR through muta the construction of high resolution chromosome maps and in tion, insertion or translocation events may even be implied the identification of individuals. in pathogenesis. For instance, the Fragile X Syndrome, the most common cause of inherited mental retardation, is partly 0004. In the past, the characterization of even a single due to an insertion of multiple CGG trinucleotides in the human gene was a painstaking process, requiring years of 5'UTR of the Fragile X mRNA resulting in the inhibition of effort. Recent developments in the areas of cloning vectors, protein Synthesis via ribosome Stalling (Feng et al., Science DNA sequencing, and computer technology have merged to 268:731-4, 1995). An aberrant mutation in regions of the greatly accelerate the rate at which human genes can be 5'UTR known to inhibit translation of the proto-oncogene isolated, Sequenced, mapped, and characterized. c-myc was shown to result in upregulation of c-myc protein 0005 Currently, two different approaches are being pur levels in cells derived from patients with multiple myelomas Sued for identifying and characterizing the genes distributed (Willis et al., Curr Top Microbiol Immunol 224:269-76, along the human genome. In one approach, large fragments 1997). In addition, the use of oligo-dT primed cDNA librar of genomic DNA are isolated, cloned, and Sequenced. Poten ies does not allow the isolation of complete 5' UTRS since tial open reading frames in these genomic Sequences are Such incomplete Sequences obtained by this process may not identified using bio-informatics Software. However, this include the first exon of the mRNA, particularly in situations approach entails Sequencing large Stretches of human DNA where the first exon is short. Furthermore, they may not which do not encode proteins in order to find the protein include Some exons, often short ones, which are located encoding Sequences Scattered throughout the genome. In upstream of Splicing Sites. Thus, there is a need to obtain addition to requiring extensive Sequencing, the bio-infor sequences derived from the 5' ends of mRNAs. US 2006/0053498A1 Mar. 9, 2006

0008 Moreover, despite the great amount of EST data techniques. In Such applications, the extracellular Secretion that large-scale Sequencing projects have yielded (Adams et of the desired protein greatly facilitates purification by al., Nature 377:174, 1996; Hillier et a..., Genome Res. 6:807 reducing the number of undesired proteins from which the 828, 1996), information concerning the biological function desired protein must be Selected. Thus, there exists a need to of the mRNAS corresponding to such obtained cDNAS has identify and characterize the 5' fragments of the genes for revealed to be limited. Indeed, whereas the knowledge of the Secretory proteins which encode Signal peptides. complete coding Sequence is absolutely necessary to inves tigate the biological function of mRNAS, ESTs yield only 0011 Sequences coding for secreted proteins may also partial coding Sequences. So far, large-scale full-length find application as therapeutics or diagnostics. In particular, cDNA cloning has been achieved only with limited success Such Sequences may be used to determine whether an because of the poor efficiency of methods for constructing individual is likely to express a detectable phenotype, Such full-length cDNA libraries. Indeed, such methods require as a disease, as a consequence of a mutation in the coding either a large amount of mRNA (Ederly et al., 1995), thus Sequence for a Secreted protein. In instances where the resulting in non representative full-length libraries when individual is at risk of Suffering from a disease or other Small amounts of tissue are available or require PCR ampli undesirable phenotype as a result of a mutation in Such a fication (Maruyama et al., 1994; CLONTECHniques, 1996) coding Sequence, the undesirable phenotype may be cor to obtain a reasonable number of clones, thus yielding rected by introducing a normal coding Sequence using gene strongly biased cDNA libraries where rare and long cDNAS therapy. Alternatively, if the undesirable phenotype results are lost. Thus, there is a need to obtain full-length cDNAS, from overexpression of the protein encoded by the coding i.e. cDNAS containing the full coding Sequence of their Sequence, expression of the protein may be reduced using corresponding mRNAS. antisense or triple helix based Strategies. 0009 While many sequences derived from human chro 0012. The secreted human polypeptides encoded by the mosomes have practical applications, approaches based on coding Sequences may also be used as therapeutics by the identification and characterization of those chromosomal administering them directly to an individual having a con Sequences which encode a protein product are particularly dition, Such as a disease, resulting from a mutation in the relevant to diagnostic and therapeutic uses. Of the 30,000 Sequence encoding the polypeptide. In Such an instance, the 120,000 protein coding genes, those genes encoding proteins condition can be cured or ameliorated by administering the which are secreted from the cell in which they are synthe polypeptide to the individual. Sized, as Well as the Secreted proteins themselves, are 0013 In addition, the Secreted human polypeptides or particularly valuable as potential therapeutic agents. Such fragments thereof may be used to generate antibodies useful proteins are often involved in cell to cell communication and in determining the tissue type or Species of origin of a may be responsible for producing a clinically relevant biological Sample. The antibodies may also be used to response in their target cells. In fact, Several Secretory determine the cellular localization of the Secreted human proteins, including tissue plasminogen activator, G-CSF, polypeptides or the cellular localization of polypeptides GM-CSF, erythropoietin, human growth hormone, insulin, which have been fused to the human polypeptides. In interferon-C, interferon-B, interferon-Y, and interleukin-2, addition, the antibodies may also be used in immunoaffinity are currently in clinical use. These proteins are used to treat chromatography techniques to isolate, purify, or enrich the a wide range of conditions, including acute myocardial human polypeptide or a target polypeptide which has been infarction, acute ischemic Stroke, anemia, diabetes, growth fused to the human polypeptide. hormone deficiency, hepatitis, kidney carcinoma, chemo therapy induced neutropenia and multiple Sclerosis. For 0014 Public information on the number of human genes these reasons, cDNAS encoding Secreted proteins or frag for which the promoters and upstream regulatory regions ments thereof represent a particularly valuable Source of have been identified and characterized is quite limited. In therapeutic agents. Thus, there is a need for the identification part, this may be due to the difficulty of isolating Such and characterization of Secreted proteins and the nucleic regulatory Sequences. Upstream regulatory Sequences Such acids encoding them. as transcription factor binding sites are typically too short to 0010. In addition to being therapeutically useful them be utilized as probes for isolating promoters from human Selves, Secretory proteins include short peptides, called genomic libraries. Recently, Some approaches have been Signal peptides, at their amino termini which direct their developed to isolate human promoters. One of them consists Secretion. These signal peptides are encoded by the Signal of making a CpG island library (Cross et al., Nature Genet Sequences located at the 5' ends of the coding Sequences of ics 6:236-244, 1994). The second consists of isolating genes encoding Secreted proteins. Because these signal human genomic DNA sequences containing Spel binding peptides will direct the extracellular Secretion of any protein sites by the use of Spel binding protein (Mortlock et al., to which they are operably linked, the Signal Sequences may Genome Res. 6:327-335, 1996). Both of these approaches be exploited to direct the efficient Secretion of any protein by have their limits due to a lack of Specificity and of compre operably linking the Signal Sequences to a gene encoding the hensiveness. Thus, there exists a need to identify and Sys protein for which Secretion is desired. In addition, fragments tematically characterize the 5' fragments of the genes. of the Signal peptides called membrane-translocating 0015 cDNAS including the 5' ends of their corresponding Sequences may also be used to direct the intracellular import mRNA may be used to efficiently identify and isolate of a peptide or protein of interest. This may prove beneficial 5'UTRs and upstream regulatory regions which control the in gene therapy Strategies in which it is desired to deliver a location, developmental Stage, rate, and quantity of protein particular gene product to cells other than the cells in which synthesis, as well as the stability of the mRNA (Theil et al., it is produced. Signal Sequences encoding Signal peptides BioFactors 4:87–93, (1993). Once identified and character also find application in Simplifying protein purification ized, these regulatory regions may be utilized in gene US 2006/0053498A1 Mar. 9, 2006 therapy or protein purification Schemes to obtain the desired ID NOs: 170-338, 456-560,785-918; (d) the epitope-bearing amount and locations of protein Synthesis or to inhibit, fragments of the polypeptides encoded by the clone inserts reduce, or prevent the Synthesis of undesirable gene prod contained in the deposited clone pool; (e) the domains of the uctS. polypeptides of SEQ ID NOs: 170-338, 456-560, 785-918; 0016. In addition, cDNAs containing the 5' ends of (f) the domains of the polypeptides encoded by the clone Secretory protein genes may include Sequences useful as inserts contained in the deposited clone pool; and (g) the probes for chromosome mapping and the identification of allelic variant polypeptides of any of the polypeptides of individuals. Thus, there is a need to identify and characterize (a)-(f). The invention further provides for fragments of the the Sequences upstream of the 5' coding Sequences of genes polypeptides of (a)-(g) above, Such as those having biologi encoding Secretory proteins. cal activity or comprising biologically functional domain(s). 0021. The present invention further includes polypep SUMMARY OF THE INVENTION tides with an amino acid sequence with at least 70% simi larity, and more preferably at least 75%, 80%, 85%, 90%, 0.017. The present invention provides a purified or iso 95%, 96%, 97%, 98%, or 99% similarity to those polypep lated polynucleotide comprising, consisting of, or consisting tides described in (a)-(g), as well as polypeptides having an essentially of a nucleotide Sequence Selected from the group amino acid Sequence at least 70% identical, more preferably consisting of: (a) the sequences of SEQ ID NOS:1-169, at least 75% identical, and still more preferably 80%, 85%, 339-455,561-784; (b) the sequences of clone inserts of the 90%, 95%, 96%, 97%, 98%, or 99% identical to those deposited clone pool; (c) the coding sequences of SEQ ID polypeptides described in (a)-(g), e.g. Over a region of at NOS:1-169,339-455, 561-784; (d) the coding sequences of least about 25, 50, 100, 150, 250, 500, 1000, or more amino the clone inserts of the deposited clone pool; (e) the acids. The invention further relates to methods of making the Sequences encoding one of the polypeptides of SEQ ID polypeptides of the present invention. NOs: 170-338, 456-560, 785-918; (f) the sequences encod ing one of the polypeptides encoded by the clone inserts of 0022. The present invention further relates to transgenic the deposited clone pool, (g) the genomic sequences coding plants or animals, wherein Said transgenic plant or animal is for the GENSET polypeptides; (h) the 5' transcriptional transgenic for a polynucleotide of the present invention and regulatory regions of GENSET genes; (i) the 3' transcrip expresses a polypeptide of the present invention. tional regulatory regions of GENSET genes; (ii) the poly 0023 The invention further relates to antibodies that nucleotides comprising the nucleotide sequence of any com specifically bind to GENSET polypeptides of the present bination of (g)-(i); (k) the variant polynucleotides of any of invention and fragments thereof as well as to methods for the polynucleotides of (a)-(); (1) the polynucleotides com producing Such antibodies and fragments thereof. prising a nucleotide Sequence of (a)-(k), wherein the poly 0024. The invention also provides kits, uses and methods nucleotide is single Stranded, double Stranded, or a portion for detecting GENSET gene expression and/or biological is single Stranded and a portion is double Stranded; (m) the activity in a biological Sample. One Such method involves polynucleotides comprising a nucleotide Sequence comple assaying for the expression of a GENSET polynucleotide in mentary to any of the Single Stranded polynucleotides of (1). a biological Sample using the polymerase chain reaction The invention further provides for fragments of the nucleic (PCR) to amplify and detect GENSET polynucleotides or acid molecules of (a)-(m) described above. Southern and Northern blot hybridization to detect GENSET 0.018 Further embodiments of the invention include puri genomic DNA, cDNA or mRNA. Alternatively, a method of fied or isolated polynucleotides that comprise, consist of, or detecting GENSET gene expression in a test Sample can be consist essentially of a nucleotide Sequence at least 70% accomplished using a compound which binds to a GENSET identical, more preferably at least 75%, and even more polypeptide of the present invention or a portion of a preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, GENSET polypeptide. or 99% identical, to any of the nucleotide sequences in 0025 The present invention also relates to diagnostic (a)-(m) above, e.g. over a region of at least about 25, 50, 100, methods and uses of GENSET polynucleotides and polypep 150, 250, 500, 1000, or more contiguous nucleotides, or a tides for identifying individuals or non-human animals hav polynucleotide which hybridizes under Stringent hybridiza ing elevated or reduced levels of GENSET gene products, tion conditions to a polynucleotide in (a)-(m) above. which individuals are likely to benefit from therapies to 0019. The present invention also relates to recombinant SuppreSS or enhance GENSET gene expression, respectively, vectors, which include the purified or isolated polynucle and to methods of identifying individuals or non-human otides of the present invention, and to host cells recombinant animals at increased risk for developing, or at present for the polynucleotides of the present invention, as well as having, certain diseases/disorders associated with GENSET to methods of making Such vectors and host cells. The polypeptide expression or biological activity. present invention further relates to the use of these recom 0026. The present invention also relates to kits, uses and binant vectors and recombinant host cells in the production methods of Screening compounds for their ability to modu of GENSET polypeptides. late (e.g. increase or inhibit) the activity or expression of 0020. The invention further provides a purified or iso GENSET polypeptides including compounds that interact lated polypeptide comprising, consisting of, or consisting with GENSET gene regulatory Sequences and compounds essentially of an amino acid Sequence Selected from the that interact directly or indirectly with a GENSET polypep group consisting of: (a) the polypeptides of SEQ ID tide. Uses of Such compounds are also within the Scope of NOs: 170-338, 456-560, 785-918; (b) the polypeptides the present invention. encoded by the clone inserts of the deposited clone pool; (c) 0027. The present invention also relates to pharmaceuti the epitope-bearing fragments of the polypeptides of SEQ cal or physiologically acceptable compositions comprising, US 2006/0053498A1 Mar. 9, 2006 an active agent, the polypeptides, polynucleotides or anti 0035) In another aspect, the present invention provides an bodies of the present invention, as well as, typically, a isolated polynucleotide, the polynucleotide comprising a pharmaceutically acceptable carrier. nucleic acid Sequence having at least about 80% identity over at least about 100 contiguous nucleotides to any one of 0028. The present invention also relates to computer the sequences shown as SEQ ID NOS:1-169, 339-455, Systems containing cDNA codes and polypeptide codes of 561-784 or any one of the sequences of the clone inserts of Sequences of the invention and to computer-related methods the deposited clone pool. of comparing Sequences, identifying homology or features using GENSET polypeptides or GENSET polynucleotide 0036). In one embodiment, the polynucleotide hybridizes Sequences of the invention. under Stringent conditions to a polynucleotide comprising any one of the sequences shown as SEQ ID NOS:1-169, 0029. In another aspect, the present invention provides an 339-455, 561-784 or any one of the sequences of the clone isolated polynucleotide, the polynucleotide comprising a inserts of the deposited clone pool. In another embodiment, nucleic acid Sequence encoding: i) a polypeptide comprising the polynucleotide comprises any one of the Sequences an amino acid Sequence having at least about 80% identity shown as SEQ ID NOS:1-169,339-455,561-784 or any one to any one of the sequences shown as SEQID NOs: 170-338, of the Sequences of the clone inserts of the deposited clone 456-560, 785-918 or any one of the sequences of polypep pool. tides encoded by the clone inserts of the deposited clone 0037. In another aspect, the present invention provides a pool; or a biologically active fragment of the polypeptide. biologically active polypeptide encoded by any of the 0.030. In one embodiment, the polypeptide comprises any herein-described polynucleotides. one of the sequences shown as SEQ ID NOs: 170-338, 0038. In another aspect, the present invention provides an 456-560, 785-918 or any one of the sequences of the isolated polypeptide or biologically active fragment thereof, polypeptides encoded by the clone inserts of the deposited the polypeptide comprising an amino acid Sequence having clone pool. In another embodiment, the polypeptide com at least about 80% sequence identity to any one of the prises a signal peptide. In another embodiment, the polypep sequences shown as SEQ ID NOs: 170-338, 456-560, 785 tide is a mature protein. In another embodiment, the nucleic 918 or any one of the Sequences of polypeptides encoded by acid Sequence has at least about 80% identity over at least the clone inserts of the deposited clone pool. about 100 contiguous nucleotides to any one of the sequences shown as SEQ ID NOS:1-169,339-455,561-784 0039. In one embodiment, the polypeptide is selectively or any one of the Sequences of the clone inserts of the recognized by an antibody raised against an antigenic deposited clone pool. In another embodiment, the polynucle polypeptide, or an antigenic fragment thereof, the antigenic otide hybridizes under Stringent conditions to a polynucle polypeptide comprising any one of the Sequences shown as otide comprising any one of the Sequences shown as SEQID SEQ ID NOs: 170-338, 456-560, 785-918 or any one of the NOS:1-169,339-455, 561-784 or any one of the sequences Sequences of polypeptides encoded by the clone inserts of of the clone inserts of the deposited clone pool. In another the deposited clone pool. In another embodiment, the embodiment, the nucleic acid Sequence comprises any one polypeptide comprises any one of the Sequences shown as SEQ ID NOs: 170-338, 456-560, 785-918 or any one of the of the sequences shown as SEQ ID NOS:1-169,339-455, Sequences of polypeptides encoded by the clone inserts of 561-784 or any one the sequences of the clone inserts of the the deposited clone pool. In another embodiment, the deposited clone pool. In another embodiment, the polynucle polypeptide comprises a signal peptide. In another embodi otide is operably linked to a promoter. ment, the polypeptide is a mature protein. 0031. In another aspect, the present invention provides an 0040. In another aspect, the present invention provides an expression vector comprising any of the herein-described antibody that specifically binds to any of ther herein-de polynucleotides, operably linked to a promoter. In another Scribed polypeptides. aspect, the present invention provides a host cell recombi nant for any of the herein-described polynucleotides. In 0041. In another aspect, the present invention provides a another aspect, the present invention provides a non-human method of determining whether a GENSET gene is expressed within a mammal, the method comprising the transgenic animal comprising the host cell. Steps of: a) providing a biological Sample from Said mam 0032. In another aspect, the present invention provides a mal; b) contacting said biological Sample with either of i) a method of making a GENSET polypeptide, the method polynucleotide that hybridizes under Stringent conditions to comprising a) providing a population of host cells compris any of the herein-described polynucleotides; or ii) a ing a herein-described polynucleotide and b) culturing the polypeptide that specifically binds to any of the herein population of host cells under conditions conducive to the described polypeptides; and c) detecting the presence or production of the polypeptide within Said host cells. absence of hybridization between the polynucleotide and an RNA species within the Sample, or the presence or absence 0033. In one embodiment, the method further comprises of binding of the polypeptide to a protein within the Sample; purifying the polypeptide from the population of host cells. wherein a detection of the hybridization or of the binding 0034. In another aspect, the present invention provides a indicates that the GENSET gene is expressed within the method of making a GENSET polypeptide, the method mammal. comprising a) providing a population of cells comprising a 0042. In one embodiment, the polynucleotide is a primer, polynucleotide encoding a herein-described polypeptide; b) and the hybridization is detected by detecting the presence culturing the population of cells under conditions conducive of an amplification product comprising the Sequence of the to the production of the polypeptide within the cells, and c) primer. In another embodiment, the polypeptide is an anti purifying the polypeptide from the population of cells. body. US 2006/0053498A1 Mar. 9, 2006

0043. In another aspect, the present invention provides a application number 60/197.873. Applicants internal desig method of determining whether a mammal has an elevated nation number (Clone ID) corresponding to each sequence or reduced level of GENSET gene expression, the method identification (SEQID) number is also provided. comprising the steps of: a) providing a biological sample from the mammal; and b) comparing the amount of any of 0052 Table II lists the putative chromosomal location of the herein-described polypeptides, or of an RNA species the polynucleotides of the present invention. The SEQ ID encoding the polypeptide, within the biological Sample with NO listed for each polynucleotide is that from the priority a level detected in or expected from a control Sample; application 60/197,873; the corresponding SEQ ID NOS for wherein an increased amount of the polypeptide or the RNA the Sequence in the present application can be determined by Species within the biological Sample compared to the level referring to Table I. detected in or expected from the control Sample indicates 0053 Table III lists the number of hits in Genset's cDNA that the mammal has an elevated level of the GENSET gene libraries of tissues and cell types for polynucleotides of the expression, and wherein a decreased amount of the polypep invention. The following abbreviations are used to refer to tide or the RNA species within the biological sample com each cell or tissue type: A=Brain; B=Fetal brain; C=Fetal pared to the level detected in or expected from the control kidney; D=Fetal liver; E=Pituitary gland; F=Liver; G=Pla Sample indicates that the mammal has a reduced level of the centa; H =Prostate; I=Salivary gland; J=Stomach/Intestine; GENSET gene expression. and K=Testis. The SEQ ID NO listed for each polynucle 0044) In another aspect, the present invention provides a otide is that from the priority application 60/197.873; the method of identifying a candidate modulator of a GENSET corresponding SEQ ID NOS for the sequence in the present polypeptide, the method comprising: a) contacting any of application can be determined by referring to Table I. the herein-described polypeptides with a test compound; and 0054 Table IV lists the number of hits in publicly avail b) determining whether the compound specifically binds to able library of tissues and cell types for polynucleotides of the polypeptide, wherein a detection that the compound the invention. The SEQ ID NO listed for each polynucle Specifically binds to the polypeptide indicates that the com otide is that from the priority application 60/197.873; the pound is a candidate modulator of the GENSET polypeptide. corresponding SEQID NOS for each sequence in the present 0.045. In one embodiment, the method further comprises application can be determined by referring to Table I. testing the biological activity of the GENSET polypeptide in 0055 Table V lists the tissues and cell types in which the the presence of the candidate modulator, wherein an alter polynucleotide Sequences of the present invention are over ation in the biological activity of the GENSET polypeptide or under-represented. The SEQ ID NO listed for each in the presence of the compound in comparison to the polynucleotide is that from the priority application 60/197, activity in the absence of Said compound indicates that the 873; the corresponding SEQ ID NOS for each sequence in compound is a modulator of the GENSET polypeptide. the present application can be determined by referring to 0046. In another aspect, the present invention provides a Table I. method for the production of a pharmaceutical composition, BRIEF DESCRIPTION OF THE SEQUENCE the method comprising a) identifying a modulator of a LISTING GENSET polypeptide using any of the herein-described methods; and b) combining the modulator with a pharma 0056 SEQ ID NOS:1-169, 339-455, 561-784 are the ceutically acceptable carrier. nucleotide Sequences of cDNAS, with open reading frames as indicated as features (CDS). When appropriate, the loca tions of the potential polyadenylation Site and polyadenyla BRIEF DESCRIPTION OF DRAWINGS tion Signal are also indicated. 0047 FIG. 1 is a block diagram of an exemplary com 0057 SEQ ID NOs: 170-338, 456-560, 785-918 are the puter System. amino acid Sequences of proteins encoded by the cDNAS of 0.048 FIG. 2 is a flow diagram illustrating one embodi SEQ ID NOs: 1-169,339-455, 561-784. ment of a process 200 for comparing a new nucleotide or 0.058 SEQ ID NOs: 1-85, 339-400, 406-407, 413-415, protein Sequence with a database of Sequences in order to 561-594, and 634-651 are the nucleotide sequences of determine the identity levels between the new Sequence and cDNAS encoding a potentially Secreted protein. The loca the Sequences in the database. tions of the ORFs and Sequences encoding Signal peptides 0049 FIG. 3 is a flow diagram illustrating one embodi are listed in the accompanying Sequence Listing. In addi ment of a proceSS 250 in a computer for determining whether tion, the Von Heijne Score of the Signal peptide computed as two Sequences are homologous. described below is listed as the “score' in the accompanying Sequence Listing. The Sequence of the Signal-peptide is 0050 FIG. 4 is a flow diagram illustrating one embodi listed as “Seq' in the accompanying Sequence Listing. The ment of an identifier process 300 for detecting the presence “f” in the Signal peptide Sequence indicates the location of a feature in a Sequence. where proteolytic cleavage of the Signal peptide occurs to generate a mature protein. When appropriate, the locations BRIEF DESCRIPTION OF THE TABLES of the first and last nucleotides of the coding Sequences, 0051 Table I provides the SEQ ID Nos in the present eventually the locations of the first and last nucleotides of application (with the SEQ ID Nos corresponding to nucleic the polyA and the locations of the first and last nucleotides acid sequences preceded by “NUC', and the SEQ ID Nos of the polyA sites are indicated. corresponding to the encoded polypeptide Sequences pre 0059 SEQ ID NOs:86-169, 401-405, 408-412,416-455, ceded by “PRT) that correspond to a SEQID NO in priority 595-633, 652-784 are the nucleotide sequences of cDNAS in US 2006/0053498A1 Mar. 9, 2006

which no Sequence encoding a Signal peptide has been DETAILED DESCRIPTION OF THE identified to date. However, it remains possible that Subse INVENTION AND PREFERRED quent analysis will identify a Sequence encoding a signal EMBODIMENTS peptide in these nucleic acids. The locations of the ORFs are listed in the accompanying Sequence Listing. When appro Definitions priate, the locations of the first and last nucleotides of the 0065. Before describing the invention in greater detail, coding Sequences, eventually the locations of the first and the following definitions are set forth to illustrate and define last nucleotides of the polyA and the locations of the first and the meaning and Scope of the terms used to describe the last nucleotides of the polyA sites are indicated. invention herein. 0060 SEQ ID NOs: 170-254, 456-517, 520-521, 527 0066. The term “GENSET gene,” when used herein, 529, 785-818, and 858-875 are the amino acid sequences of encompasses genomic, mRNA and cDNA sequences encod polypeptides which contain a signal peptide. These polypep ing a GENSET polypeptide, including the 5' and 3' untrans tides are encoded by the cDNAs of SEQ ID NOs: 1-85, lated regions of Said Sequences. 339-400, 406-407, 413-415, 561-594, and 634-651. The location of the Signal peptide is listed in the accompanying 0067. The term “GENSET polypeptide biological activ Sequence Listing. ity” or “GENSET biological activity” is intended for polypeptides exhibiting any activity Similar, but not neces 0061 SEQ ID NOs:255-338, 517-519, 522-526, 530 sarily identical, to an activity of a GENSET polypeptide of 560, 819-857, 876-918 are the amino acid sequences of the invention. The GENSET polypeptide biological activity polypeptides in which no signal peptide has been identified of a given polypeptide may be assessed using any Suitable to date. However, it remains possible that Subsequent analy biological assay, a number of which are known to those sis will identify a Signal peptide in these polypeptides. These skilled in the art. In contrast, the term “biological activity” polypeptides are encoded by the nucleic acids of SEQ ID refers to any activity that any polypeptide may have. NOs: 86-169, 401-405, 408-412, 416-455, 595-633, 652 0068. The term “corresponding mRNA” refers to mRNA 784. which was or can be a template for cDNA synthesis for 0.062. In accordance with the regulations relating to producing a cDNA of the present invention. Sequence Listings, the following codes have been used in 0069. The term “corresponding genomic DNA” refers to the Sequence Listing to describes nucleotide Sequences. The genomic DNA which encodes an mRNA of interest, e.g. code “r” in the Sequences indicates that the nucleotide may corresponding to a cDNA of the invention, which genomic be a guanine or an adenine. The code “y” in the Sequences DNA includes the sequence of one of the strands of the indicates that the nucleotide may be a thymine or a cytosine. mRNA, in which thymidine residues in the sequence of the The code "m” in the Sequences indicates that the nucleotide genomic DNA (or cDNA) are replaced by uracil residues in may be an adenine or a cytosine. The code "k” in the the mRNA. Sequences indicates that the nucleotide may be a guanine or 0070 The term “deposited clone pool” is used herein to a thymine. The code "s' in the Sequences indicates that the refer to the pool of clones entitled cDNA-8-2000, deposited nucleotide may be a guanine or a cytosine. The code “w” in with the ATCC on Sep. 27, 2000, or the pool of clones the Sequences indicates that the nucleotide may be an entitled cDNA-11-2000, deposited with the ATCC on Nov. adenine or an thymine. In addition, all instances of the 27, 2000, or any other deposited clone pool containing a Symbol “n” in the nucleic acid Sequences mean that the clone corresponding to any of the herein-described nucleotide can be adenine, guanine, cytosine or thymine. Sequences. 0.063. In Some instances, the polypeptide Sequences in the 0071. The term "heterologous', when used herein, is Sequence Listing contain the symbol “Xaa.” These “Xaa’ intended to designate any polynucleotide or polypeptide Symbols indicate either (1) a residue which cannot be other than a GENSET polynucleotide or GENSET polypep identified because of nucleotide sequence ambiguity or (2) a tide of the invention, respectively. Stop codon in the determined Sequence where applicants 0072 “Providing with respect to, e.g. a biological believe one should not exist (if the sequence were deter Sample, population of cells, etc. indicates that the Sample, mined more accurately). In Some instances, several possible population of cells, etc. is Somehow used in a method or identities of the unknown amino acids may be Suggested by procedure. Significantly, “providing a biological Sample or the genetic code. population of cells does not require that the Sample or cells 0064. In the case of secreted proteins, it should be noted are specifically isolated or obtained for the purposes of the that, in accordance with the regulations governing Sequence invention, but can instead refer, for example, to the use of a Listings, in the appended Sequence Listing the encoded biological Sample obtained by another individual, for protein (i.e. the protein containing the signal peptide and the another purpose. mature protein or part thereof) extends from an amino acid 0073. An “amplification product” refers to a product of residue having a negative number through a positively any amplification reaction, e.g. PCR, RT-PCR, LCR, etc. numbered amino acid residue. Thus, the first amino acid of the mature protein resulting from cleavage of the Signal 0074. A “modulator of a protein or other compound peptide is designated as amino acid number 1, and the first refers to any agent that has a functional effect on the protein, amino acid of the Signal peptide is designated with the including physical binding to the protein, alterations of the appropriate negative number. quantity or quality of expression of the protein, altering any US 2006/0053498A1 Mar. 9, 2006 measurable or detectable activity, property, or behavior of involves the creation of a synthetic substance (cDNA) and the protein, or in any way interacts with the protein or pure individual cDNA clones can be isolated from the compound. synthetic library by clonal selection. Thus, creating a cDNA library from messenger RNA and Subsequently isolating 0075 “A test compound” can be any molecule that is individual clones from that library results in an approxi evaluated for its ability to modulate a protein or other compound. mately 10-10 fold purification of the native message. 007.9 The term “purified” is further used herein to 0.076 An antibody or other compound that specifically describe a polypeptide or polynucleotide of the invention binds to a polypeptide or polynucleotide of the invention is which has been Separated from other compounds including, also said to “selectively recognize” the polypeptide or but not limited to, polypeptides or polynucleotides, carbo polynucleotide. hydrates, lipids, etc. The term “purified” may be used to Specify the Separation of monomeric polypeptides of the 0077. The term “isolated” with respect to a molecule invention from oligomeric forms Such as homo- or hetero requires that the molecule be removed from its original dimers, trimers, etc. The term “purified” may also be used to environment (e.g., the natural environment if it is naturally Specify the separation of covalently closed (i.e. circular) occurring). For example, a naturally-occurring polynucle polynucleotides from linear polynucleotides. A polynucle otide or polypeptide present in a living animal is not otide is substantially pure when at least about 50%, prefer isolated, but the same polynucleotide or DNA or polypep ably 60 to 75% of a sample exhibits a single polynucleotide tide, Separated from Some or all of the coexisting materials Sequence and conformation (linear versus covalently close). in the natural System, is isolated. Such polynucleotide could A Substantially pure polypeptide or polynucleotide typically be part of a vector and/or Such polynucleotide or polypeptide comprises about 50%, preferably 60 to 90% weight/weight could be part of a composition, and Still be isolated in that of a polypeptide or polynucleotide Sample, respectively, the vector or composition is not part of its natural environ more usually about 95%, and preferably is over about 99% ment. For example, a naturally-occurring polynucleotide pure. Polypeptide and polynucleotide purity, or homogene present in a living animal is not isolated, but the same ity, is indicated by a number of means well known in the art, polynucleotide, Separated from Some or all of the coexisting Such as agarose or polyacrylamide gel electrophoresis of a materials in the natural System, is isolated. Specifically Sample, followed by Visualizing a single band upon Staining excluded from the definition of “isolated” are: naturally the gel. For certain purposes higher resolution can be occurring chromosomes (Such as chromosome spreads), provided by using HPLC or other means well known in the artificial chromosome libraries, genomic libraries, and art. AS an alternative embodiment, purification of the cDNA libraries that exist either as an in vitro nucleic acid polypeptides and polynucleotides of the present invention preparation or as a transfected/transformed host cell prepa may be expressed as “at least a percent purity relative to ration, wherein the host cells are either an in vitro hetero heterologous polypeptides and polynucleotides (DNA, RNA geneous preparation or plated as a heterogeneous population or both). As a preferred embodiment, the polypeptides and of Single colonies. Also specifically excluded are the above polynucleotides of the present invention are at least, 10%, libraries wherein a specified polynucleotide makes up leSS 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, than 5% of the number of nucleic acid inserts in the vector 96%, 98%, 99%, or 100% pure relative to heterologous molecules. Further specifically excluded are whole cell polypeptides and polynucleotides, respectively. As a further genomic DNA or whole cell RNA preparations (including preferred embodiment the polypeptides and polynucleotides Said whole cell preparations which are mechanically sheared have a purity ranging from any number, to the thousandth or enzymatically digested). Further specifically excluded are position, between 90% and 100% (e.g., a polypeptide or the above whole cell preparations as either an in Vitro polynucleotide at least 99.995% pure) relative to either preparation or as a heterogeneous mixture Separated by heterologous polypeptides or polynucleotides, respectively, electrophoresis (including blot transfers of the same) or as a weight/weight ratio relative to all compounds and wherein the polynucleotide of the invention has not further molecules other than those existing in the carrier. Each been Separated from the heterologous polynucleotides in the number representing a percent purity, to the thousandth electrophoresis medium (e.g., further separating by excising position, may be claimed as individual species of purity. a single band from a heterogeneous band population in an agarose gel or nylon blot). 0080. As used interchangeably herein, the terms “nucleic acid molecule(s)”, “oligonucleotide(s)', and “polynucle 0078. The term “purified” does not require absolute otide(s)" include RNA or DNA (either single or double purity; rather, it is intended as a relative definition. Purifi Stranded, coding, complementary or antisense), or RNA/ cation of Starting material or natural material to at least one DNA hybrid sequences of more than one nucleotide in either order of magnitude, preferably two or three orders, and more Single chain or duplex form (although each of the above preferably four or five orders of magnitude is expressly Species may be particularly specified). The term “nucle contemplated. As an example, purification from 0.1% con otide' is used herein as an adjective to describe molecules centration to 10% concentration is two orders of magnitude. comprising RNA, DNA, or RNA/DNA hybrid sequences of To illustrate, individual cDNA clones isolated from a cDNA any length in Single-Stranded or duplex form. More pre library have been conventionally purified to electrophoretic cisely, the expression “nucleotide Sequence” encompasses homogeneity. The Sequences obtained from these clones the nucleic material itself and is thus not restricted to the could not be obtained directly either from the library or from Sequence information (i.e. the Succession of letters chosen total human DNA. The cDNA clones are not naturally among the four base letters) that biochemically characterizes occurring as Such, but rather are obtained via manipulation a specific DNA or RNA molecule. The term “nucleotide' is of a partially purified naturally occurring Substance (mes also used herein as a noun to refer to individual nucleotides senger RNA). The conversion of mRNA into a cDNA library or varieties of nucleotides, meaning a molecule, or indi US 2006/0053498A1 Mar. 9, 2006

vidual unit in a larger nucleic acid molecule, comprising a nucleotides may be prepared as described in U.S. Pat. No. purine or pyrimidine, a ribose or deoxyribose Sugar moiety, 5,023,243, which disclosure is hereby incorporated by ref and a phosphate group, or phosphodiester linkage in the case erence in its entirety. Borano phosphate oligonucleotides of nucleotides within an oligonucleotide or polynucleotide. may be prepared as described in U.S. Pat. Nos. 5,130,302 The term “nucleotide' is also used herein to encompass and 5,177,198 which disclosures are hereby incorporated by “modified nucleotides” which comprise at least one modi reference in their entireties. fication Such as (a) an alternative linking group, (b) an 0081. The term “upstream” is used herein to refer to a analogous form of purine, (c) an analogous form of pyrimi location which is toward the 5' end of the polynucleotide dine, or (d) an analogous Sugar. For examples of analogous from a Specific reference point. linking groups, purine, pyrimidines, and Sugars, See, for example, PCT publication No. WO95/04064, which disclo 0082) The terms “base paired” and “Watson & Crick base Sure is hereby incorporated by reference in its entirety. paired” are used interchangeably herein to refer to nucle Preferred modifications of the present invention include, but otides which can be hydrogen bonded to one another by are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorou Virtue of their Sequence identities in a manner like that found racil, 5-iodouracil, hypoxanthine, Xantine, 4-acetylcytosine, in double-helical DNA with thymine or uracil residues 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylami linked to adenine residues by two hydrogen bonds and nomethyl-2-thiouridine, 5-carboxymethylaminomethylu cytosine and guanine residues linked by three hydrogen racil, dihydrouracil, beta-D-galactosylcqueosine, inosine, bonds (see Stryer, 1995, which disclosure is hereby incor N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, porated by reference in its entirety). 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 0083. The terms “complementary' or “complement 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-meth thereof are used herein to refer to the Sequences of poly ylguanine, 5-methylaminomethyluracil, 5-methoxyaminom nucleotides which is capable of forming Watson & Crick ethyl-2-thiouracil, beta-D-mannosylcqueosine, 5'-methoxy base pairing with another specified polynucleotide through carboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6 out the entirety of the complementary region. For the isopentenyladenine, uracil-5-oxyacetic acid (v) purpose of the present invention, a first polynucleotide is ybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-me deemed to be complementary to a Second polynucleotide thyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, when each base in the first polynucleotide is paired with its uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, complementary base. Complementary bases are, generally, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) A and T (or A and U), or C and G. “Complement” is used uracil, and 2,6-diaminopurine. The polynucleotide herein as a Synonym from “complementary polynucleotide', Sequences of the invention may be prepared by any known “complementary nucleic acid” and “complementary nucle method, including Synthetic, recombinant, ex vivo genera otide Sequence'. These terms are applied to pairs of poly tion, or a combination thereof, as well as utilizing any nucleotides based Solely upon their Sequences and not any purification methods known in the art. Methylenemeth particular Set of conditions under which the two polynucle ylimino linked oligonucleosides as well as mixed backbone otides would actually bind. Unless otherwise stated, all compounds having, may be prepared as described in U.S. complementary polynucleotides are fully complementary on Pat. Nos. 5,378,825; 5,386,023; 5,489,677; 5,602,240; and the whole length of the considered polynucleotide. 5,610,289, which disclosures are hereby incorporated by reference in their entireties. Formacetal and thioformacetal 0084. The terms “polypeptide' and “protein', used inter linked oligonucleosides may be prepared as described in changeably herein, refer to a polymer of amino acids without U.S. Pat. Nos. 5,264,562 and 5,264,564, which disclosures regard to the length of the polymer; thus, peptides, oligopep are hereby incorporated by reference in their entireties. tides, and proteins are included within the definition of Ethylene oxide linked oligonucleosides may be prepared as polypeptide. This term also does not specify or exclude described in U.S. Pat. No. 5,223,618, which disclosure is chemical or post-expression modifications of the polypep hereby incorporated by reference in its entirety. Phosphinate tides of the invention, although chemical or post-expression oligonucleotides may be prepared as described in U.S. Pat. modifications of these polypeptides may be included No. 5,508,270, which disclosure is hereby incorporated by excluded as Specific embodiments. Therefore, for example, reference in its entirety. Alkyl phosphonate oligonucleotides modifications to polypeptides that include the covalent may be prepared as described in U.S. Pat. No. 4,469,863, attachment of glycosyl groups, acetyl groups, phosphate which disclosure is hereby incorporated by reference in its groups, lipid groups and the like are expressly encompassed entirety. 3'-Deoxy-3-methylene phosphonate oligonucle by the term polypeptide. Further, polypeptides with these otides may be prepared as described in U.S. Pat. Nos. modifications may be specified as individual Species to be 5,610,289 or 5,625,050 which disclosures are hereby incor included or excluded from the present invention. The natural porated by reference in their entireties. Phosphoramidite or other chemical modifications, Such as those listed in oligonucleotides may be prepared as described in U.S. Pat. examples above can occur anywhere in a polypeptide, No. 5,256,775 or U.S. Pat. No. 5,366,878 which disclosures including the peptide backbone, the amino acid Side-chains are hereby incorporated by reference in their entireties. and the amino or carboxyl termini. It will be appreciated that Alkylphosphonothioate oligonucleotides may be prepared as the same type of modification may be present in the same or described in published PCT applications WO 94/17093 and varying degrees at Several Sites in a given polypeptide. Also, WO94/02499 which disclosures are hereby incorporated by a given polypeptide may contain many types of modifica reference in their entireties. 3'-Deoxy-3'-amino phosphora tions. Polypeptides may be branched, for example, as a midite oligonucleotides may be prepared as described in result of ubiquitination, and they may be cyclic, with or U.S. Pat. No. 5,476,925, which disclosure is hereby incor without branching. Modifications include acetylation, acy porated by reference in its entirety. Phosphotriester oligo lation, ADP-ribosylation, amidation, covalent attachment of US 2006/0053498A1 Mar. 9, 2006 flavin, covalent attachment of a heme moiety, covalent 0088 As used herein, the term “non-human animal' attachment of a nucleotide or nucleotide derivative, covalent refers to any non-human animal, including insects, birds, attachment of a lipid or lipid derivative, covalent attachment rodents and more usually mammals. Preferred non-human of phosphotidylinositol, cross-linking, cyclization, disulfide animals include: primates, farm animals Such as Swine, bond formation, demethylation, formation of covalent croSS goats, sheep, donkeys, cattle, horses, chickens, rabbits, and links, formation of cysteine, formation of pyroglutamate, rodents, preferably rats or mice. AS used herein, the term formylation, gamma-carboxylation, glycosylation, GPI "animal' is used to refer to any species in the animal anchor formation, hydroxylation, iodination, methylation, kingdom, preferably vertebrates, including birds and fish, myristoylation, oxidation, pegylation, proteolytic proceSS and more preferable a mammal. Both the terms “animal' and ing, phosphorylation, prenylation, racemization, Selenoyla “mammal’ expressly embrace human Subjects unless pre tion, Sulfation, transfer-RNA mediated addition of amino ceded with the term “non-human'. acids to proteins Such as arginylation, and ubiquitination. (See, for instance Creighton (1993); Seifter et al., (1990); 0089. The term “domain” refers to an amino acid frag Rattan et al., (1992)). Also included within the definition are ment with Specific biological properties. This term encom polypeptides which contain one or more analogs of an amino passes all known Structural and linear biological motifs. acid (including, for example, non-naturally occurring amino Examples of such motifs include but are not limited to acids, amino acids which only occur naturally in an unre leucine Zippers, helix-turn-helix motifs, glycosylation sites, lated biological System, modified amino acids from mam ubiquitination sites, alpha helices, and beta sheets, Signal malian Systems, etc.), polypeptides with Substituted link peptides which direct the Secretion of proteins, Sites for ages, as well as other modifications known in the art, both post-translational modification, enzymatic active Sites, Sub naturally occurring and non-naturally occurring. Strate binding sites, and enzymatic cleavage Sites. 0085. As used herein, the terms “recombinant polynucle 0090 Although each of these terms has a distinct mean otide' and “polynucleotide construct” are used interchange ing, the terms “comprising”, “consisting of and “consisting ably to refer to linear or circular, purified or isolated poly essentially of may be interchanged for one another through nucleotides that have been artificially designed and which out the instant application. The term "having has the same comprise at least two nucleotide Sequences that are not meaning as “comprising” and may be replaced with either found as contiguous nucleotide Sequences in their initial the term “consisting of or “consisting essentially of. natural environment. In particular, these terms mean that the 0091 Unless otherwise specified in the application, polynucleotide or cDNA is adjacent to “backbone' nucleic nucleotides and amino acids of polynucleotides and acid to which it is not adjacent in its natural environment. polypeptides, respectively, of the present invention are con Additionally, to be “enriched” the cDNAs will represent 5% tiguous and not interrupted by heterologous Sequences. or more of the number of nucleic acid inserts in a population of nucleic acid backbone molecules. Backbone molecules Identity Between Nucleic Acids Or Polypeptides according to the present invention include nucleic acids Such 0092. The terms “percentage of sequence identity” and as expression vectors, Self-replicating nucleic acids, Viruses, "percentage homology' are used interchangeably herein to integrating nucleic acids, and other vectors or nucleic acids refer to comparisons among polynucleotides and polypep used to maintain or manipulate a nucleic acid insert of tides, and are determined by comparing two optimally interest. Preferably, the enriched cDNAs represent 15% or aligned Sequences over a comparison window, wherein the more of the number of nucleic acid inserts in the population portion of the polynucleotide or polypeptide Sequence in the of recombinant backbone molecules. More preferably, the comparison window may comprise additions or deletions enriched cDNAs represent 50% or more of the number of (i.e., gaps) as compared to the reference Sequence (which nucleic acid inserts in the population of recombinant back does not comprise additions or deletions) for optimal align bone molecules. In a highly preferred embodiment, the ment of the two Sequences. The percentage is calculated by enriched cDNAs represent 90% or more (including any determining the number of positions at which the identical number between 90 and 100%, to the thousandth position, nucleic acid base or amino acid residue occurs in both e.g., 99.5%) of the number of nucleic acid inserts in the Sequences to yield the number of matched positions, divid population of recombinant backbone molecules. ing the number of matched positions by the total number of 0.086 The term “recombinant polypeptide' is used herein positions in the window of comparison and multiplying the to refer to polypeptides that have been artificially designed result by 100 to yield the percentage of Sequence identity. and which comprise at least two polypeptide Sequences that Homology is evaluated using any of the variety of Sequence are not found as contiguous polypeptide Sequences in their comparison algorithms and programs known in the art. Such initial natural environment, or to refer to polypeptides which algorithms and programs include, but are by no means have been expressed from a recombinant polynucleotide. limited to, TBLASTN, BLASTP, FASTA, TEASTA, CLUSTALW. FASTDB (Pearson and Lipman, 1988; Alts 0.087 As used herein, the term “operably linked” refers to chul et al., 1990; Thompson et al., 1994; Higgins et al., a linkage of polynucleotide elements in a functional rela 1996; Altschulet al., 1990; Altschul et al., 1993; Brutlaget tionship. A sequence which is "operably linked' to a regu al, 1990), the disclosures of which are incorporated by latory Sequence Such as a promoter means that Said regula reference in their entireties. tory element is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase 0093. In a particularly preferred embodiment, protein and initiation and expression of the nucleic acid of interest. For nucleic acid Sequence homologies are evaluated using the instance, a promoter or enhancer is operably linked to a Basic Local Alignment Search Tool (“BLAST) which is coding Sequence if it affects the transcription of the coding well known in the art (see, e.g., Karlin and Altschul, 1990; Sequence. Altschulet al., 1990, 1993, 1997), the disclosures of which US 2006/0053498A1 Mar. 9, 2006

are incorporated by reference in their entireties. In particular, FASTDB program does not account for 5' and 3' truncations five specific BLAST programs are used to perform the of the Subject Sequence when calculating percent identity. following task: For Subject Sequences truncated at the 5' or 3' ends, relative to the query Sequence, the percent identity is corrected by 0094) (1) BLASTP and BLAST3 compare an amino acid calculating the number of bases of the query Sequence that query Sequence against a protein Sequence database; are 5' and 3' of the Subject Sequence, which are not matched/ 0.095 (2) BLASTN compares a nucleotide query aligned, as a percent of the total bases of the query Sequence. Sequence against a nucleotide Sequence database; Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage 0096 (3) BLASTX compares the six-frame conceptual is then Subtracted from the percent identity, calculated by the translation products of a query nucleotide Sequence (both above FASTDB program using 10, the specified parameters, Strands) against a protein Sequence database; to arrive at a final percent identity Score. This corrected Score 0097 (4) TBLASTN compares a query protein sequence is what is used for the purposes of the present invention. against a nucleotide Sequence database translated in all Six Only nucleotides outside the 5' and 3' nucleotides of the reading frames (both Strands); and Subject Sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query Sequence, are 0098 (5) TBLASTX compares the six-frame translations calculated for the purposes of manually adjusting the percent of a nucleotide query Sequence against the Six-frame trans identity Score. For example, a 90 nucleotide Subject lations of a nucleotide Sequence database. Sequence is aligned to a 100 nucleotide query Sequence to 0099] The BLAST programs identify homologous determine percent identity. The deletions occur at the 5' end Sequences by identifying Similar Segments, which are of the subject sequence and therefore, the FASTDB align referred to herein as “high-scoring Segment pairs,” between ment does not show a matched/alignment of the first 10 a query amino or nucleic acid Sequence and a test Sequence nucleotides at 5' end. The 10 unpaired nucleotides represent which is preferably obtained from a protein or nucleic acid 10% of the sequence (number of nucleotides at the 5' and 3' Sequence database. High-scoring Segment pairs are prefer ends not matched/total number of nucleotides in the query ably identified (i.e., aligned) by means of a Scoring matrix, Sequence) So 10% is Subtracted from the percent identity many of which are known in the art. Preferably, the scoring score calculated by the FASTDB program. If the remaining matrix used is the BLOSUM62 matrix (Gonnet et al., 1992; 90 nucleotides were perfectly matched the final percent Henikoff and Henikoff, 1993, the disclosures of which are identity would be 90%. In another example, a 90 nucleotide incorporated by reference in their entireties). Less prefer Subject Sequence is compared with a 100 nucleotide query ably, the PAM or PAM250 matrices may also be used (see, Sequence. This time the deletions are internal deletions. So e.g., Schwartz and Dayhoff, eds., 1978, the disclosure of that there are no nucleotides on the 5' or 3' of the subject which is incorporated by reference in its entirety). The Sequence which are not matched/aligned with the query. In BLAST programs evaluate the Statistical Significance of all this case the percent identity calculated by FASTDB is not high-scoring Segment pairs identified, and preferably Selects manually corrected. Once again, only nucleotides 5' and 3 those Segments which Satisfy a user-specified threshold of of the Subject Sequence which are not matched/aligned with Significance, Such as a user-specified percent homology. the query Sequence are manually corrected. No other manual Preferably, the Statistical significance of a high-scoring corrections are made for the purposes of the present inven Segment pair is evaluated using the Statistical Significance tion. formula of Karlin (see, e.g., Karlin and Altschul, 1990), the 0101 Another preferred method for determining the best disclosure of which is incorporated by reference in its overall match between a query amino acid sequence (a entirety. The BLAST programs may be used with the default Sequence of the present invention) and a Subject sequence, parameters or with modified parameters provided by the also referred to as a global Sequence alignment, can be USC. determined using the FASTDB computer program based on 0100 Another preferred method for determining the best the algorithm of Brutlag et al. (1990). In a sequence align overall match between a query nucleotide sequence (a ment the query and Subject Sequences are both amino acid Sequence of the present invention) and a Subject sequence, Sequences. The result of Said global Sequence alignment is in also referred to as a global Sequence alignment, can be percent identity. Preferred parameters used in a FASTDB determined using the FASTDB computer program based on amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mis the algorithm of Brutlag et al. (1990), the disclosure of match Penalty=1, Joining Penalty=20, Randomization which is incorporated by reference in its entirety. In a Group Length=0, Cutoff Score=1, Window Size=sequence Sequence alignment the query and Subject Sequences are length, Gap Penalty=5, Gap Size Penalty=0.05, Window both DNA sequences. An RNA sequence can be compared Size=500 or the length of the Subject amino acid Sequence, by first converting Us to T's. The result of said global whichever is shorter. If the Subject Sequence is shorter than Sequence alignment is in percent identity. Preferred param the query Sequence due to N-or C-terminal deletions, not eters used in a FASTDB alignment of DNA sequences to because of internal deletions, the results, in percent identity, calculate percent identity are: Matrix=Unitary, k-tuple=4, must be manually corrected. This is because the FASTDB Mismatch Penalty=1, Joining Penalty=30, Randomization program does not account for N- and C-terminal truncations Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size of the Subject Sequence when calculating global percent Penalty=0.05, Window Size=500 or the length of the subject identity. For Subject Sequences truncated at the N- and nucleotide Sequence, whichever is shorter. If the Subject C-termini, relative to the query Sequence, the percent iden Sequence is shorter than the query Sequence because of 5" or tity is corrected by calculating the number of residues of the 3' deletions, not because of internal deletions, a manual query Sequence that are N- and C-terminal of the Subject correction must be made to the results. This is because the Sequence, which are not matched/aligned with a correspond US 2006/0053498A1 Mar. 9, 2006 ing Subject residue, as a percent of the total bases of the homologues of the GENSET genes. Procedures known in query Sequence. Whether a residue is matched/aligned is the art can be used to obtain full-length genes and cDNAS, determined by results of the FASTDB sequence alignment. allelic variants, Splice variants, full-length coding portions, This percentage is then Subtracted from the percent identity, orthologs, and/or species homologues of genes and cDNAS calculated by the above FASTDB program using the speci corresponding to a nucleotide Sequence Selected from the fied parameters, to arrive at a final percent identity Score. group consisting of sequences of SEQ ID NOS:1-169,339 This final percent identity score is what is used for the 455,561-784 and sequences of clone inserts of the deposited purposes of the present invention. Only residues to the N clone pool, using information from the Sequences disclosed and C-termini of the Subject Sequence, which are not herein or the clone pool deposited with the ATCC or other matched/aligned with the query Sequence, are considered for depositary authority. For example, allelic variants, orthologs the purposes of manually adjusting the percent identity and/or Species homologues may be isolated and identified by Score. That is, only query amino acid residues outside the making Suitable probes or primers from the Sequences farthest N- and C-terminal residues of the Subject Sequence. provided herein and Screening a Suitable nucleic acid Source For example, a 90 amino acid residue Subject Sequence is for allelic variants and/or the desired homologue using any aligned with a 100-residue query Sequence to determine technique known to those skilled in the art including those percent identity. The deletion occurs at the N-terminus of the described into the section entitled “To find similar subject sequence and therefore, the FASTDB alignment Sequences. does not match/align with the first residues at the N-termi 0105. In a specific embodiment, the polynucleotides of nus. The 10 unpaired residues represent 10% of the sequence the invention are at least 15, 30, 50, 100, 125, 500, or 1000 (number of residues at the N- and C-termini not matched/ continuous nucleotides. In another embodiment, the poly total number of residues in the query Sequence) So 10% is nucleotides are less than or equal to 300 kb, 200 kb, 100 kb, Subtracted from the percent identity Score calculated by the 50 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2 kb, 1.5 kb, or 1 kb in FASTDB program. If the remaining 90 residues were per length. In a further embodiment, polynucleotides of the fectly matched the final percent identity would be 90%. In invention comprise a portion of the coding Sequences, as another example, a 90-residue Subject Sequence is compared disclosed herein, but do not comprise all or a portion of any with a 100-residue query Sequence. This time the deletions intron. In another embodiment, the polynucleotides com are internal So there are no residues at the N- or C-termini prising coding Sequences do not contain coding Sequences of of the Subject Sequence, which are not matched/aligned with a genomic flanking gene (i.e., 5' or 3' to the gene of interest the query. In this case the percent identity calculated by in the genome). In other embodiments, the polynucleotides FASTDB is not manually corrected. Once again, only resi of the invention do not contain the coding Sequence of more due positions outside the N- and C-terminal ends of the than 1000, 500, 250, 100, 75, 50, 25, 20, 15, 10, 5, 4, 3, 2, Subject Sequence, as displayed in the FASTDB alignment, or 1 naturally occurring genomic flanking gene(s). which are not matched/aligned with the query Sequence are manually corrected. No other manual corrections are made Deposited Clone Pool of the Invention for the purposes of the present invention. 0106 Expression of GENSET genes has been shown to lead to the production of at least one mRNA species per 0102) The term “percentage of sequence similarity” GENSET gene, which cDNA sequence is set forth in the refers to comparisons between polypeptide Sequences and is appended sequence listing as SEQ ID NOS:1-169,339-455, determined by comparing two optimally aligned Sequences 561-784. The cDNAs (SEQ ID NOS:1-169,339-455,561 over a comparison window, wherein the portion of the 784) corresponding to these GENSET mRNA species were polypeptide Sequence in the comparison window may com cloned either in the vector pBluescriptiISK (Stratagene) or prise additions or deletions (i.e., gaps) as compared to the in a vector called pPT. Cells containing the cloned cDNAS reference Sequence (which does not comprise additions or of the present invention are maintained in permanent deposit deletions) for optimal alignment of the two sequences. The by the inventors at Genset, S.A., 24 Rue Royale, 75008 percentage is calculated by determining the number of Paris, France. Each cDNA can be removed from the Blue positions at which an identical or equivalent amino acid script vector in which it was inserted by performing a Not residue occurs in both Sequences to yield the number of Pst I double digestion, or from the pPT vector by performing matched positions, dividing the number of matched posi a Mun HindIII double digestion, to produce the appropriate tions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the fragment for each clone, provided the cDNA sequence does percentage of Sequence Similarity. Similarity is evaluated not contain any of the corresponding restriction Sites within using any of the variety of Sequence comparison algorithms its Sequence. Alternatively, other restriction enzymes of the and programs known in the art, including those described multicloning site of the Vector may be used to recover the above in this Section. Equivalent amino acid residues are desired insert as indicated by the manufacturer. defined herein in the “Mutated polypeptides' section. 0107 Pools of cells containing certain cDNAs of the invention, from which the cells containing a particular Polynucleotides of the Invention polynucleotide is obtainable, have also been deposited with 0103) The present invention concerns GENSET genomic the American Tissue Culture Collection (ATCC), 10801 and cDNA sequences. The present invention encompasses University Boulevard, Manassas, Va. 20110-2209, United GENSET genes, polynucleotides comprising GENSET States. These cDNA clones have been transfected into genomic and cDNA sequences, as well as fragments and Separate bacterial cells (E-coli) for these composite deposits. variants thereof. These polynucleotides may be purified, 0.108 Bacterial cells containing a particular clone can be isolated, or recombinant. obtained from the composite deposit as follows: 0104. Also encompassed by the present invention are 0109) An oligonucleotide probe or probes should be allelic variants, orthologs, Splice variants, and/or Species designed to the Sequence that is known for that particular US 2006/0053498A1 Mar. 9, 2006 clone. This sequence can be derived from the Sequences 0117. Alternatively, to recover cDNA inserts from the provided herein, or from a combination of those Sequences. pool of bacteria, a PCR can be performed on plasmid DNA The design of the oligonucleotide probe should preferably isolated using Standard procedures and primerS designed at follow these parameters: both ends of the cDNA insertion, including primerS designed in the multicloning site of the vector. If a specific cDNA of 0110 (a) It should be designed to an area of the sequence interest is to be recovered, primerS may be designed in order which has the fewest ambiguous bases (“N’s”), if any; to be specific for the 5' end and the 3' end of this cDNA using 0111 (b) Preferably, the probe is designed to have a Tm Sequence information available from the appended Sequence of approximately 80 degrees Celsius (assuming 2 degrees listing. The PCR product which corresponds to the cDNA of for each A or T and 4 degrees for each G or C). However, interest can then be manipulated using Standard cloning probes having melting temperatures between 40 degrees techniques familiar to those skilled in the art. Celsius and 80 degrees Celsius may also be used provided that specificity is not lost. 0118. Therefore, an object of the invention is an isolated, purified, or recombinant polynucleotide comprising a nucle 0112 The oligonucleotide should preferably be labeled otide Sequence Selected from the group consisting of cDNA with gamma PATP (specific activity 6000 Ci/mmole) and inserts of the deposited clone pool. Moreover, preferred T4 polynucleotide kinase using commonly employed tech polynucleotides of the invention include purified, isolated, niques for labeling oligonucleotides. Other labeling tech or recombinant GENSET cDNAs consisting of, consisting niques can also be used. Unincorporated label should pref essentially of, or comprising a nucleotide Sequence Selected erably be removed by gel filtration chromatography or other from the group consisting of cDNA inserts of the deposited established methods. The amount of radioactivity incorpo clone pool. rated into the probe should be quantified by measurement in a scintillation counter. Preferably, specific activity of the cDNA Sequences of the Invention resulting probe should be approximately 4x10 dpm/pmole. 0119) Another object of the invention is a purified, iso 0113. The bacterial culture containing the pool of full lated, or recombinant polynucleotide comprising a nucle length clones should preferably be thawed and 100 ul of the otide Sequence Selected from the group consisting of Stock used to inoculate a sterile culture flask containing 25 sequences of SEQ ID NOS:1-169, 339-455, 561-784, ml of sterile L-broth containing amplicillin at 100 ug/ml. The complementary Sequences thereto, and fragments thereof. culture should preferably be grown to saturation at 37 Moreover, preferred polynucleotides of the invention degrees Celsius, and the Saturated culture should preferably include purified, isolated, or recombinant GENSET cDNAS be diluted in fresh L-broth. Aliquots of these dilutions consisting of, consisting essentially of, or comprising a should preferably be plated to determine the dilution and Sequence Selected from the group consisting of SEQ ID volume which will yield approximately 5000 distinct and NOs: 1-169,339-455, 561-784. well-Separated colonies on Solid bacteriological media con 0120 Accordingly, the coding sequence (CDS) or open taining L-broth containing amplicillin at 100 ug/ml and agar reading frame (ORF) of each cDNA of the invention refers at 1.5% in a 150 mm petri dish when grown overnight at 37 to the nucleotide Sequence beginning with the first nucle degrees Celsius. Other known methods of obtaining distinct, otide of the Start codon and ending with the last nucleotide well-Separated colonies can also be employed. of the stop codon. Similarly, the 5' untranslated region (or 0114 Standard colony hybridization procedures should 5'UTR) of each cDNA of the invention refers to the nucle then be used to transfer the colonies to nitrocellulose filters otide Sequence Starting at nucleotide 1 and ending at the and lyse, denature and bake them. nucleotide immediately 5' to the first nucleotide of the start 0115 The filter is then preferably incubated at 65 degrees codon. The 3' untranslated region (or 3'UTR) of each cDNA Celsius for 1 hour with gentle agitation in 6xSSC (20x stock of the invention refers to the nucleotide Sequence Starting at is 175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH the nucleotide immediately 3' to the last nucleotide of the 7.0 with NaOH) containing 0.5% SDS, 100 pg/ml of yeast Stop codon and ending at the last nucleotide of the cDNA. RNA, and 10 mM EDTA (approximately 10 ml per 150 mm Untranslated Regions filter). Preferably, the probe is then added to the hybridiza tion mix at a concentration greater than or equal to 1x10' 0121. In addition, the invention concerns a purified, iso dpm/ml. The filter is then preferably incubated at 65 degrees lated, and recombinant nucleic acid comprising a nucleotide Celsius with gentle agitation overnight. The filter is then Sequence Selected from the group consisting of the 5' UTRS preferably washed in 500 ml of 2xSSC/0.1% SDS at room of sequences of SEQ ID NOS:1-169,339-455,561-784 and temperature with gentle Shaking for 15 minutes. A third Sequences of clone inserts of the deposited clone pool, wash with 0.1xSSC/0.5% SDS at 65 degrees Celsius for 30 Sequences complementary thereto, and allelic variants minutes to 1 hour is optional. The filter is then preferably thereof. The invention also concerns a purified, isolated, dried and Subjected to autoradiography for Sufficient time to and/or recombinant nucleic acid comprising a nucleotide visualize the positives on the X-ray film. Other known Sequence Selected from the group consisting of the 3'UTRS hybridization methods can also be employed. of sequences of SEQ ID NOS:1-169,339-455,561-784 and 0116. The positive colonies are picked, grown in culture, Sequences of clone inserts of the deposited clone pool, and plasmid DNA isolated using Standard procedures. The Sequences complementary thereto, and allelic variants clones can then be verified by restriction analysis, hybrid thereof. ization analysis, or DNA sequencing. The plasmid DNA 0.122 These polynucleotides may be used to detect the obtained using these procedures may then be manipulated presence of GENSET mRNA species in a biological sample using Standard cloning techniques familiar to those skilled in using either hybridization or RT-PCR techniques well the art. known to those skilled in the art. US 2006/0053498A1 Mar. 9, 2006

0123. In addition, these polynucleotides may be used as either the expression Signals contained in the regulatory regulatory molecules able to affect the processing and matu regions in the GENSET genes of the invention or, in ration of any polynucleotide including them (either a contrast, the Signals may be exogenous regulatory nucleic GENSET polynucleotide or an heterologous polynucle Sequences. Such a polynucleotide, when placed under the otide), preferably the localization, stability and/or transla Suitable expression signals, may also be inserted in a vector tion of Said polynucleotide including them (for a review on for its expression and/or amplification. UTRs see Decker and Parker, 1995, Derrigo et al., 2000). In particular, 3'UTRS may be used in order to control the 0128. Further included in the present invention are poly stability of heterologous mRNAS in recombinant vectors nucleotides encoding the polypeptides of the present inven using any methods known to those skilled in the art includ tion that are fused in frame to the coding Sequences for ing Makrides (1999) Protein Expr Purif November 1999; additional heterologous amino acid Sequences. Also 17(2):183-202), U.S. Pat. Nos. 5,925,564; 5,807,707 and included in the present invention are nucleic acids encoding 5,756.264, which disclosures are hereby incorporated by polypeptides of the present invention together with addi tional, non-coding Sequences, including, but not limited to, reference in their entireties. non-coding 5' and 3' sequences, vector Sequence, Sequences Coding Sequences used for purification, probing, or priming. For example, 0.124. Another object of the invention is an isolated, heterologous Sequences include transcribed, untranslated purified or recombinant polynucleotide comprising the cod Sequences that may play a role in transcription and mRNA ing Sequence of a Sequence Selected from the group con processing, Such as ribosome binding and Stability of sisting of sequences of SEQ ID NOS:1-169,339-455, 561 mRNA. The heterologous Sequences may alternatively com 784, clone inserts of the deposited clone pool, and variants prise additional coding Sequences that provide additional thereof. functionalities. Thus, a nucleotide Sequence encoding a polypeptide may be fused to a tag Sequence, Such as a 0.125 A further object of the invention is an isolated, Sequence encoding a peptide that facilitates purification or purified or recombinant polynucleotide encoding a polypep detection of the fused polypeptide. In certain preferred tide comprising a sequence Selected from the group consist embodiments of this aspect of the invention, the tag amino ing of sequences of SEQ ID NOs: 170-338, 456-560, acid Sequence is a hexa-histidine peptide, Such as the tag 785-918 and allelic variants thereof. Another object of the provided in a pCE vector (QIAGEN), or in any of a number invention is an isolated, purified or recombinant polynucle of additional, commercially available vectors. For instance, otide encoding a polypeptide comprising a Sequence hexa-histidine provides for the convenient purification of the Selected from the group consisting of polypeptides encoded fusion protein (see, Gentz et al., 1989, Proc Natl Acad Sci by cDNA inserts of the deposited clone pool and allelic USA Feb;86(3):821-4, the disclosure of which is incorpo variants thereof. rated by reference in its entirety). The “HA' tag is another 0126. It will be appreciated that should the extent of the peptide useful for purification which corresponds to an coding Sequence differ from that indicated in the appended epitope derived from the influenza hemagglutinin protein Sequence listing as a result of a Sequencing error, reverse (see, Wilson et al., 1984, Cell Jul;37(3):767-78, the disclo transcription or amplification error, mRNA splicing, post Sure of which is incorporated by reference in its entirety). AS translational modification of the encoded protein, enzymatic discussed below, other Such fusion proteins include a cleavage of the encoded protein, or other biological factors, GENSET polypeptide fused to Fc at the N- or C-terminus. one skilled in the art would be readily able to identify the 0.129 Suitable recombinant vectors that contain a poly extent of the coding Sequences in the Sequences of SEQ ID nucleotide Such as described herein are disclosed elsewhere NOS:1-169,339-455, 561-784. Accordingly, the scope of in the specification. Expression vectors encoding GENSET any claims herein relating to nucleic acids containing the polypeptides or fragments thereof are described in the coding sequence of one of SEQ ID NOS:1-169,339-455, section entitled “Preparation of the polypeptides”. 561-784 is not to be construed as excluding any readily identifiable variations from or equivalents to the coding Regulatory Sequences of the Invention Sequences described in the appended Sequence listing. Equivalents includes any alterations in a nucleotide coding 0.130. As mentioned, the genomic sequence of GENSET Sequence that does not result in an amino acid change, or that genes contain regulatory Sequences in the non-coding results in a conservative amino acid Substitution, as defined 5'-flanking region and possibly in the non-coding 3'-flanking below, in the polypeptide encoded by the nucleotide region that border the GENSET polypeptide coding regions Sequence. Similarly, should the extent of the polypeptides containing the exons of these genes. differ from those indicated in the appended Sequence listing 0131 Polynucleotides derived from GENSET polynucle as a result of any of the preceding factors, the Scope of otide 5' and 3' regulatory regions are useful in order to detect claims relating to polypeptides comprising the amino acid the presence of at least a copy of a genomic nucleotide sequence of the polypeptides of SEQ ID NOs: 170-338, sequence of the GENSET gene or a fragment thereof in a test 456-560, 785-918 is not to be construed as excluding any Sample. readily identifiable variations from or equivalents to the Sequences described in the appended Sequence listing. Preferred Regulatory Sequences 0127. The above-disclosed polynucleotides that contain 0132 Polynucleotides carrying the regulatory elements the coding sequence of the GENSET genes may be located at the 5' end and at the 3' end of GENSET polypep expressed in a desired host cell or a desired host organism, tide coding regions may be advantageously used to control, when this polynucleotide is placed under the control of e.g., the transcriptional and translational activity of a heter Suitable expression signals. The expression Signals may be ologous polynucleotide of interest. US 2006/0053498A1 Mar. 9, 2006

0.133 Thus, the present invention also concerns a purified tory active fragments or variants thereof. More preferred or isolated nucleic acid comprising a polynucleotide which 5'-regulatory polynucleotides of the invention include is Selected from the group consisting of the 5' and 3 Sequences Selected from the group consisting of 5'-UTRS of GENSET polynucleotide regulatory regions, Sequences sequences of SEQ ID NOS:1-169, 339-455, 561-784, complementary thereto, regulatory active fragments and 5'-UTRs of clone inserts of the deposited clone pool, regu variants thereof. The invention also pertains to a purified or latory active fragments and variants thereof. isolated nucleic acid comprising a polynucleotide having at least 95% nucleotide identity with a polynucleotide selected 0140 Preferred 3'-regulatory polynucleotide of the from the group consisting of GENSET polynucleotide 5' and invention include 3'-UTRs of GENSET cDNAs, or regula 3' regulatory regions, advantageously 99% nucleotide iden tory active fragments or variants thereof. More preferred tity, preferably 99.5% nucleotide identity and most prefer 3'-regulatory polynucleotides of the invention include ably 99.8% nucleotide identity with a polynucleotide Sequences Selected from the group consisting of 3'-UTRS of selected from the group consisting of GENSET polynucle sequences of SEQ ID NOS:1-169, 339-455, 561-784, otide 5' and 3' regulatory regions, Sequences complementary 3'-UTRs of clone inserts of the deposited clone pool, regu thereto, Variants and regulatory active fragments thereof. latory active fragments and variants thereof. 0134) Another object of the invention consists of purified, 0.141. A further object of the invention consists of a isolated or recombinant nucleic acids comprising a poly purified or isolated nucleic acid comprising: nucleotide that hybridizes, under the Stringent hybridization 0.142 a) a polynucleotide comprising a 5' regulatory conditions defined herein, with a polynucleotide Selected nucleotide Sequence Selected from the group consisting of: from the group consisting of the nucleotide Sequences of GENSET polynucleotide 5' and 3' regulatory regions, 0.143 (i) a nucleotide sequence comprising a polynucle Sequences complementary thereto, Variants and regulatory otide of a GENSET polynucleotide 5' regulatory region or a active fragments thereof. complementary Sequence thereto; 0135 Preferred fragments of 5" regulatory regions have a 0144 (ii) a nucleotide sequence comprising a polynucle length of about 1500 or 1000 nucleotides, preferably of otide having at least 95% of nucleotide identity with the about 500 nucleotides, more preferably about 400 nucle nucleotide sequence of a GENSET polynucleotide 5' regu otides, even more preferably 300 nucleotides and most latory region or a complementary Sequence thereto; preferably about 200 nucleotides. 0145 (iii) a nucleotide sequence comprising a polynucle 0.136 Preferred fragments of 3' regulatory regions are at otide that hybridizes under Stringent hybridization condi least 20, 50, 100, 150, 200, 300 or 400 bases in length. tions with the nucleotide sequence of a GENSET polynucle “Regulatory active” polynucleotide derivatives of the 5' or 3' otide 5' regulatory region or a complementary Sequence regulatory region are polynucleotides comprising or alter natively consisting of a fragment of Said polynucleotide thereto, and which is functional as a regulatory region for expressing a 0146 (iv) a regulatory active fragment or variant of the recombinant polypeptide or a recombinant polynucleotide in polynucleotides in (i), (ii) and (iii); a recombinant cell host. It could act either as an enhancer or as a repressor. For the purpose of the invention, a nucleic 0147 b) a nucleic acid molecule encoding a desired acid or polynucleotide is “functional” as a regulatory region polypeptide or a nucleic acid molecule of interest, wherein for expressing a recombinant polypeptide or a recombinant Said nucleic acid molecule is operably linked to the poly polynucleotide if Said regulatory polynucleotide contains nucleotide defined in (a); and nucleotide Sequences which contain transcriptional and 0.148 c) optionally, a polynucleotide comprising a translational regulatory information, and Such Sequences are 3'-regulatory polynucleotide, preferably a 3'-regulatory “operably linked' to nucleotide Sequences which encode the polynucleotide of a GENSET gene. desired polypeptide or the desired polynucleotide. 0149. In a specific embodiment, the nucleic acid defined 0.137 The regulatory polynucleotides of the invention above includes the 5'-UTR of a GENSET cDNA, or a may be prepared from the nucleotide sequence of GENSET regulatory active fragment or variant thereof. genomic or cDNA sequence, for example, by cleavage using suitable restriction enzymes, or by PCR. The regulatory 0150. In a second specific embodiment, the nucleic acid polynucleotides may also be prepared by digestion of a defined above includes the 3'-UTR of a GENSET cDNA, or GENSET gene-containing genomic clone by an exonuclease a regulatory active fragment or variant thereof. enzyme, such as Bal31 (Wabiko et al., DNA 5(4):305-14 0151. The regulatory polynucleotide of the 5' regulatory (1986), the disclosure of which is incorporated by reference region, or its regulatory active fragments or variants, is in its entirety). These regulatory polynucleotides can also be operably linked at the 5'-end of the nucleic acid molecule prepared by nucleic acid chemical Synthesis, as described encoding the desired polypeptide or nucleic acid molecule of elsewhere in the Specification. interest. 0.138. The regulatory polynucleotides according to the invention may be part of a recombinant expression vector 0152 The regulatory polynucleotide of the 3' regulatory that may be used to express a coding Sequence in a desired region, or its regulatory active fragments or variants, is host cell or host organism. The recombinant expression advantageously operably linked at the 3'-end of the nucleic vectors according to the invention are described elsewhere acid molecule encoding the desired polypeptide or nucleic in the Specification. acid molecule of interest. 0139 Preferred 5'-regulatory polynucleotides of the 0153. The desired polypeptide encoded by the above invention include 5'-UTRs of GENSET cDNAs, or regula described nucleic acid may be of various nature or origin, US 2006/0053498A1 Mar. 9, 2006 encompassing proteins of prokaryotic viral or eukaryotic variants' throughout the instant application. That is, all origin. Among the polypeptides expressed under the control possible polynucleotide sequences that encode the GENSET of a GENSET polynucleotide regulatory region include polypeptides of the present invention are contemplated. This bacterial, fungal or viral antigens. Also encompassed are includes the genetic code and Species-specific codon pref eukaryotic proteins Such as intracellular proteins, Such as erences known in the art. Thus, it would be routine for one “house-keeping proteins, membrane-bound proteins, Such skilled in the art to generate the degenerate variants as mitochondrial membrane-bound proteins and cell Surface described above, for instance, to optimize codon expression receptors, and Secreted proteins Such as endogenous media for a particular host (e.g., change codons in the human torS Such as cytokines. The desired polypeptide may be an mRNA to those preferred by other mammalian or bacterial heterologous polypeptide or a GENSET polypeptide, espe host cells). cially a protein with an amino acid Sequence Selected from the group consisting of sequences of SEQ ID NOs: 170-338, 0159. Nucleotide changes present in a variant polynucle 456-560, 785-918, fragments and variants thereof. otide may be Silent, which means that they do not alter the amino acids encoded by the polynucleotide. However, 0154) The desired nucleic acids encoded by the above nucleotide changes may also result in amino acid Substitu described polynucleotides, usually an RNA molecule, may tions, additions, deletions, fusions and truncations in the be complementary to a desired coding polynucleotide, for polypeptide encoded by the reference Sequence. The Substi example to a GENSET coding Sequence, and thus useful as tutions, deletions or additions may involve one or more an antisense polynucleotide. Such a polynucleotide may be nucleotides. The variants may be altered in coding or included in a recombinant expression vector in order to non-coding regions or both. Alterations in the coding express the desired polypeptide or the desired nucleic acid in regions may produce conservative or non-conservative host cell or in a host organism. Suitable recombinant vectors amino acid Substitutions, deletions or additions. In the that contain a polynucleotide Such as described herein are context of the present invention, preferred embodiments are disclosed elsewhere in the Specification. those in which the polynucleotide variants encode polypep tides which retain Substantially the same biological proper Polynucleotide Variants ties or activities as the GENSET protein. More preferred O155 The invention also relates to variants of the poly polynucleotide variants are those containing conservative nucleotides described herein and fragments thereof. “Vari Substitutions. ants of polynucleotides, as the term is used herein, are polynucleotides that differ from a reference polynucleotide. Similar Polynucleotides Generally, differences are limited So that the nucleotide 0160 Other embodiments of the present invention pro Sequences of the reference and the variant are closely similar vide a purified, isolated or recombinant polynucleotide overall and, in many regions, identical. The present inven which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or tion encompasses both allelic variants and degenerate Vari 99% identical to a polynucleotide selected from the group antS. consisting of sequences of SEQ ID NOS:1-169,339-455, 0156 Examples of variant sequences of polynucleotides 561-784 and the clone inserts of the deposited clone pool. of the invention are given in the appended Sequence listing. The above polynucleotides are included regardless of Specifically, Table I includes Sequences for which a plurality whether they encode a polypeptide having a GENSET of closely related Sequences, e.g. Variants, are provided. biological activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having Allelic Variants activity, one of skill in the art would still know how to use O157. A variant of a polynucleotide may be a naturally the nucleic acid molecule, for instance, as a hybridization occurring variant Such as a naturally occurring allelic Vari probe or primer. Uses of the nucleic acid molecules of the ant, or it may be a variant that is not known to occur present invention that do not encode a polypeptide having naturally. By an “allelic variant' is intended one of several GENSET activity include, inter alia, isolating a GENSET alternate forms of a gene occupying a given locus on a gene or allelic variants thereof from a DNA library, and chromosome of an organism (see Lewin, 1990), the disclo detecting GENSET mRNA expression in biological samples Sure of which is incorporated by reference in its entirety. suspected of containing GENSET mRNA or DNA, e.g., by Diploid organisms may be homozygous or heterozygous for Northern Blot or PCR analysis. an allelic form. Non-naturally occurring variants of the 0.161 The present invention is further directed to poly polynucleotide may be made by art-known mutagenesis nucleotides having sequences at least 50%. 60%, 70%, 80%, techniques, including those applied to polynucleotides, cells 90%, 95%, 96%, 97%, 98% or 99% identity to a polynucle or organisms. See, for example, Table I, which includes otide Selected from the group consisting of Sequences of Sequences for which a plurality of closely related Sequences, SEQ ID NOS:1-169,339-455, 561-784 and clone inserts of e.g. allelic variants of a single gene, are provided. the deposited clone pool, where said polynucleotide do, in fact, encode a polypeptide having a GENSET biological Degenerate Variant activity. Of course, due to the degeneracy of the genetic 0158. In addition to the isolated polynucleotides of the code, one of ordinary skill in the art will immediately present invention, and fragments thereof, the invention recognize that a large number of the polynucleotides at least further includes polynucleotides which comprise a sequence 50%. 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% substantially different from those described above but identical to a polynucleotide Selected from the group con which, due to the degeneracy of the genetic code, Still sisting of sequences of SEQ ID NOS:1-169,339-455, 561 encode a GENSET polypeptide of the present invention. 784 and clone inserts of the deposited clone pool will encode These polynucleotide variants are referred to as “degenerate a polypeptide having biological activity. In fact, Since US 2006/0053498A1 Mar. 9, 2006 degenerate variants of these nucleotide Sequences all encode tary to a sequence Selected from the group consisting of the same polypeptide, this will be clear to the skilled artisan sequences of SEQ ID NOS:1-169, 339-455, 561-784, even without performing the above described comparison Sequences of clone inserts of the deposited clone pool and assay. It will be further recognized in the art that, for Such fragments thereof. Such isolated molecules, particularly nucleic acid molecules that are not degenerate variants, a DNA molecules, are useful as probes for gene mapping and reasonable number will also encode a polypeptide having for identifying GENSET mRNA in a biological sample, for biological activity. This is because the skilled artisan is fully instance, by PCR or Northern blot analysis. aware of amino acid Substitutions that are either less likely or not likely to significantly affect protein function (e.g., Polynucleotide Fragments replacing one aliphatic amino acid with a Second aliphatic amino acid), as further described below. By a polynucleotide 0166 The present invention is further directed to poly having a nucleotide Sequence at least, for example, 95% nucleotides encoding portions or fragments of the nucleotide “identical” to a reference nucleotide Sequence of the present -Sequences described herein. Uses for the polynucleotide invention, it is intended that the nucleotide Sequence of the fragments of the present invention include probes, primers, polynucleotide is identical to the reference Sequence except molecular weight markers and for expressing the polypep that the polynucleotide Sequence may include up to five tide fragments of the present invention. Fragments include point mutations per each 100 nucleotides of the reference portions of polynucleotides Selected from the group consist nucleotide Sequence encoding the GENSET polypeptide. In ing of a) the sequences of SEQ ID NOs: 1-169,339-455, other words, to obtain a polynucleotide having a nucleotide 561-784, b) genomic GENSET sequences, c) the polynucle Sequence at least 95% identical to a reference nucleotide otides encoding a polypeptide Selected from the group Sequence, up to 5% of the nucleotides in the reference consisting of the sequences of SEQ ID NOs: 170-338, 456 Sequence may be deleted, inserted, or Substituted with 560, 785-918, d) the sequences of clone inserts of the another nucleotide. The query Sequence may be an entire deposited clone pool, and e) the polynucleotides encoding Sequence Selected from the group consisting of Sequences of the polypeptides encoded by the clone inserts of the depos SEQ ID NOS:1-169,339-455, 561-784 and sequences of ited clone pool. Particularly included in the present inven clone inserts of the deposited clone pool, or the ORF (open tion is a purified or isolated polynucleotide comprising at reading frame) of a polynucleotide sequence Selected from least 8 consecutive bases of a polynucleotide of the present Said group, or any fragment Specified as described herein. invention. In one aspect of this embodiment, the polynucle Hybridizing Polynucleotides otide comprises at least 10, 12, 15, 18, 20, 25, 28, 30,35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 800, 1000, 1500, or 0162. In another aspect, the invention provides an iso 2000 consecutive nucleotides of a polynucleotide of the lated or purified nucleic acid molecule comprising a poly present invention. nucleotide which hybridizes under stringent hybridization conditions to any polynucleotide of the present invention 0167. In addition to the above preferred polynucleotide using any methods known to those skilled in the art includ sizes, further preferred Sub-genuses of polynucleotides com ing those disclosed herein and in particular in the “To find prise at least 8 nucleotides, wherein “at least 8” is defined as Similar Sequences' Section. Also contemplated are nucleic any integer between 8 and the integer representing the 3' acid molecules that hybridize to the polynucleotides of the most nucleotide position as Set forth in the Sequence listing present invention at lower Stringency hybridization condi or elsewhere herein. Further included as preferred poly tions, preferably at moderate or low Stringency conditions as nucleotides of the present invention are polynucleotide defined herein. Such hybridizing polynucleotides may be of fragments at least 8 nucleotides in length, as described at least 15, 18, 20, 23, 25, 28, 30, 35, 40, 50, 75, 100, 200, above, that are further specified in terms of their 5' and 3' position. The 5' and 3' positions are represented by the 300, 500 or 1000 nucleotides in length. position numbers Set forth in the appended Sequence listing. 0163 Of particular interest are polynucleotides hybrid For allelic, degenerate and other variants, position 1 is izing to any polynucleotide of the invention and encoding defined as the 5' most nucleotide of the ORF, i.e., the GENSET polypeptides, particularly GENSET polypeptides nucleotide “A” of the start codon with the remaining nucle exhibiting a GENSET biological activity. otides numbered consecutively. Therefore, every combina 0164. Of course, a polynucleotide which hybridizes only tion of a 5' and 3' nucleotide position that a polynucleotide to poly A+ Sequences (Such as any 3' terminal polyA+ tract fragment of the present invention, at least 8 contiguous of a cDNA shown in the Sequence listing), or to a 5' nucleotides in length, could occupy on a polynucleotide of complementary stretch of T (or U) residues, would not be the invention is included in the invention as an individual included in the definition of “polynucleotide,” since such a Species. The polynucleotide fragments specified by 5' and 3 polynucleotide would hybridize to any nucleic acid mol positions can be immediately envisaged and are therefore ecule containing a poly(A) stretch or the complement not individually listed Solely for the purpose of not unnec thereof (e.g., practically any double-Stranded cDNA clone essarily lengthening the Specification. generated using oligo dT as a primer). 0.168. It is noted that the above species of polynucleotide Complementary Polynucleotides fragments of the present invention may alternatively be described by the formula "a to b”; where “a” equals the 5' 0.165. The invention further provides isolated nucleic most nucleotide position and “b' equals the 3' most nucle acid molecules having a nucleotide Sequence fully comple otide position of the polynucleotide; and further where “a” mentary to any polynucleotide of the invention. The present equals an integer between 1 and the number of nucleotides invention encompasses a purified, isolated or recombinant of the polynucleotide Sequence of the present invention polynucleotide having a nucleotide Sequence complemen minus 8, and where “b' equals an integer between 9 and the US 2006/0053498A1 Mar. 9, 2006 number of nucleotides of the polynucleotide Sequence of the Sequences, the cDNA sequences and the Sequences fully present invention; and where “a” is an integer Smaller then complementary thereto. Another object of the invention is a “b” by at least 8. purified, isolated, or recombinant polynucleotide comprising 0169. Therefore, the present invention encompasses iso the nucleotide Sequence of a Sequence Selected from the lated, purified, or recombinant polynucleotides which con group consisting of the sequences of SEQ ID NOS:1-169, sist of, consist essentially of, or comprise a contiguous span 339-455, 561-784, sequences of clone inserts of the depos of at least 8, 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, ited clone pool, Sequences fully complementary thereto, 100, 150, 200, 300, 400, 500, 1000 or 2000 nucleotides of allelic variants thereof, and fragments thereof. Moreover, a Sequence Selected from the group consisting of the preferred probes and primers of the invention include puri sequences of SEQ ID NOS:1-169,339-455, 561-784 and fied, isolated, or recombinant GENSET cDNAs consisting Sequences fully complementary thereto. of, consisting essentially of, or comprising the Sequences of SEQ ID NOS:1-169,339-455, 561-784 and sequences of 0170 Other preferred fragments of the invention are clone inserts of the deposited clone pool. Particularly pre polynucleotides comprising polynucleotides encoding ferred probes and primers of the invention include isolated, domains of polypeptides. Such fragments may be used to purified, or recombinant polynucleotides comprising a con obtain other polynucleotides encoding polypeptides having tiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, similar domains using hybridization or RT-PCR techniques. 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of a Alternatively, these fragments may be used to express a Sequence Selected from the group consisting of the polypeptide domain which may have a specific biological sequences of SEQ ID NOS:1-169,339-455,561-784 and the property. Thus, another object of the invention is an isolated, Sequences fully complementary thereto. purified or recombinant polynucleotide encoding a polypep tide consisting of, consisting essentially of, or comprising a Design of Primers and Probes contiguous span of at least 5, 6, 8, 10, 12, 15, 20, 25, 30, 35, 0.174. A probe or a primer according to the invention has 40, 50, 60, 75, 100, 150 or 200 consecutive amino acids of between 8 and 1000 nucleotides in length, or is specified to a Sequence Selected from the group consisting of the be at least 12, 15, 18, 20, 25, 35, 40, 50, 60, 70, 80, 100, 250, sequences of SEQ ID NOs: 170-338, 456-560, 785-918, to 500 or 1000 nucleotides in length. More particularly, the the extent that a contiguous span of these lengths is consis length of these probes and primers can range from 8, 10, 15, tent with the lengths of Said Selected Sequence, where said 20, or 30 to 100 nucleotides, preferably from 10 to 50, more contiguous span comprises at least 1, 2, 3, 5, or 10 of the preferably from 15 to 30 nucleotides. Shorter probes and amino acid positions of a domain of Said Selected Sequence. primers tend to lack Specificity for a target nucleic acid The present invention also encompasses isolated, purified or Sequence and generally require cooler temperatures to form recombinant polynucleotides encoding a polypeptide com sufficiently stable hybrid complexes with the template. prising a contiguous span of at least 5, 6, 8, 10, 12, 15, 20, Longer probes and primers are expensive to produce and can 25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 consecutive amino sometimes self-hybridize to form hairpin structures. The acids of a Sequence Selected from the group consisting of appropriate length for primers and probes under a particular sequences of SEQ ID NOs: 170-338, 456-560, 785-918, to Set of assay conditions may be empirically determined by the extent that a contiguous span of these lengths is consis one of skill in the art. The formation of stable hybrids tent with the lengths of Said Selected Sequence, where said depends on the melting temperature (Tm) of the DNA. The contiguous span is a domain of Said Selected Sequence. The Tm depends on the length of the primer or probe, the ionic present invention also encompasses isolated, purified or strength of the solution and the G+C content. The higher the recombinant polynucleotides encoding a polypeptide com G+C content of the primer or probe, the higher is the melting prising a domain of a Sequence Selected from the group temperature because G:C pairs are held by three H bonds consisting of the sequences of SEQ ID NOs: 170-338, 456 whereas A:T pairs have only two. The GC content in the 560, 785-918. probes of the invention usually ranges between 10 and 75%, 0171 The present invention further encompasses any preferably between 35 and 60%, and more preferably combination of the polynucleotide fragments listed in this between 40 and 55%. Section. 0.175 For amplification purposes, pairs of primers with approximately the same Tm are preferable. PrimerS may be Oligonucleotide Primers and Probes designed using the OSP software (Hillier and Green, 1991), 0172 The present invention also encompasses fragments the disclosure of which is incorporated by reference in its of GENSET polynucleotides for use as primers and probes. entirety, based on GC content and melting temperatures of Polynucleotides derived from the GENSET genomic and oligonucleotides, or using PC-Rare (http://bioinformatics cDNA sequences are useful in order to detect the presence weizmann.ac.il/software/PC-Rare/doc/manuel.html) based of at least a copy of a GENSET polynucleotide or fragment, on the octamer frequency disparity method (Griffais et al., complement, or variant thereof in a test Sample. 1991), the disclosure of which is incorporated by reference in its entirety. DNA amplification techniques are well known Structural Definition to those skilled in the art. Amplification techniques that can 0173 Any polynucleotide of the invention may be used be used in the context of the present invention include, but as a primer or probe. Particularly preferred probes and are not limited to, the ligase chain reaction (LCR) described primers of the invention include isolated, purified, or recom in EP-A-320 308, WO 9320227 and EP-A-439 182, the binant polynucleotides comprising a contiguous span of at polymerase chain reaction (PCR, RT-PCR) and techniques least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, Such as the nucleic acid Sequence based amplification 150, 200, 500, or 1000 nucleotides of a sequence selected (NASBA) described in Guatelliet al.(1990) and in Compton from the group consisting of the GENSET genomic (1991), Q-beta amplification as described in European US 2006/0053498A1 Mar. 9, 2006

Patent Application No 4544610, strand displacement ampli sites. PCR has further been described in several patents fication as described in Walker et al. (1996) and EPA 684 including U.S. Pat. Nos. 4,683,195; 4,683.202; and 4,965, 315 and, target mediated amplification as described in PCT 188, the disclosures of which are incorporated herein by Publication WO 9322461, the disclosures of which are reference in their entireties. incorporated by reference in their entireties. Preparation of Primers and Probes 0176 LCR and Gap LCR are exponential amplification techniques, both depending on DNA ligase to join adjacent 0179 Primers and probes can be prepared by any suitable primerS annealed to a DNA molecule. In Ligase Chain method, including, for example, cloning and restriction of Reaction (LCR), probe pairs are used which include two appropriate Sequences and direct chemical Synthesis by a primary (first and Second) and two Secondary (third and method Such as the phosphodiester method of Narang et fourth) probes, all of which are employed in molar excess to al.(1979), the phosphodiester method of Brown et al.(1979), target. The first probe hybridizes to a first segment of the the diethylphosphoramidite method of Beaucage et al.(1981) target Strand and the Second probe hybridizes to a Second and the solid support method described in EP 0 707 592, Segment of the target Strand, the first and Second Segments which disclosures are hereby incorporated by reference in being contiguous So that the primary probes abut one their entireties. another in 5" phosphate-3'hydroxyl relationship, and So that 0180 Detection probes are generally nucleic acid a ligase can covalently fuse or ligate the two probes into a Sequences or uncharged nucleic acid analogs Such as, for fused product. In addition, a third (Secondary) probe can example peptide nucleic acids which are disclosed in Inter hybridize to a portion of the first probe and a fourth national Patent Application WO92/20702, morpholino ana (Secondary) probe can hybridize to a portion of the Second logs which are described in U.S. Pat. Nos. 5,185,444; probe in a Similar abutting fashion. Of course, if the target 5,034,506 and 5,142,047, which disclosures are hereby is initially double Stranded, the Secondary probes also will incorporated by reference in their entireties. The probe may hybridize to the target complement in the first instance. Once have to be rendered “non-extendable' in that additional the ligated Strand of primary probes is separated from the dNTPs cannot be added to the probe. In and of themselves target strand, it will hybridize with the third and fourth analogs usually are non-extendable and nucleic acid probes probes, which can be ligated to form a complementary, can be rendered non-extendable by modifying the 3' end of Secondary ligated product. It is important to realize that the the probe Such that the hydroxyl group is no longer capable ligated products are functionally equivalent to either the of participating in elongation. For example, the 3' end of the target or its complement. By repeated cycles of hybridiza probe can be functionalized with the capture or detection tion and ligation, amplification of the target Sequence is label to thereby consume or otherwise block the hydroxyl achieved. A method for multiplex LCR has also been group. Alternatively, the 3' hydroxyl group Simply can be described (WO9320227), the disclosure of which is incor cleaved, replaced or modified, U.S. patent application Ser. porated by reference in its entirety. Gap LCR (GLCR) is a No. 07/049,061 filed Apr. 19, 1993, which disclosure is version of LCR where the probes are not adjacent but are hereby incorporated by reference in its entirety, describes Separated by 2 to 3 bases. modifications, which can be used to render a probe non 0177 For amplification of mRNAS, it is within the scope extendable. Labeling of Probes of the present invention to reverse transcribe mRNA into 0181 Any of the polynucleotides of the present invention cDNA followed by polymerase chain reaction (RT-PCR); or, can be labeled, if desired, by incorporating any label known to use a single enzyme for both Steps as described in U.S. in the art to be detectable by Spectroscopic, photochemical, Pat. No. 5,322,770 or, to use Asymmetric Gap LCR (RT biochemical, immunochemical, or chemical means. For AGLCR) as described by Marshall et al.(1994), the disclo example, useful labels include radioactive Substances Sures of which are incorporated by reference in its entireties. (including, P, S, H, °I), fluorescent dyes (including, AGLCR is a modification of GLCR that allows the ampli 5-bromodesoxyuridin, fluorescein, acetylaminofluorene, fication of RNA. digoxigenin) or biotin. Preferably, polynucleotides are 0.178 PCR technology is the preferred amplification labeled at their 3' and 5' ends. Examples of non-radioactive technique used in the present invention. A variety of PCR labeling of nucleic acid fragments are described in the techniques are familiar to those skilled in the art. For a French patent No. FR-7810975 or by Urdea et al (1988) or review of PCR technology, see White (1997), Erlich (1992) Sanchez-Pescador et al (1988), which disclosures are hereby and the publication entitled “PCR Methods and Applica incorporated by reference in their entireties. In addition, the tions” (1991) Cold Spring Harbor Laboratory Press), the probes according to the present invention may have struc disclosures of which are incorporated by reference in their tural characteristics Such that they allow the Signal ampli entireties. In each of these PCR procedures, PCR primers on fication, Such structural characteristics being, for example, either Side of the nucleic acid Sequences to be amplified are branched DNA probes as those described by Urdea et al. in added to a Suitably prepared nucleic acid Sample along with 1991 or in the European patent No. EP 0 225807 (Chiron), dNTPs and a thermostable polymerase such as Taq poly which disclosures are hereby incorporated by reference in merase, Pfu polymerase, Tth polymerase or Vent poly their entireties. merase. The nucleic acid in the Sample is denatured and the 0182. The detectable probe may be single stranded or PCR primers are specifically hybridized to complementary double Stranded and may be made using techniques known nucleic acid Sequences in the Sample. The hybridized prim in the art, including in Vitro transcription, nick translation, or erS are extended. Thereafter, another cycle of denaturation, kinase reactions. A nucleic acid Sample containing a hybridization, and extension is initiated. The cycles are Sequence capable of hybridizing to the labeled probe is repeated multiple times to produce an amplified fragment contacted with the labeled probe. If the nucleic acid in the containing the nucleic acid Sequence between the primer Sample is double Stranded, it may be denatured prior to US 2006/0053498A1 Mar. 9, 2006

contacting the probe. In Some applications, the nucleic acid which is insoluble, or can be made insoluble by a Subsequent Sample may be immobilized on a Surface Such as a nitro reaction. The Solid Support can be chosen for its intrinsic cellulose or nylon membrane. The nucleic acid Sample may ability to attract and immobilize the capture reagent. Alter comprise nucleic acids obtained from a variety of Sources, natively, the Solid phase can retain an additional receptor including genomic DNA, cDNA libraries, RNA, or tissue which has the ability to attract and immobilize the capture Samples. reagent. The additional receptor can include a charged Substance that is oppositely charged with respect to the 0183 Procedures used to detect the presence of nucleic capture reagent itself or to a charged Substance conjugated acids capable of hybridizing to the detectable probe include to the capture reagent. As yet another alternative, the recep well known techniques such as Southern blotting, Northern tor molecule can be any specific binding member which is blotting, dot blotting, colony hybridization, and plaque immobilized upon (attached to) the Solid Support and which hybridization. In Some applications, the nucleic acid capable has the ability to immobilize the capture reagent through a of hybridizing to the labeled probe may be cloned into Specific binding reaction. The receptor molecule enables the vectorS Such as expression vectors, Sequencing vectors, or in indirect binding of the capture reagent to a Solid Support Vitro transcription vectors to facilitate the characterization material before the performance of the assay or during the and expression of the hybridizing nucleic acids in the performance of the assay. The Solid phase thus can be a Sample. For example, Such techniques may be used to isolate plastic, derivatized plastic, magnetic or non-magnetic metal, and clone Sequences in a genomic library or cDNA library glass or Silicon Surface of a test tube, microtiter well, sheet, which are capable of hybridizing to the detectable probe as bead, microparticle, chip, sheep (or other Suitable animals) described herein. red blood cells, duracytes(E) and other configurations known Immobilization of Probes to those of ordinary skill in the art. The polynucleotides of 0184. A label can also be used to capture the primer, so the invention can be attached to or immobilized on a Solid as to facilitate the immobilization of either the primer or a Support individually or in groups of at least 2, 5, 8, 10, 12, primer eXtension product, Such as amplified DNA, on a Solid 15, 20, or 25 distinct polynucleotides of the invention to a Support. A capture label is attached to the primers or probes Single Solid Support. In addition, polynucleotides other than and can be a specific binding member which forms a binding those of the invention may be attached to the same Solid pair with the Solid phase reagent's Specific binding member Support as one or more polynucleotides of the invention. (e.g. biotin and Streptavidin). Therefore depending upon the Oligonucleotide Array type of label carried by a polynucleotide or a probe, it may be employed to capture or to detect the target DNA. Further, 0187. A substrate comprising a plurality of oligonucle it will be understood that the polynucleotides, primerS or otide primerS or probes of the invention may be used either probes provided herein, may, themselves, Serve as the cap for detecting or amplifying targeted Sequences in GENSET ture label. For example, in the case where a Solid phase genes, may be used for detecting mutations in the coding or reagent's binding member is a nucleic acid Sequence, it may in the non-coding Sequences of GENSET genes, and may be selected Such that it binds a complementary portion of a also be used to determine GENSET gene expression in primer or probe to thereby immobilize the primer or probe different contexts. Such as in different tissues, at different to the Solid phase. In cases where a polynucleotide probe Stages of a process (embryo development, disease treat itself Serves as the binding member, those skilled in the art ment), and in patients versus healthy individuals as will recognize that the probe will contain a Sequence or described elsewhere in the application. “tail” that is not complementary to the target. In the case 0188 As used herein, the term “array' means a one where a polynucleotide primer itself Serves as the capture dimensional, two dimensional, or multidimensional arrange label, at least a portion of the primer will be free to hybridize ment of nucleic acids of Sufficient length to permit specific with a nucleic acid on a Solid phase. DNA Labeling tech detection of gene expression. For example, the array may niques are well known to the skilled technician. contain a plurality of nucleic acids derived from genes 0185. The probes of the present invention are useful for whose expression levels are to be assessed. The array may a number of purposes. They can notably be used in Southern include a GENSET genomic DNA, a GENSET cDNA, hybridization to genomic DNA. The probes can also be used Sequences complementary thereto or fragments thereof. to detect PCR amplification products. They may also be used Preferably, the fragments are at least 12, 15, 18, 20, 25, 30, to detect mismatches in the GENSET gene or mRNA using 35, 40 or 50 nucleotides in length. More preferably, the other techniques. They may also be used for in situ hybrid fragments are at least 100 nucleotides in length. Even more ization. preferably, the fragments are more than 100 nucleotides in length. In Some embodiments the fragments may be more 0186. Any of the polynucleotides, primers and probes of than 500 nucleotides in length. the present invention can be conveniently immobilized on a Solid Support. The Solid Support is not critical and can be 0189 Any polynucleotide provided herein may be Selected by one skilled in the art. Thus, lateX particles, attached in overlapping areas or at random locations on the microparticles, magnetic beads, non-magnetic beads Solid Support. Alternatively the polynucleotides of the inven (including polystyrene beads), membranes (including nitro tion may be attached in an ordered array wherein each cellulose Strips), plastic tubes, walls of microtiter wells, polynucleotide is attached to a distinct region of the Solid glass or Silicon chips, sheep (or other Suitable animal's) red Support which does not overlap with the attachment Site of blood cells and duracytes are all Suitable examples. Suitable any other polynucleotide. Preferably, Such an ordered array methods for immobilizing nucleic acids on Solid phases of polynucleotides is designed to be “addressable” where the include ionic, hydrophobic, covalent interactions and the distinct locations are recorded and can be accessed as part of like. A Solid Support, as used herein, refers to any material an assay procedure. Addressable polynucleotide arrays typi US 2006/0053498A1 Mar. 9, 2006 2O cally comprise a plurality of different oligonucleotide probes Synthesized either enzymatically using techniques well that are coupled to a Surface of a Substrate in different known known to those skilled in the art including amplification or locations. The knowledge of the precise location of each hybridization-based methods as described herein, or chemi polynucleotides location makes these "addressable' arrayS cally. particularly useful in hybridization assayS. Any addressable array technology known in the art can be employed with the 0193 A variety of chemical methods of synthesizing polynucleotides of the invention. One particular embodi nucleic acids are known to those skilled in the art. In many ment of these polynucleotide arrays is known as the Gene of these methods, Synthesis is conducted on a Solid Support. chipsTM, and has been generally described in U.S. Pat. No. These included the 3' phosphoramidite methods in which the 5,143,854; PCT publications WO 90/15070 and 92/10092, 3' terminal base of the desired oligonucleotide is immobi which disclosures are hereby incorporated by reference in lized on an insoluble carrier. The nucleotide base to be added their entireties. These arrayS may generally be produced is blocked at the 5’ hydroxyl and activated at the 3' hydroxyl using mechanical Synthesis methods or light directed Syn So as to cause coupling with the immobilized nucleotide thesis methods which incorporate a combination of photo base. Deblocking of the new immobilized nucleotide com lithographic methods and Solid phase oligonucleotide Syn pound and repetition of the cycle will produce the desired thesis (Fodor et al., 1991), which disclosure is hereby polynucleotide. Alternatively, polynucleotides may be pre incorporated by reference in its entirety. The immobilization pared as described in U.S. Pat. No. 5,049,656, which dis of arrays of oligonucleotides on Solid Supports has been closure is hereby incorporated by reference in its entirety. In rendered possible by the development of a technology Some embodiments, Several polynucleotides prepared as generally identified as “Very Large Scale Immobilized Poly described above are ligated together to generate longer mer Synthesis” (VLSIPSTM) in which, typically, probes are polynucleotides having a desired Sequence. immobilized in a high density array on a Solid Surface of a chip. Examples of VLSIPSTM technologies are provided in Polypeptides of the Invention U.S. Pat. Nos. 5,143,854; and 5,412,087 and in PCT Pub 0194 The term “GENSET polypeptides” is used herein lications WO 90/15070, WO 92/10092 and WO95/11995, to embrace all of the proteins and polypeptides of the present which disclosures are hereby incorporated by reference in invention. The present invention encompasses GENSET their entireties, which describe methods for forming oligo polypeptides, including recombinant, isolated or purified nucleotide arrays through techniqueS Such as light-directed GENSET polypeptides consisting of, consisting essentially Synthesis techniques. In designing Strategies aimed at pro of, or comprising a Sequence Selected from the group Viding arrays of nucleotides immobilized on Solid Supports, consisting of SEQ ID NOs: 170-338, 456-560, 785-918 and further presentation Strategies were developed to order and the polypeptides encoded by human cDNAS contained in the display the oligonucleotide arrays on the chips in an attempt deposited clones. Other objects of the invention are polypep to maximize hybridization patterns and Sequence informa tides encoded by the polynucleotides of the invention as well tion. Examples of Such presentation Strategies are disclosed as fusion polypeptides comprising Such polypeptides. in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256, the disclosures of which are Polypeptide Variants incorporated herein by reference in their entireties. 0.195 The present invention further provides for 0.190 Consequently, the invention concerns an array of GENSET polypeptides encoded by allelic and splice vari nucleic acid molecules comprising at least one polynucle ants, orthologs, and/or Species homologues. Procedures otide of the invention, particularly a probe or primer as known in the art can be used to obtain, allelic variants, Splice described herein. Preferably, the invention concerns an array variants, orthologs, and/or species homologues of poly of nucleic acids comprising at least two polynucleotides of nucleotides encoding by polypeptides of the group consist the invention, particularly probes or primers as described ing of SEQ ID NOs: 170-338, 456-560, 785-918 and herein. Preferably, the invention concerns an array of nucleic polypeptides encoded by the clone inserts of the deposited acids comprising at least five polynucleotides of the inven clone pool, using information from the Sequences disclosed tion, particularly probes or primers as described herein. herein or the clones deposited with the ATCC. 0191) A preferred embodiment of the present invention is 0196. The polypeptides of the present invention also an array of polynucleotides of at least 12, 15, 18, 20, 25, 30, include polypeptides having an amino acid Sequence at least 35, 40, 50, 100 or 500 nucleotides in length which includes 50% identical, more preferably at least 60% identical, and at least 1, 2, 5, 10, 15, 20, 35, 50 or 100 sequences selected still more preferably 70%, 80%, 90%, 95%, 96%, 97%, 98% from the group consisting of the Sequences of SEQ ID or 99% identical to a polypeptide selected from the group NOS:1-169, 339-455, 561-784 and sequences of clone consisting of the sequences of SEQ ID NOs: 170-338, 456 inserts of the deposited clone pool, Sequences fully comple 560, 785-918 and those encoded by the clone inserts of the mentary thereto, and fragments thereof. deposited clone pool. By a polypeptide having an amino acid Methods of Making the Polynucleotides of the Invention Sequence at least, for example, 95% “identical’ to a query amino acid Sequence of the present invention, it is intended 0.192 The present invention also comprises methods of that the amino acid Sequence of the Subject polypeptide is making the polynucleotides of the invention, including the identical to the query Sequence except that the Subject polynucleotides of SEQ ID NOS:1-169,339-455,561-784, polypeptide Sequence may include up to five amino acid genomic DNA obtainable therefrom, or fragments thereof. alterations per each 100 amino acids of the query amino acid These methods comprise Sequentially linking together Sequence. In other words, to obtain a polypeptide having an nucleotides to produce the nucleic acids having the preced amino acid Sequence at least 95% identical to a query amino ing Sequences. Polynucleotides of the invention may be acid sequence, up to 5% (5 of 100) of the amino acid US 2006/0053498A1 Mar. 9, 2006 residues in the Subject Sequence may be inserted, deleted, 0201 Consequently, the present invention also comprises (indels) or Substituted with another amino acid. methods of making the polypeptides of the invention, par 0197) Further polypeptides of the present invention ticularly polypeptides encoded by the cDNAs of SEQ ID include polypeptides which have at least 90% similarity, NOS:1-169,339-455,561-784 or by the clone inserts of the more preferably at least 95% similarity, and still more deposited clone pool, genomic DNA obtainable therefrom, preferably at least 96%, 97%, 98% or 99% similarity to or fragments thereof and methods of making the polypep those described above. By a polypeptide having an amino tides of SEQ ID NOs: 170-338, 456-560, 785-918 or frag acid Sequence at least, for example, 95% "similar to a query ments thereof. The methods comprise Sequentially linking amino acid Sequence of the present invention, it is intended together amino acids to produce the nucleic polypeptides that the amino acid Sequence of the Subject polypeptide is having the preceding Sequences. In Some embodiments, the Similar (i.e. contains identical or equivalent amino acid polypeptides made by these methods are 150 amino acids or residues) to the query sequence except that the Subject leSS in length. In other embodiments, the polypeptides made polypeptide Sequence may include up to five amino acid by these methods are 120 amino acids or less in length. alterations per each 100 amino acids of the query amino acid Isolation Sequence. In other words, to obtain a polypeptide having an amino acid Sequence at least 95% similar to a query amino From Natural Sources acid sequence, up to 5% (5 of 100) of the amino acid 0202) The GENSET proteins of the invention may be residues in the Subject Sequence may be inserted, deleted, isolated from natural Sources, including bodily fluids, tissues (indels) or Substituted with another non-equivalent amino and cells, whether directly isolated or cultured cells, of acid. humans or non-human animals. Methods for extracting and purifying natural proteins are known in the art, and include 0198 These alterations of the reference sequence may the use of detergents or chaotropic agents to disrupt particles occur at the amino or carboxy terminal positions of the followed by differential extraction and separation of the reference amino acid Sequence or anywhere between those polypeptides by ion exchange chromatography, affinity terminal positions, interspersed either individually among chromatography, Sedimentation according to density, and residues in the reference Sequence or in one or more con gel electrophoresis. See, for example, "Methods in Enymol tiguous groups within the reference Sequence. The query ogy, Academic Press, 1993 for a variety of methods for Sequence may be an entire amino acid Sequence Selected purifying proteins, which disclosure is hereby incorporated from the group consisting of sequences of SEQID NOs: 170 by reference in its entirety. Polypeptides of the invention 338, 456-560, 785-918 and those encoded by the clone also can be purified from natural Sources using antibodies inserts of the deposited clone pool or any fragment Specified directed against the polypeptides of the invention, Such as as described herein. those described herein, in methods which are well known in 0199 The variant polypeptides described herein are the art of protein purification. included in the present invention regardless of whether they have their normal biological activity. This is because even From Recombinant Sources where a particular polypeptide molecule does not have 0203 Preferably, the GENSET polypeptides of the inven biological activity, one of skill in the art would still know tion are recombinantly produced using routine expression how to use the polypeptide, for instance, as a vaccine or to methods known in the art. The polynucleotide encoding the generate antibodies. Other uses of the polypeptides of the desired polypeptide is operably linked to a promoter into an present invention that do not have GENSET biological expression vector Suitable for any convenient host. Both activity include, inter alia, as epitope tags, in epitope map eukaryotic and prokaryotic host Systems are used in forming ping, and as molecular weight markers on SDS-PAGE gels recombinant polypeptides. The polypeptide is then isolated or on molecular Sieve gel filtration columns using methods from lysed cells or from the culture medium and purified to known to those of skill in the art. As described below, the the extent needed for its intended use. polypeptides of the present invention can also be used to raise polyclonal and monoclonal antibodies, which are use 0204 Any GENSET polynucleotide, including those ful in assays for detecting GENSET protein expression or as described in SEQID NOS:1-169,339-455,561-784, those of agonists and antagonists capable of enhancing or inhibiting clone inserts of the deposited clone pool, and allelic variants thereof may be used to express GENSET polypeptides. The GENSET protein function. Further, such polypeptides can nucleic acid encoding the GENSET polypeptide to be be used in the yeast two-hybrid system to “capture” expressed is operably linked to a promoter in an expression GENSET protein binding proteins, which are also candidate vector using conventional cloning technology. The agonists and antagonists according to the present invention GENSET insert in the expression vector may comprise the (see, e.g., Fields et al. 1989, which disclosure is hereby full coding sequence for the GENSET protein or a portion incorporated by reference in its entirety). thereof. For example, the GENSET derived insert may Preparation of the Polypeptides of the Invention encode a polypeptide comprising at least 6, 8, 10, 12, 15, 20, 0200. The polypeptides of the present invention can be 25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 consecutive amino prepared in any Suitable manner. Such polypeptides include acids of a GENSET protein selected from the group con isolated naturally occurring polypeptides, recombinantly sisting of sequences of SEQ ID NOs: 170-338, 456-560, produced polypeptides, Synthetically produced polypep 785-918 and polypeptides encoded by the clone inserts of tides, or polypeptides produced by a combination of these the deposited clone pool. methods. The polypeptides of the present invention are 0205 Consequently, a further embodiment of the present preferably provided in an isolated form, and may be partially invention is a method of making a polypeptide comprising or preferably substantially purified. a protein Selected from the group consisting of Sequences of US 2006/0053498A1 Mar. 9, 2006 22

SEQ ID NOs: 170-338, 456-560, 785-918 and polypeptides poly A Signal. The purified fragment obtained from the encoded by the clone inserts of the deposited clone pool, resulting PCR reaction is digested with Pst, blunt ended Said method comprising the Steps of: with an exonuclease, digested with Bgl II, purified and 0206 a) obtaining a cDNA comprising a sequence ligated to pXTl, now containing a poly A Signal and digested Selected from the group consisting of i) the sequences SEQ with BglII. ID NOs: 1-169,339-455,561-784, ii) the sequences of clone 0212. In another embodiment, it is often advantageous to inserts of the deposited clone pool one, iii) Sequences add to the recombinant polynucleotide additional nucleotide encoding one of the polypeptide of SEQ ID NOs: 170-338, Sequence which codes for Secretory or leader Sequences, 456-560, 785-918, and iv) sequences of polynucleotides pro-Sequences, Sequences which aid in purification, Such as encoding a polypeptide which is encoded by one of the clone multiple histidine residues, or an additional Sequence for insert of the deposited clone pool; Stability during recombinant production. 0207 b) inserting said cDNA in an expression vector 0213 As a control, the expression vector lacking a cDNA such that the cDNA is operably linked to a promoter; and insert is introduced into host cells or organisms. 0208 c) introducing said expression vector into a host 0214) Transfection of a GENSET expression vector into cell whereby Said host cell produces said polypeptide. mouse NTH 3T3 cells is but one embodiment of introducing polynucleotides into host cells. Introduction of a polynucle 0209. In one aspect of this embodiment, the method otide encoding a polypeptide into a host cell can be effected further comprises the Step of isolating the polypeptide. by calcium phosphate transfection, DEAE-dextran mediated Another embodiment of the present invention is a polypep transfection, cationic lipid-mediated transfection, electropo tide obtainable by the method described in the preceding ration, transduction, infection, or other methods. Such meth paragraph. ods are described in many Standard laboratory manuals, Such 0210. The expression vector is any of the mammalian, as Davis et al. (1986), which disclosure is hereby incorpo yeast, insect or bacterial expression Systems known in the rated by reference in its entirety. It is specifically contem art. Commercially available vectors and expression Systems plated that the polypeptides of the present invention may in are available from a variety of Suppliers including Genetics fact be expressed by a host cell lacking a recombinant Institute (Cambridge, Mass.), Stratagene (La Jolla, Calif.), VectOr. Promega (Madison, Wis.), and Invitrogen (San Diego, 0215 Recombinant cell extracts, or proteins from the Calif.). If desired, to enhance expression and facilitate culture medium if the expressed polypeptide is Secreted, are proper protein folding, the codon context and codon pairing then prepared and proteins Separated by gel electrophoresis. of the Sequence is optimized for the particular expression If desired, the proteins may be ammonium Sulfate precipi organism in which the expression vector is introduced, as tated or Separated based on size or charge prior to electro explained in U.S. Pat. No. 5,082,767, which disclosure is phoresis. The proteins present are detected using techniques hereby incorporated by reference in its entirety. Such as Coomassie or Silver Staining or using antibodies against the protein encoded by the GENSET cDNA of 0211. In one embodiment, the entire coding sequence of interest. Coomassie and Silver Staining techniques are famil a GENSET cDNA and the 3'UTR through the polyA signal iar to those skilled in the art. of the cDNA is operably linked to a promoter in the expression vector. Alternatively, if the nucleic acid encoding 0216) Proteins from the host cells or organisms contain a portion of the GENSET protein lacks a methionine to serve ing an expression vector which contains the GENSET cDNA as the initiation Site, an initiating methionine can be intro or a fragment thereof are compared to those from the control duced next to the first codon of the nucleic acid using cells or organism. The presence of a band from the cells conventional techniques. Similarly, if the insert from the containing the expression vector which is absent in control GENSET cDNA lacks a poly A signal, this sequence can be cells indicates that the GENSET cDNA is expressed. Gen added to the construct by, for example, Splicing out the Poly erally, the band corresponding to the protein encoded by the A signal from pSG5 (Stratagene) using BglI and Sal restric GENSET cDNA will have a mobility near that expected tion endonuclease enzymes and incorporating it into the based on the number of amino acids in the open reading mammalian expression vector pXT1 (Stratagene). pXT1 frame of the cDNA. However, the band may have a mobility contains the LTRS and a portion of the gag gene from different than that expected as a result of modifications Such Moloney Murine Leukemia Virus. The position of the LTRs as glycosylation, ubiquitination, or enzymatic cleavage. in the construct allow efficient stable transfection. The 0217. Alternatively, the GENSET polypeptide to be vector includes the Herpes Simplex Thymidine Kinase pro expressed may also be a product of transgenic animals, i.e., moter and the Selectable neomycin gene. The nucleic acid as a component of the milk of transgenic cows, goats, pigs encoding the GENSET protein or a portion thereof is or sheep which are characterized by Somatic or germ cells obtained by PCR from a vector containing a GENSET containing a nucleotide Sequence encoding the protein of cDNA Selected from the group consisting of the Sequences interest. of SEQ ID NOS:1-169,339-455, 561-784 and the clone inserts of the deposited clone pool using oligonucleotide 0218. A polypeptide of this invention can be recovered primers complementary to the GENSET cDNA or portion and purified from recombinant cell cultures by well-known thereof and containing restriction endonuclease Sequences methods including differential extraction, ammonium Sul for Pst I incorporated into the 5' primer and BglII at the 5' fate or ethanol precipitation, acid extraction, anion or cation end of the corresponding cDNA 3’ primer, taking care to eXchange chromatography, phosphocellulose chromatogra ensure that the sequence encoding the GENSET protein or phy, hydrophobic interaction chromatography, affinity chro a portion thereof is positioned properly with respect to the matography, hydroxylapatite chromatography and lectin US 2006/0053498A1 Mar. 9, 2006 23 chromatography. See, for example, "Methods in Enzymol translation initiation codon generally is removed with high ogy', Supra for a variety of methods for purifying proteins. efficiency from any protein after translation in all eukaryotic Most preferably, high performance liquid chromatography cells. While the N-terminal methionine on most proteins also (“HPLC) is employed for purification. A recombinantly is efficiently removed in most prokaryotes, for Some pro produced version of a GENSET polypeptide can be substan teins, this prokaryotic removal proceSS is inefficient, depend tially purified using techniques described herein or other ing on the nature of the amino acid to which the N-terminal wise known in the art, Such as, for example, by the one-step methionine is covalently linked. method described in Smith and Johnson (1988), which disclosure is hereby incorporated by reference in its entirety. From Chemical Synthesis Polypeptides of the invention also can be purified from 0223) In addition, polypeptides of the invention, espe recombinant Sources using antibodies directed against the cially short protein fragments, can be chemically Synthe polypeptides of the invention, Such as those described sized using techniques known in the art (See, e.g., Creigh herein, in methods which are well known in the art of protein ton, 1983; and Hunkapiller et al., 1984), which disclosures purification. are hereby incorporated by reference in their entireties. For 0219 Preferably, the recombinantly expressed GENSET example, a polypeptide corresponding to a fragment of a polypeptide is purified using Standard immunochromatog polypeptide Sequence of the invention can be Synthesized by raphy techniques Such as the one described in the Section use of a peptide Synthesizer. A variety of methods of making entitled “Immunoaffinity Chromatography'. In such proce polypeptides are known to those skilled in the art, including dures, a Solution containing the protein of interest, Such as methods in which the carboxyl terminal amino acid is bound the culture medium or a cell extract, is applied to a column to polyvinyl benzene or another Suitable resin. The amino having antibodies against the protein attached to the chro acid to be added possesses blocking groups on its amino matography matrix. The recombinant protein is allowed to moiety and any Side chain reactive groups So that only its bind the immunochromatography column. Thereafter, the carboxyl moiety can react. The carboxyl group is activated column is washed to remove non-specifically bound pro with carbodiimide or another activating agent and allowed to teins. The Specifically bound Secreted protein is then couple to the immobilized amino acid. After removal of the released from the column and recovered using Standard blocking group, the cycle is repeated to generate a polypep techniques. tide having the desired Sequence. Alternatively, the methods described in U.S. Pat. No. 5,049,656, which disclosure is 0220. If antibody production is not possible, the hereby incorporated by reference in its entirety, may be used. GENSET cDNA sequence or fragment thereof may be 0224 Furthermore, if desired, nonclassical amino acids incorporated into expression vectors designed for use in or chemical amino acid analogs can be introduced as a purification Schemes employing chimeric polypeptides. In Substitution or addition into the polypeptide Sequence. Non such strategies the coding sequence of the GENSET cDNA classical amino acids include, but are not limited to, to the or fragment thereof is inserted in frame with the gene D-isomers of the common amino acids, 2,4-diaminobutyric encoding the other half of the chimera. The other half of the acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, chimera may be beta-globin or a nickel binding polypeptide 2-aminobutyric acid, g-Abu, e-AhX, 6-amino hexanoic acid, encoding Sequence. A chromatography matrix having anti Aib, 2-amino isobutyric acid, 3-amino propionic acid, orni body to beta-globin or nickel attached thereto is then used to thine, norleucine, norvaline, hydroxyproline, Sarcosine, cit purify the chimeric protein. Protease cleavage Sites may be rulline, homocitrulline, cysteic acid, t-butylglycine, t-buty engineered between the beta-globin gene or the nickel lalanine, phenylglycine, cyclohexylalanine, b-alanine, binding polypeptide and the GENSET cDNA or fragment fluoroamino acids, designer amino acids Such as b-methyl thereof. Thus, the two polypeptides of the chimera may be amino acids, Ca-methyl amino acids, Na-methyl amino Separated from one another by protease digestion. acids, and amino acid analogs in general. Furthermore, the 0221) One useful expression vector for generating beta amino acid can be D (dextrorotary) or L (levorotary). globin chimerics is pSG5 (Stratagene), which encodes rabbit beta-globin. Intron II of the rabbit beta-globin gene facili Modifications tates Splicing of the expressed transcript, and the polyade 0225. The invention encompasses polypeptides which are nylation signal incorporated into the construct increases the differentially modified during or after translation, e.g., by level of expression. These techniques as described are well glycosylation, acetylation, phosphorylation, amidation, known to those skilled in the art of molecular biology. derivatization by known protecting/blocking groups, pro Standard methods are published in methods texts. Such as teolytic cleavage, linkage to an antibody molecule or other Davis et al., (1986) and many of the methods are available cellular ligand, etc. Any of numerous chemical modifica from Stratagene, Life Technologies, Inc., or Promega. tions may be carried out by known techniques, including but Polypeptide may additionally be produced from the con not limited, to specific chemical cleavage by cyanogen Struct using in vitro translation Systems. Such as the In vitro bromide, trypsin, chymotrypsin, papain, V8 protease, ExpressTM Translation Kit (Stratagene). NaBH4, acetylation, formylation, oxidation, reduction; 0222 Depending upon the host employed in a recombi metabolic Synthesis in the presence of tunicamycin; etc. nant production procedure, the polypeptides of the present 0226. Additional post-translational modifications encom invention may be glycosylated or may be non-glycosylated. passed by the invention include, for example, e.g., N-linked In addition, polypeptides of the invention may also include or O-linked carbohydrate chains, processing of N-terminal an initial modified methionine residue, in Some cases as a or C-terminal ends), attachment of chemical moieties to the result of host-mediated processes. Thus, it is well known in amino acid backbone, chemical modifications of N-linked or the art that the N-terminal methionine encoded by the O-linked carbohydrate chains, and addition or deletion of an US 2006/0053498A1 Mar. 9, 2006 24

N-terminal methionine residue as a result of prokaryotic host nally pegylated protein. The method of obtaining the N-ter cell expression. The polypeptides may also be modified with minally pegylated preparation (i.e., separating this moiety a detectable label, Such as an enzymatic, fluorescent, isoto from other monopegylated moieties if necessary) may be by pic or affinity label to allow for detection and isolation of the purification of the N-terminally pegylated material from a protein. population of pegylated protein molecules. Selective pro teins chemically modified at the N-terminus modification 0227. Also provided by the invention are chemically may be accomplished by reductive alkylation, which modified derivatives of the polypeptides of the invention exploits differential reactivity of different types of primary which may provide additional advantages Such as increased amino groups (lysine versus the N-terminal) available for Solubility, Stability and circulating time of the polypeptide, derivatization in a particular protein. Under the appropriate or decreased immunogenicity. See U.S. Pat. No. 4,179,337. reaction conditions, Substantially Selective derivatization of The chemical moieties for derivatization may be Selected the protein at the N-terminus with a carbonyl group con See U.S. Pat. No. 4,179,337, which disclosure is hereby taining polymer is achieved. incorporated by reference in its entirety. The chemical moieties for derivatization may be selected from water Multimerization Soluble polymerS Such as polyethylene glycol, ethylene 0231. The polypeptides of the invention may be in mono glycol?propylene glycol copolymers, carboxymethylcellu mers or multimers (i.e., dimers, trimers, tetramers and higher lose, dextran, polyvinyl alcohol and the like. The polypep multimers). Accordingly, the present invention relates to tides may be modified at random positions within the monomers and multimers of the polypeptides of the inven molecule, or at predetermined positions within the molecule tion, their preparation, and compositions containing them. In and may include one, two, three or more attached chemical Specific embodiments, the polypeptides of the invention are moieties. monomers, dimers, trimerS or tetramers. In additional 0228. The polymer may be of any molecular weight, and embodiments, the multimers of the invention are at least may be branched or unbranched. For polyethylene glycol, dimers, at least trimers, or at least tetramers. the preferred molecular weight is between about 1 kDa and 0232 Multimers encompassed by the invention may be about 100 kDa (the term “about” indicating that in prepa homomers or heteromers. AS used herein, the term rations of polyethylene glycol, Some molecules will weigh “homomer', refers to a multimer containing only polypep more, Some less, than the Stated molecular weight) for ease tides corresponding to the amino acid Sequences of SEQ ID in handling and manufacturing. Other sizes may be used, NOs: 170-338, 456-560, 785-918 or encoded by the clone depending on the desired therapeutic profile (e.g., the dura inserts of the deposited clone pool (including fragments, tion of Sustained release desired, the effects, if any on variants, Splice variants, and fusion proteins, corresponding biological activity, the ease in handling, the degree or lack to these polypeptides as described herein). These homomers of antigenicity and other known effects of the polyethylene may contain polypeptides having identical or different glycol to a therapeutic protein or analog). amino acid Sequences. In a specific embodiment, a homomer 0229. The polyethylene glycol molecules (or other of the invention is a multimer containing only polypeptides chemical moieties) should be attached to the protein with having an identical amino acid Sequence. In another specific consideration of effects on functional or antigenic domains embodiment, a homomer of the invention is a multimer of the protein. There are a number of attachment methods containing polypeptides having different amino acid available to those skilled in the art, e.g., EPO 401 384, Sequences. In specific embodiments, the multimer of the (coupling PEG to G-CSF), and Malik et al. (1992) (reporting invention is a homodimer (e.g., containing polypeptides pegylation of GM-CSF using tresyl chloride), which disclo having identical or different amino acid Sequences) or a Sures are hereby incorporated by reference in their entireties. homotrimer (e.g., containing polypeptides having identical For example, polyethylene glycol may be covalently bound and/or different amino acid Sequences). In additional through amino acid residues via a reactive group, Such as, a embodiments, the homomenc multimer of the invention is at free amino or carboxyl group. Reactive groups are those to least a homodimer, at least a homotrimer, or at least a which an activated polyethylene glycol molecule may be homotetramer. bound. The amino acid residues having a free amino group 0233 AS used herein, the term “heteromer” refers to a may include lysine residues and the N-terminal amino acid multimer containing one or more heterologous polypeptides residues; those having a free carboxyl group may include (i.e., polypeptides of different proteins) in addition to the aspartic acid residues glutamic acid residues and the C-ter polypeptides of the invention. In a specific embodiment, the minal amino acid residue. Sulfhydryl groups may also be multimer of the invention is a heterodimer, a heterotrimer, or used as a reactive group for attaching the polyethylene a heterotetramer. In additional embodiments, the hetero glycol molecules. Preferred for therapeutic purposes is meric multimer of the invention is at least a heterodimer, at attachment at an amino group, Such as attachment at the least a heterotrimer, or at least a heterotetramer. N-terminus or lysine group. 0234) Multimers of the invention may be the result of 0230. One may specifically desire proteins chemically hydrophobic, hydrophilic, ionic and/or covalent associations modified at the N-terminus. Using polyethylene glycol as an and/or may be indirectly linked, by for example, liposome illustration of the present composition, one may select from formation. Thus, in one embodiment, multimers of the a variety of polyethylene glycol molecules (by molecular invention, Such as, for example, homodimerS or homotrim weight, branching, etc.), the proportion of polyethylene ers, are formed when polypeptides of the invention contact glycol molecules to protein (polypeptide) molecules in the one another in Solution. In another embodiment, heteromul reaction mix, the type of pegylation reaction to be per timers of the invention, Such as, for example, heterotrimers formed, and the method of obtaining the selected N-termi or heterotetramers, are formed when polypeptides of the US 2006/0053498A1 Mar. 9, 2006 25 invention contact antibodies to the polypeptides of the described in Hoppe et al. (1994) and in U.S. patent appli invention (including antibodies to the heterologous polypep cation Ser. No. 08/446,922, which disclosure is hereby tide sequence in a fusion protein of the invention) in incorporated by reference in its entirety. Other peptides Solution. In other embodiments, multimers of the invention derived from naturally occurring trimeric proteins may be are formed by covalent associations with and/or between the employed in preparing trimeric polypeptides of the inven polypeptides of the invention. Such covalent associations tion. In another example, proteins of the invention are may involve one or more amino acid residues contained in asSociated by interactions between Flag(E) polypeptide the polypeptide sequence (e.g., that recited in the Sequence Sequence contained in fusion proteins of the invention listing, or contained in the polypeptide encoded by a depos containing Flag(E) polypeptide Sequence. In a further ited clone). In one instance, the covalent associations are croSS-linking between cysteine residues located within the embodiment, associations proteins of the invention are asso polypeptide Sequences, which interact in the native (i.e., ciated by interactions between heterologous polypeptide naturally occurring) polypeptide. In another instance, the Sequence contained in Flag(E) fusion proteins of the inven covalent associations are the consequence of chemical or tion and anti Flag(R) antibody. recombinant manipulation. Alternatively, Such covalent 0238. The multimers of the invention may be generated asSociations may involve one or more amino acid residues using chemical techniques known in the art. For example, contained in the heterologous polypeptide Sequence in a polypeptides desired to be contained in the multimers of the fusion protein of the invention. invention may be chemically croSS-linked using linker mol 0235. In one example, covalent associations are between ecules and linker molecule length optimization techniques the heterologous Sequence contained in a fusion protein of known in the art (see, e.g., U.S. Pat. No. 5,478,925, which the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Addi disclosure is hereby incorporated by reference in its tionally, multimers of the invention may be generated using entirety). In a specific example, the covalent associations are techniques known in the art to form one or more inter between the heterologous Sequence contained in an Fc molecule croSS-links between the cysteine residues located fusion protein of the invention (as described herein). In within the Sequence of the polypeptides desired to be another Specific example, covalent associations of fusion contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, proteins of the invention are between heterologous polypep which is herein incorporated by reference in its entirety). tide Sequence from another protein that is capable of form Further, polypeptides of the invention may be routinely ing covalently associated multimerS, Such as for example, modified by the addition of cysteine or biotin to the C oseteoprotegerin (See, e.g., International Publication No: terminus or N-terminus of the polypeptide and techniques WO98/49305, the contents of which are herein incorporated known in the art may be applied to generate multimers by reference in its entirety). In another embodiment, two or containing one or more of these modified polypeptides (see, more polypeptides of the invention are joined through e.g., U.S. Pat. No. 5,478,925, which is herein incorporated peptide linkers. Examples include those peptide linkers by reference in its entirety). Additionally, other techniques described in U.S. Pat. No. 5,073,627 (hereby incorporated known in the art may be applied to generate liposomes by reference). Proteins comprising multiple polypeptides of containing the polypeptide components desired to be con the invention Separated by peptide linkerS may be produced tained in the multimer of the invention (See, e.g., U.S. Pat. using conventional recombinant DNA technology. No. 5,478,925, which is herein incorporated by reference in 0236 Another method for preparing multimer polypep its entirety). tides of the invention involves the use of polypeptides of the invention fused to a leucine Zipper or isoleucine Zipper 0239 Alternatively, multimers of the invention may be polypeptide Sequence. Leucine Zipper and isoleucine Zipper generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in domains are polypeptides that promote multimerization of multimers of the invention are produced recombinantly the proteins in which they are found. Leucine Zippers were using fusion protein technology described herein or other originally identified in Several DNA-binding proteins, and wise known in the art (see, e.g., U.S. Pat. No. 5,478,925, have since been found in a variety of different proteins which is herein incorporated by reference in its entirety). In (Landschulz et al., 1988). Among the known leucine Zippers a specific embodiment, polynucleotides coding for a are naturally occurring peptides and derivatives thereof that homodimer of the invention are generated by ligating a dimerize or trimerize. Examples of leucine Zipper domains polynucleotide Sequence encoding a polypeptide of the Suitable for producing Soluble multimeric proteins of the invention to a Sequence encoding a linker polypeptide and invention are those described in PCT application WO then further to a Synthetic polynucleotide encoding the 94/10308, hereby incorporated by reference. Recombinant translated product of the polypeptide in the reverse orien fusion proteins comprising a polypeptide of the invention tation from the original C-terminus to the N-terminus (lack fused to a polypeptide Sequence that dimerizes or trimerizes ing the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, in Solution are expressed in Suitable host cells, and the which is herein incorporated by reference in its entirety). In resulting Soluble multimeric fusion protein is recovered another embodiment, recombinant techniques described from the culture Supernatant using techniques known in the herein or otherwise known in the art are applied to generate art. recombinant polypeptides of the invention which contain a 0237 Trimeric polypeptides of the invention may offer transmembrane domain (or hydrophobic or signal peptide) the advantage of enhanced biological activity. Preferred and which can be incorporated by membrane reconstitution leucine Zipper moieties and isoleucine moieties are those techniques into liposomes (see, e.g., U.S. Pat. No. 5,478, that preferentially form trimers. One example is a leucine 925, which is herein incorporated by reference in its Zipper derived from lung surfactant protein D (SPD), as entirety). US 2006/0053498A1 Mar. 9, 2006 26

Mutated Polypeptides reference in its entirety). The first method relies on the 0240. To improve or alter the characteristics of GENSET process of evolution, in which mutations are either accepted polypeptides of the present invention, protein engineering or rejected by natural Selection. may be employed. Recombinant DNA technology known to 0244. The Second approach uses genetic engineering to those skilled in the art can be used to create novel mutant introduce amino acid changes at Specific positions of a proteins or muteins including Single or multiple amino acid cloned gene and Selections or Screens to identify Sequences Substitutions, deletions, additions, or fusion proteins. Such that maintain functionality. These Studies have revealed that modified polypeptides can Show, e.g., increased/decreased proteins are Surprisingly tolerant of amino acid Substitutions. biological activity or increased/decreased Stability. In addi The Studies indicate which amino acid changes are likely to tion, they may be purified in higher yields and Show better be permissive at a certain position of the protein. For Solubility than the corresponding natural polypeptide, at example, most buried amino acid residues require nonpolar least under certain purification and Storage conditions. Fur Side chains, whereas few features of Surface Side chains are ther, the polypeptides of the present invention may be generally conserved. Other Such phenotypically Silent Sub produced as multimers including dimers, trimerS and tet Stitutions are described by Bowie et al. (Supra) and the ramers. Multimerization may be facilitated by linkers or references cited therein. recombinantly though heterologous polypeptides Such as Fc regions. 0245 Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino N- and C-terminal Deletions acids Ala, Val, Leu and Phe, interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues ASp 0241. It is known in the art that one or more amino acids and Glu, Substitution between the amide residues ASn and may be deleted from the N-terminus or C-terminus without Gln, eXchange of the basic residues LyS and Arg and Substantial loSS of biological function. For instance, Ron et replacements among the aromatic residues Phe, Tyr. Thus, al. (1993), reported modified KGF proteins that had heparin the fragment, derivative, analog, or homologue of the binding activity even if 3, 8, or 27 N-terminal amino acid polypeptide of the present invention may be, for example: (i) residues were missing. Accordingly, the present invention one in which one or more of the amino acid residues are provides polypeptides having one or more residues deleted Substituted with a conserved or non-conserved amino acid from the amino terminus of the polypeptides of SEQ ID residue (preferably a conserved amino acid residue) and NOs: 170-338, 456-560, 785-918 or that encoded by the Such Substituted amino acid residue may or may not be one clone inserts of the deposited clone pool. Similarly, many encoded by the genetic code: or (ii) one in which one or examples of biologically functional C-terminal deletion more of the amino acid residues includes a Substituent mutants are known. For instance, Interferon gamma ShowS group: or (iii) one in which the GENSET polypeptide is up to ten times higher activities by deleting 810 amino acid fused with another compound, Such as a compound to residues from the C-terminus of the protein (See, e.g., increase the half-life of the polypeptide (for example, poly Dobeli, et al. 1988), which disclosure is hereby incorporated ethylene glycol): or (iv) one in which the additional amino by reference in its entirety. Accordingly, the present inven acids are fused to the above form of the polypeptide, Such as tion provides polypeptides having one or more residues an IgG Fc fusion region peptide or leader or Secretory deleted from the carboxy terminus of the polypeptides Sequence or a Sequence which is employed for purification shown of SEQ ID NOs: 170-338, 456-560, 785-918 or encoded by the clone inserts of the deposited clone pool. The of the above form of the polypeptide or a pro-protein invention also provides polypeptides having one or more Sequence. Such fragments, derivatives and analogs are amino acids deleted from both the amino and the carboxyl deemed to be within the scope of those skilled in the art from termini as described below. the teachings herein. Other Mutations 0246 Thus, the GENSET polypeptides of the present invention may include one or more amino acid Substitutions, 0242 Other mutants in addition to N- and C-terminal deletions, or additions, either from natural mutations or deletion forms of the protein discussed above are included in human manipulation. AS indicated, changes are preferably of the present invention. It also will be recognized by one of a minor nature, Such as conservative amino acid Substitu ordinary skill in the art that Some amino acid Sequences of tions that do not significantly affect the folding or activity of the GENSET polypeptides of the present invention can be the protein. The following groups of amino acids generally varied without significant effect of the Structure or function represent equivalent changes: (1) Ala, Pro, Gly, Glu, Asp, of the protein. If Such differences in Sequence are contem Gin, ASn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, Ile, Leu, plated, it should be remembered that there will be critical Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Trp, His. areas on the protein which determine activity. Thus, the invention further includes variations of the GENSET 0247 A specific embodiment of a modified GENSET polypeptides which show substantial GENSET polypeptide peptide molecule of interest according to the present inven activity. Such mutants include deletions, insertions, inver tion, includes, but is not limited to, a peptide molecule which Sions, repeats, and Substitutions Selected according to gen is resistant to proteolysis, is a peptide in which the eral rules known in the art so as to have little effect on -CONH- peptide bond is modified and replaced by a (CH2NH) reduced bond, a (NHCO) retro inverso bond, a activity. For example, guidance concerning how to make (CH2-O) methylene-oxy bond, a (CH2-S) thiomethylene phenotypically Silent amino acid Substitutions is provided. bond, a (CH2CH2) carba bond, a (CO-CH2) cetomethyl 0243 There are two main approaches for studying the ene bond, a (CHOH-CH2) hydroxyethylene bond), a tolerance of an amino acid sequence to change (see, Bowie (N-N) bound, a E-alcene bond or also a -CH=CH et al. 1994, which disclosure is hereby incorporated by bond. The invention also encompasses a human GENSET US 2006/0053498A1 Mar. 9, 2006 27 polypeptide or a fragment or a variant thereof in which at C-terminal position. That is, every combination of a N-ter least one peptide bond has been modified as described minal and C-terminal position that a fragment at least 6 above. contiguous amino acid residues in length could occupy, on any given amino acid Sequence of the Sequence listing or of 0248 Amino acids in the GENSET proteins of the the present invention is included in the present invention present invention that are essential for function can be identified by methods known in the art, Such as site-directed 0252) The present invention also provides for the exclu mutagenesis or alanine-Scanning mutagenesis (See, e.g., Sion of any fragment Species Specified by N-terminal and Cunningham et al. 1989, which disclosure is hereby incor C-terminal positions or of any fragment Sub-genus Specified porated by reference in its entirety). The latter procedure by size in amino acid residues as described above. Any introduces Single alanine mutations at every residue in the number of fragments specified by N-terminal and C-terminal molecule. The resulting mutant molecules are then tested for positions or by size in amino acid residues as described biological activity using assays appropriate for measuring above may be excluded as individual species. the function of the particular protein. Of Special interest are 0253) The above polypeptide fragments of the present Substitutions of charged amino acids with other charged or invention can be immediately envisaged using the above neutral amino acids which may produce proteins with highly description and are therefore not individually listed Solely desirable improved characteristics, Such as leSS aggregation. for the purpose of not unnecessarily lengthening the Speci Aggregation may not only reduce activity but also be fication. Moreover, the above fragments need not have a problematic when preparing pharmaceutical formulations, GENSET biological activity, although polypeptides having because aggregates can be immunogenic, (see, e.g., Pinck these activities are preferred embodiments of the invention, ard et al., 1967; Robbins, et al., 1987; and Cleland, et al., Since they would be useful, for example, in immunoassays, 1993). in epitope mapping, epitope tagging, as Vaccines, and as 0249. A further embodiment of the invention relates to a molecular weight markers. The above fragments may also be polypeptide which comprises the amino acid Sequence of a used to generate antibodies to a particular portion of the GENSET polypeptide having an amino acid Sequence which polypeptide. These antibodies can then be used in immu contains at least one conservative amino acid Substitution, noassays well known in the art to distinguish between but not more than 50 conservative amino acid Substitutions, human and non-human cells and tissueS or to determine not more than 40 conservative amino acid Substitutions, not whether cells or tissues in a biological Sample are or are not more than 30 conservative amino acid Substitutions, and not of the same type which express the polypeptides of the more than 20 conservative amino acid Substitutions. Also present invention. provided are polypeptides which comprise the amino acid 0254. It is noted that the above species of polypeptide Sequence of a GENSET polypeptide, having at least one, but fragments of the present invention may alternatively be not more than 10,9,8,7,6, 5, 4, 3, 2 or 1 conservative amino described by the formula "a to b”; where “a” equals the acid Substitutions. N-terminal most amino acid position and “b' equals the Polypeptide Fragments C-terminal most amino acid position of the polynucleotide; and further where “a” equals an integer between 1 and the Structural Definition number of amino acids of the polypeptide Sequence of the 0250) The present invention is further directed to frag present invention minus 6, and where “b' equals an integer ments of the amino acid Sequences described herein Such as between 7 and the number of amino acids of the polypeptide the polypeptides of SEQ ID NOs: 170-338, 456-560, 785 Sequence of the present invention; and where “a” is an 918 or those encoded by the clone inserts of the deposited integer smaller then “b' by at least 6. clone pool. More specifically, the present invention embod 0255 The present invention also provides for the exclu ies purified, isolated, and recombinant polypeptides com Sion of any species of polypeptide fragments of the present prising at least 6, preferably at least 8 to 10, more preferably invention specified by 5' and 3' positions or Sub-genuses of 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 125, 150, 175, polypeptides Specified by Size in amino acids as described 200, 225, 250, 275, or 300 consecutive amino acids of a above. Any number of fragments specified by 5' and 3' polypeptide Selected from the group consisting of the positions or by Size in amino acids, as described above, may sequences of SEQ ID NOs: 170-338, 456-560, 785-918, the be excluded. polypeptides encoded by the clone inserts of the deposited clone pool, and other polypeptides of the present invention. Functional Definition 0251. In addition to the above polypeptide fragments, Domains further preferred Sub-genuses of polypeptides comprise at 0256 Preferred polynucleotide fragments of the inven least 6 amino acids, wherein “at least 6' is defined as any tion are domains of polypeptides of the invention. Such integer between 6 and the integer representing the C-termi domains may eventually comprise linear or Structural motifs nal amino acid of the polypeptide of the present invention and Signatures including, but not limited to, leucine Zippers, including the polypeptide Sequences of the Sequence listing helix-turn-helix motifs, post-translational modification Sites below. Further included are species of polypeptide frag Such as glycosylation sites, ubiquitination sites, alpha heli ments at least 6 amino acids in length, as described above, ces, and beta sheets, Signal Sequences encoding Signal that are further specified in terms of their N-terminal and peptides which direct the Secretion of the encoded proteins, C-terminal positions. However, included in the present Sequences implicated in transcription regulation Such as invention as individual Species are all polypeptide frag homeoboxes, acidic Stretches, enzymatic active sites, Sub ments, at least 6 amino acids in length, as described above, Strate binding Sites, and enzymatic cleavage Sites. Such and may be particularly Specified by a N-terminal and domains may present a particular biological activity Such as US 2006/0053498A1 Mar. 9, 2006 28

DNA or RNA-binding, secretion of proteins, transcription noted that although a particular epitope may not be immu regulation, enzymatic activity, Substrate binding activity, nogenic, it is nonetheless useful since antibodies can be etc. made to both immunogenic and antigenic epitopes. 0257. A domain has a size generally comprised between 0261) An epitope can comprise as few as 3 amino acids 3 and 1000 amino acids. In a preferred embodiment, in a Spatial conformation, which is unique to the epitope. domains comprise a number of amino acids that is any integer between 6 and 200. Domains may be synthesized Generally an epitope consists of at least 6 Such amino acids, using any methods known to those skilled in the art, includ and more often at least 8-10 Such amino acids. In preferred ing those disclosed herein, particularly in the Section entitled embodiment, antigenic epitopes comprise a number of “Preparation of the polypeptides of the invention'. Methods amino acids that is any integer between 3 and 50. Fragments for determining the amino acids which make up a domain which function as epitopes may be produced by any con with a particular biological activity include mutagenesis ventional means (See, e.g., Houghten, 1985), also further Studies and assays to determine the biological activity to be described in U.S. Pat. No. 4,631,21, which disclosures are tested. hereby incorporated by reference in their entireties. Methods for determining the amino acids which make up an epitope 0258 Alternatively, the polypeptides of the invention include X-ray crystallography, 2-dimensional nuclear mag may be Scanned for motifs, domains and/or signatures in databases using any computer method known to those netic resonance, and epitope mapping, e.g., the PepScan skilled in the art. Searchable databases include ProSite method described by Geysen et al. (1984); PCT Publication (Hofmann et al., 1999; Bucher and Bairoch 1994), Pfam No. WO 84/03564; and PCT Publication No. WO 84/03506, (Sonnhammer et al., 1997; Henikoffet al., 2000; Bateman et which disclosures are hereby incorporated by reference in al., 2000), Blocks (Henikoffet al., 2000), Print (Attwood et their entireties. Another example is the algorithm of Jame al., 1996), Prodom (Sonnhammer and Kahn, 1994; Corpet et son and Wolf, (1988) (said reference incorporated by refer al. 2000), Sbase (Pongor et al., 1993; Murvai et al., 2000), ence in its entirety). The Jameson-Wolf antigenic analysis, Smart (Schultz et al., 1998), Dali/FSSP (Holm and Sander, for example, may be performed using the computer program 1996, 1997 and 1999), HSSP (Sander and Schneider 1991), PROTEAN, using default parameters (Version 4.0 Win CATH (Orengo et al., 1997; Pearl et al., 2000), SCOP dows, DNASTAR, Inc., 1228 South Park Street Madison, (Murzin et al., 1995; Lo Conte et al., 2000), COG (Tatusov Wis. et al., 1997 and 2000), specific family databases and deriva 0262 The epitope-bearing fragments of the present tives thereof (Nevill-Manning et al., 1998; Yona et al., 1999; invention preferably comprise 6 to 50 amino acids (i.e. any Attwood et al., 2000), each of which disclosures are hereby integer between 6 and 50, inclusive) of a polypeptide of the incorporated by reference in their entireties. For a review on present invention. Also, included in the present invention are available databases, see issue 1 of volume 28 of Nucleic antigenic fragments between the integers of 6 and the full Acid Research (2000), which disclosure is hereby incorpo length GENSET Sequence of the Sequence listing. All com rated by reference in its entirety. binations of Sequences between the integers of 6 and the 0259. The domains of the present invention preferably full-length sequence of a GENSET polypeptide are comprises 6 to 200 amino acids (i.e. any integer between 6 included. The epitope-bearing fragments may be specified and 200, inclusive) of a polypeptide of the present invention. by either the number of contiguous amino acid residues (as Also, included in the present invention are domain frag a Sub-genus) or by specific N-terminal and C-terminal ments between the integers of 6 and the full length GENSET positions (as species) as described above for the polypeptide Sequence of the Sequence listing. All combinations of fragments of the present invention. Any number of epitope Sequences between the integers of 6 and the full-length bearing fragments of the present invention may also be sequence of a GENSET polypeptide are included. The excluded in the same manner. domain fragments may be specified by either the number of 0263 Antigenic epitopes are useful, for example, to raise contiguous amino acid residues (as a Sub-genus) or by antibodies, including monoclonal antibodies that Specifi Specific N-terminal and C-terminal positions (as species) as cally bind the epitope (see, Wilson et al., 1984; and Sutcliffe, described above for the polypeptide fragments of the present et al., 1983, which disclosures are hereby incorporated by invention. Any number of domain fragments of the present reference in their entireties). The antibodies are then used in invention may also be excluded in the same manner. various techniques Such as diagnostic and tissue/cell iden Epitopes and Antibody Fusions: tification techniques, as described herein, and in purification methods Such as immunoaffinity chromatography. 0260 A preferred embodiment of the present invention is directed to epitope-bearing polypeptides and epitope-bear 0264. Similarly, immunogenic epitopes can be used to ing polypeptide fragments. These epitopes may be “anti induce antibodies according to methods well known in the genic epitopes' or both an "antigenic epitope' and an art (See, Sutcliffe et al., Supra; Wilson et al., Supra; Chow et “immunogenic epitope'. An "immunogenic epitope' is al.(1985); and Bittle, et al., (1985), which disclosures are defined as a part of a protein that elicits an antibody response hereby incorporated by reference in their entireties). A in Vivo when the polypeptide is the immunogen. On the preferred immunogenic epitope includes the natural other hand, a region of polypeptide to which an antibody GENSET protein. The immunogenic epitopes may be pre binds is defined as an "antigenic determinant' or “antigenic Sented together with a carrier protein, Such as an albumin, to epitope.” The number of immunogenic epitopes of a protein an animal System (such as rabbit or mouse) or, if it is long generally is less than the number of antigenic epitopes (See, enough (at least about 25 amino acids), without a carrier. e.g., Geysen, et al., 1984), which disclosure is hereby However, immunogenic epitopes comprising as few as 8 to incorporated by reference in its entirety. It is particularly 10 amino acids have been shown to be Sufficient to raise US 2006/0053498A1 Mar. 9, 2006 29 antibodies capable of binding to, at the very least, linear Pat. Nos.: 5,605,793; 5,811,238; 5,834,252; 5,837,458; and epitopes in a denatured polypeptide (e.g., in Western blot Patten, et al., (1997); Harayama, (1998); Hansson, et al ting.). (1999); and Lorenzo and Blasco, (1998). (Each of these 0265 Epitope-bearing polypeptides of the present inven documents are hereby incorporated by reference). In one tion are used to induce antibodies according to methods well embodiment, one or more components, motifs, Sections, known in the art including, but not limited to, in vivo parts, domains, fragments, etc., of coding polynucleotides of immunization, in vitro immunization, and phage display the invention, or the polypeptides encoded thereby may be methods (See, e.g., Sutcliffe, et al., Supra; Wilson, et al., recombined with one or more components, motifs, Sections, Supra, and Bittle, et al., Supra). If in Vivo immunization is parts, domains, fragments, etc. of one or more heterologous used, animals may be immunized with free peptide; how molecules. ever, anti-peptide antibody titer may be boosted by coupling 0268. The present invention further encompasses any of the peptide to a macromolecular carrier, Such as keyhole combination of the polypeptide fragments listed in this limpet hemacyanin (KLH) or tetanus toxoid. For instance, Section. peptides containing cysteine residues may be coupled to a Antibodies carrier using a linker Such as -maleimidobenzoyl-N-hydrox ySuccinimide ester (MBS), while other peptides may be Definitions coupled to carriers using a more general linking agent Such 0269. The present invention further relates to antibodies as glutaraldehyde. Animals. Such as rabbits, rats and mice are and T-cell antigen receptors (TCR), which specifically bind immunized with either free or carrier-coupled peptides, for the polypeptides, and more Specifically, the epitopes of the instance, by intraperitoneal and/or intradermal injection of polypeptides of the present invention. The antibodies of the emulsions containing about 100 lugs of peptide or carrier present invention include IgG (including IgG1, IgG2, IgG3, protein and Freund's adjuvant. Several booster injections and IgG4), IgA (including IgA1 and IgA2), Ig), IgE, or may be needed, for instance, at intervals of about two weeks, IgM, and IgY. The term “antibody” (Ab) refers to a polypep to provide a useful titer of anti-peptide antibody, which can tide or group of polypeptides which are comprised of at least be detected, for example, by ELISA assay using free peptide one binding domain, where a binding domain is formed from adsorbed to a Solid Surface. The titer of anti-peptide anti the folding of variable domains of an antibody molecule to bodies in Serum from an immunized animal may be form three-dimensional binding Spaces with an internal increased by Selection of anti-peptide antibodies, for Surface shape and charge distribution complementary to the instance, by adsorption to the peptide on a Solid Support and features of an antigenic determinant of an antigen, which elution of the Selected antibodies according to methods well allows an immunological reaction with the antigen. AS used known in the art. herein, the term “antibody' is meant to include whole antibodies, including Single-chain whole antibodies, and 0266. As one of skill in the art will appreciate, and antigen binding fragments thereof. In a preferred embodi discussed above, the polypeptides of the present invention ment the antibodies are human antigen binding antibody comprising an immunogenic or antigenic epitope can be fragments of the present invention include, but are not fused to heterologous polypeptide Sequences. For example, limited to, Fab, Fab'F(ab)2 and F(ab')2, Fd, single-chain Fvs the polypeptides of the present invention may be fused with (scFv), single-chain antibodies, disulfide-linked FVs (sdFv) the constant domain of immunoglobulins (IgA, IgE, IgG, and fragments comprising either a V or V domain. The IgM), or portions thereof (CH1, CH2, CH3, any combina antibodies may be from any animal origin including birds tion thereof including both entire domains and portions and mammals. Preferably, the antibodies are human, murine, thereof) resulting in chimeric polypeptides. These fusion rabbit, goat, guinea pig, camel, horse, or chicken. proteins facilitate purification, and Show an increased half 0270 Antigen-binding antibody fragments, including life in Vivo. This has been shown, e.g., for chimeric proteins Single-chain antibodies, may comprise the variable region(s) consisting of the first two domains of the human CD4 alone or in combination with the entire or partial of the polypeptide and various domains of the constant regions of following: hinge region, CH1, CH2, and CH3 domains. Also the heavy or light chains of mammalian immunoglobulins included in the invention are any combinations of variable (See, e.g., EPA 0,394,827; and Traunecker et al., 1988, region(s) and hinge region, CH1, CH2, and CH3 domains. which disclosures are hereby incorporated by reference in The present invention further includes chimeric, humanized, their entireties). Fusion proteins that have a disulfide-linked and human monoclonal and polyclonal antibodies, which dimeric Structure due to the IgG portion can also be more Specifically bind the polypeptides of the present invention. efficient in binding and neutralizing other molecules than The present invention further includes antibodies that are monomeric polypeptides or fragments thereof alone (See, anti-idiotypic to the antibodies of the present invention. e.g., Fountoulakis et al., 1995, which disclosure is hereby 0271 The antibodies of the present invention may be incorporated by reference in its entirety). Nucleic acids monospecific, bispecific, and trispecific or have greater encoding the above epitopes can also be recombined with a multispecificity. Multispecific antibodies may be specific for gene of interest as an epitope tag to aid in detection and different epitopes of a polypeptide of the present invention purification of the expressed polypeptide. or may be specific for both a polypeptide of the present 0267 Additional fusion proteins of the invention may be invention as well as for heterologous compositions, Such as generated through the techniques of gene-shuffling, motif a heterologous polypeptide or Solid Support material. See, Shuffling, exon-shuffling, or codon-Shuffling (collectively e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO referred to as “DNA shuffling”). DNA shuffling may be 92/05793; Tutt, et al. (1991); U.S. Pat. Nos. 5,573,920, employed to modulate the activities of polypeptides of the 4,474,893, 5,601,819, 4,714,681, 4,925,648; Kostelny et al. present invention thereby effectively generating agonists (1992), which disclosures are hereby incorporated by refer and antagonists of the polypeptides. See, for example, U.S. ence in their entireties. US 2006/0053498A1 Mar. 9, 2006 30

0272 Antibodies of the present invention may be GENSET protein'. For example, a polypeptide of the described or specified in terms of the epitope(s) or epitope present invention or an antigenic fragment thereof can be bearing portion(s) of a polypeptide of the present invention, administered to an animal in order to induce the production which are recognized or Specifically bound by the antibody. of Sera containing "polyclonal antibodies'. AS used herein, The antibodies may specifically bind a complete protein the term “monoclonal antibody' is not limited to antibodies encoded by a nucleic acid of the present invention, or a produced through hybridoma technology but it rather refers fragment thereof. Therefore, the epitope(s) or epitope bear to an antibody that is derived from a single clone, including ing polypeptide portion(s) may be specified as described eukaryotic, prokaryotic, or phage clone, and not the method herein, e.g., by N-terminal and C-terminal positions, by size by which it is produced. Monoclonal antibodies can be in contiguous amino acid residues, or otherwise described prepared using a wide variety of techniques known in the art herein (including the Sequence listing). Antibodies which including the use of hybridoma, recombinant, and phage Specifically bind any epitope or polypeptide of the present display technology. invention may also be excluded as individual Species. There fore, the present invention includes antibodies that Specifi 0277 Hybridoma techniques include those known in the cally bind Specified polypeptides of the present invention, art (See, e.g., Harlow et al. 1988; Hammerling, et al., 1981). (Said references incorporated by reference in their entire and allows for the exclusion of the Same. ties). Fab and F(ab')2 fragments may be produced, for 0273 Thus, another embodiment of the present invention example, from hybridoma-produced antibodies by pro is a purified or isolated antibody capable of Specifically teolytic cleavage, using enzymes Such as papain (to produce binding to a polypeptide comprising a Sequence Selected Fab fragments) or pepsin (to produce F(ab')2 fragments). from the group consisting of the Sequences of SEQ ID NOs: 170-338, 456-560, 785-918 and the sequences of the 0278 Alternatively, antibodies of the present invention clone inserts of the deposited clone pool. In one aspect of can be produced through the application of recombinant this embodiment, the antibody is capable of binding to an DNA technology or through Synthetic chemistry using meth epitope-containing polypeptide comprising at least 6 con ods known in the art. For example, the antibodies of the secutive amino acids, preferably at least 8 to 10 consecutive present invention can be prepared using various phage amino acids, more preferably at least 12, 15, 20, 25, 30, 40, display methods known in the art. In phage display methods, 50, or 100 consecutive amino acids of a Sequence Selected functional antibody domains are displayed on the Surface of from the group consisting of SEQ ID NOs: 170-338, 456 a phage particle, which carries polynucleotide Sequences 560, 785-918 and sequences of the clone inserts of the encoding them. Phage with a desired binding property are deposited clone pool. Selected from a repertoire or combinatorial antibody library (e.g. human or murine) by Selecting directly with antigen, 0274 Antibodies of the present invention may also be typically antigen bound or captured to a Solid Surface or described or Specified in terms of their croSS-reactivity. bead. Phage used in these methods are typically filamentous Antibodies that do not specifically bind any other analog, phage including fl and M13 with Fab, Fv or disulfide ortholog, or homologue of the polypeptides of the present stabilized FV antibody domains recombinantly fused to invention are included. Antibodies that do not bind polypep either the phage gene III or gene VIII protein. Examples of tides with less than 95%, less than 90%, less than 85%, less phage display methods that can be used to make the anti than 80%, less than 75%, less than 70%, less than 65%, less bodies of the present invention include those disclosed in than 60%, less than 55%, and less than 50% identity (as Brinkman et al. (1995); Ames, et al. (1995); Kettleborough, calculated using methods known in the art and described et al. (1994); Persic, et al. (1997); Burton et al. (1994); herein, e.g., using FASTDB and the parameterS Set forth PCT/GB91/01134; WO 90/02809; WO 91/10737; WO herein) to a polypeptide of the present invention are also 92/01047; WO 92/18619; WO 93/11236; WO95/15982; included in the present invention. Further included in the WO95/20401; and U.S. Pat. Nos. 5,698,426, 5,223,409, present invention are antibodies, which only bind polypep 5,403.484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, tides encoded by polynucleotides, which hybridize to a 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and polynucleotide of the present invention under Stringent 5,733,743 (said references incorporated by reference in their hybridization conditions (as described herein). Antibodies of the present invention may also be described or Specified in entireties). terms of their binding affinity. Preferred binding affinities 0279. As described in the above references, after phage include those with a isSociation constant or Kd less than Selection, the antibody coding regions from the phage can be 5x10M, 10M, 5x10-7M, 107M, 5x10-8M, 10-8M, isolated and used to generate whole antibodies, including 5x10M, 10M, 5x100M, 100M, 5x10M, 10M, human antibodies, or any other desired antigen binding 5x10-12M, 10-12M, 5x10-13M, 10-13M, 5x101*M, fragment, and expressed in any desired host including mam 10'M, 5x10M, and 10 M. malian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques to recombinantly produce Fab, Fab' 0275. The invention also concerns a purified or isolated F(ab)2 and F(ab')2 fragments can also be employed using antibody capable of Specifically binding to a mutated methods known in the art Such as those disclosed in WO GENSET protein or to a fragment or variant thereof com 92/22324; Mullinax et al. (1992); and Sawai et al. (1995); prising an epitope of the mutated GENSET protein. and Better et al. (1988) (said references incorporated by Preparation of Antibodies reference in their entireties). 0276 The antibodies of the present invention may be 0280 Examples of techniques which can be used to prepared by any Suitable method known in the art. Some of produce Single-chain FVS and antibodies include those these methods are described in more detail in the example described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston entitled “Preparation of Antibody Compositions to the et al. (1991); Shu et al. (1993); and Skerra et al. (1988), US 2006/0053498A1 Mar. 9, 2006 which disclosures are hereby incorporated by reference in the polypeptides of the present invention to antibody por their entireties. For Some uses, including in Vivo use of tions are known in the art. See e.g., U.S. Pat. Nos. 5,336,603, antibodies in humans and in Vitro detection assays, it may be 5,622,929, 5,359,046, 5,349,053, 5,447.851, 5,112,946; EP preferable to use chimeric, humanized, or human antibodies. 0 307434, EPO 367 166; WO 96/04388, WO 91/06570; Methods for producing chimeric antibodies are known in the Ashkenazi et al. (1991); Zheng et al. (1995); and Vilet al. art. See e.g., Morrison, (1985); Oi et al., (1986); Gillies et al. (1992) (said references incorporated by reference in their (1989); and U.S. Pat. No. 5,807,715, which disclosures are entireties). hereby incorporated by reference in their entireties. Anti 0283 Non-human animals or mammals, whether wild bodies can be humanized using a variety of techniques type or transgenic, which express a different species of including CDR-grafting (EPO 239 400; WO91/09967; U.S. GENSET than the one to which antibody binding is desired, Pat. No. 5,530,101; and 5,585,089), veneering or resurfac and animals which do not express GENSET (i.e. a GENSET ing, (EPO 592 106; EP 0519596; Padlan, 1991; Studnicka knockout animal as described herein) are particularly useful et al., 1994; Roguska et al., 1994), and chain shuffling (U.S. for preparing antibodies. GENSET knock out animals will Pat. No. 5,565,332), which disclosures are hereby incorpo recognize all or most of the exposed regions of a GENSET rated by reference in their entireties. Human antibodies can protein as foreign antigens, and therefore produce antibodies be made by a variety of methods known in the art including with a wider array of GENSET epitopes. Moreover, Smaller phage display methods described above. See also, U.S. Pat. polypeptides with only 10 to 30 amino acids may be useful Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; WO in obtaining specific binding to any one of the GENSET 98/46645; WO 98/50433; WO 98/24893; WO 96/34096; proteins. In addition, the humoral immune System of animals WO 96/33735; and WO 91/10741 (said references incorpo which produce a species of GENSET that resembles the rated by reference in their entireties). antigenic Sequence will preferentially recognize the differ 0281 Further included in the present invention are anti ences between the animal's native GENSET species and the bodies recombinantly fused or chemically conjugated antigen Sequence, and produce antibodies to these unique (including both covalently and non-covalently conjugations) Sites in the antigen Sequence. Such a technique will be to a polypeptide of the present invention. The antibodies particularly useful in obtaining antibodies that specifically may be specific for antigens other than polypeptides of the bind to any one of the GENSET proteins. present invention. For example, antibodies of the present invention may be recombinantly fused or conjugated to 0284. The antibodies of the invention may be labeled by, molecules useful as labels in detection assays and effector e.g., any one of the radioactive, fluorescent or enzymatic molecules Such as heterologous polypeptides, drugs, or labels known in the art. toxins. See, e.g., WO 92/08495; WO 91/14438; WO Uses of Polynucleotides 89/12624; U.S. Pat. No. 5,314,995; and EPO 396387, which disclosures are hereby incorporated by reference in their Uses of Polynucleotides as Reagents entireties. Fused antibodies may also be used to target the 0285) The polynucleotides of the present invention, par polypeptides of the present invention to particular cell types, ticularly those described in the “Oligonucleotide primers either in Vitro or in Vivo, by fusing or conjugating the and probes' Section, may be used as reagents in isolation polypeptides of the present invention to antibodies Specific procedures, diagnostic assays, and forensic procedures. For for particular cell Surface receptors. Antibodies fused or example, sequences from the GENSET polynucleotides of conjugated to the polypeptides of the present invention may the invention may be detectably labeled and used as probes also be used in vitro immunoassays and purification methods to isolate other Sequences capable of hybridizing to them. In using methods known in the art (See e.g., Harbor et al. Supra; addition, sequences from the GENSET polynucleotides of WO93/21232; EPO 439 095; Naramura, M. et al. 1994; U.S. the invention may be used to design PCR primers to be used Pat. No. 5,474,981; Gillies et al., 1992; Fell et al., 1991) in isolation, diagnostic, or forensic procedures. (Said references incorporated by reference in their entire ties). In Forensic Analyses 0282. The present invention further includes composi 0286 PCR primers may be used in forensic analyses, tions comprising the polypeptides of the present invention Such as the DNA fingerprinting techniques described below. fused or conjugated to antibody domains other than the Such analyses may utilize detectable probes or primers variable regions. For example, the polypeptides of the based on the Sequences of the polynucleotides of the inven present invention may be fused or conjugated to an antibody tion. Consequently, the present invention encompasses Fc region, or portion thereof. The antibody portion fused to methods of identification of an individual using the poly a polypeptide of the present invention may comprise the nucleotides of the invention in forensic analyses, wherein hinge region, CH1 domain, CH2 domain, and CH3 domain Said method includes the Steps of or any combination of whole domains or portions thereof. The polypeptides of the present invention may be fused or 0287 a) obtaining a biological Sample containing nucleic conjugated to the above antibody portions to increase the in acid material from an individual; Vivo half-life of the polypeptides or for use in immunoassays 0288 b) obtaining an identification pattern for this indi using methods known in the art. The polypeptides may also vidual using the polynucleotides of the invention, particu be fused or conjugated to the above antibody portions to larly using GENSET primers and probes; form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers 0289 c) comparing said identification pattern with a through disulfide bonding between the Fc portions. Higher reference identification pattern; and multimeric forms can be made by fusing the polypeptides to 0290 d) determining whether said identification pattern portions of IgA and IgM. Methods for fusing or conjugating is identical to Said reference identification pattern. US 2006/0053498A1 Mar. 9, 2006 32

0291. In one embodiment of this method, the identifica tains 200 amplified sequences. This PCR-generated DNA is tion pattern consists in Sequences of amplicons obtained then digested with one or a combination of, preferably, four using GENSET primers as explained in the sections entitled base specific restriction enzymes. Such enzymes are com “Forensic Matching by DNA Sequencing” and “Positive mercially available and known to those of skill in the art. Identification by DNA Sequencing”. After digestion, the resultant gene fragments are size Sepa 0292. In another embodiment, the identification pattern rated in multiple duplicate Wells on an agarose gel and consists in unique band or dot patterns obtained using any transferred to nitrocellulose using Southern blotting tech method described in the sections entitled “Southern Blot niques well known to those with skill in the art. For a review Forensic Identification”, “Dot Blot Identification Procedure” of Southern blotting see Davis et al. (1986), which disclo and “Alternative “Fingerprint Identification Technique”. Sure is hereby incorporated by reference in its entirety. 0293 Table I provides sets of related cDNAs of the 0297. A panel of probes based on the sequences of the invention, e.g. Sequences that represent allelic variants of a polynucleotides of the invention, or fragments thereof of at Single Sequence. Such variants are especially useful for the least 10 bases, are radioactively or calorimetrically labeled herein-described forensic analyses, and are also useful as using methods known in the art, Such as nick translation or polymorphic markers to examine, e.g. associations between end labeling, and hybridized to the Southern blot using the herein-discussed GENSET genes and various diseases or techniques known in the art. Preferably, the probe comprises conditions. at least 12, 15, or 17 consecutive nucleotides from the Forensic Matching by DNA Sequencing polynucleotide of the invention. More preferably, the probe 0294. In one exemplary method, DNA samples are iso comprises at least 20-30 consecutive nucleotides from the lated from forensic Specimens of, for example, hair, Semen, polynucleotide of the invention. In Some embodiments, the blood or skin cells by conventional methods. A panel of PCR probe comprises more than 30 nucleotides from the poly primerS designed from different polynucleotides of the nucleotide of the invention. In other embodiments, the probe invention using any technique known to those skilled in the comprises at least 40, at least 50, at least 75, at least 100, at art including those described herein, is then utilized to least 150, or at least 200 consecutive nucleotides from the amplify DNA of approximately 100-200 bases in length polynucleotide of the invention. from the forensic specimen. Corresponding Sequences are 0298 Preferably, at least 5 to 10 of these labeled probes obtained from a test subject. Each of these identification are used, and more preferably at least about 20 or 30 are used DNAS is then Sequenced using Standard techniques, and a to provide a unique pattern. The resultant bands appearing Simple database comparison determines the differences, if from the hybridization of a large Sample of polynucleotide any, between the Sequences from the Subject and those from of the invention will be a unique identifier. Since the the Sample. Statistically significant differences between the restriction enzyme cleavage will be different for every Suspect's DNA sequences and those from the Sample con individual, the band pattern on the Southern blot will also be clusively prove a lack of identity. This lack of identity can unique. Increasing the number of cDNA probes will provide be proven, for example, with only one Sequence. Identity, on a Statistically higher level of confidence in the identification the other hand, should be demonstrated with a large number Since there will be an increased number of Sets of bands used of Sequences, all matching. Preferably, a minimum of 50 for identification. Statistically identical Sequences of 100 bases in length are used to prove identity between the Suspect and the Sample. Dot Blot Identification Procedure Positive Identification by DNA Sequencing 0299 Another technique for identifying individuals using 0295) The “Forensic Matching by DNA Sequencing” the polynucleotide Sequences disclosed herein utilizes a dot technique described herein may also be used on a larger blot hybridization technique. Scale to provide a unique fingerprint-type identification of 0300 Genomic DNA is isolated from nuclei of Subject to any individual. In this technique, primers are prepared from be identified. Oligonucleotide probes of approximately 30 a large number of polynucleotides of the invention. Prefer bp in length are Synthesized that correspond to at least 10, ably, 20 to 50 different primers are used. These primers are preferably 50 sequences from the polynucleotide of the used to obtain a corresponding number of PCR-generated invention. The probes are used to hybridize to the genomic DNA segments from the individual in question. Each of DNA through conditions known to those in the art. The these DNA segments is Sequenced. The database of oligonucleotides are end labeled with P. using polynucle Sequences generated through this procedure uniquely iden otide kinase (Pharmacia). Dot Blots are created by spotting tifies the individual from whom the Sequences were the genomic DNA onto nitrocellulose or the like using a obtained. The same panel of primerS may then be used at any vacuum dot blot manifold (BioRad, Richmond Calif.). The later time to absolutely correlate tissue or other biological nitrocellulose filter containing the genomic Sequences is Specimen with that individual. baked or UV linked to the filter, prehybridized and hybrid ized with labeled probe using techniques known in the art Southern Blot Forensic Identification (Davis et al. 1986). The P labeled DNA fragments are 0296) The “Positive Identification by DNA Sequencing” Sequentially hybridized with Successively Stringent condi procedure described herein is repeated to obtain a panel of tions to detect minimal differences between the 30 bp at least 10 amplified Sequences from an individual and a sequence and the DNA. Tetramethylammonium chloride is Specimen. Preferably, the panel contains at least 50 ampli useful for identifying clones containing Small numbers of fied Sequences. More preferably, the panel contains 100 nucleotide mismatches (Wood et al., 1985). A unique pattern amplified Sequences. In Some embodiments, the panel con of dots distinguishes one individual from another individual. US 2006/0053498A1 Mar. 9, 2006 33

Alternative “Fingerprint Identification Technique different restriction enzyme which has a 6 base recognition 0301 In a representative alternative fingerprinting pro Site and leaves a blunt end. Following digestion, oligonucle cedure, the probes are derived from cDNAS. Preferably, a otide adapters are ligated to each end of the resulting plurality of probes having Sequences from different genes genomic DNA fragments. are used as follows. Polynucleotides containing at least 10 0307 For each of the five genomic DNA libraries, a first consecutive bases from these Sequences can be used as probes. Preferably, the probe comprises at least 12, 15, or 17 PCR reaction is performed according to the manufacturer's consecutive nucleotides from the polynucleotide of the instructions (which are incorporated herein by reference) invention. More preferably, the probe comprises at least using an Outer adaptor primer provided in the kit and an 20-30 consecutive nucleotides from the polynucleotide of outer gene Specific primer. The gene Specific primer should the invention. In Some embodiments, the probe comprises be selected to be specific for the polynucleotide of the more than 30 nucleotides from the polynucleotide of the invention of interest and should have a melting temperature, invention. In other embodiments, the probe comprises at length, and location in the polynucleotide of the invention least 40, at least 50, at least 75, at least 100, at least 150, or which is consistent with its use in PCR reactions. Each first at least 200 consecutive nucleotides from the polynucleotide PCR reaction contains 5 ng of genomic DNA, 5 ul of 10xTth of the invention. reaction buffer, 0.2 mM of each dNTP, 0.2M each of outer adaptor primer and Outer gene Specific primer, 1.1 mM of 0302 Oligonucleotides, generally 20-mers, are prepared Mg(OAc), and 1ul of the Tth polymerase 50x mix in a total from a large number, e.g. 50, 100, or 200, of polynucleotides volume of 50 ul. The reaction cycle for the first PCR reaction of the invention using commercially available oligonucle is as follows: 1 min at 94 degrees Celsius/2 sec at 94 degree otide Services Such as Genset (Paris, France). Cell Samples Celsius, 3 min at 72 degrees Celsius (7 cycles)/2 sec at 94 from the test Subject are processed for DNA using tech degrees Celsius, 3 min at 67 degrees Celsius (32 cycles)/5 niques well known to those with skill in the art. The nucleic min at 67 degrees Celsius. acid is digested with restriction enzymes Such as EcoRI and Xbal. Following digestion, Samples are applied to Wells for 0308 The product of the first PCR reaction is diluted and electrophoresis. The procedure, as known in the art, may be used as a template for a Second PCR reaction according to modified to accommodate polyacrylamide electrophoresis, the manufacturer's instructions using a pair of nested prim however in this example, Samples containing 5 ug of DNA erS which are located internally on the amplicon resulting are loaded into wells and separated on 0.8% agarose gels. from the first PCR reaction. For example, 5 ul of the reaction The gels are transferred onto nitrocellulose using Standard product of the first PCR reaction mixture may be diluted 180 Southern blotting techniques. times. Reactions are made in a 50 ul Volume having a composition identical to that of the first PCR reaction except 0303) 10 ng of each of the oligonucleotides are pooled the nested primers are used. The first nested primer is and end-labeled with P. The nitrocellulose is prehybrid Specific for the adaptor, and is provided with the Genom ized with blocking solution and hybridized with the labeled eWalkerTM kit. The second nested primer is specific for the probes. Following hybridization and washing, the nitrocel particular polynucleotide of the invention for which the lulose filter is exposed to X-Omat AR X-ray film. The promoter is to be cloned and should have a melting tem resulting hybridization pattern will be unique for each perature, length, and location in the polynucleotide of the individual. invention which is consistent with its use in PCR reactions. 0304. It is additionally contemplated within this example The reaction parameters of the second PCR reaction are as that the number of probe Sequences used can be varied for follows: 1 min at 94 degrees Celsius/2 sec at 94 degrees additional accuracy or clarity. Celsius, 3 min at 72 degrees Celsius (6 cycles)/2 sec at 94 degrees Celsius, 3 min at 67 degrees Celsius (25 cycles)/5 To Find Corresponding Genomic DNA Sequences min at 67 degrees Celsius 0305) The GENSET cDNAs of the invention may also be 0309 The product of the second PCR reaction is purified, used to clone Sequences located upstream of the cDNAS of cloned, and Sequenced using Standard techniques. Alterna the invention on the corresponding genomic DNA. Such tively, two or more human genomic DNA libraries can be upstream Sequences may be capable of regulating gene constructed by using two or more restriction enzymes. The expression, including promoter Sequences, enhancer digested genomic DNA is cloned into vectors which can be Sequences, and other upstream Sequences which influence converted into Single Stranded, circular, or linear DNA. A transcription or translation levels. Once identified and biotinylated oligonucleotide comprising at least 15 nucle cloned, these upstream regulatory Sequences may be used in otides from the polynucleotide of the invention Sequence is expression vectors designed to direct the expression of an hybridized to the single stranded DNA. Hybrids between the inserted gene in a desired Spatial, temporal, developmental, biotinylated oligonucleotide and the single stranded DNA or quantitative fashion. containing the polynucleotide of the invention Sequence are Use of cDNAS or Fragments Thereof to Clone Upstream isolated as described herein. Thereafter, the Single Stranded Sequences from Genomic DNA DNA containing the polynucleotide of the invention Sequence is released from the beads and converted into 0306 Sequences derived from polynucleotides of the double Stranded DNA using a primer Specific for the poly inventions may be used to isolate the promoters of the nucleotide of the invention Sequence or a primer correspond corresponding genes using chromosome walking tech ing to a Sequence included in the cloning vector. The niques. In one chromosome walking technique, which ulti resulting double stranded DNA is transformed into bacteria. lizes the GenomeWalkerTM kit available from Clontech, five DNAS containing the GENSET polynucleotide sequences complete genomic DNA Samples are each digested with a are identified by colony PCR or colony hybridization. US 2006/0053498A1 Mar. 9, 2006 34

Identification of Promoters in Cloned Upstream Sequences Nos. 5,698,389; 5,643,746; 5,502,176; and 5,266,488; the disclosures of which are incorporated by reference herein in 0310. Once the upstream genomic sequences have been their entirety. cloned and Sequenced as described above, prospective pro moters and transcription Start Sites within the upstream 0313 The strength and the specificity of the promoter of Sequences may be identified by comparing the Sequences each GENSET gene can be assessed through the expression upstream of the polynucleotides of the inventions with levels of a detectable polynucleotide operably linked to the GENSET promoter in different types of cells and tissues. databases containing known transcription Start Sites, tran The detectable polynucleotide may be either a polynucle Scription factor binding Sites, or promoter Sequences. otide that specifically hybridizes with a predefined oligo 0311. In addition, promoters in the upstream Sequences nucleotide probe, or a polynucleotide encoding a detectable may be identified using promoter reporter vectors as fol protein, including a GENSET polypeptide or a fragment or lows. The expression of the reporter gene will be detected a variant thereof. This type of assay is well known to those when placed under the control of regulatory active poly skilled in the art and is described in U.S. Pat. Nos. 5,502, nucleotide fragments or variants of the GENSET promoter 176; and 5,266,488; the disclosures of which are incorpo region located upstream of the first exon of the GENSET rated by reference herein in their entirety. Some of the gene. Suitable promoter reporter vectors, into which the methods are discussed in more detail elsewhere in the GENSET promoter sequences may be cloned include application. pSEAP-Basic, pSEAP-Enhancer, p?3gal-Basic, p?3gal-En 0314. The promoters and other regulatory sequences hancer, or pEGFP-1 Promoter Reporter vectors available located upstream of the polynucleotides of the inventions from Clontech, or pGL2-basic or pGL3-basic promoterless may be used to design expression vectors capable of direct luciferase reporter gene vector from Promega. Briefly, each ing the expression of an inserted gene in a desired Spatial, of these promoter reporter vectors include multiple cloning temporal, developmental, or quantitative manner. A pro Sites positioned upstream of a reporter gene encoding a moter capable of directing the desired Spatial, temporal, readily assayable protein Such as Secreted alkaline phos developmental, and quantitative patterns may be Selected phatase, luciferase, beta-galactosidase, or green fluorescent using the results of the expression analysis described herein. protein. The sequences upstream the GENSET coding For example, if a promoter which confers a high level of region are inserted into the cloning Sites upstream of the expression in muscle is desired, the promoter Sequence reporter gene in both orientations and introduced into an upstream of a polynucleotide of the invention derived from appropriate host cell. The level of reporter protein is assayed an mRNA which is expressed at a high level in muscle may and compared to the level obtained from a vector which be used in the expression vector. Such vectors are described lacks an insert in the cloning site. The presence of an in more detail elsewhere in the application. elevated expression level in the vector containing the insert with respect to the control vector indicates the presence of 0315 Preferably, the desired promoter is placed near a promoter in the insert. If necessary, the upstream multiple restriction Sites to facilitate the cloning of the Sequences can be cloned into Vectors which contain an desired insert downstream of the promoter, Such that the enhancer for increasing transcription levels from weak pro promoter is able to drive expression of the inserted gene. The moter Sequences. A significant level of expression above that promoter may be inserted in conventional nucleic acid observed with the vector lacking an insert indicates that a backbones designed for extrachromosomal replication, inte promoter Sequence is present in the inserted upstream gration into the host chromosomes or transient expression. Sequence. Suitable backbones for the present expression vectors include retroviral backbones, backbones from eukaryotic 0312 Promoter sequence within the upstream genomic episomes such as SV40 or Bovine Papilloma Virus, back DNA may be further defined by site directed mutagenesis, bones from bacterial episomes, or artificial chromosomes. linker Scanning analysis, or other techniques familiar to those skilled in the art. For example, the boundaries of 0316 Preferably, the expression vectors also include a promoters may be further investigated by constructing polyA signal downstream of the multiple restriction sites for nested 5' and/or 3' deletions in the upstream DNA using directing the polyadenylation of mRNA transcribed from the conventional techniques Such as Exonuclease III or appro gene inserted into the expression vector. priate restriction endonuclease digestion. The resulting dele tion fragments can be inserted into the promoter reporter To Find Similar Sequences vector to determine whether the deletion has increased, 0317 Polynucleotides of the invention may be used to reduced or illuminated promoter activity, Such as described, isolate and/or purify nucleic acids similar thereto using any for example, by Coles et al. (1998), the disclosure of which methods well known to those skilled in the art including the is incorporated herein by reference in its entirety. In this techniques based on hybridization or on amplification way, the boundaries of the promoters may be defined. If described in this section. These methods may be used to desired, potential individual regulatory Sites within the pro obtain the genomic DNAS which encode the mRNAS from moter may be identified using site directed mutagenesis or which the GENSET cDNAs are derived, mRNAs corre linker Scanning to obliterate potential transcription factor sponding to GENSET cDNAs, or nucleic acids which are binding sites within the promoter individually or in combi homologous to GENSET cDNAS or fragments thereof, such nation. The effects of these mutations on transcription levels as variants, Species homologues or orthologs. Thus, a plu may be determined by inserting the mutations into cloning rality of cDNAS similar to GENSET polynucleotides may be Sites in promoter reporter vectors. This type of assay is well provided as cDNA libraries for Subsequent evaluation of the known to those skilled in the art and is described in WO encoded proteins or used in diagnostic assays as described 97/17359, U.S. Pat. No. 5,374,544; EP 582 796; U.S. Pat. herein. cDNAS prepared by any method described therein US 2006/0053498A1 Mar. 9, 2006 35 may be Subsequently engineered to obtain nucleic acids 0324 For probes between 14 and 70 nucleotides in length which include desired fragments of the cDNA using con the melting temperature (Tm) is calculated using the for ventional techniques Such as Subcloning, PCR, or in vitro mula: Tm=81.5+16.6(log (Na+))+0.41(fraction G+C)-600/ oligonucleotide Synthesis. For example, nucleic acids which N) where N is the length of the probe. include only the coding Sequences may be obtained using 0325 If the hybridization is carried out in a solution techniques known to those skilled in the art. Similarly, containing formamide, the melting temperature may be nucleic acids containing any other desired fragment of the calculated using the equation: Tm=81.5+16.6(log (Na+))+ coding Sequences for the encoded protein may be obtained. 0.41(fraction G+C)–(0.63% formamide)-(600/N) where N 0318 Indeed, cDNAs of the present invention or frag is the length of the probe. ments thereof may be used to isolate nucleic acids similar to 0326 Prehybridization may be carried out in 6xSSC, 5x cDNAS from a cDNA library or a genomic DNA library. Denhardt's reagent, 0.5% SDS, 100 ug denatured frag Such cDNA libraries or genomic DNA libraries may be mented salmon sperm DNA or 6xSSC, 5x Denhardt's obtained from a commercial Source or made using tech reagent, 0.5% SDS, 100 ug denatured fragmented salmon niques familiar to those skilled in the art Such as those sperm DNA, 50% formamide. The formulas for SSC and described in PCT publication WO 00/37491, which disclo Sure is hereby incorporated by reference in its entirety. Denhardt's Solutions are listed in Sambrook et al., 1986. Examples of methods for obtaining nucleic acids similar to 0327) Hybridization is conducted by adding the detect GENSET polynucleotides are described below. able probe to the prehybridization solutions listed above. Where the probe comprises double stranded DNA, it is Hybridization-Based Methods denatured before addition to the hybridization solution. The filter is contacted with the hybridization solution for a 03.19 Techniques for identifying cDNA clones in a sufficient period of time to allow the probe to hybridize to cDNA library which hybridize to a given probe sequence are nucleic acids containing Sequences complementary thereto disclosed in Sambrook et al., (1989) and in Hames and or homologous thereto. For probes over 200 nucleotides in Higgins (1985), the disclosures of which are incorporated length, the hybridization may be carried out at 15-25 C. herein by reference in their entireties. The same techniques below the Tm. For shorter probes, such as oligonucleotide may be used to isolate genomic DNAS. probes, the hybridization may be conducted at 15-25 C. 0320 Briefly, cDNA or genomic DNA clones which below the Tm. Preferably, for hybridizations in 6xSSC, the hybridize to the detectable probe are identified and isolated hybridization is conducted at approximately 68 C. Prefer for further manipulation as follows. Any polynucleotide ably, for hybridizations in 50% formamide containing solu fragment of the invention may be used as a probe, in tions, the hybridization is conducted at approximately 42 C. particular those defined in the “Oligonucleotide primers and 0328. Following hybridization, the filter is washed in probes' Section. A probe comprising at least 10 consecutive 2xSSC, 0.1% SDS at room temperature for 15 minutes. The nucleotides from a GENSET cDNA or fragment thereof is filter is then washed with 0.1XSSC, 0.5% SDS at room labeled with a detectable label Such as a radioisotope or a temperature for 30 minutes to 1 hour. Thereafter, the solu fluorescent molecule. Preferably, the probe comprises at tion is washed at the hybridization temperature in 0.1xSSC, least 12, 15, or 17 consecutive nucleotides from the cDNA 0.5% SDS. A final wash is conducted in 0.1XSSC at room or fragment thereof. More preferably, the probe comprises temperature. 20 to 30 consecutive nucleotides from the cDNA or frag ment thereof. In Some embodiments, the probe comprises 0329 Nucleic acids which have hybridized to the probe more than 30 nucleotides from the cDNA or fragment are identified by autoradiography or other conventional thereof. techniques. 0321 Techniques for labeling the probe are well known Low and Moderate Conditions and include phosphorylation with polynucleotide kinase, 0330 Changes in the stringency of hybridization and nick translation, in Vitro transcription, and non radioactive Signal detection are primarily accomplished through the techniques. The cDNAS or genomic DNAS in the library are manipulation of formamide concentration (lower percent transferred to a nitrocellulose or nylon filter and denatured. ages of formamide result in lowered Stringency); Salt con After blocking of non Specific Sites, the filter is incubated ditions, or temperature. The above procedure may thus be with the labeled probe for an amount of time sufficient to modified to identify nucleic acids having decreasing levels allow binding of the probe to cDNAS or genomic DNAS of identity to the probe Sequence. For example, the hybrid containing a Sequence capable of hybridizing thereto. ization temperature may be decreased in increments of 5 C. from 68 C. to 42 C. in a hybridization buffer having a 0322 By varying the stringency of the hybridization Sodium concentration of approximately 1M. Following conditions used to identify cDNAS or genomic DNAS which hybridization, the filter may be washed with 2xSSC, 0.5% hybridize to the detectable probe, cDNAS or genomic DNAS SDS at the temperature of hybridization. These conditions having different levels of identity to the probe can be are considered to be “moderate” conditions above 50 C. and identified and isolated as described below. “low” conditions below 50° C. Alternatively, the hybridiza Stringent Conditions tion may be carried out in buffers, such as 6xSSC, contain ing formamide at a temperature of 42 C. In this case, the 0323 “Stringent hybridization conditions” are defined as concentration of formamide in the hybridization buffer may conditions in which only nucleic acids having a high level of be reduced in 5% increments from 50% to 0% to identify identity to the probe are able to hybridize to said probe. clones having decreasing levels of identity to the probe. These conditions may be calculated as follows: Following hybridization, the filter may be washed with US 2006/0053498A1 Mar. 9, 2006 36

6xSSC, 0.5% SDS at 50° C. These conditions are considered Scriptase used. Preferred oligo(dT) primers for priming to be “moderate' conditions above 25% formamide and reverse transcription of mRNAS are oligonucleotides con “low” conditions below 25% formamide. cDNAS or taining a stretch of thymidine residues of Sufficient length to genomic DNAS which have hybridized to the probe are hybridize specifically to the polyA tail of mRNAS, prefer identified by autoradiography or other conventional tech ably of 12 to 18 thymidine residues in length. More pref niques. erably, Such oligo(T) primers comprise an additional 0331 Note that variations in the above conditions may be Sequence upstream of the poly(dT) stretch in order to allow accomplished through the inclusion and/or Substitution of the addition of a given Sequence to the 5' end of all first alternate blocking reagents used to SuppreSS background in cDNA strands which may then be used to facilitate Subse hybridization experiments. Typical blocking reagents quent manipulation of the cDNA. Preferably, this added include Denhardt's reagent, BLOTTO, heparin, denatured Sequence is 8 to 60 residues in length. For instance, the Salmon Sperm DNA, and commercially available proprietary addition of a restriction site in 5' of cDNAS facilitates formulations. The inclusion of Specific blocking reagents subcloning of the obtained cDNA. Alternatively, such an may require modification of the hybridization conditions added 5' end may also be used to design primers of PCR to described above, due to problems with compatibility. specifically amplify cDNA clones of interest. 0339) The first cDNA strand is then hybridized to a 0332 Consequently, the present invention encompasses Second primer. Any polynucleotide fragment of the inven methods of isolating nucleic acids Similar to the polynucle tion may be used, and in particular those described in the otides of the invention, comprising the Steps of “Oligonucleotide primers and probes' Section. This Second 0333 a) contacting a collection of cDNA or genomic primer contains at least 10 consecutive nucleotides of a DNA molecules with a detectable probe comprising at least polynucleotide of the invention. Preferably, the primer com 12, 15, 18, 20, 23, 25, 28, 30, 35, 40 or 50 consecutive prises at least 10, 12, 15, 17, 18, 20, 23, 25, or 28 consecu nucleotides of a Sequence Selected from the group consisting tive nucleotides of a polynucleotide of the invention. In of the sequences of SEQ ID NOs: 1-169,339-455,561-784, Some embodiments, the primer comprises more than 30 the Sequences of clones inserts of the deposited clone pool nucleotides of a polynucleotide of the invention. If it is and Sequences complementary thereto under Stringent, mod desired to obtain cDNAS containing the full protein coding erate or low conditions which permit said probe to hybridize Sequence, including the authentic translation initiation site, to at least a cDNA or genomic DNA molecule in said the Second primer used contains Sequences located upstream collection; of the translation initiation site. The second primer is extended to generate a Second cDNA Strand complementary 0334 b) identifying said cDNA or genomic DNA mol to the first cDNA strand. Alternatively, RT-PCR may be ecule which hybridizes to said detectable probe; and performed as described above using primers from both ends 0335 c) isolating said cDNA or genomic DNA molecule of the cDNA to be obtained. which hybridized to said probe. 0340. The double stranded cDNAs made using the meth PCR-Based Methods ods described above are isolated and cloned. The cDNAS 0336. In addition to the above described methods, other may be cloned into vectorS Such as plasmids or viral vectors protocols are available to obtain homologous cDNAS using capable of replicating in an appropriate host cell. For GENSET cDNA of the present invention or fragment thereof example, the host cell may be a bacterial, mammalian, avian, as outlined in the following paragraphs. or insect cell. 0341 Techniques for isolating mRNA, reverse transcrib 0337 cDNAS may be prepared by obtaining mRNA from ing a primer hybridized to mRNA to generate a first cDNA the tissue, cell, or organism of interest using mRNA prepa Strand, extending a primer to make a Second cDNA Strand ration procedures utilizing polyA Selection procedures or complementary to the first cDNA Strand, isolating the other techniques known to those skilled in the art. A first double stranded cDNA and cloning the double stranded primer capable of hybridizing to the polyA tail of the mRNA cDNA are well known to those skilled in the art and are is hybridized to the mRNA and a reverse transcription described in Current Protocols in Molecular Biology, John reaction is performed to generate a first cDNA Strand. Wiley & Sons, Inc. 1997 and Sambrook et al., 1989. 0338. The term “capable of hybridizing to the polyA tail 0342 Consequently, the present invention encompasses of said mRNA refers to and embraces all primers contain ing stretches of thymidine residues, So-called oligo(dT) methods of making cDNAs. In a first embodiment, the primers, that hybridize to the 3' end of eukaryotic poly(A)+ method of making a cDNA comprises the Steps of mRNAS to prime the synthesis of a first cDNA strand. 0343 a) contacting a collection of mRNA molecules Techniques for generating Said oligo (dT) primers and from human cells with a primer comprising at least 12, 15, hybridizing them to mRNA to Subsequently prime the 18, 20, 23, 25, 28, 30, 35, 40, or 50 consecutive nucleotides reverse transcription of Said hybridized mRNA to generate a of a Sequence Selected from the group consisting of the first cDNA strand are well known to those skilled in the art sequences complementary to SEQ ID NOS:1-169,339-455, and are described in Current Protocols in Molecular Biology, 561-784 and Sequences complementary to a clone insert of John Wiley and Sons, Inc. 1997 and Sambrook et al., 1989. the deposited clone pool; Preferably, said oligo (dT) primers are present in a large excess in order to allow the hybridization of all mRNA 3’ 0344 b) hybridizing said primer to an mRNA in said ends to at least one oligo (dT) molecule. The priming and collection; reverse transcription Steps are preferably performed between 0345 c) reverse transcribing said hybridized primer to 37 C. and 55 C. depending on the type of reverse tran make a first cDNA strand from said mRNA; US 2006/0053498A1 Mar. 9, 2006 37

0346 d) making a second cDNA strand complementary forming a polymerase chain reaction with Said Second and to said first cDNA strand; and third primers to generate Said Second cDNA Strand. 0347 e) isolating the resulting cDNA comprising said 0361 Alternatively, the second cDNA strand may be first cDNA strand and said second cDNA strand. made by: 0348 Another embodiment of the present invention is a 0362) a) contacting said first cDNA strand with a second purified cDNA obtainable by the method of the preceding primer comprising at least 12, 15, 18, 20, 23, 25, 28, 30, 35, paragraph. In one aspect of this embodiment, the cDNA 40, or 50 consecutive nucleotides of a Sequence Selected encodes at least a portion of a human polypeptide. from the group consisting of SEQ ID NOS:1-169,339-455, 561-784 and sequences of clone inserts of the deposited 0349. In a second embodiment, the method of making a clone pool; cDNA comprises the steps of 0363 b) hybridizing said second primer to said first 0350 a) contacting a collection of mRNA molecules strand cDNA; and from human cells with a first primer capable of hybridizing to the polyA tail of said mRNA; 0364 c) extending said hybridized second primer to generate Said Second cDNA Strand. 0351 b) hybridizing said first primer to said polyA tail; 0365 Another embodiment of the present invention is a 0352 c) reverse transcribing said mRNA to make a first purified cDNA obtainable by a method of making a cDNA cDNA strand; of the invention. In one aspect of this embodiment, Said 0353 d) making a second cDNA strand complementary cDNA encodes at least a portion of a human polypeptide. to Said first cDNA Strand using at least one primer compris Other Protocols ing at least 12, 15, 18, 20, 23, 25, 28, 30, 35, 40, or 50 consecutive nucleotides of a Sequence Selected from the 0366 Alternatively, other procedures may be used for group consisting of SEQ ID NOS:1-169,339-455, 561-784 obtaining homologous cDNAS. In one approach, cDNAS are and Sequences of clone inserts of the deposited clone pool; prepared from mRNA and cloned into double stranded and phagemids as follows. The cDNA library in the double Stranded phagemids is then rendered Single Stranded by 0354) e) isolating the resulting cDNA comprising said treatment with an endonuclease, Such as the Gene II product first cDNA strand and said second cDNA strand. of the phage F1 and an exonuclease (Chang et al., 1993, which disclosure is hereby incorporated by reference in its 0355. In another aspect of this method the second cDNA entirety). A biotinylated oligonucleotide comprising the Strand is made by sequence of a fragment of a known GENSET cDNA, 0356) a) contacting said first cDNA strand with a second genomic DNA or fragment thereof is hybridized to the single primer comprising at least 12, 15, 18, 20, 23, 25, 28, 30, 35, Stranded phagemids. Preferably, the fragment comprises at 40, or 50 consecutive nucleotides of a Sequence Selected least 10, 12, 15, 17, 18, 20, 23, 25, or 28 consecutive from the group consisting of SEQ ID NOS:1-169,339-455, nucleotides of a Sequence Selected from the group consisting 561-784 and sequences of clone inserts of the deposited of the sequences of SEQ ID NOS:1-169,339-455,561-784 clone pool, and a third primer which Sequence is fully and Sequences of clone inserts of the deposited clone pool. included within the Sequence of Said first primer; 0367 Hybrids between the biotinylated oligonucleotide 0357 b) performing a first polymerase chain reaction and phagemids are isolated by incubating the hybrids with with said second and third primers to generate a first PCR Streptavidin coated paramagnetic beads and retrieving the product; beads with a magnet (Fry et al., 1992, which disclosure is hereby incorporated by reference in its entirety). Thereafter, 0358 c) contacting said first PCR product with a fourth the resulting phagemids are released from the beads and primer, comprising at least 12, 15, 18, 20, 23, 25, 28.30, 35, converted into double Stranded DNA using a primer Specific 40, or 50 consecutive nucleotides of Said Sequence Selected for the GENSET cDNA or fragment used to design the from the group consisting of SEQ ID NOS:1-169,339-455, biotinylated oligonucleotide. Alternatively, protocols Such 561-784 and sequences of clone inserts of the deposited as the Gene Trapper kit (Gibco BRL), which disclosure is clone pool, and a fifth primer, which Sequence is fully which disclosure is hereby incorporated by reference in its included within the Sequence of Said third primer, wherein entirety, may be used. The resulting double stranded DNA is said fourth and fifth hybridize to sequences within said first transformed into bacteria. Homologous cDNAS to the PCR product; and GENSET cDNA or fragment thereof sequence are identified 0359 d) performing a second polymerase chain reaction, by colony PCR or colony hybridization. thereby generating a Second PCR product. As a Chromosome Marker 0360 Alternatively, the second cDNA strand may be 0368 Chromosomal localization of the cDNA of the made by contacting Said first cDNA Strand with a Second present invention were determined using information from primer comprising at least 12, 15, 18, 20, 23, 25, 28, 30, 35, public and proprietary databases. Table II lists the putative 40, or 50 consecutive nucleotides of a Sequence Selected chromosomal location of the polynucleotides of the present from the group consisting of SEQ ID NOS:1-169,339-455, invention. Column one lists the Sequence identification 561-784 and sequences of clone inserts of the deposited number with the corresponding chromosomal location listed clone pool, and a third primer which Sequence is fully in column two. Thus, the present invention also relates to included within the Sequence of Said first primer and per methods and compositions using the chromosomal location US 2006/0053498A1 Mar. 9, 2006 38 of the polynucleotides of the invention to construct a human is performed in a microplate thermocycler (Techne) under high resolution map or to identify a given chromosome in a the following conditions: 30 cycles of 94 degrees Celsius, Sample using any techniques known to those skilled in the 1.4 min; 55 degrees Celsius, 2 min; and 72 degrees Celsius, art including those disclosed below. 2 min; with a final extension at 72 degrees Celsius for 10 min. The amplified products are analyzed on a 6% poly 0369 GENSET polynucleotides may also be mapped to acrylamide Sequencing gel and Visualized by autoradiogra their chromosomal locations using any methods or tech phy. If the length of the resulting PCR product is identical niques known to those skilled in the art including radiation to the distance between the ends of the primer Sequences in hybrid (RH) mapping, PCR-based mapping and Fluores the cDNA from which the primers are derived, then the PCR cence in situ hybridization (FISH) mapping described reaction is repeated with DNA templates from two panels of below. human-rodent somatic cell hybrids, BIOS PCRable DNA Radiation Hybrid Mapping (BIOS Corporation) and NIGMS Human-Rodent Somatic 0370 Radiation hybrid (RH) mapping is a somatic cell Cell Hybrid Mapping Panel Number 1 (NIGMS, Camden, genetic approach that can be used for high resolution map N.J.). ping of the human genome. In this approach, cell lines 0374 PCR is used to screen a series of somatic cell containing one or more human chromosomes are lethally hybrid cell lines containing defined Sets of human chromo irradiated, breaking each chromosome into fragments whose somes for the presence of a given cDNA or genomic DNA. Size depends on the radiation dose. These fragments are DNA is isolated from the somatic hybrids and used as rescued by fusion with cultured rodent cells, yielding Sub Starting templates for PCR reactions using the primer pairs clones containing different fragments of the human genome. from the GENSET cDNAs or genomic DNAs. Only those This technique is described by Benham et al. (1989) and Cox Somatic cell hybrids with chromosomes containing the et al., (1990), which disclosures are hereby incorporated by human gene corresponding to the GENSET cDNA or reference in their entireties. The random and independent genomic DNA will yield an amplified fragment. The nature of the Subclones permits efficient mapping of any GENSET cDNAS or genomic DNAS are assigned to a human genome marker. Human DNA isolated from a panel chromosome by analysis of the Segregation pattern of PCR of 80-100 cell lines provides a mapping reagent for ordering products from the somatic hybrid DNA templates. The GENSET cDNAS or genomic DNAS. In this approach, the Single human chromosome present in all cell hybrids that frequency of breakage between markers is used to measure give rise to an amplified fragment is the chromosome distance, allowing construction of fine resolution maps as containing that GENSET cDNA or genomic DNA. For a has been done using conventional ESTs (Schuler et al., review of techniques and analysis of results from Somatic 1996), which disclosure is hereby incorporated by reference cell gene mapping experiments, see Ledbetter et al., (1990), in its entirety. which disclosure is hereby incorporated by reference in its 0371 RH mapping has been used to generate a high entirety. resolution whole genome radiation hybrid map of human Mapping of cDNAS to Chromosomes Using Fluorescence in chromosome 17q22-q25.3 acroSS the genes for growth hor situ Hybridization mone (GH) and thymidine kinase (TK) (Foster et al., 1996), the region Surrounding the Gorlin Syndrome gene (Ober 0375 Fluorescence in situ hybridization (FISH) allows mayr et al., 1996), 60 loci covering the entire short arm of the GENSET cDNA or genomic DNA to be mapped to a chromosome 12 (Raeymaekers et al., 1995), the region of particular location on a given chromosome. The chromo human chromosome 22 containing the neurofibromatosis Somes to be used for fluorescence in Situ hybridization type 2 locus (Frazer et al., 1992) and 13 loci on the long arm techniques may be obtained from a variety of Sources of chromosome 5 (Warrington et al., 1991), which disclo including cell cultures, tissues, or whole blood. Sures are hereby incorporated by reference in their entireties. 0376. In a preferred embodiment, chromosomal localiza Mapping of cDNAS to Human Chromosomes using PCR tion of a GENSET cDNA or genomic DNA is obtained by Techniques FISH as described by Cherif et al. (1990), which disclosure 0372 GENSET cDNAs and genomic DNAS may be is hereby incorporated by reference in its entirety. assigned to human chromosomes using PCR based meth Metaphase chromosomes are prepared from phytohemag odologies. In Such approaches, oligonucleotide primer pairs glutinin (PHA)-stimulated blood cell donors. PHA-stimu are designed from the cDNA sequence to minimize the lated lymphocytes from healthy males are cultured for 72 h chance of amplifying through an intron. Preferably, the in RPMI-1640 medium. For synchronization, methotrexate oligonucleotide primers are 18-23 bp in length and are (10 uM) is added for 17 h, followed by addition of 5-bro designed for PCR amplification. The creation of PCR prim modeoxyuridine (5-BudR, 0.1 mM) for 6 h. Colcemid (1 erS from known Sequences is well known to those with Skill ug/ml) is added for the last 15 min before harvesting the in the art. For a review of PCR technology see Erlich (1992), cells. Cells are collected, washed in RPMI, incubated with which disclosure is hereby incorporated by reference in its a hypotonic solution of KCl (75 mM) at 37 degrees Celsius entirety. for 15 min and fixed in three changes of methanol:acetic acid (3:1). The cell Suspension is dropped onto a glass slide and 0373 The primers are used in polymerase chain reactions air dried. The GENSET cDNA or genomic DNA is labeled (PCR) to amplify templates from total human genomic with biotin-16 dUTP by nick translation according to the DNA. PCR conditions are as follows: 60 ng of genomic manufacturer's instructions (Bethesda Research Laborato DNA is used as a template for PCR with 80 ng of each ries, Bethesda, Md.), purified using a Sephadex G-50 col oligonucleotide primer, 0.6 unit of Taq polymerase, and 1 umn (Pharmacia, UpSSala, Sweden) and precipitated. Just uCu of a P-labeled deoxycytidine triphosphate. The PCR prior to hybridization, the DNA pellet is dissolved in hybrid US 2006/0053498A1 Mar. 9, 2006 39 ization buffer (50% formamide, 2xSSC, 10% dextran sul Identification of Genes Associated with Hereditary Diseases fate, 1 mg/ml Sonicated salmon sperm DNA, pH 7) and the or Drug Response probe is denatured at 70 degrees Celsius for 5-10 min. 0380 This example illustrates an approach useful for the 0377 Slides kept at -20 degrees Celsius are treated for 1 association of GENSET cDNAS or genomic DNAS with h at 37 degrees Celsius with RNase A (100 ug/ml), rinsed particular phenotypic characteristics. In this example, a three times in 2xSSC and dehydrated in an ethanol series. particular GENSET cDNA or genomic DNA is used as a test Chromosome preparations are denatured in 70% formamide, probe to associate that GENSET cDNA or genomic DNA 2xSSC for 2 min at 70 degrees Celsius, then dehydrated at with a particular phenotypic characteristic. 4 degrees Celsius. The Slides are treated with proteinase K (10 ug/100 ml in 20 mM Tris-HCl, 2 mM CaCl) at 37 0381 GENSET cDNAS or genomic DNAS are mapped to degrees Celsius for 8 min and dehydrated. The hybridization a particular location on a human chromosome using tech mixture containing the probe is placed on the Slide, covered niques Such as those described herein or other techniques with a coverslip, Sealed with rubber cement and incubated known in the art. A search of Mendelian Inheritance in Man overnight in a humid chamber at 37 degrees Celsius. After (V. McKusick, Mendelian Inheritance in Man; available on hybridization and post-hybridization washes, the biotiny line through Johns Hopkins University Welch Medical lated probe is detected by avidin-FITC and amplified with Library) reveals the region of the human chromosome which additional layers of biotinylated goat anti-avidin and avidin contains the GENSET cDNA or genomic DNA to be a very FITC. For chromosomal localization, fluorescent R-bands gene rich region containing Several known genes and Several are obtained as previously described (Cherif et al., 1990). diseases or phenotypes for which genes have not been The slides are observed under a LEICA fluorescence micro identified. The gene corresponding to this GENSET cDNA scope (DMRXA). Chromosomes are counterstained with or genomic DNA thus becomes an immediate candidate for propidium iodide and the fluorescent Signal of the probe each of these genetic diseases. appears as two Symmetrical yellow-green spots on both 0382 Cells from patients with these diseases or pheno chromatids of the fluorescent R-band chromosome (red). types are isolated and expanded in culture. PCR primers Thus, a particular GENSET cDNA or genomic DNA may be from the GENSET cDNA or genomic DNA are used to localized to a particular cytogenetic R-band on a given screen genomic DNA, mRNA or cDNA obtained from the chromosome. patients. GENSET cDNAS or genomic DNAS that are not Use of cDNAS to Construct or Expand Chromosome Maps amplified in he patients can be positively associated with a particular disease by further analysis. Alternatively, the PCR 0378. Once the GENSET cDNAS or genomic DNAS have been assigned to particular chromosomes using any tech analysis may yield fragments of different lengths when the nique known to those skilled in the art those skilled in the Samples are derived from an individual having the pheno art, particularly those described herein, they may be utilized type associated with the disease than when the Sample is to construct a high resolution map of the chromosomes on derived from a healthy individual, indicating that the gene which they are located or to identify the chromosomes in a containing the cDNA may be responsible for the genetic Sample. disease. 0379 Chromosome mapping involves assigning a given Uses of Polynucleotides in Recombinant Vectors unique Sequence to a particular chromosome as described 0383. The present invention also relates to recombinant above. Once the unique Sequence has been mapped to a vectors including the isolated polynucleotides of the present given chromosome, it is ordered relative to other unique invention, and to host cells recombinant for a polynucleotide Sequences located on the Same chromosome. One approach of the invention, Such as the above vectors, as well as to to chromosome mapping utilizes a Series of yeast artificial methods of making Such vectors and host cells and for using chromosomes (YACs) bearing Several thousand long inserts them for production of GENSET polypeptides by recombi derived from the chromosomes of the organism from which nant techniques. the GENSET cDNAs or genomic DNAS are obtained. This approach is described in Nagaraja et al. (1997), which Recombinant Vectors disclosure is hereby incorporated by reference in its entirety. 0384. The term “vector” is used herein to designate either Briefly, in this approach each chromosome is broken into a circular or a linear DNA or RNA molecule, which is either overlapping pieces which are inserted into the YAC vector. double-Stranded or Single-Stranded, and which comprise at The YAC inserts are screened using PCR or other methods least one polynucleotide of interest that is Sought to be to determine whether they include the GENSET cDNA or transferred in a cell host or in a unicellular or multicellular genomic DNA whose position is to be determined. Once an host organism. The present invention encompasses a family insert has been found which includes the GENSET cDNA or of recombinant vectors that comprise a regulatory poly genomic DNA, the insert can be analyzed by PCR or other nucleotide and/or a coding polynucleotide derived from methods to determine whether the insert also contains other either the GENSET genomic sequence or the cDNA Sequences known to be on the chromosome or in the region Sequence. Generally, a recombinant vector of the invention from which the GENSET cDNA or genomic DNA was may comprise any of the polynucleotides described herein, derived. This process can be repeated for each insert in the including regulatory Sequences, coding Sequences and poly YAC library to determine the location of each of the nucleotide constructs, as well as any GENSET primer or GENSET cDNA or genomic DNA relative to one another probe as defined herein. and to other known chromosomal markers. In this way, a high resolution map of the distribution of numerous unique 0385) In a first preferred embodiment, a recombinant markerS along each of the organisms chromosomes may be vector of the invention is used to amplify the inserted obtained. polynucleotide derived from a GENSET genomic sequence US 2006/0053498A1 Mar. 9, 2006 40 or a GENSET cDNA, for example any cDNA selected from tide, Said Structural or coding Sequence being operably the group consisting of sequences of SEQ ID NOS:1-169, linked to the regulatory elements described in (1); and 339-455, 561-784, sequences of clone inserts of the depos 0392 (3) appropriate transcription initiation and termi ited clone pool, Variants and fragments thereof in a Suitable nation Sequences. Structural units intended for use in yeast cell host, this polynucleotide being amplified at every time or eukaryotic expression Systems preferably include a leader that the recombinant vector replicates. Sequence enabling extracellular Secretion of translated pro 0386 A second preferred embodiment of the recombi tein by a host cell. Alternatively, when a recombinant protein nant vectors according to the invention comprises expres is expressed without a leader or transport Sequence, it may Sion vectors comprising either a regulatory polynucleotide include a N-terminal residue. This residue may or may not or a coding nucleic acid of the invention, or both. Within be Subsequently cleaved from the expressed recombinant certain embodiments, expression vectors are employed to protein to provide a final product. express a GENSET polypeptide which can be then purified 0393 Generally, recombinant expression vectors will and, for example be used in ligand Screening assays or as an include origins of replication, Selectable markers permitting immunogen in order to raise Specific antibodies directed transformation of the host cell, and a promoter derived from against the GENSET protein. In other embodiments, the a highly expressed gene to direct transcription of a down expression vectors are used for constructing transgenic Stream Structural Sequence. The heterologous structural animals and also for gene therapy. Expression requires that Sequence is assembled in appropriate phase with translation appropriate Signals are provided in the vectors, Said Signals initiation and termination Sequences, and preferably a leader including various regulatory elements, Such as enhancerS/ Sequence capable of directing Secretion of the translated promoters from both viral and mammalian Sources that drive protein into the periplasmic Space or the extracellular expression of the genes of interest in host cells. Dominant medium. In a specific embodiment wherein the vector is drug Selection markers for establishing permanent, stable adapted for transfecting and expressing desired Sequences in cell clones expressing the products are generally included in mammalian host cells, preferred vectors will comprise an the expression vectors of the invention, as they are elements origin of replication in the desired host, a Suitable promoter that link expression of the drug Selection markers to expres and enhancer, and also any necessary ribosome binding Sion of the polypeptide. Sites, polyadenylation signals, Splice donor and acceptor 0387 More particularly, the present invention relates to Sites, transcriptional termination Sequences, and 5'-flanking expression vectors which include nucleic acids encoding a non-transcribed Sequences. DNA sequences derived from GENSET protein, preferably a GENSET protein with an the SV40 viral genome, for example SV40 origin, early amino acid Sequence Selected from the group consisting of promoter, enhancer, Splice and polyadenylation Signals may sequences of SEQ ID NOs: 170-338, 456-560, 785-918, be used to provide the required non-transcribed genetic Sequences of polypeptides encoded by the clone inserts of elements. the deposited clone pool, Variants and fragments thereof. 0394 The in vivo expression of a GENSET polypeptide The polynucleotides of the present invention may be used to of the present invention may be useful in order to correct a express an encoded protein in a host organism to produce a genetic defect related to the expression of the native gene in beneficial effect. In Such procedures, the encoded protein a host organism, for the treatment or prevention of any may be transiently expressed in the host organism or stably disease or condition that can be treated or prevented by expressed in the host organism. The encoded protein may increasing the level of GENSET polypeptide expression, or have any of the activities described herein. The encoded to the production of a biologically inactive GENSET pro protein may be a protein which the host organism lacks or, tein. Consequently, the present invention also comprises alternatively, the encoded protein may augment the existing recombinant expression vectors mainly designed for the in levels of the protein in the host organism. vivo production of a GENSET polypeptide the present invention by the introduction of the appropriate genetic 0388. Some of the elements which can be found in the material in the organism or the patient to be treated. This vectors of the present invention are described in further genetic material may be introduced in vitro in a cell that has detail in the following Sections. been previously extracted from the organism, the modified General Features of the Expression Vectors of the Invention cell being Subsequently reintroduced in the Said organism, directly in Vivo into the appropriate tissue. 0389. A recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Regulatory Elements Chromosome), a BAC (Bacterial Artificial Chromosome), a 0395. The Suitable promoter regions used in the expres phage, a phagemid, a coSmid, a plasmid or even a linear Sion vectors according to the present invention are chosen DNA molecule which may comprise a chromosomal, non taking into account the cell host in which the heterologous chromosomal, semi-synthetic and synthetic DNA. Such a gene has to be expressed. The particular promoter employed recombinant vector can comprise a transcriptional unit com to control the expression of a nucleic acid Sequence of prising an assembly of interest is not believed to be important, So long as it is capable of directing the expression of the nucleic acid in the 0390 (1) a genetic element or elements having a regu targeted cell. Thus, where a human cell is targeted, it is latory role in gene expression, for example promoters or preferable to position the nucleic acid coding region adja enhancers. Enhancers are cis-acting elements of DNA, usu cent to and under the control of a promoter that is capable ally from about 10 to 300 bp in length that act on the of being expressed in a human cell, Such as, for example, a promoter to increase the transcription. human or a viral promoter. 0391 (2) a structural or coding sequence which is tran 0396 A Suitable promoter may be heterologous with Scribed into mRNA and eventually translated into a polypep respect to the nucleic acid for which it controls the expres US 2006/0053498A1 Mar. 9, 2006

Sion or alternatively can be endogenous to the native poly (Qiagen), pbs, pID10, phagescript, psiX174, pblueScript SK, nucleotide containing the coding Sequence to be expressed. pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); Additionally, the promoter is generally heterologous with ptrc99a, pKK223-3, pKK233-3, pIDR540, pRIT5 (Pharma respect to the recombinant vector Sequences within which cia); pWLNEO, pSV2CAT, p0G44, pXT1, pSG (Strat the construct promoter/coding Sequence has been inserted. agene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pCE-30 0397) Promoter regions can be selected from any desired (QIAexpress). gene using, for example, CAT (chloramphenicol transferase) Bacteriophage Vectors vectors and more preferably pKK232-8 and pCM7 vectors. 04.04 The P1 bacteriophage vector may contain large 0398 Preferred bacterial promoters are the LacI, LacZ, inserts ranging from about 80 to about 100 kb. The con the T3 or T7 bacteriophage RNA polymerase promoters, the struction of P1 bacteriophage vectors such as p158 or gpt, lambda PR, PL and trp promoters (EP 0036776), the p158/neo8 are notably described by Sternberg (1992, 1994), polyhedrin promoter, or the p10 protein promoter from which disclosure is hereby incorporated by reference in its baculovirus (Kit Novagen), (Smith et al., 1983; O'Reilly et entirety. Recombinant P1 clones comprising GENSET al., 1992; which disclosures are hereby incorporated by nucleotide Sequences may be designed for inserting large reference in their entireties), the lambda PR promoter or also polynucleotides of more than 40 kb (See Linton et al., 1993), the trc promoter. which disclosure is hereby incorporated by reference in its entirety. To generate P1 DNA for transgenic experiments, a 0399 Eukaryotic promoters include CMV immediate preferred protocol is the protocol described by McCormick early, HSV thymidine kinase, early and late SV40, LTRs et al. (1994), which disclosure is hereby incorporated by from retrovirus, and mouse metallothionein-L. Selection of reference in its entirety. Briefly, E. coli (preferably strain a convenient vector and promoter is well within the level of NS3529) harboring the P1 plasmid are grown overnight in a ordinary skill in the art. The choice of a promoter is well Suitable broth medium containing 25 ug/ml of kanamycin. within the ability of a person skilled in the field of genetic The P1 DNA is prepared from the E. coli by alkaline lysis engineering. For example, one may refer to the book of using the Qiagen Plasmid Maxi kit (Qiagen, Chatsworth, Sambrook et al., (1989) or also to the procedures described Calif., USA), according to the manufacturer's instructions. by Fuller et al., (1996), which disclosures are hereby incor The P1 DNA is purified from the bacterial lysate on two porated by reference in their entireties. Qiagen-tip 500 columns, using the Washing and elution Other Regulatory Elements buffers contained in the kit. A phenol/chloroform extraction 04.00 Where a cDNA insert is employed, one will typi is then performed before precipitating the DNA with 70% cally desire to include a polyadenylation Signal to effect ethanol. After solubilizing the DNA in TE (10 mM Tris-HCl, proper polyadenylation of the gene transcript. The nature of pH 7.4, 1 mM EDTA), the concentration of the DNA is the polyadenylation Signal is not believed to be crucial to the assessed by Spectrophotometry. Successful practice of the invention, and any Such Sequence 04.05) When the goal is to express a P1 clone comprising may be employed such as human growth hormone and SV40 GENSET polypeptide-encoding nucleotide Sequences in a polyadenylation Signals. Also contemplated as an element of transgenic animal, typically in transgenic mice, it is desir the expression cassette is a terminator. These elements can able to remove vector sequences from the P1 DNA fragment, Serve to enhance message levels and to minimize read for example by cleaving the P1 DNA at rare-cutting sites through from the cassette into other Sequences. within the P1 polylinker (Sfil, NotI or Sall). The P1 insert is then purified from vector Sequences on a pulsed-field aga Selectable Markers rose gel, using methods Similar to those originally reported 0401 Selectable markers confer an identifiable change to for the isolation of DNA from YACs (See e.g., Schedl et al., the cell permitting easy identification of cells containing the 1993a; Peterson et al., 1993), which disclosures are hereby expression construct. The Selectable marker genes for Selec incorporated by reference in their entireties. At this stage, tion of transformed host cells are preferably dihydrofolate the resulting purified insert DNA can be concentrated, if reductase or neomycin resistance for eukaryotic cell culture, necessary, on a Millipore Ultrafree-MC Filter Unit (Milli TRP1 for S. cerevisiae or tetracycline, rifampicin or ampi pore, Bedford, Mass., USA-30,000 molecular weight cillin resistance in E. Coli, or levan Saccharase for myco limit) and then dialyzed against microinjection buffer (10 bacteria, this latter marker being a negative Selection marker. mM Tris-HCl, pH 7.4; 250 uM EDTA) containing 100 mM NaCl, 30 uM spermine, 70 uM spermidine on a micrody Preferred Vectors alisis membrane (type VS, 0.025 uM from Millipore). The Bacterial Vectors intactness of the purified P1 DNA insert is assessed by 0402. As a representative but non-limiting example, use electrophoresis on 1% agarose (Sea Kem GTG.; FMC Bio ful expression vectors for bacterial use can comprise a products) pulse-field gel and staining with ethidium bro Selectable marker and a bacterial origin of replication mide. derived from commercially available plasmids comprising Viral Vectors genetic elements of pBR322 (ATCC 37017). Such commer 0406. In one specific embodiment, the vector is derived cial vectors include, for example, pKK223-3 (Pharmacia, from an adenovirus. Preferred adenovirus vectors according Uppsala, Sweden), and pGEM1 (Promega Biotec, Madison, to the invention are those described by Feldman and Steg. Wis., USA). (1996), or Ohno et al., (1994), which disclosures are hereby 0403 Large numbers of other suitable vectors are known incorporated by reference in their entireties. Another pre to those of Skill in the art, and commercially available, Such ferred recombinant adenovirus according to this specific as the following bacterial vectors: pCE70, pCE60, pGE-9 embodiment of the present invention is the human adenovi US 2006/0053498A1 Mar. 9, 2006 42 rus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal cloning site is flanked by two Not I Sites, permitting cloned origin (French patent application No. FR-93.05954), which Segments to be excised from the vector by Not I digestion. disclosure is hereby incorporated by reference in its entirety. Alternatively, the DNA insert contained in the p3eloBACll vector may be linearized by treatment of the BAC vector 04.07 Retrovirus vectors and adeno-associated virus vec with the commercially available enzyme lambda terminase tors are generally understood to be the recombinant gene that leads to the cleavage at the unique coSN Site, but this delivery Systems of choice for the transfer of exogenous cleavage method results in a full length BAC clone contain polynucleotides in Vivo, particularly to mammals, including ing both the insert DNA and the BAC sequences. humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably inte Baculovirus grated into the chromosomal DNA of the host. Particularly preferred retroviruses for the preparation or construction of 0410 Another specific suitable host vector system is the retroviral in vitro or in vitro gene delivery vehicles of the pVL1392/1393 baculovirus transfer vector (Pharmingen) present invention include retroviruses Selected from the that is used to transfect the SF9 cell line (ATCC No. CRL group consisting of Mink-Cell Focus Inducing Virus, 1711) which is derived from Spodoptera frugiperda. Other Murine Sarcoma Virus, Reticuloendotheliosis virus and suitable vectors for the expression of the GENSET polypep Rous Sarcoma virus. Particularly preferred Murine Leuke tide of the present invention in a baculovirus expression mia Viruses include the 4070A and the 1504A viruses, system include those described by Chai et al., (1993), Vlasak Abelson (ATCC No VR-999), Friend (ATCC No VR-245), et al., (1983), and Lenhard et al., (1996), which disclosures Gross (ATCC No VR-590), Rauscher (ATCC No VR-998) are hereby incorporated by reference in their entireties. and Moloney Murine Leukemia Virus (ATCC No VR-190; PCT Application No WO 94/24298). Particularly preferred Delivery of the Recombinant Vectors Rous Sarcoma Viruses include Bryan high titer (ATCC Nos 0411 To effect expression of the polynucleotides and VR-334, VR-657, VR-726, VR-659 and VR-728). Other polynucleotide constructs of the invention, the constructs preferred retroviral vectors are those described in Roth et al. must be delivered into a cell. This delivery may be accom (1996), PCT Application No WO 93/25234, PCT Applica plished in vitro, as in laboratory procedures for transforming tion No WO 94/06920, Roux et al., (1989), Julan et al., cell lines, or in Vivo or eX Vivo, as in the treatment of certain (1992), and Neda et al., (1991), which disclosures are hereby diseases States. One mechanism is viral infection where the incorporated by reference in their entireties. expression construct is encapsulated in an infectious viral particle. 0408. Yet another viral vector system that is contem plated by the invention comprises the adeno-associated virus 0412 Several non-viral methods for the transfer of poly (AAV). The adeno-associated virus is a naturally occurring nucleotides into cultured mammalian cells are also contem defective virus that requires another virus, Such as an plated by the present invention, and include, without being adenovirus or a herpes virus, as a helper virus for efficient limited to, calcium phosphate precipitation (Graham et al., replication and a productive life cycle (Muzyczka et al., 1973; Chen et al., 1987); DEAE-dextran (Gopal, 1985); 1992), which disclosure is hereby incorporated by reference electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984); in its entirety. It is also one of the few viruses that may direct microinjection (Harland et al., 1985); DNA-loaded integrate its DNA into non-dividing cells, and exhibits a high liposomes (Nicolau et al., 1982; Fraley et al., 1979); and frequency of stable integration (Flotte et al. 1992; Samulski receptor-mediated transfection. (Wu and Wu, 1987, 1988), et al., 1989; McLaughlin et al., 1989), which disclosures are which disclosures are hereby incorporated by reference in hereby incorporated by reference in their entireties. One their entireties. Some of these techniques may be Success advantageous feature of AAV derives from its reduced fully adapted for in vivo or ex vivo use. efficacy for transducing primary cells relative to transformed 0413. Once the expression polynucleotide has been deliv cells. ered into the cell, it may be stably integrated into the genome BAC Vectors of the recipient cell. This integration may be in the cognate location and orientation via homologous recombination 04.09 The bacterial artificial chromosome (BAC) cloning (gene replacement) or it may be integrated in a random, system (Shizuya et al., 1992), which disclosure is hereby non-specific location (gene augmentation). In yet further incorporated by reference in its entirety, has been developed embodiments, the nucleic acid may be stably maintained in to stably maintain large fragments of genomic DNA (100 the cell as a separate, episomal Segment of DNA. Such 300 kb) in E. coli. A preferred BAC vector comprises a nucleic acid Segments or “episomes' encode Sequences pBeloBACll vector that has been described by Kim et al. Sufficient to permit maintenance and replication independent (1996), which disclosure is hereby incorporated by reference of or in Synchronization with the host cell cycle. in its entirety. BAC libraries are prepared with this vector using Size-Selected genomic DNA that has been partially 0414. One specific embodiment for a method for deliv digested using enzymes that permit ligation into either the ering a protein or peptide to the interior of a cell of a Bam HI or HindIII sites in the vector. Flanking these cloning vertebrate in Vivo comprises the Step of introducing a sites are T7 and SP6 RNA polymerase transcription initia preparation comprising a physiologically acceptable carrier tion sites that can be used to generate end probes by either and a naked polynucleotide operatively coding for the RNA transcription or PCR methods. After the construction polypeptide of interest into the interstitial Space of a tissue of a BAC library in E. coli, BAC DNA is purified from the comprising the cell, whereby the naked polynucleotide is host cell as a Supercoiled circle. Converting these circular taken up into the interior of the cell and has a physiological molecules into a linear form precedes both size determina effect. This is particularly applicable for transfer in vitro but tion and introduction of the BACs into recipient cells. The it may be applied to in Vivo as well. US 2006/0053498A1 Mar. 9, 2006 43

0415 Compositions for use in vitro and in vivo compris group of signal sequences of SEQ ID NOs: 1-85,339-400, ing a “naked” polynucleotide are described in PCT appli 406-407, 413-415, 561-594, and 634-651 and signal cation No. WO 90/11092 (Vical Inc.) and also in PCT Sequences of clone inserts of the deposited clone pool is application No. WO95/11307 (Institut Pasteur, INSERM, operably linked to the promoter such that the mRNA tran Université d’Ottawa) as well as in the articles of Tascon et scribed from the promoter will direct the translation of the al. (1996) and of Huygen et al., (1996), which disclosures Signal peptide. The host cell, tissue, or organism may be any are hereby incorporated by reference in their entireties. cell, tissue, or organism which recognizes the Signal peptide 0416) In still another embodiment of the invention, the encoded by the signal sequence in the GENSET cDNA or transfer of a naked polynucleotide of the invention, includ genomic DNA. Suitable hosts include mammalian cells, ing a polynucleotide construct of the invention, into cells tissueS or organisms, avian cells, tissues, or organisms, may be accomplished with particle bombardment (biolistic), insect cells, tissueS or organisms, or yeast. Said particles being DNA-coated microprojectiles acceler 0424. In addition, the Secretion vector contains cloning ated to a high Velocity allowing them to pierce cell mem Sites for inserting genes encoding the proteins which are to branes and enter cells without killing them, Such as be Secreted. The cloning sites facilitate the cloning of the described by Klein et al., (1987), which disclosure is hereby insert gene in frame with the Signal Sequence Such that a incorporated by reference in its entirety. fusion protein in which the Signal peptide is fused to the 0417. In a further embodiment, the polynucleotide of the protein encoded by the inserted gene is expressed from the invention may be entrapped in a liposome (Ghosh and mRNA transcribed from the promoter. The signal peptide Bacchawat, 1991; Wong et al., 1980; Nicolau et al., 1987, directs the extracellular Secretion of the fusion protein. which disclosures are hereby incorporated by reference in 0425 The secretion vector may be DNA or RNA and may their entireties). integrate into the chromosome of the host, be Stably main 0418. In a specific embodiment, the invention provides a tained as an extrachromosomal replicon in the host, be an composition for the in vivo production of the GENSET artificial chromosome, or be transiently present in the host. polypeptides described herein. It comprises a naked poly Preferably, the secretion vector is maintained in multiple nucleotide operatively coding for this polypeptide, in Solu copies in each host cell. AS used herein, multiple copies tion in a physiologically acceptable carrier, and Suitable for means at least 2, 5, 10, 20, 25, 50 or more than 50 copies per introduction into a tissue to cause cells of the tissue to cell. In Some embodiments, the multiple copies are main express the said protein or polypeptide. tained extrachromosomally. In other embodiments, the mul tiple copies result from amplification of a chromosomal 0419. The amount of vector to be injected to the desired Sequence. host organism varies according to the site of injection. AS an indicative dose, it will be injected between 0.1 and 100 lug 0426 Many nucleic acid backbones suitable for use as of the vector in an animal body, preferably a mammal body, Secretion vectors are known to those skilled in the art, for example a mouse body. including retroviral vectors, SV40 vectors, Bovine Papil loma Virus vectors, yeast integrating plasmids, yeast episo 0420. In another embodiment of the vector according to mal plasmids, yeast artificial chromosomes, human artificial the invention, it may be introduced in vitro in a host cell, chromosomes, P element vectors, baculovirus vectors, or preferably in a host cell previously harvested from the bacterial plasmids capable of being transiently introduced animal to be treated and more preferably a Somatic cell Such into the host. as a muscle cell. In a Subsequent Step, the cell that has been transformed with the vector coding for the desired GENSET 0427. The secretion vector may also contain a poly A polypeptide or the desired fragment thereof is reintroduced Signal Such that the polyA signal is located downstream of into the animal body in order to deliver the recombinant the gene inserted into the Secretion vector. protein within the body either locally or systemically. 0428. After the gene encoding the protein for which Secretion Vectors Secretion is desired is inserted into the Secretion vector, the Secretion vector is introduced into the host cell, tissue, or 0421) Some of the GENSET cDNAS or genomic DNAS organism using calcium phosphate precipitation, DEAE of the invention may also be used to construct Secretion Dextran, electroporation, liposome-mediated transfection, vectors capable of directing the Secretion of the proteins viral particles or as naked DNA. The protein encoded by the encoded by genes inserted in the vectors. Such Secretion inserted gene is then purified or enriched from the Superna vectors may facilitate the purification or enrichment of the tant using conventional techniques Such as ammonium Sul proteins encoded by genes inserted therein by reducing the fate precipitation, immunoprecipitation, immunochromatog number of background proteins from which the desired raphy, Size exclusion chromatography, ion exchange protein must be purified or enriched. Exemplary Secretion chromatography, and hplc. Alternatively, the Secreted pro vectors are described below. tein may be in a Sufficiently enriched or pure State in the 0422 The secretion vectors of the present invention Supernatant or growth media of the host to permit it to be include a promoter capable of directing gene expression in used for its intended purpose without further enrichment. the host cell, tissue, or organism of interest. Such promoters 0429 The signal sequences may also be inserted into include the Rous Sarcoma Virus promoter, the SV40 pro vectors designed for gene therapy. In Such vectors, the Signal moter, the human cytomegalovirus promoter, and other Sequence is operably linked to a promoter Such that mRNA promoters familiar to those skilled in the art. transcribed from the promoter encodes the Signal peptide. A 0423) A signal sequence from a polynucleotide of the cloning Site is located downstream of the Signal Sequence invention, preferably a signal Sequences Selected from the Such that a gene encoding a protein whose Secretion is US 2006/0053498A1 Mar. 9, 2006 44 desired may readily be inserted into the vector and fused to niques known in the art may be used to operably associate the Signal Sequence. The vector is introduced into an appro heterologous control regions (e.g., promoter and/or priate host cell. The protein expressed from the promoter is enhancer) and endogenous polynucleotide sequences via Secreted extracellularly, thereby producing a therapeutic homologous recombination, See, e.g., U.S. Pat. No. 5,641, effect. 670, issued Jun. 24, 1997; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publica Cell Hosts tion No. WO94/12650, published Aug. 4, 1994; Koller et 0430. Another object of the invention comprises a host al., (1989); and Zijlstra et al. (1989) (the disclosures of each cell that has been transformed or transfected with one of the of which are incorporated by reference in their entireties). polynucleotides described herein, and in particular a poly nucleotide either comprising a GENSET polypeptide-encod 0435 The present invention further relates to a method of ing polynucleotide regulatory Sequence or the polynucle making a homologously recombinant host cell in Vitro or in otide coding for a GENSET polypeptide. Also included are Vivo, wherein the expression of a targeted gene not normally host cells that are transformed (prokaryotic cells) or that are expressed in the cell is altered. Preferably the alteration transfected (eukaryotic cells) with a recombinant vector causes expression of the targeted gene under normal growth Such as one of those described above. However, the cell conditions or under conditions Suitable for producing the hosts of the present invention can comprise any of the polypeptide encoded by the targeted gene. The method polynucleotides of the present invention. In a preferred comprises the steps of: (a) transfecting the cell in vitro or in embodiment, host cells contain a polynucleotide Sequence Vivo with a polynucleotide construct, Said polynucleotide comprising a Sequence Selected from the group consisting of construct comprising: (i) a targeting Sequence; (ii) a regu sequences of SEQ ID NOS:1-169, 339-455, 561-784, latory sequence and/or a coding sequence; and (iii) an Sequences of clone inserts of the deposited clone pool, unpaired Splice donor Site, if necessary, thereby producing a variants and fragments thereof. Preferred host cells used as transfected cell; and (b) maintaining the transfected cell in recipients for the expression vectors of the invention are the Vitro or in Vivo under conditions appropriate for homologous following: recombination. 0431) a) Prokaryotic host cells: Escherichia coli strains 0436 The present invention further relates to a method of (I.E.DH5-C. strain), Bacillus Subtilis, Salmonella typhimu altering the expression of a targeted gene in a cell in Vitro or rium, and Strains from Species like Pseudomonas, Strepto in Vivo wherein the gene is not normally expressed in the myces and StaphylococcuS. cell, comprising the Steps of: (a) transfecting the cell in Vitro or in Vivo with a polynucleotide construct, Said polynucle 0432 b) Eukaryotic host cells: HeLa cells (ATCC otide construct comprising: (i) a targeting Sequence; (ii) a No.CCL2; No.CCL2.1; No.CCL2.2), Cv 1 cells (ATCC regulatory Sequence and/or a coding Sequence; and (iii) an No.CCL70), COS cells (ATCC No.CRL1650; unpaired Splice donor Site, if necessary, thereby producing a No.CRL1651), Sf-9 cells (ATCC No.CRL1711), C127 cells transfected cell; and (b) maintaining the transfected cell in (ATCC No. CRL-1804), 3T3 (ATCC No. CRL-6361), CHO Vitro or in Vivo under conditions appropriate for homologous (ATCC No. CCL-61), human kidney 293. (ATCC No. recombination, thereby producing a homologously recom 45504; No. CRL-1573) and BHK (ECACC No. 84100501; binant cell; and (c) maintaining the homologously recom No. 84111301). binant cell in vitro or in Vivo under conditions appropriate for expression of the gene. c) Other Mammalian Host Cells 0437. The present invention further relates to a method of 0433. The present invention also encompasses primary, making a polypeptide of the present invention by altering the Secondary, and immortalized homologously recombinant expression of a targeted endogenous gene in a cell in Vitro host cells of Vertebrate origin, preferably mammalian origin or in Vivo wherein the gene is not normally expressed in the and particularly human origin, that have been engineered to: cell, comprising the steps of: a) transfecting the cell in vitro a) insert exogenous (heterologous) polynucleotides into the with a polynucleotide construct, Said polynucleotide con endogenous chromosomal DNA of a targeted gene, b) delete Struct comprising: (i) a targeting Sequence; (ii) a regulatory endogenous chromosomal DNA, and/or c) replace endog Sequence and/or a coding Sequence; and (iii) an unpaired enous chromosomal DNA with exogenous polynucleotides. Splice donor Site, if necessary, thereby producing a trans Insertions, deletions, and/or replacements of polynucleotide fected cell; (b) maintaining the transfected cell in vitro or in Sequences may be to the coding Sequences of the targeted Vivo under conditions appropriate for homologous recom gene and/or to regulatory regions, Such as promoter and bination, thereby producing a homologously recombinant enhancer Sequences, operably associated with the targeted cell; and c) maintaining the homologously recombinant cell gene. in vitro or in Vivo under conditions appropriate for expres Sion of the gene thereby making the polypeptide. 0434 In addition to encompassing host cells containing the vector constructs discussed herein, the invention also 0438. The present invention further relates to a poly encompasses primary, Secondary, and immortalized host nucleotide construct which alters the expression of a tar cells of vertebrate origin, particularly mammalian origin, geted gene in a cell type in which the gene is not normally that have been engineered to delete or replace endogenous expressed. This occurs when the polynucleotide construct is genetic material (e.g., coding Sequence), and/or to include inserted into the chromosomal DNA of the target cell, genetic material (e.g., heterologous polynucleotide wherein said polynucleotide construct comprises: a) a tar Sequences) that is operably associated with the polynucle geting Sequence; b) a regulatory Sequence and/or coding otides of the invention, and which activates, alters, and/or Sequence; and c) an unpaired splice-donor Site, if necessary. amplifies endogenous polynucleotides. For example, tech Further included are a polynucleotide construct, as described US 2006/0053498A1 Mar. 9, 2006 45 above, wherein Said polynucleotide construct further com ture shift or chemical induction, and cells are cultivated for prises a polynucleotide which encodes a polypeptide and is an additional period. Cells are typically harvested by cen in-frame with the targeted endogenous gene after homolo trifugation, disrupted by physical or chemical means, and gous recombination with chromosomal DNA. the resulting crude extract retained for further purification. Microbial cells employed in the expression of proteins can 0439. The compositions may be produced, and methods be disrupted by any convenient method, including freeze performed, by techniques known in the art, Such as those thaw cycling, Sonication, mechanical disruption, or use of described in U.S. Pat. NOS: 6,054,288; 6,048,729; 6,048, cell lysing agents. Such methods are well known by the 724; 6,048,524; 5,994,127; 5,968,502; 5,965,125; 5,869, skilled artisan. 239; 5,817,789; 5,783,385; 5,733,761; 5,641,670; 5,580, 734; International Publication NOs: WO96/29411, WO Transgenic Animals 94/12650; and scientific articles described by Koller et al., 0445. The terms “transgenic animals” or “host animals” (1994). (The disclosures of each of which are incorporated are used herein to designate animals that have their genome by reference in their entireties). genetically and artificially manipulated So as to include one 0440 GENSET gene expression in mammalian cells, of the nucleic acids according to the invention. Preferred preferably human cells, may be rendered defective, or animals are non-human mammals and include those belong alternatively may be altered by replacing endogenous ing to a genus Selected from MuS (e.g. mice), Rattus (e.g. GENSET polypeptide-encoding genes in the genome of an rats) and Oryctogalus (e.g. rabbits) which have their genome animal cell by a GENSET polypeptide-encoding polynucle artificially and genetically altered by the insertion of a otide according to the invention. These genetic alterations nucleic acid according to the invention. In one embodiment, may be generated by homologous recombination using the invention encompasses non-human host mammals and previously described specific polynucleotide constructs. animals comprising a recombinant vector of the invention or a GENSET gene disrupted by homologous recombination 0441 Mammal Zygotes, Such as murine Zygotes may be with a knock out vector. used as cell hosts. For example, murine Zygotes may undergo microinjection with a purified DNA molecule of 0446. The transgenic animals of the invention all include interest, for example a purified DNA molecule that has within a plurality of their cells a cloned recombinant or previously been adjusted to a concentration ranging from 1 Synthetic DNA sequence, more Specifically one of the puri ng/ml-for BAC inserts-to 3 ng/ul-for P1 bacteriophage fied or isolated nucleic acids comprising a GENSET inserts—in 10 mM Tris-HCl, pH 7.4, 250 uM EDTA con polypeptide coding sequence, a GENSET polynucleotide taining 100 mM NaCl, 30 uM spermine, and 70 uM spermi regulatory Sequence, a polynucleotide construct, or a DNA dine. When the DNA to be microinjected has a large size, Sequence encoding an antisense polynucleotide Such as polyamines and high Salt concentrations can be used in order described in the present Specification. to avoid mechanical breakage of this DNA, as described by 0447 Generally, a transgenic animal according the Schedl et al (1993b), which disclosure is hereby incorpo present invention comprises any of the polynucleotides, the rated by reference in its entirety. recombinant vectors and the cell hosts described in the 0442. Any one of the polynucleotides of the invention, present invention. In a first preferred embodiment, these including the polynucleotide constructs described herein, transgenic animals may be good experimental models in may be introduced in an embryonic stem (ES) cell line, order to Study the diverse pathologies related to the dyS preferably a mouse ES cell line. ES cell lines are derived regulation of the expression of a given GENSET gene, in from pluripotent, uncommitted cells of the inner cell mass of particular the transgenic animals containing within their pre-implantation blastocysts. Preferred ES cell lines are the genome one or Several copies of an inserted polynucleotide following: ES-E14TG2a (ATCC No.CRL-1821), ES-D3 encoding a native GENSET polypeptide, or alternatively a (ATCC No.CRL1934 and No. CRL-11632), YS001 (ATCC mutant GENSET polypeptide. No. CRL-11776), 36.5 (ATCCNo. CRL-11116). ES cells are 0448. In a second preferred embodiment, these transgenic maintained in an uncommitted State by culture in the pres animals may express a desired polypeptide of interest under ence of growth-inhibited feeder cells which provide the the control of the regulatory polynucleotides of the appropriate Signals to preserve this embryonic phenotype GENSET gene, leading to high yields in the synthesis of this and serve as a matrix for ES cell adherence. Preferred feeder protein of interest, and eventually to tissue specific expres cells are primary embryonic fibroblasts that are established Sion of the protein of interest. from tissue of day 13-day 14 embryos of virtually any mouse Strain, that are maintained in culture, Such as described by 0449 The design of the transgenic animals of the inven Abbondanzo et al. (1993) and are growth-inhibited by tion may be made according to the conventional techniques irradiation, such as described by Robertson (1987), or by the well known from the one skilled in the art. For more details presence of an inhibitory concentration of LIF, Such as regarding the production of transgenic animals, and Specifi described by Pease and Williams (1990), which disclosures cally transgenic mice, it may be referred to U.S. Pat. No. are hereby incorporated by reference in their entireties. 4,873,191, issued Oct. 10, 1989; U.S. Pat. No. 5,464,764 issued Nov. 7, 1995; and U.S. Pat. No. 5,789,215, issued 0443) The constructs in the host cells can be used in a Aug. 4, 1998; these documents being herein incorporated by conventional manner to produce the gene product encoded reference to disclose methods producing transgenic mice. by the recombinant Sequence. 0450 Transgenic animals of the present invention are 0444. Following transformation of a suitable host and produced by the application of procedures which result in an growth of the host to an appropriate cell density, the Selected animal with a genome that has incorporated exogenous promoter is induced by appropriate means, Such as tempera genetic material. The procedure involves obtaining the US 2006/0053498A1 Mar. 9, 2006 46 genetic material which encodes either a GENSET polypep 0455 As the frequency of transgene incorporation is tide coding Sequence, a GENSET polynucleotide regulatory often low, the detection of transgene integration in pre Sequence, or a DNA sequence encoding a GENSET poly implantation embryoS is often desirable using any of the nucleotide antisense Sequence, or a portion thereof, Such as herein-described methods. Any of a number of methods can described in the present Specification. A recombinant poly be used to detect the presence of a transgene in a pre nucleotide of the invention is inserted into an embryonic or ES stem cell line. The insertion is preferably made using implantation embryo. For example, one or more cells may electroporation, such as described by Thomas et al. (1987), be removed from the pre-implantation embryo, and the which disclosure is hereby incorporated by reference in its presence or absence of the transgene in the removed cell or entirety. The cells Subjected to electroporation are Screened cells can be detected using any Standard method e.g. PCR. (e.g. by selection via selectable markers, by PCR or by Alternatively, the presence of a transgene can be detected in Southern blot analysis) to find positive cells which have utero or post partum using Standard methods. integrated the exogenous recombinant polynucleotide into their genome, preferably via an homologous recombination 0456. In a particularly preferred embodiment of the event. An illustrative positive-negative Selection procedure present invention, transgenic mammals are generated that that may be used according to the invention is described by secrete recombinant GENSET polypeptides in their milk. As Mansour et al. (1988), which disclosure is hereby incorpo the mammary gland is a highly efficient protein-producing rated by reference in its entirety. organ, Such methods can be used to produce protein con centrations in the gram per liter range, and often signifi 0451. The positive cells are then isolated, cloned and cantly more. Preferably, expression in the mammary gland is injected into 3.5 days old blastocysts from mice, Such as accomplished by operably linking the polynucleotide encod described by Bradley (1987), which disclosure is hereby ing the GENSET polypeptide to a mammary gland Specific incorporated by reference in its entirety. The blastocysts are promoter and, optionally, other regulatory elements. Suit then inserted into a female host animal and allowed to grow to term. Alternatively, the positive ES cells are brought into able promoters and other elements include, but are not contact with embryos at the 2.5 days old 8-16 cell stage limited to, those derived from mammalian short and long (morulae) such as described by Wood et al. (1993), or by WAP, alpha, beta, and kappa, casein, alpha and beta lacto Nagy et al. (1993), which disclosures are hereby incorpo globulin, beta-CN 5' genes, as well as the the mouse rated by reference in their entireties, the ES cells being mammary tumor virus (MMTV) promoter. Such promoters internalized to colonize extensively the blastocyst including and other elements may be derived from any mammal, the cells which will give rise to the germ line. including, but not limited to, cows, goats, sheep, pigs, mice, rabbits, and guinea pigs. Promoter and other regulatory 0452. The offspring of the female host are tested to Sequences, vectors, and other relevant teachings are pro determine which animals are transgenic e.g. include the vided, e.g., by Clark (1998) J Mammary Gland Biol Neo inserted exogenous DNA sequence and which ones are wild plasia 3:337-50; Jost et al. (1999) Nat. Biotechnol 17:1604; type. U.S. Pat. Nos. 5,994,616; 6,140,552; 6,013,857; Sohn et al. 0453 Thus, the present invention also concerns a trans (1999) DNA Cell Biol. 18:845-52; Kim et al. (1999) J. genic animal containing a nucleic acid, a recombinant Biochem. (Japan) 126:320-5; Soulier et al. (1999) Euro. J. expression vector or a recombinant host cell according to the Biochem. 260:533-9, Zhang et al. (1997) Chin. J. Biotech. invention. 13:271-6; Rijnkels et al. (1998) Transgen. Res. 7:5-14; Korhonen et al. (1997) Euro. J. Biochem. 245:482-9; Uusi 0454. In another embodiment, transgenic animals are produced by microinjecting polynucleotides ares microin Oukari et al. (1997) Transgen. Res. 6:75-84; Hitchin et al. jected into a fertilized oocyte. Typically, fertilized oocytes (1996) Prot. Expr. Purif. 7:247-52; Platenburg et al. (1994) are microinjected using Standard techniques, and then cul Transgen. Res. 3:99-108; Heng-Cherl et al. (1993) Animal tured in vitro until a “pre-implantation embryo” is obtained. Biotech. 4:89-107; and Christa et al. (2000) Euro. J. Bio Such pre-implantation embryoS preferably contain approxi chem. 267:1665-71; the entire disclosures of each of which mately 16 to 150 cells. Methods for culturing fertilized is herein incorporated by reference. oocytes to the pre-implantation Stage are described, e.g., by 0457. In another embodiment, the polypeptides of the Gordon et al. (1984) Methods in Enzymology, 101, 414); invention can be produced in milk by introducing polynucle Hogan et al. (1986) in Manipulating the mouse embryo. A otides encoding the polypeptides into Somatic cells of the Laboratory Manual. Cold Spring Harbor Laboratory Press, mammary gland in Vivo, e.g. mammary Secreting epithelial Cold Spring Harbor, N.Y) (for the mouse embryo); Hammer et al. (1985) Nature, 315, 680) (for rabbit and porcine cells. For example, plasmid DNA can be infused through the embryos); Gandolfi et al. (1987).J. Reprod. Fert. 81, 23-28); nipple canal, e.g. in association with DEAE-dextran (see, Rexroad et al. (1988) J. Anim. Sci. 66,947-953) (for ovine e.g., Hens et al. (2000) Biochim. Biophys. Acta 1523:161 embryos); and Eyestone et al. (1989) J. Reprod. Fert. 85, 171), in association with a ligand that can lead to receptor 715-720); Camous et al. (1984) J. Reprod. Fert. 72, 779 mediated endocytosis of the construct (see, e.g., Sobolev et 785); and Heyman et al. (1987) Theriogenology 27, 5968) al. (1998) 273:7928-33), or in a viral vector such as a (for bovine embryos); the disclosures of each of which are retroviral vector, e.g. the Gibbon ape leukemia virus (see, incorporated herein in their entireties. Pre-implantation e.g., Archer et al. (1994) PNAS 91:6840-6844). In any of embryos are then transferred to an appropriate female by these embodiments, the polynucleotide may be operably Standard methods to permit the birth of a transgenic or linked to a mammary gland Specific promoter, as described chimeric animal, depending upon the Stage of development above, or, alternatively, any Strongly expressing promoter when the transgene is introduced. Such as CMV or MoMLV LTR US 2006/0053498A1 Mar. 9, 2006 47

0458. The Suitability of any vector, promoter, regulatory Protein of SEQ ID NO:255 (internal designation element, etc. for use in the present invention can be assessed 500762786 255-24-5-0-A2-R 104) beforehandby transfecting cells Such as mammary epithelial cells, e.g. MacT cells (bovine mammary epithelial cells) or 0466. The cDNA of clone 500762786 255-24-5-0-A2 GME cells (goat mammary epithelial cells), in vitro and R 104 (SEQ ID NO:86) encodes the human EDR4 protein assessing the efficiency of transfection and expression of the LFPAPAPPPAPAFAPPPKVPSPERS transgene in the cells. APRVPLPSPOPSYPFRPAASGGTPPPA CLPPAOPCOGSP AMNLFRFLGDLSHLLAIILLLLKI 0459 For in vivo administration, the polynucleotides can WKSRSCAAHPOLPLSFCLSVCLSVSLSLSXSLSLSFSV be administered in any Suitable formulation, at any of a SKKKKK (SEQ ID NO:255). It will be appreciated that all range of concentrations (e.g. 1-500 tug/ml, preferably 50-100 characteristics and uses of the polynucleotides of SEQ ID Aug/ml), at any Volume (e.g. 1-100 ml, preferably 1 to 20 ml), NO:86 and polypeptides of SEQ ID NO:355, described and can be administered any number of times (e.g. 1, 2, 3, throughout the present application also pertain to the human 5, or 10 times), at any frequency (e.g. every 1, 2, 3, 5, 10, cDNA of clone 500762786 255-24-5-0-A2-R 104 and or any number of days). Suitable concentrations, frequen polypeptides encoded thereby. Polypeptide fragments hav cies, modes of administration, etc. will depend upon the ing a biological activity described herein and polynucle particular polynucleotide, Vector, animal, etc., and can otides encoding the same are included in the present inven readily be determined by one of skill in the art. tion. Related polynucleotide and polypeptide Sequences 0460. In a preferred embodiment, a retroviral vector Such included in the present invention are SEQ ID NOS:406 and as as Gibbon ape leukemia viral vector is used, as described 52O. in Archer et al. (1994) PNAS 91:6840-6844). As retroviral 0467. The normal functioning of the eukaryotic cell infection typically requires cell division, cell division in the requires that all newly Synthesized proteins be correctly mammary glands can be Stimulated in conjunction with the folded, modified, and delivered to Specific inter and extra administration of the vector, e.g. using a factor Such as cellular Sites. Newly Synthesized membrane and Secretory estrodiol benzoate, progesterone, reSerpine, or dexametha proteins enter a cellular Sorting and distribution network Sone. Further, retroviral and other methods of infection can during or immediately after Synthesis (cotranslationally or be facilitated using accessory compounds Such as polybrene. posttranslationally) and are routed to specific locations 0461). In any of the herein-described methods for obtain inside and outside of the cell. The initial compartment in this ing GENSET polypeptides from milk, the quantity of milk process is the endoplasmic reticulum (ER) where proteins obtained, and thus the quantity of GENSET polypeptides undergo modifications Such as glycosylation, disulfide bond produced, can be enhanced using any Standard method of formation, and assembly into oligomers. The proteins are lacation induction, e.g. using hexestrol, estrogen, and/or then transported through an additional Series of membrane progesterOne. bound compartments which include the various cistemae of the Golgi complex, where further carbohydrate modifica 0462. The polynucleotides used in Such embodiments can tions occur. Transport between compartments occurs by either encode a full-length GENSET protein or a GENSET means of vesicles that bud and fuse in a specific manner; fragment. Typically, the encoded polypeptide will include a once within the Secretory pathway, proteins do not have to Signal Sequence to ensure the Secretion of the protein into the croSS a membrane to reach the cell Surface. milk. 0468. The complexity of this system has advantages for Recombinant Cell Lines Derived From the Transgenic Ani the cell because it allows proteins to fold and mature in mals of the Invention closed compartments that contain the appropriate enzyme 0463 A further object of the invention comprises recom catalysts. It is, however, dependent on Sorting mechanisms binant host cells obtained from a transgenic animal that position the enzymes correctly and maintain them in described herein. In one embodiment the invention encom place. passes cells derived from non-human host mammals and 0469 The first organelle in this system, the ER, contains animals comprising a recombinant vector of the invention or multiple enzymes involved in protein Structure modifica a GENSET gene disrupted by homologous recombination tions. Among these are BiP (binding protein) which directs with a knock out vector. the correct folding of proteins and, PDI (protein disulfide 0464 Recombinant cell lines may be established in vitro isomerase) and a homologue of the 90 kDa heat-shock from cells obtained from any tissue of a transgenic animal protein, both of which catalyze the formation and rearrange according to the invention, for example by transfection of ment of disulfide bonds (Gething, M. J. and Sambrook, J. primary cell cultures with vectors expressing onc-genes Such (1992) Nature 355:33-45). These abundant soluble proteins as SV40 large T antigen, as described by Chou (1989), and must be retained in the ER and must be distinguished from Shay et al. (1991), which disclosures are hereby incorpo the newly Synthesized Secretory proteins which are rapidly rated by reference in their entireties. transported to the Golgi apparatus. The Signal for retention in the ER in mammalian cells consists of the tetrapeptide Uses of Polypeptides of the Invention Sequence, KDEL, located at the carboxy terminus of pro teins. This Sequence was first identified when the Sequences 0465. The polypeptides and polynucleotides of the of rat BiP and PDI were compared and it was subsequently present invention can be used in any of a large number of found at the carboxy terminus of other luminal ER proteins ways, including numerous in vitro and in Vivo uses. Specific from a number of species (Munro, S. (1986) Cell 46:291 uses for many of the herein-described polypeptides and 300; Pelham, H. R. (1989) Ann. Rev. Cell. Biol. 5:1-23). polynucleotides are described in detail below. Proteins containing this Sequence leave the ER but are US 2006/0053498A1 Mar. 9, 2006 48 quickly retrieved from the early Golgi compartment and smaller peptides which can then bind to MHC molecules and returned to the ER, while proteins without this signal con be released for presentation at the cell Surface. (Kakiuchi, T. tinue through the distribution pathway. (1991) J. Immunol. 147:3289-3295). 0470 Two endoplasmic retrieval receptors were first 0474. The discovery of polynucleotides encoding a novel identified in S. cereveSiae, two human endoplasmic retrieval human KDEL receptor, and the molecules themselves, pro receptors were Subsequently isolated by the use of degen vides the means to further investigate the regulation of the erate PCR primers based on the S. cerevesiae sequences cellular protein Secretory pathway. Discovery of molecules (Hardwick, K. G. (1990) EMBO J. 9:623-630; Semenza, J. related to a novel human KDEL receptor Satisfies a need in C. (1990) Cell 61:1349-1357; Lewis, M.J. and Pelham, H. the art by providing a means or a tool for the Study of this R. (1990) Nature 348:162-163; Lewis, M.J. and Pelham, H. pathway and the diseases that involve the dysfunction of this R. (1992) J. Mol. Biol. 226:913-916). Comparisons of these pathway. Sequences shows that they consist of a conserved 7-trans 0475. In an embodiment of the present invention, ERD4 membrane domain Structure with only short loops in the cell polypeptides of the present invention are used to purify cytoplasm and the ER lumen. Studies with these endoplas KDEL containing proteins and other homologous proteins mic retrieval receptorS Show that ligand binding controls the with similar signals for ER retention such as “HDEL', movement of the receptor; when expressed in COS cells, the “DDEL”, “ADEL”, “SDEL", “RDEL”, “KEEL”, “OEDL", human receptor is normally concentrated in the Golgi, but “HIEL”, “HTEL" and “KQDL". This may be carried out by moves to the ER when bound to a ligand such as KDEL covalently or non-covalently attached the EDR4 polypep tagged hen lysozyme (Lewis, M.J. and Pelham, H. R. (1992) tides of the present invention to a column or other Solid Cell 68:353-364). Support using techniques well known in the art (e.g., affinity 0471. The ER retrieval function of these molecules serves chromatography, panning, etc.). Once bound to the ERD4 to maintain the pool of enzymes in the ER that are necessary polypeptide, the complex is washed to remove contami to perform protein Structure modifications, retains newly nants. The target protein is released using increasing Salt synthesized proteins in the ER until they have been correctly concentrations either in a gradient or Step type purification. modified, and regulates the Structure of the Golgi apparatus. The bound target protein may also be released from the Saccharomyces cerevisiae cells that lack an ER retrieval ERD4 polypeptide by a single Step up in Salt concentration. receptor (Erd2) have a defective Golgi apparatus and fail to 0476. In another embodiment of the present invention, grow. Analysis of yeast Erd2 mutants Suggests that their the EDR4 polypeptides of the present invention are used to growth requires both the retention of multiple proteins in the detect KDEL containing proteins and other homologous ER and the selective removal of specific proteins from the Sequences as described above by methods comprising the Golgi (Townsley, F. M. (1994) J. Cell Biol. 127:21-28). Steps of contacting KDEL or other homologous Sequences Overexpression of a human ER retrieval receptor in COS with an EDR4 polypeptide under conditions that allow cells results in hyperactive retrograde traffic from the Golgi binding to Said Sequence, and detecting the presence of to the ER leading to a loSS of the Golgi Structure and the bound EDR4. The presence of bound EDR4 can be detected breakdown of the secretory pathway (Hsu V.W. (1992) Cell using methods known in the art, Such as by labeling EDR4 69:625-635). directly or indirectly. Bound EDR4 can be detected, for 0472. Disruptions in the cellular secretory pathway have example, by using an antibody that Specifically binds to been implicated in Several human diseases. In familial EDR4 or another EDR4-binding compound that is detect hypercholesterolemia the low density lipoprotein receptors able directly or indirectly. remain in the ER, rather than moving to the cell Surface 0477 Preferred ERD4 polypeptides for binding KDEL (Pathak, R. K. (1988) J. Cell Biol. 106:1831-1841). A form containing proteins and other homologous Sequences of congenital hypothyroidism is produced by a deficiency of described above comprise the amino acid sequence -KIWK thyroglobulin, the thyroid prohormone. In this disease the or -MNLFRFLGDLSHLLAIILLLLKIWKSRSCA thyroglobulin is incorrectly folded and is therefore retained in the ER (Kim, P. S. (1996) J.Cell Biol. 133:517-527). 0478. The present invention is further directed to a trans Mutant forms of proteolipid protein (PLP) have been exam formant comprising the following expression units in a ined as they play a role in generating dysmyelinating or co-expressible State: an expression unit containing a gene coding for an ERD4 polypeptide which is capable of binding hypomyelinating diseases. In this case, the mutations that to a protein localizing in the endoplasmic reticulum and result in disease are mutations that arrest transport of PLP in having a signal for Staying therein; an expression unit the ER and the early Golgi, the Subsequent accumulation of containing a gene coding for Said protein localizing in PLP in the ER results in rapid oligodendrocyte death (Gow, endoplasmic reticulum; and an expression unit containing a A. (1994) J. Neurosci. Res. 37:574-583). foreign gene coding for a polypeptide which is a Subject of 0473. The human ER retrieval receptor function is nec function of Said protein localizing in endoplasmic reticulum, essary for processing and presentation of Specific antigens to and to a transformant comprising, in a co-expressible State, T cells. Many antigens must be processed intracellularly a fusion gene which is composed of a DNA fragment coding before they can be presented, in association with major for a human Serum albumin prepro-Sequence and a foreign histocompatability complex (MHC) molecules at the cell gene coding for a useful polypeptide. The present invention Surface, for recognition by the antigen-Specific receptor of T is also directed to a process for producing Said polypeptide cells. Disruption of the ER retrieval receptor function with by co-expressing Said genes in Said transformant Such that an antibiotic, Brefeldin A, abolishes the ability of a cell to the polypeptide is predominantly Secreted out of the trans present these specific antigen complexes to T cells. These formant cell. Consequently, the invention has an advantage antigenic proteins must be retained in the ER for cleavage to of improving the productivity of Said polypeptide. US 2006/0053498A1 Mar. 9, 2006 49

0479 More particularly, the invention relates to: A trans comprising contacting Said cells with a contracting effective formed yeast cell comprising the following expression units amount of an SMPE polypeptide. Further included in the integrated on a yeast chromosome in a co-expressible State: present invention is a method of inhibiting angiogenesis a first expression unit containing a gene coding for a receptor comprising contacting vascular endothelial cells with an for an endoplasmic reticulum retention signal, wherein the angiogenesis inhibiting effective amount of an SMPE receptor is the receptor protein ERD4 or a fragment thereof polypeptide. The SMPE anti-angiogenic affect can be mea which is capable of binding to a retention signal Selected Sured using assays known in the art. For example, the from the group consisting of “KDEL”, “HDEL”, “DDEL", anti-angiogenic effect in Vivo can be assayed by using the “ADEL”, “SDEL”, “RDEL”, “KEEL”, “OEDL”, “HIEL", 10-day-old embryo chick chorioallantoic membrane model. “HTEL and “KQDL’. and a second expression unit con 0482 SMPE binds with a high affinity to both ileum and taining a gene encoding a protein disulfide isomerase, brain membranes. Thefore, as a further embodiment of the wherein Said isomerase comprises an endoplasmic reticulum present invention is a method of binding an SMPE polypep retention signal, or a gene encoding a fusion protein com tide to ileum or brain membranes. The method can be further prising the amino acid Sequence of Said isomerase and a used as a method of detecting ileum or brain membranes human Serum albumin prepro-Sequence. These methods can comprising the Steps of contacting ileum or brain mem be carried out using methods known in the art or described branes with an SMPE polypeptide under conditions that in U.S. Pat. No. 5,578,466, incorporated herein by reference allow binding to Said membranes, and detecting the presence in its entirety. of SMPE. The presence of SMPE can be detected using Proteins of SEQ ID NO:193 and 194 (internal designation methods known in the art, such as by labeling SMPE directly 585770 215-16-5-0-E8-F and 123996 140-002-5-0-B4-F) or indirectly. Bound SMPE can be detected, for example, by using an antibody that specifically binds to SMPE or another 0480. The cDNA of clones 585770 215-16-5-0-E8-F SMPE-binding compound that is detectable directly or indi (SEQ ID NO:24) and 123996 140-002-5-0 B4-F (SEQ ID rectly. NO:25) encode the human Smooth Muscle and Pain Effector (SMPE) proteins: MRGATRVSIMLLLVTVSDCAVIT 0483 SMPE is also expressed in spermatocytes. There GACERDVOCGAGTCCAISLWLRGLRMCT fore, a further embodiment of the present invention is a PLGRXGEEC HPGSHKIPFFRKRKHHTCPCLPNLLCS method of detecting testes or Spermatocytes by detecting an RFPDGRYRCSMDLKNINF (SEQ ID NO: 193) and SMPE polypeptide or nucleic acid. An SMPE polypeptide MRGATRVSIMLLLVTVSDCAVITGACER can be detected using anti-SMPE antibodies or other SMPE DVOCGAGTCCAISLWLRGLRMCTPLGREGEEC HPG binding compounds. SMPE polynucleotides, such as SHKIPFFRKRKHHTCPCLPNLLCSRFP mRNA, can be detected using methods known in the art Such DGRYRCSMDLKNINF (SEQID NO: 194), respectively. It as PCR (RT-PCR), hybridization (Northern blot analysis), will be appreciated that all characteristics and uses of the etc. polynucleotides of SEQID NOS:24 and 25 and polypeptides 0484 SMPE elicits hyperalgesia when it contacts the of SEQ ID NO: 193 and 194, described throughout the CNS, e.g., the brain. Therefore, the present invention present application also pertain to the human cDNA of includes a method of causing hyperalgesia comprising con clones 585770 215-16-5-0-E8-F and 123996 140-002-5- tacting the CNS with a hyperalgesia effecting amount of an 0-B4-F, and the polypeptides encoded thereby. Polypeptide SMPE polypeptide. SMPE can be delivered to the CNS fragments having a biological 25 activity described herein using methods well known in the art including those and polynucleotides encoding the same are also included in described in PCT application WO9906060, incorporated the present invention. Related polynucleotide and polypep herein by reference in its entirety. Using the methods of tide sequences included in the present invention are SEQ ID WO9906060, the TGF-alpha or other polypeptide that binds NOS:360 and 447. the epidermal growth factor (EGF) receptor, is substituted 0481 SMPE contracts longitudinal ileal muscle and dis with an SMPE polypeptide of the present invention. tal colon, and relaxes the proximal colon. SMPE binds with a high affinity to both ileum and brain membranes. There 0485. Further included in the present invention are meth fore, included as embodiments of the present invention is a ods of inhibiting the above SMPE activities using an inhibi method of causing gastrointestinal Smooth muscle cells to tor of SMPE. A preferred inhibitor of SMPE is an anti contract, in vitro or in Vivo, comprising the Steps of con SMPE antibody. Thus, an embodiment of the present tacting Said cells with a contracting effective amount of an invention is a method of inhibiting Smooth muscle contrac SMPE polypeptide. Preferrably, the gastrointestinal smooth tion (bladder, gastrointestional cells, uterine) or pain com muscle cells are those of the longitudinal ileal or distal prising the Step of contacting Said cells with an effective colon. A further embodiments of the present invention is a contractive or pain inhibiting amount of an anti-SMPE method of causing gastrointestinal Smooth muscle cells to antibody or other SMPE inhibitor. relax comprising the Steps of contacting Said cells with a 0486 The invention further relates to a method of screen relaxing effective amount of an SMPE polypeptide. Prefer ing for test compounds that bind and/or inhibit an SMPE rably, the gastrointestinal Smooth muscle cells are proximal activity above comprising the Steps of contacting an SMPE colon cells. SMPE can also be used in the same manner to polypeptide with Said test compound and detecting or mea contract uterine cells. Therefore, included in the present Suring whether said test compound binds said SMPE invention is a method of causing uterine Smooth muscle cells polypeptide. Alternatively, the method comprises the Steps to contract comprising contacting Said cells with a contract of contacting an SMPE polypeptide with a binding target ing effective amount of an SMPE polypeptide. Further (e.g., smooth muscle cells or brain cells) of said SMPE included in the present invention is a method of causing polypeptide in the presence of a test compound, and detect Smooth muscle cells (e.g., bladder, vascular) to contract ing or measuring the binding of the SMPE polypeptide to US 2006/0053498A1 Mar. 9, 2006 50

Said binding target, wherein a difference in the amount of 0490 VAMP-10, like other VAMPs, has a three domain Said binding in the presence of Said test compound relative organization. The domains include a variable proline-rich, to the amount of binding in the absence of the test compound N-terminal Sequence, a highly conserved central hydrophilic indicates that the test compound modulates, preferably core of amino acids, and a hydrophobic Sequence of amino inhibits, the binding of Said polypeptide to Said binding acids presumed to be the membrane anchor. target. The method may alternatively comprise the Steps of 0491 In one aspect, the invention includes a VAMP-10 contacting an SMPE polypeptide with a binding target in the polypeptide composition for use in delivering a Second presence of a test compound, wherein the binding of Said composition, preferably nucleic acids, polypeptides, or SMPE polypeptide with said binding target elicits or causes Small molecules Such as therapeutic drugs, to target biologi a biological activity (e.g., activities described above) which cal cells either in vitro or in Vivo. The composition com is detected or measured, and further wherein a difference in prises a VAMP-10 polypeptide as a first molecule and a the level of Said biological activity in the presence of the test Second molecule. The Second molecule may, if desirable, be compound relative to the amount of biological activity in the covalently or non-covalently attached or fused to the VAMP absence of the test compound indicates that the test com 10 polypeptide. The VAMP-10 polypeptide composition pound modulates, preferably inhibits or activates, the bio may further comprise artificial lipids to facilitate delivery of logical activity of said SMPE polypeptide. the second molecule by lipisomes or lipid vesicles. Methods 0487. Preferred SMPE polypeptides for use in the meth for using VAMP-10 polypeptides in these methods are ods described herein include the amino acid Sequences known in the art and include U.S. Pat. Nos. 6,074,844, -AVITGACERDVOCGAGTCCAISLWLRGL 6,203,794 and 6,099,857, incorporated by reference in their RMCTPLGREGEECHPGSHKIPFFRKRKHH- or -TGAC entireties. In a preferred embodiment, VAMP-10 polypep ERDVOCGAGTCCAISLWLRGLRMCT of SEO ID NO: tides are used to faciliate delivery of a Second composition, 193 or 194. e.g., lipisome mediated DNA transfection, to cells in culture, Protein of SEQ ID NO:305 (internal designation preferably neuronal cells, and further preferably to the 500691428 255-2-5-0-D4-R 104) presynaptic membrane. 0492 VAMP-10 polypeptides are also useful in methods 0488. The human cDNA of clone 500691428 255-2-5- of inhibiting the release of neurotransmitters by preventing 0-D4-R 104 (SEQ ID NO:136) encodes the human the docking and/or fusing of a presynaptic vesicle to the VESICLE-ASSOCIATED MEMBRANE PROTEIN 10 or presynaptic membrane. These polypeptides may be referred VAMP-10 protein: MSATAATAPPAAPAGEGGPPAPPPN to as excitation-secretion uncoupling peptides (ESUPs). LTSNRRLOOTOAOVDEVVDIMRVNVDKVLERDOKL Fragments of VAMP-10 having this blocking activity can be SELDDRADALOAGPSOFETSAAKLKRKY identified using methods known in the art (See e.g., U.S. Pat. WWKNLKMMIILGVICAIILIIIIVYFST (SEQ ID Nos. 6,090,631 and 6,169,074 incorporated by reference in NO:305). It will be appreciated that all characteristics and their entireties). ESUPs of the present invention comprise uses of the polynucleotides of SEQID NO:136 and polypep synthetic and purified VAMP-10 peptide fragments which tides of SEQ ID NO:305 described throughout the present correspond in primary Structure to peptides which Serve as application also pertain to the human cDNA of clone binding domains for the assembly of a ternary protein 500691428 255-2-5-0-D4-R 104 and the polypeptides complex ("docking complex”) which is critical to neuronal encoded thereby. Polypeptide fragments having a biological vesicle docking with the cellular plasma membrane prior to activity described herein and polynucleotides encoding the neurotransmitter Secretion. Preferably, the primary Sequence Same are also included in the present invention. Related of the ESUPS of the invention also includes amino acids polynucleotide and polypeptide Sequences included in the which are identical in sequence to the VAMP-10 peptide present invention are SEQ ID NOS:432 and 546. products of BoTX and TeTX proteolytic cleavage in neuronal 0489 VAMP-10 is an integral membrane protein cells, or fragments thereof (“proteolytic products”). For involved in the movement of vesicles from the plasmale optimal activity, ESUPs of the invention have a minimum mma of one cell, acroSS the Synapse, to the plasma mem length of about 20 amino acids and a maximal length of brane of the receptive neuron. This regulated vesicle traf about 28 amino acids, although they may be larger or ficking pathway and the endocytotic proceSS may be blocked smaller. Preferably, the ESUPS correspond in primary struc by the highly specific action of cloStridial, tetanus toxin ture to binding domains in the docking complex, most (TeTx) and botulinum toxin (BoNT) and other metalloen preferably the region of Such binding domains that are doprotease neurotoxins which prevents neurotransmitter involved in the formation of a coiled-coil structure in the release by cleaving VAMPs. VAMP-10 is important in native docking complex proteins. ESUPS may also be used membrane trafficking. It participates in axon extension via as pharmaceutical carriers as part of fusion proteins to exocytosis during development, in the release of neurotrans deliver Substances of interest into neural cells in a targeted mitters and modulatory peptides, and in endocytosis. The manner. Preferred VAMP-10, or ESUP, polypeptides for use tightly-regulated Synaptic vesicle cycle at the nerve terminal in inhibiting the release of neurotransmitters include those consists of the formation of Synaptic vesicles, the docking of comprising -NRRLQQTOAQVDEVVDIMRVNVDKV vesicles comprising VAMP-10 to the presynaptic plasma LERDOKLSELDDRADALOAGPSOFETSAAKLKRK- of membrane, the fusion of these membranes and consequent SEQ ID NO:305. More preferred ESUP polypeptides com neurotransmitter release, endocytosis of the empty vesicles prise an amino acid sequence portion of SEQ ID NO:305 and the regeneration of fresh vesicles. Endocytotic vesicular selected from the group consisting of RVNVDKVLER transport includes Such intracellular events as the fusions DOKLSELDD; KVLERDOKLSELDDRA; VNVDKV and fissions of the nuclear membrane, endoplasmic reticu LERDOKLSELDDRA; DIMRVNVDKVLER lum, Golgi apparatus, and various inclusion bodies Such as DOKLSELDDRADAL; DEVVDIMRVNVD; peroxisomes or lySOSomes. OAOVDEVVDIMRVNVD; LOOTOAOVDEVVDIM US 2006/0053498A1 Mar. 9, 2006

RVNVD; OOTOAOVDEVVD; binds a cleavage product that is either the N-terminal or NRRLOOTOAOVDEVVD; and NLTSNR C-terminal portion of the VAMP-10, and the assay may RLOOTOAOVDEVVD. comprise the Steps of: 0493) The ESUPS above may be used to inhibit or treat 0504 (i) combining a test compound that may contain or pain according to U.S. Pat. Nos. 6,113,915 or 5,989,545 consist of the toxin with the Solid-phase peptide to form an (incorporated by reference herein in their entireties) by assay mixture, Substituting the polypeptides of the present invention for 0505 (ii) Subsequently or simultaneously combining the BoTX type A. assay mixture with the antibody, and 0494 Because VAMP-10 is a component of vesicles, 0506 (iii) Subsequently or simultaneously determining antibodies to VAMP-10 are useful in the detection of whether there has been formed any conjugate between the vesicles, preferably neuronal vesicles transporting neu antibody and the cleavage product. rotransmitters. VAMP-10 can be used during purification of vesicles as a marker for vesicles or vesicles can be detected 0507 Preferably, the step (i) of the assay is carried out in using antibodies to VAMP-10 in assays such as immuno the presence of a zinc compound and a VAMP-10 polypep histochemistry. Following exocytosis of Vesicles, a portion tide. of the VAMP-10 inserted in the vesicle appears on the surface of the axon, thus making VAMP-10 useful for the 0508. In this embodiment, the assay comprises: detection and monitoring of exocytosis of Synaptic vesicles. 0509 (i) combining the test compound with a solid phase 0495) Detection of VAMP-10 expression (mRNA or pro comprising a VAMP-10 polypeptide, tein) levels or mutated forms of VAMP-10 is further useful 0510 (ii) washing the test compound from the solid in the determination or diagnosing of whether Someone is at phase, risk of developing or has a neurological disorder, Such as mood disorders Selected from depression, bipolar disorder, 0511 (iii) combining the solid phase with an antibody Schizophrenia, etc.), wherein a decreased level in expression adapted for binding Selectively with peptide cleaved by of VAMP-10, mRNA or protein, as compared to an indi toxin, and vidual without a neurological disorder indicates the indi 0512 (iv) detecting a conjugate of the antibody with vidual has the disorder or is as risk of having the disorder in cleaved peptide. the future. 0513. In another embodiment, the assay comprises: 0496 The present invention further includes a novel assay System for toxins, Such as cloStridial, tetanus toxin 0514 (i) adding a test Solution to an assay plate com (TeTx) and botulinum toxin (BoNT), using novel reagents. prising immobilized peptide, the peptide being a VAMP-10 Preferably, methods of U.S. Pat. No. 6,043,042, incorpo polypeptide; rated by reference in its entirety, are used to perform the 0515 (ii) incubating the assay plate, assay, wherein a VAMP-10 polypeptide is the substrate cleaved by the test compound. More specifically, the assay 0516 (iii) washing the plate with a buffer, comprises the Steps of: 0517 (iv) adding to the plate an antibody solution, said 0497. The invention relates to an assay for botulinum Solution comprising an antibody adapted Selectively to bind toxin or tetanus toxin comprising the Steps of to a peptide Selected from the group consisting of (1) the 50 C-terminal amino acid residues SEQ ID NO:305, the 30 0498 (a) combining a test compound with a substrate and C-terminal amino acid residues SEO ID NO:305, and the 20 with antibody, wherein the Substrate has a cleavage Site for C-terminal amino acid residues SEQ ID NO:305 (any other the toxin and when cleaved by toxin forms a product, and VAMP-10 polypeptide of the present invention may also be wherein the antibody binds to the product but not to the Selected). substrate; and wherein the substrate is a VAMP-10 polypep 0518 (2) a peptide the N-terminal end of which is tide; and selected from the group consisting of: (1) the 50 N-terminal 0499 (b) testing for the presence of antibody bound to amino acid residues SEQ ID NO:305, the 30 N-terminal the product, which product is attached to a Solid phase assay amino acid residues SEQ ID NO:305, and the 25 N-terminal component. amino acid residues SEQ ID NO:305 (any other VAMP-10 0500 Preferably, in the practice of this invention, the polypeptide of the present invention may also be selected). VAMP-10 polypeptide is cleaved by the toxin to generate 0519 (v) incubating the assay plate, new peptides having N- and C-terminal ends. In addition, the peptide Substrate is attached to a Solid phase component 0520 (vi) washing the plate with a buffer, and of the assay. 0521 (vii) measuring the presence of antibody on the 0501) The assay according to the invention may utilize assay plate. assay components (a) and (b): 0522. In this embodiment, the antibody may be linked to 0502 (a) a peptide linked to a solid-phase, the peptide an enzyme and the presence of antibody on the plate is being cleavable by the toxin-to generate a cleavage product, measured by adding an enzyme Substrate and measuring the conversion of the substrate into detectable product. The 0503 (b) an antibody that binds to the cleavage product detectable product may be colored and measured by absor but not to the uncleaved polypeptide or an antibody that bance at a Selected wavelength. US 2006/0053498A1 Mar. 9, 2006 52

0523. In the practice of the invention, the inactive toxin 0531. It will be appreciated that all characteristics and present in the test compound may be converted to active uses of the polynucleotides of SEQ ID NO:2 and SEQ ID toxin. This may be accomplished by adding a protease to the NO:340 and polypeptides of SEQ ID NO:171 and 457 test compound. described throughout the present application also pertain to 0524. The antibody-peptide conjugate may be detected the human cDNA of clone 589115. using a further antibody Specific to the first antibody and Proteins of SEQ ID NO:302 (Internal Designation Clone linked to an enzyme. ID:1000853793) and Related Protein of SEQ ID NO:543. Proteins of SEQ ID NO:171 (Internal Designation Clone 0532) MRLFLSLPVLVVVLSIVLEGPAPAQGTP ID:589115) and Related Protein of SEQ ID NO:457. DVSSALDKLKEFGNTLEDKARELISRIKO SELSAKM 0525) The polynucleotides of SEQ ID NO:2 and SEQ ID REWFSETFOKVKDKLKIDS NO:340 and polypeptides of SEQ ID NO: 171 and 457 0533. The polynucleotides of SEQ ID NO:133 and SEQ encode a C-terminal variant of Apollipoprotein A1, herein ID NO:429 encode human apolipoprotein CI (ApoCI) referred to as Apo AI-CTV. An embodiment of the invention polypeptide of SEQ ID NO:302 and SEQ ID NO:543, includes compositions of SEQ ID NO:2, 340, 171, and 457 respectively. The ApoCI of the invention differs by 1 amino which encode for this novel variant of the apolipoprotein acid comprising the amino acid Sequence FOKVKDKLKI, family of lipid transporting proteins. Specifically, ApoAI where aspartate (D at position 77 of SEQ ID NO:302) CTV is a component of high density lipoprotein which replaces a glutamate (E) of the ApoCI of GENPEP accession functions to remove cholesterol from circulation and thus X00570, AF050154, and M20902. ApoCI is a member of the providing protection against the development of atheroscle apolipoprotein family of lipid binding and transporting rosis, coronary atherOSclerotic lesions and Subsequent proteins Specifically functioning to transport cholesterol microvascular and cardiovascular disease. eSterS. 0526 Preferred polynucleotides of the invention are 0534. It will be appreciated that all characteristics and compositions of the novel portion of the cDNA from bases uses of the polynucleotides of SEQ ID NO:133 and SEQ ID 465 to 521 of SEQ ID NO:2 including the nucleic acids NO:429 and polypeptides of SEQ ID NO:302 and SEQ ID comprising the sequences -GCAGCTTTCTTAACTATC NO:543, described throughout the present application also CTAACAAGCCTTGGACCAAATG pertain to the human cDNA of clone 1000853793, and the GAAATAAAGCTTTTTGA-, -GAAGGCAGCTTTCT polypeptides encoded thereby. TAACTATCCTAACAAGCCTTGGACCAAATGGAAATA AAGCTTTGA-, O -AGCTCTACCGCCAGAAG Proteins of SEQ ID NO:295 (Internal Designation Clone GCAGCTTTCTTAACTATCCTAACAAGC ID:642948), SEQ ID NO:296 (Internal Designation Clone CTFGGACCAAATGG AAATAAAGCTTTTTGAT ID:638743), and SEQ ID NO:539. GAAAAAA- of SEO ID NO:2 and 340. 0535 MEASALTSSAVTSVAKWRVASGSAVVL PLARIATVVIGGWAMAAVPMVLS AMGFTAA 0527 Preferred polypeptides of the invention are com GIASSSIAAKMMSAAA positions of the novel C-terminal portion comprising the IANGGGVASGSLVATLOSLGATGLSGLT amino acid sequence -AAFLTILTSLGPNGNKAF, -ME KFILGSIGSAIAAVIARFY LYROKAAFLTILTSLGPNGNKAF, or -OKKWOEEME LYROKAAFLTILTSLGPNGNKAF of SEO ID NO: 171 0536 The polynucleotides of SEQ ID NO:126, 127, and and SEO ID NO:457. 425 and the polypeptides of SEQ ID NO:295, 296 and 539 encode human transmembrane, alpha-interferon-inducible 0528. Further preferred polypeptides of the invention polypeptides, alNFIP-1, alNFIP-1, and alNFIP-3, respec include the compositions comprising the apolipoprotein tively. Preferred polynucleotides and polypeptides of the domain KAAVLTLAVLFLTGSOARHFWOODEP POSPWDRVKDLATVYVDVLKDSGRDYVS OFEG invention comprise the nucleic acid Sequences of SEQ ID SALGKOLNLKLLDNWDSVTSAFSNL NO:126, 127, and 425 and amino acid sequences of SEQ ID REOLGPVTOEXWDNLEKETEGLROEMSKDLE NO:295, 296 and 539. EVKAKVOPYLDDFOKKWOEEMELYRO 0537) Preferred polypeptides of SEQ ID NO:295 and KAAFLTILTSLGPNGNKAof SEQ ID NO: 171 and 457, or SEO ID NO:539 for use in the methods described herein the amino acid residue positions -17 to +141 of SEQ ID include the amino acid Sequences comprising -VLSAMG NO: 171. FTAAGIASSSIAAKMMSAAAIANGGGVASG-, -SSIAAKMMSAAAIANGGGVASGSLVATLOSLGAT-, or 0529) An embodiment of the invention includes a method -VIGGVVAMAAVPMVLSAMGFTAA for treatment of atherosclerosis or cardiovascular diseases, GIASSSIAAKMMSAAAIANGGGVASGSLVATLOSLG comprising administering to an individual a therapeutically ATGLSGLTK effective amount of apoAI-CTV or variants or mixtures thereof to lower total plasma cholesterol at least 5% of 0538 Preferred polypeptides of SEQ ID NO:296 for use pretreatment levels. in the methods described herein include the amino acid 0530. Further utility of the polypeptides of the present Sequence -AAAIANXGGVASGSLVATLOSLGATGLS invention may be further confirmed by methods of produc GLTKF- or -LSAMGFTAAGIASSSIAAKMMSAAA tion and use of other apolipoproteins by those skilled in the IANXGGVASGSLVATLOSLGATGLS-. art or as described by Ageland et al in U.S. Pat. No. 0539. It will be appreciated that all characteristics and 5.990,081, which disclosure is hereby incorporated by ref uses of the polynucleotides of SEQ ID NOS:126, 127, and erence in its entirety. 425 and the polypeptides of SEQ ID NOS:295, 296 and 539, US 2006/0053498A1 Mar. 9, 2006 53 described throughout the present application also pertain to thereof to treat and/or prevent the ill-effect of bacterial the human cDNA of Clone ID:642948 and Clone infection. In a preferred embodiment, the protein of the ID:638743, and the polypeptides encoded thereby. invention may be used to counteract the effects of the 0540 Sites of glycine myristylation within a polypeptide bacterial endotoxin lipopolysaccharide (LPS). The methods function to modulate the activity and compartmentalization for using Such compositions is described in DZiegielewska and Andersen, Biol. Neonate, 74:372-5 (1998), the disclo of the protein (Resh, M. D. Biochim Biophys Acta 1451:1- Sure of which is incorporated herein by reference in its 16 (1999)). entirety. 0541. Preferred polypeptides of the invention include fragments comprising the Sites of N-myristylation. Preferred 0547. Furthermore, the alNFIP polypeptides or fragments amino acids of Said sites within SEO ID NO:295 and SEO thereof may be used to identify specific molecules with ID NO:539 include GGVVAM (positions 39-44), GIASSS which it binds Such as agonists, antagonists or inhibitors. (positions 60-65), GGGVAS (positions 79-84), GSLVAT Another embodiment of the present invention relates to (positions 85-90), and GSIGSX (positions 108-113). Further methods of using the alNFIP polypeptides or fragments preferred are amino acids within 6 residues preceding or 6 thereof to identify and/or quantify cytokines of the inter residues following Said amino acid Sequences. Further pre feron family as well as other cytokines such as IL 10 and ferred amino acids include Sequences comprising the Sites of tumor antigens, which may interact with the alNFIP N-myristylation in the polypeptides of SEQ ID NO:296. polypeptides of the invention. Preferred amino acids of Said sites within SEO ID NO:296 0548. The alNFIP polypeptides of the invention or frag include IATVVIGGVVAMAAVPMV, MGFTAA ments thereof are included in pharmaceutical preparations GIASSSIAAKMM, AAIANXGGVASGSLVATL, NXG for treatment, prevention or alleviation of cancers. In GVASGSLVATLOSLGA, and LTKFILGSIGSAIAAVIAR. another embodiment of the present invention, the alNFIP 0542 Interferons (IFNs) are a part of the group of inter polypeptides of the invention or fragments thereof are used cellular messenger proteins known as cytokines and are part included in pharmaceutical preparations for treatment, pre of the body's natural defense to viruses and tumors. Type I vention or alleviation of viral or bacterial infections. In IFNs (alpha and beta interferons) are produced in a variety another embodiment of the present invention, the alNFIP of cells types and their biosynthesis is stimulated by viruses polypeptides of the invention or fragments thereof are used and other pathogens, and by various cytokines and growth to inhibit and/or modulate the effect of cytokines and related factors. Both C- and Y-IFNs are immunomodulators and molecule such as Il-2, TNF alpha, CTLA4, CD28, and anti-inflammatory agents, activating macrophages, T-cells others, by preventing the binding of the endogenous cytok and natural killer cells (reviewed in Jonasch and Halluska, ine to their natural receptors, thereby blocking cell prolif Oncologist 6(1):34 (2001)). As part of the body's natural eration or inhibitory signals generated by the ligand-receptor defense to viruses and tumors, INFS affect the function of the binding event. immune System and have direct action on pathogens and tumor cells. IFNs mediate these multiple effects by inducing 0549. In another embodiment of the present invention, the Synthesis of cellular proteins, including the polypeptides the alNFIP polypeptides of the invention or fragments thereof are useful to correct defects in in vivo models of of the present invention, alNFIP-1, alNFIP-1, and alNFIP-3. disease Such as autoimmune, inflammation and tumor mod 0543 Antiviral activity of the alNFIP polypeptides are els, by injecting the protein either intra peritoneally intra assayed according to conventional methods (Tovey et al., venously, Subcutaneously or directly in the diseased tissue. Proc. Soc. Exp. Biol. and Med., 1974 146:809-815). Pre ferred polypeptides of SEQ ID NO:295, 296 and 539 and 0550 The polynucleotides of SEQ ID NO:126, 127, and fragments thereof include those which possess antiviral 425 or fragments thereof is useful in diagnostic assays for function, where preferred antiviral activity is against herpes aINFIP-1, alNFIP-2, or alNFIP-3 gene expression in in vitro Simplex virus and hepatitis virus C, alone or in combination models or in conditions associated with expression of the with known antiviral treatments Such as interferon alpha. aINFIP polypeptides of the invention. The diagnostic assay is useful to distinguish between absence, presence, and 0544 The antitumor activity the alNFIP polypeptides of exceSS expression of the gene and to monitor regulation of the invention can be demonstrated by Similar methods using levels of the gene of the invention during therapeutic inter tumor cell lines rather than treatment of cells with virus as vention. The DNA may also be incorporated into effective used to test antiviral activity. Tumor cell lines examples eukaryotic expression vectors and directly targeted to a include MCF-7 (human breast cancer derived), NOS-1 Specific tissue, organ, or cell population for use in gene (human oral primary Squamous cell carcinoma derived), and therapy to treat the above mentioned conditions, including MedB-1 (human primary mediastinal large B-cell lym tumors and/or to correct disease- or genetic-induced defects phoma derived). in any of the above mentioned proteins including the protein 0545) Further utility of the polypeptides of the present of the invention. invention may be further confirmed by methods of interferon inducible proteins in the inhibition of viral functions such as Protein of SEQ ID NO: 170 (Internal Designation Clone cell penetration, uncoating, RNA and protein Synthesis, ID:502084) and Related Protein of SEQ ID NO:456. assembly and release described in Hardman et al., Pharma 0551) The polynucleotides of SEQ ID NO:339 and cological Basis of Therapeutics, McGraw-Hill, New York polypeptides of SEQ ID NO:456 encode neutrophil stimu N.Y. pp 1211-1214, 25 (1996), disclosure of which is hereby lating protein 2, previously described in WO 9006321 incorporated by reference in its entirety. (GENPEP accession A01319) as a novel factor having 0546) Another embodiment of the present invention neutrophil-stimulating activity. The polynucleotide of SEQ relates to the use of alNFIP polypeptides or fragments ID NO:1 encodes a novel polypeptide variant, neutrophil US 2006/0053498A1 Mar. 9, 2006 54

Stimulating protein 2V, comprising the amino acid Sequence Proteins of SEQ ID NO: 227 (Internal Designation Clone of SEQ ID NO: 170 in which an aspartate (D) residue is ID: 166601) and Related Protein of SEQ ID NO:502. located at position +16 of SEQ ID NO: 170 rather than a glutamate (E). Preferred compositions of the invention 0557 Polynucleotides of SEQ ID NO:58 and SEQ ID include the polypeptides of SEQ ID NO: 170. Further NO:385 encode the polypeptides of SEQ ID NO:227 and preferred amino acids of SEQ ID NO: 170 comprise the SEQ ID NO:502, respectively, with amino acid sequence sequence LAKGKDESLDS, QXKRNLAKGKDESLDSD MAAAAVPSLLLSLPPHOGLTFSNKIOPF LYAE, or SSTKGOXKRNLAKGKDESLDSDLYAEL GAOGVLHPEPGLRDWLLPTCSROLRVALPEKGS RCMCIKTTSGIHPKNIOSLEVIGKGTHCNOVEVIA EGSLCOTOLPATPCFLPSNTVRT. It will be appreciated TLKDGRKICLDPDAPRIKKIVOKKLAGDESAD. It will that all characteristics and uses of the polynucleotides of be appreciated that all characteristics and uses of the poly SEQ ID NOS:58 and 385 and polypeptides of SEQ ID nucleotides of SEO ID NO:1 and SEO ID NO:339 and NO:227 and 502, described throughout the present applica polypeptides of SEQ ID NO:170 and SEQ ID NO:456, tion also pertain to the human cDNA of clone 166601, and described throughout the present application also pertain to the polypeptides encoded thereby. the human cDNA of clone 502084, and the polypeptides 0558) The polynucleotides of SEQ ID NO:58 and 385 encoded thereby. and polypeptides of SEQ ID NO:227 and 502 encode a 0552) A preferred embodiment of the invention includes transcriptional regulatory protein. Preferred polynucleotides use of the novel neutrophilstimulating protein 2v of SEQID of the invention include the nucleic acid Sequences com NO: 170 in a method to stimulate wound healing by con prising Clone 166601, the polynucleotides comprising SEQ tacting the wound area with effective amount of polypeptide ID NO:58 and the polynucleotides comprising SEQ ID of SEO ID NO:170 or further use as described in U.S. Pat. NO:385. Preferred polypeptides of the invention include the No. 5,804,176, which disclosure is hereby incorporated by amino acid Sequences derived from the nucleic acid reference in its entirety. A further preferred includes use of Sequence comprising Clone 166601, the polypeptides com neutrophil Stimulating protein 2V in the enhancement of prising the amino acid sequences of SEQID NO:227 and the angiogenesis for revascularization after injury Such follow polypeptides comprising the amino acid Sequences of SEQ ing myocardial infarction, wherein Site of injury is contacted ID NO:5O2. with effective amount of polypeptide of SEQ ID NO:170 or 0559). In an embodiment of the invention, preferred use as further described in U.S. Pat. No. 5,871,723, which polypeptides include the portion comprising the Site of disclosure is hereby incorporated by reference in its entirety. protein kinase C phosphorylation or the amino acid Antibodies against neutrophil Stimulating protein 2v, by sequences comprising SNK or TVR of SEQID NO:227 and preventing or blocking the deposition of connective tissue 5O2. matrix, are useful in the treatment of fibrotic disorders by 0560. In another embodiment, preferred polypeptides of contacting the polypeptides of SEQ ID NO:170 with fibrotic the invention include the portion of the amino acid Sequence tissue, Such as in Scleroderma, liver cirrhosis, and myelofi comprising Sites of myristylation or the amino acids com brosis. prising the sequence GLTFSN or GSEGSL of SEQ ID 0553 An embodiment of the invention includes frag NO:227 or 5O2. ments of SEQ ID NO:170 which comprise domains which impart function to this cytokine. Preferred fragments include Proteins of SEQ ID NO:268 (Internal Designation Clone the amino acid Sequence comprising the IL8 domain, DSD ID:211056) and Related Protein of SEQ ID NO:530. LYAELRCMCIKTTSGIHPK 0561. The polynucleotides of SEQ ID NO:99 and SEQ NIOSLEVIGKGTHCNOVEVI ID NO:416 and polypeptides of SEQ ID 35 NO:268 and ATLKDGRKICLDPDAPRIKKIVQKKL. Further preferred SEQ ID NO:530, respectively, encode a novel human tryp amino acids include the Small cytokines (intercrine/chemok tophan hydroxylase, including the amino acid Sequence ine) C-X-C Subfamily signature of the amino acid sequence hereafter referred to as nhTOH. Tryptophan is taken up by comprising CMCIKTTSGIHPKNIOSLEVIGKGTHCN active transport into the neurons where it is hydroxylated to OVEVIATLKDGRKICLD. 5-hydroxytryptophan (5HTP). The latter is then decarboxy lated to Serotonin, a neurotransmitter involved in central 0554 Further preferred polypeptides include portions nervous disorders, especially mood disorders, Sleep disor comprising Sites of Protein Kinase C phosphorylation ders, and eating disorders. Activity of the polypeptide of the including amino acid residues 2 to 4, residues 13 to 15, invention increaseS production of Serotonin levels and residues 36 to 38 and residues 97 to 99 of SEO ID NO: or increase the metabolism of tryptophan. Thus polypeptides of amino acids Sequence comprising SLR, SAR, STK, and the invention are useful in the in vitro production of the TLK. Further preferred polypeptides include portions of the Serotonin and metabolism f tryptophan. AS example, an amino acid Sequence comprising Sites of Casein kinase II expression vector containing the polynucleotides of SEQID phosphorylation including amino acid residues 97 to 100 or NO:99 or SEO ID NO:416 can be introduced into a cell line the amino acid Sequence comprising TLKD. by methods known in the art Such as by calcium precipita 0555. Further preferred polypeptides include portions of tion; tryptophan can be Supplied in the media; and Serotonin the amino acid Sequence comprising Sites of N-myristylation produced by the cells can be extracted by known methods. or the amino acid residues comprising GTHCNO. 0562. The invention further relates to a method of screen 0556 Further preferred polypeptides include the small ing for test compounds that bind hnTOH comprising the cytokines (intercrine/chemokine) C-X-C Subfamily signature Steps of contacting a hnTOH polypeptide with Said test of the amino acid sequence comprising CMCIKTTSGIHP compound and detecting or measuring whether said test KNIOSLEVIGKGTHCNOVEVIATLKDGRKICLD. compound binds said hnTOH polypeptide. The invention US 2006/0053498A1 Mar. 9, 2006 55 further relates to a method of Screening for test compounds invention may be further confirmed by methods described, that activate hnTOH comprising the Steps of contacting a for example, by Bosslet et al (U.S. Pat. No. 5,643,731) in hnTOH polypeptide with Said test compound and detecting which use of a pair of leucine ZipperS for in vitro diagnosis, or measuring whether said test compound activates Said in particular for the immunochemical detection and deter hnTOH polypeptide, for example by measuring Serotonin mination of an analyte in a biological liquid; by TSO et al production or tryptophan depletion. (U.S. Pat. No. 5,932,448) in which use of leucine zippers for 0563 Another embodiment includes physiologically producing bispecific antibody heterodimers, by Conrad et al acceptable compositions of test compounds found to (U.S. Pat. No. 5,965,712), Ciardelli et al (U.S. Pat. No. increase Serotonin production, referred to as activators, in a 5,837.816), and Spriggs et al (WO9410308) in which meth Screen. Further embodiments include methods to use acti ods of preparing Soluble oligomeric proteins using leucine Vators that have been identified in a Screen or previously Zippers have been described; and by Pelletier et al known in the art in the preparation of physiological accept (WO9834120) in which methods to use leucine zipper able formulations for use in in vivo. Further preferred are forming Sequences in protein fragment complementation methods to use activators in a physiologically acceptable assays to detect biomolecular interactions has been formulation in the treatment of CNS disorders in which described, all examples which disclosures are hereby incor tryptophan and Serotonin levels are aberrant, particularly porated by reference in their entireties. depression, anxiety disorder, bipolar disorder, and eating disorders. 0567 The multimerization activity of the polypeptides of 0564. It will be appreciated that all characteristics and the present invention containing leucine Zipper domains may uses of the polynucleotides of SEQ ID NO:99 and SEQ ID be assayed using any of the assays known to those skilled in NO:416 and polypeptides of SEQ ID NO:268 and SEQ ID the art including circular dichroism Spectrum and thermal NO:530, described throughout the present application also melting analyses as described in U.S. Pat. No. 5,942,433, pertain to the human cDNA of clone 211056, and the which disclosures are hereby incorporated by reference in polypeptides encoded thereby. their entirety. Alternatively, the leucine Zipper motif in LZP could be used by those skilled in art as a “bait protein' in a Proteins of SEQ ID NO: 190 (Internal Designation Clone well established yeast double hybridization system to iden ID: 147648) and Related Protein of SEQ ID NO:474. tify its interacting protein partners in vivo from cDNA 0565. The polynucleotides of SEQ ID NO:21 and SEQ library derived from different tissues or cell types of a given ID NO:357 and polypeptides of SEQ ID NO:190 and SEQ organism. Alternatively, LZP or part thereof could be used ID NO:474 encode a novel DNA binding polypeptide con by those skilled in art in mammalian cell transfection taining a leucine Zipper pattern multimerization domain, experiments. When fused to a Suitable peptide tag Such as thereafter referred to as LZP, also known as bZIP transcrip His), tag in a protein expression vector and introduced into tion factor basic domain signature (Hai et al., Genes Dev. culture cells, this expressed fusion protein can be immuno 3:2083(1989)). An embodiment of the present invention precipitated with its potential interacting proteins by using includes the polynucleotides, polypeptides and fragments anti-tag peptide antibody. This method could be chosen thereof comprising the sequences of SEQ ID NO:21, 357, either to identify the associated partner or to confirm the 190, and 474 of the invention. Preferred polypeptides of the present invention are directed to the amino acid Sequences results obtained by other methods Such as those just men which comprise the leucine Zipper domain Selected from the tioned. following amino acids of SEQID NO:190 and 474 including 0568. In a preferred embodiment, the invention relates to LAAGAVTLGIGFFALASALWFL; PKGFFNYLTYFLAA compositions and methods of using the LZP polynuceotides GAVTLGIG; or FFALASALWFLICKRREIFONS. It will and polypeptides of SEQ ID NO: and SEQ ID NO: or be appreciated that all characteristics and uses of-the poly fragment thereof for preparing Soluble multimeric proteins, nucleotides of SEO ID NO:21 and SEO ID NO:357 and which consist in multimers of fusion proteins containing a polypeptides of SEQ ID NO:190 and SEQ ID NO:474, leucine Zipper fused to a protein of interest, using any described throughout the present application also pertain to technique known to those skilled in the art including those the human cDNA of clone 147648, and the polypeptides described in international patent WO9410308, which dis encoded thereby. closure is hereby incorporated by reference in its entirety. In 0566 Leucine-zippers permit dimerization of various another preferred embodiment, LZP or derivative thereof is cytoplasmic hormone receptors and enzymes (Forman, et used to produce bispecific antibody heterodimers as al., Mol Endocrinol, 3, 1610-1626 (1989)). Leucine zippers described in U.S. Pat. No. 5,932,448, which disclosure is are also a common feature of transcription factors, where hereby incorporated by reference in its entirety. Briefly, they permit homo- or heterodimerization resulting in tight leucine ZipperS capable of forming heterodimers are respec binding to DNA strands (for reviews, see Abel, et al., Nature tively linked to epitope binding components with different 341, 24-25 (1989); Jones, et al., Cell 61, 9-11 (1990); Lamb, Specificities. Bispecific antibodies are formed by pairwise et al., Trends in Biochemical Sciences 16, 417-422 (1991)). asSociation of the leucine Zippers, forming an heterodimer Therefore, preferred polypeptides of the present invention which links two distinct epitope binding components. In Still are useful tools in Several areas of biotechnology, especially another preferred embodiment, LZP or part thereof or in protein engineering, where their ability to mediate derivative thereof is used for detection and determination of homodimerization or hetero-dimerization has found Several an analyte in a biological liquid as described in U.S. Pat. No. applications, including but not limited to immunochemistry, 5,643,731, which disclosure is hereby incorporated by ref antibody generation, preparation of Soluble oligomeric pro erence in its entirety. Briefly, a first leucine Zipper is immo teins, complementation assasyS. The utility of the present bilized on a Solid Support and the Second leucine Zipper is US 2006/0053498A1 Mar. 9, 2006 56 coupled to a specific binding partner for an analyte in a Ausubel et al., Supra). Alternatively, another method for the biological fluid. The two peptides are then brought into detection of protein interaction in vivo, the two-hybrid contact thereby immobilizing the binding partner on the System, may be used. Solid phase. The biological Sample is then contacted with the immobilized binding partner and the amount of analyte in Proteins of SEQID NO:318 (Internal Designation Clone ID: the Sample bound to the binding partner determined. In Still 124608) and Related Protein of SEQ ID NO:556. another preferred embodiment, the LZP or part thereof may 0572 The polynucleotides of SEQ ID NO:149 and SEQ be used to Synthesize novel nucleic acid binding proteins ID NO:442 and polypeptides of SEQ ID NO:318 and SEQ which are able to multimerize with proteins of interest, for ID NO:556 encode an RNA-binding protein, hgRBP, which example to inhibit and/or control cellular growth using any functions in RNA processing and protein expression. The genetic engineering technique known to those skilled in the preferred composition of SEQ ID NO:318 and 556 include MERPDKAALNALOPPEFRNESSLASTLK art including the ones described in the U.S. Pat. No. 5,942, TLLFFTALMITVPIGLYFTTKSYIFEGALGMSNR DSY 433, which disclosure is hereby incorporated by reference in FYAAIVAVVAVHVVLALFVYVAWNEG its entirety. SROWREGKOD. 0569. In another embodiment, the invention relates to 0573. Further preferred polypeptides include those of compositions and methods using the LZP or part thereof or SEQ ID NO:318 or SEQ ID NO:556 comprising an derivative thereof in protein fragment complementation N-myristoylation site or the amino acid Sequence at posi assays to detect biomolecular interactions in Vivo and in tions 43-48 or comprising the amino acid sequence GLYFTT vitro as described in international patent WO9834120, which targets the protein to the membrane of the endoplas which disclosures is hereby incorporated by reference in its mic reticulum for function of hgRBP in translation of entirety. Such assays may be used to Study the equilibrium cellular mRNA into protein. and kinetic aspects of molecular interactions including pro 0574. It will be appreciated that all characteristics and tein-protein, protein-nucleic acid, protein-carbohydrate and uses of the polynucleotides of SEQ ID NO: 149 and SEQ ID protein-Small molecule interactions, for Screening cDNA NO:442 and polypeptides of SEQ ID NO:318 and SEQ ID libraries for binding to a target protein with unknown NO:556, described throughout the present application also proteins or libraries of Small organic molecules for biologi pertain to the human cDNA 124608of clone , and the cal activity. polypeptides encoded thereby. 0570 Still, another object of the present invention relates Protein of SEQ ID NO:337 (Internal Designation Clone to the use of the LZP or part thereof for identifying new ID: 113448) leucine Zipper domains using any techniques for detecting protein-protein interaction known to those skilled in the art. 0575. The polynucleotides of SEQ ID NO:168 and Among the traditional methods which may be employed are related SEQ ID NO:454 and polypeptides of SEQ ID co-immunoprecipitation, crosslinking and co-purification NO:337 encode a novel human RNA-binding protein through gradients or chromatographic columns of cell involved in RNA processing and protein expression which is lysates. Once isolated as a protein interacting with the LZP, related to Clone ID:183902 and Clone ID:635993. Such an intracellular protein can be identified (e.g. its amino 0576. It will be appreciated that all characteristics and acid sequence determined) and can, in turn, be used, in uses of the The polynucleotides of SEQ ID NO:168 and conjunction with Standard techniques, to identify other pro related SEQ ID NO:454 and polypeptides of SEQ ID teins with which it interacts. The amino acid Sequence thus NO:337, described throughout the present application also obtained may be used as a guide for the generation of pertain to the human cDNA of clone 113448, and the oligonucleotide mixtures that can be used to Screen for gene polypeptides encoded thereby. Sequences encoding Such intracellular proteins. Screening may be accomplished, for example, by Standard hybridiza Protein of SEQID NO:328 (Internal Designation Clone ID: tion or PCR techniques. Techniques for the generation of 183902) oligonucleotide mixtures and the Screening are well-known. 0577 Polynucleotides of SEQ ID NO: 159 and related (See, e.g., Ausubel el al., eds., Current Protocols in Molecu SEQ ID NO:450 and polypeptides of SEQ ID NO:328 lar Biology, J.Wiley and Sons (New York, N.Y. 1993) and encode a novel human RNA-binding protein involved in PR Protocols: A Guide to Methods and Applications, 1990, RNA processing and protein expression which is related to Innis, M. et al., eds. Academic Press, Inc., New York). Clone ID: 113448 and Clone ID:635993. 0571 Alternatively, methods may be employed which 0578. It will be appreciated that all characteristics and result in the Simultaneous identification of genes which uses of the The polynucleotides of SEQ ID NO:159 and encode the intracellular proteins that can dimerize with the related SEQ ID NO:450 and polypeptides of SEQ ID LZP or part thereof using any technique known to those NO:328, described throughout the present application also skilled in the art. These methods include, for example, pertain to the human cDNA of clone 183902, and the probing cDNA expression libraries, in a manner Similar to polypeptides encoded thereby. the well known technique of antibody probing of lamb da.gt11 libraries, using as a probe a labeled version of the Protein of SEQ ID NO:329 (Internal Designation Clone LZP or part thereof, or fusion protein, e.g., the LZP or part ID:635993) thereof fused to a marker (e.g., an enzyme, fluor, lumines 0579 Polynucleotides of SEQ ID NO:160 and related cent protein, or dye), or an Ig-Fc domain (for technical SEQ ID NO:451 and polypeptides of SEQ ID NO:329 details on Screening of cDNA expression libraries, See encode a novel human RNA-binding protein involved in US 2006/0053498A1 Mar. 9, 2006 57

RNA processing and protein expression which is related to encode human RNA-associated polypeptides which act as Clone ID:183902 and Clone ID: 113448. It will be appreci Splicing factors. It will be appreciated that all characteristics ated that all characteristics and uses of the The polynucle and uses of the polynucleotides of SEQ ID NOS:79, 80, 81 otides of SEO ID NO:160 and related SEO ID NO:451 and and 401 and polypeptides of SEQID NOS:248,249,250 and polypeptides of SEQ ID NO:329, described throughout the 518, described throughout the present application also per present application also pertain to the human cDNA of clone tain to the human cDNA of clones 199782, 821212, and 635993, and the polypeptides encoded thereby. 202863, and the polypeptides encoded thereby. 0580. Many eukaryotic proteins that bind single-stranded RNA contain one or more copies of a putative RNA-binding 0584) The translation of genetic information into protein domain of about 90 amino acids. This is known as the depends on RNA and the first step in this process is the eukaryotic RNA-binding region, RNP-1 signature or RNA transcription of DNA into RNA while retaining all the recognition motif (RRM) (Bandziulis et al. Genes Dev. genetic information encoded in DNA. The RNA transcript 3:431 (1989); Swanson et al. Trends Biochem. Sci. 13: undergoes various processing Steps which include splicing 86-91 (1988)). RRMs are found in a variety of RNA binding and polyadenylation. The mature RNA transcript is trans proteins, including heterogeneous nuclear ribonucleopro lated into protein by the ribosomal machinery. Nascent RNA teins (hnRNPs), proteins implicated in regulation of alter transcripts are spliced in the nucleus by the Spliceosomal native splicing, and protein components of Small nuclear complex which catalyzes the removal of introns and the ribonucleoproteins (snRNPs). The polypeptides of SEQ ID rejoining of exons. At least 40 Splicing factors have been NO:337, 328, and 329 encode novel human RNA binding identified and interaction of these factors are important in protein, hereafter referred to as ghRBP which contains one the conformational changes needed for the enzymatic copy of an RRM. Further characteristic of a protein which removal of introns and religation of the exons. Both protein binds to nucleic acids, ghRBP contains a Zinc finger motif and RNA components are involved in the Spliceosome comprising the amino acid Sequence. Preferred polynucle otides of the invention include polynucleotides comprising assembly and the Splicing reaction. There are 2 distinct the nucleic acids of SEQ ID NO:159, 160, 168, 450,451,and catalytic Steps involved in the RNA splicing reaction with 454. Preferred polypeptides of the invention are polypep distinct proteins and RNA species. Alternative Splicing tides comprising the amino acids of SEQ ID NO:337,328 factors include developmentally regulated proteins that play and 329. It will be appreciated that all characteristics and key roles in developmental processes Such as pattern for uses of the polynucleotides of SEQ ID NO:159, 160, 168, mation and sex determination, respectively (Hodgkin, J. et 450, 451 and 454 described throughout the present appli al. (1994) Development 120:3681-3689). Alternate splicing cation also pertain to the human cDNA of Clone ID:183902, is also involved in the tissue specific expression of isoforms Clone ID:635993 and Clone ID: 113448. of proteins, including Structural proteins and enzymes. 0581 Preferred amino acids of the invention are residues 0585 An embodiment of the present invention relates to which comprise the RNA-binding domain or portion compositions of the polynucleotides of SEQ ID NO:79, 80, thereof. Preferred amino acid Sequences are Selected from 81 and 401 and polypeptides of SEQ ID NO:248, 249, 250 the following set of sequences including AFVRRXP and 518. Preferred amino acids of the invention comprise the WTAASSOLKEHFAOFGHVRRCILPFD Zinc finger region or fragment thereof and are Selected from KETGFHRGLGWVOFSSEEGLRNALOOE NHI the following sequences of amino acids from SEQ ID IDGVKVOV; NO:248, 249, 250 and 518 including GACENCGAMTH SINOPVAFVRRXPWTAASSOLKEHFAOF KKKDCFE; NSIITKYRKGACENCGAM; or THKKKD GHVRRCILPFDKETGFHRGLGWVOFSSEEGLRN CFERPRRVGAKF. ALOOENHIID; PWTAASSOLKEHFAOFGHVR RCILPFDKETGFHRGLGWVOFSSEEGLR 0586. The polypeptides of SEQID NO:248,249,250 and NALQQENHIIDGV KVOVHTRRP. Further preferred are 518 are involved in the SpliceSome complex and have polypeptides of the invention include any fragment of SEQ function in the processing of RNA processing. Alternatively, ID NO: which binds to RNA. the polypeptides of the present invention are involved in 0582 An embodiment of the invention relates to methods RNA processing and thus involved in protein expression. A of using the polypeptides of the invention to bind to RNA preferred embodiment of the invention relates to a method of molecules in Vitro by techniques that are known in the art. using the polynucleotides of polynucleotides of SEQ ID Preferred use of the polypeptides of the invention includes NO:79, 80, 81 and 401 in vitro. A preferred method of use extraction of RNA from biological Samples, chemical relates to introduction of Said polypeptides or fragments reagents, cell homogenates and tissue homogenates. Further thereof into cells by techniques known in the art Such as utility of the polypeptides of the present invention or part transfection or microinjection. Further preferred are meth ods to use the polynucleotides of the invention to alter thereof may be further confirmed by binding methods protein expression in the given cell. Alternately, polypep described in Trifillis, et al., RNA 5(8): 1071-82 (1999) and tides of the present invention can be used in combination U.S. Pat. No. 6,107,029, which disclosures are hereby with reagents known in the art to alter protein expression in incorporated by reference in their entireties. cell free expression Systems, mammalian expression Sys Proteins of SEQ ID NO:248 (Internal Designation Clone tems, insect expression Systems, or bacterial expression ID: 199782). SEQ ID NO:249 (Internal Designation Clone Systems. Furthermore, methods to use the polynucleotides or ID:82.1212). SEQ ID NO:250 (Internal Designation Clone polypeptides the present invention to increase or decrease ID:202863) and Related Protein of SEQ ID NO:518. protein expression is preferred. The utility of the polypep 0583. The polynucleotides of SEQID NOS:79,80, 81 and tides of the invention or part thereof may be further con 401 and polypeptides of SEQID NOS:248,249,250 and 518 firmed using methods described in U.S. Pat. No. 6,020,164 US 2006/0053498A1 Mar. 9, 2006 58 and Chua and Reed, Gene Devel 13:841-850 (1999), which plasmic latent forms of STAT proteins via tyrosine phos disclosures are hereby incorporated by reference in their phorylation on a specific tyrosine residue near the SH2 entireties. domain (Ihle et al., Trends Genet., 11: 69 (1995); Darnell, Science 277(5332):1630 (1997); Johnston et al., Nature, 0587. In another embodiment, methods to screen for 370: 1513 (1994)). Tyrosine phosphorylated STAT proteins inhibitors and activators of the polypeptides of the invention dimerize through Specific reciprocal SH2-phosphotyrosine are preferred. In another embodiment, molecules or com pounds which are identified in Such a Screen are further interactions and translocate from the cytoplasm to the preferred. Further preferred are compounds which activate nucleus where they Stimulate the transcription of Specific or inhibit activity of the polypeptides of the current inven target genes by binding to response elements in their pro tion. Activity of the the polypeptides of the invention is moters (Leonard, Nature Medicine, 2:968 (1996); Zhong et modified by phosphorylation at cAMP and coMP dependent al., PNAS USA,91:4806 (1994) Darnell, Science, 277:1630 phosphorylation sites (including 3-6:55-58; 107-110) and (1997)). casein kinase 11 phosphorylation sites (including 33-36:58 0591. In an embodiment of the present invention, com 61: 126-129). Preferred activators include but are not limited positions of the polynucleotides and polypeptides or frag to compounds which promote accumulation of intracellular ments thereof SEQ ID NO:87, 88 and 407 and SEQ ID cAMP and coMP. Further preferred activators include those NO:256, 257 and 521, respectively are included. Further compounds which activate casein kinase II. Preferred inhibi preferred are polypeptides of the present invention which tors are those compounds which inhibit intracellular cAMP interact with activated STAT3, but may also interact with and coMP accumulation, or those compounds which pro STAT1, STAT2 or other STAT homologues. Preferred mote cAMP and coMP degradation. Further preferred polypeptides of the invention act to inhibit or decrease the inhibitors include compounds which promote the deactiva activity of STATs. Further preferred amino acids of SEQ ID tion of casein kinase II. Furthermore, inhibitors and activa NO:256, 257 and 521 include the SAP domain VSS tors of the polypeptides of the present invention include FRVSELOVLLGFAGRNKSGRKHDLLMRALHLL. Acti compounds known in the art as well as compounds to be Vation of cytokine receptors by their cognate ligands activate identified by the method of Screening. Furthermore, com JAKs which in turn, activate STATS. Therefore cytokines pounds that inhibit or activate the activity of the polypep and other hormones which Signal through cytokine-like tides of the present invention by means other than phospho receptors may be modulated by polypeptides or polynucle rylation or dephosphorylation are also preferred. otides of the present invention. Cytokines and other hor 0588. In another embodiment, a method for the use of the mones which can thus be modulated by the present invention polynucleotides or polypeptides of the present invention in include but are not limited to interferons, interleukins, the treatment, prevention, attenuation or diagnosis of disor prolactin, and growth hormone. The utility of the polypep ders of RNA processing or protein processing are preferred. tides of the present invention or part thereof may be further Such disorders are Selected from a group which includes but confirmed using the methods described in WIPO Publication is not limited to cancerS Such as adenocarcinoma, leukemia, W09928465 which disclosure is hereby incorporated by Sarcoma, teratocarcinoma, and any disorder associated with reference in its entirety. cell growth and differentiation, embryogenesis, and mor 0592. The polypeptides of this invention can be used in a phogenesis involving any tissue, organ, or System, e.g., the method of inhibiting the activity of STAT proteins in a cell brain, adrenal gland, or reproductive System. in Vitro, the method comprising introducing a nucleic acid into the cell, wherein the nucleic acid comprises a nucleotide Proteins of SEQ ID NO:256 (Internal Designation Clone Sequence encoding the amino acid Sequence of SEQ ID ID:822794). SEQ ID NO:257 (Internal Designation Clone NO:256,257 and 521 or the amino acid sequence of SEQ ID ID:337572) and Related Protein of SEQ ID NO:521. NO:256, 257 and 521 with one or more conservative amino 0589 The polynucleotides of SEQ ID NO:87,88 and 407 acid alterations, and wherein the nucleic acid expresses the and polypeptides of SEQ ID NO:256, 257 and 521 encode amino acid Sequence in an amount and for a time Sufficient human nuclear polypeptides which interact with transcrip for the amino acid sequence to specifically bind to STAT tion factors of the Signal Transducers and Activators of proteins and to decrease STAT activity, thereby decreasing Transcription (STAT) family of proteins involved in the STAT activity in the cell. regulation of cell division. It will be appreciated that all characteristics and uses of the polynucleotides of SEQ ID 0593 Suitable compositions of polypeptides or poly NO:87, 88 and 407 and polypeptides of SEQ ID NO:256, nucleotides of the present invention are useful as a method 257 and 521, described throughout the present application of treatment of pathologies Such as diseases, Syndromes, or other undesirable conditions resulting from defects in cell also pertain to the human cDNA of clones 822794 and cycle progression. Such cell cycle defects may result from 337572, and the polypeptides encoded thereby. defects in the regulation of activated STAT or an upstream 0590 STATs are pleiotropic transcription factors which factor Such as activated JNKS or activated cytokine recep mediate cytokine-Stimulated gene expression in multiple tors. Alternatively, polypeptides or polynucleotides of the cell populations (Levy, Cytokine Growth Factor Rev., 8:81 present invention may be used in a method of treating (1997)). All STAT proteins contain a DNA binding domain, pathologies resulting from defects in cell cycle progression a Src homology 2 (SH2) domain, and a transactivation due to defects in a step “downstream” of STAT regulation of domain necessary for transcriptional activation of target cell cycle progression. In preferred embodiments, agonists gene expression. Janus kinases (JAK), including JAK1, of polypeptides or polynucleotides of the present invention JAK2, Tyk, and JAK3, are cytoplasmic protein tyrosine are useful in the treatment of pathologies Such as but not kinases (PTKs) which play pivotal roles in initiation of limited to hyperproliferative diseases Such as cancer (e.g., cytokine-triggered Signaling events by activating the cyto leukemia, lymphoma, breast cancer, colon cancer, prostate US 2006/0053498A1 Mar. 9, 2006 59 cancer, Wilms tumor), coronary artery disease, pulmonary proteins in vitro or in Vivo. Further preferred are polypep vascular obstructive disease, either primary or as a feature of tides of the invention which prevent or reverse ubiquitina Eisenmenger's Syndrome, and other disorders of abnormal tion of extracellular proteins in Vitro or in Vivo. cellular proliferation. Cells to be treated include but are not limited to hyperproliferative cells, cancer cells, Vascular 0599. The polynucleotides of SEQ ID NO:161 and SEQ ID NO:452 encode polypeptides of SEQ ID NO:330 which Smooth muscle cells, endothelial cells, and gametes. contain protein domains or motifs including but not limited 0594. In some embodiments of the invention, antagonists to a Protein kinase C phosphorylation site comprising the of the polypeptides or polynucleotides of the present inven amino acid fragment SAR, and an N-myristylation Site tion are used to Stimulate, promote, or facilitate progression comprising amino acid fragment GLNMSE. Further pre through the cell cycle, Such as in the cellular regeneration of ferred amino acids of SEQ ID NO:330 include the Ubiquitin terminally differentiated cardiac myocytes or tissues, e.g., carboxyl-terminal hydrolases family 2 Signature or amino striated muscle myocytes. For example, this could allow acid sequence YDLIAVSNHYGAMGVGHY. restoration of damaged myocardium after cardiac injury, myocardial infarction, myocarditis, cardiomyopathy, 0600. It will be appreciated that all characteristics and trauma, as a consequence of cardiac Surgery, etc., or reple uses of the polynucleotides of SEQ ID NO:161 and SEQ ID tion of Striated muscle exhausted by muscular dystrophy. NO:452 and polypeptides of SEQ ID NO:330, described 0595. In further embodiments, expression of the polypep throughout the present application also pertain to the human tides encoded by the nucleic acids is expected to prevent, cDNA of clone 398.703, and the polypeptides encoded ameliorate, or lessen the cell cycle defect of the host cell, or thereby. to restore normal cell cycle progression of the host cell. 0601 The utility of the polynucleotides and polypeptides Whether provided via nucleic acid or polypeptides delivered of the present invention or part thereof may be further directly to cells, the therapeutic formulations of the inven confirmed using methods which assess activity or function tion can also be used as adjuncts to other forms of therapy, of deubiquitinating enzymes described in United States including but not limited to chemotherapy, and radiation Patents 5391490 and 5565352 which disclosures are hereby therapy. incorportated by reference in their entireties. Protein of SEQ ID NO:330 (Internal Designation Clone ID:398703) and Related Protein of SEQ ID NO:330. Proteins of SEQ ID NO:277 (Internal Designation Clone 0596) The polynucleotides of SEQ ID NO:161 and SEQ ID:653966) and Related Protein of SEQ ID NO:535. ID NO:452 and polypeptides of SEQ ID NO:330 encode a 0602) The polynucleotides of SEQ ID NO:108 and SEQ novel human deubiquitinating enzyme (GNP:AF017306). ID NO:421 and polypeptides of SEQ ID NO:277 and SEQ Deubiquitinating enzymes Serve a number of functions ID NO:535 encode human liver fatty acid binding protein (Hochstrasser Cur Opin Cell Biol 4:1024 (1992); Rose, In: (L-FABP) comprising the amino acid sequence of SEQ ID Ubiguitin, Plenum Press, New York (1988)). First, ubiquitin NO:277 and 535. The amino acid sequence of SEQ ID must be cleaved from a Set of biosyntheticprecursors, which NO:277 and 535 are the same as human L-FABP (Genbank occur either as a Series of ubiquitin monomers in head-to-tail accession GNP:M10617: Lowe et al., JBC 260:3413-17 linkage or as fusions to certain ribosomal proteins (Finley & (1985)) and homologous to human FABP (Genbank acces Chau, Annu Rev Cell Biol 7, 25-69 (1991)). Secondly, sion GNP:M10050). The polypeptides of the present inven ubiquitin must be recycled from intracellular conjugates, tion belong to the FABP/P2/CRBP/CRABP family of trans both to maintain adequate pools of free ubiquitin and, in porters and functionally binds to free fatty acids and principle at least, to reverse the modification of inappropri derivatives thereof. L-FABP is normally expressed in the ately targeted proteins. Finally, deubiquitinating reactions cytoplasm of hepatocytes, but preferred embodiments may be integral to the degradation of ubiquitinated proteins include use of the polypeptides of the present invention as by the 26S proteasome, a complex ATP-dependent enzyme extracellular polypeptides. Further preferred embodiments whose exact composition and range of activities remain include use of the polypeptides of the present invention as poorly characterized (Hershko & Ciechanover, Annu Rev Serum or plasma polypeptides. Further preferred embodi Biochem 61, 761-807 (1992); Hadari et al., J Biol Chem ments use polypeptides of the invention in vitro. Still further 267, 719-727 (1992); Murakami et al., Nature 360, 597-9 preferred embodiments include use of the polypeptides of (1992); Rechsteiner, J. Biol. Chem. 268, 6065-6068 (1993)). the present invention in Vivo. 0597 An embodiment of the invention includes preferred 0603 Preferred amino acids of the invention include the polypeptides with ubiquitin-Specific protease activity with a lipocalin domain, from 2 to 127 or polypeptides comprising novel N-terminus of Clone ID:3987.03 comprising the amino the amino acid sequence SFSGKYOLOSQENFEAFM acid sequence MCTTSLPCPIIMEPWGLATTKAAYVL KAIGLPEELIOKGKDIKGVSEIVONGKH FYORRDDEFYKTPSLSSSGSSDGGTRPSSSOOGFGD FKFTITAGSKVIONEFT VGEECELETMTGEKVKTV DEACSMDTN encoded by ATGTGTACGACCTCATTGC VOLEGDNKLVTTFKNIKSVTELNGDIITNTMTLGDIV CGTGTCCAATCATTATGGAGC FKRISKR I. The polypeptides of SEQ ID NO:277 and SEQ CATGGGGGTTGGCCACTAC TAAAGCAGCTTATGT ID NO:535 contain a cytosolic fatty-acid binding protein GCTATTTTACCAACGTCGAGATGATGAATTTTATAA Signature comprising the amino acid Sequence GACACCTT CACTTAGCAGTTCTGGTTCCTCTGATG GKYOLOSOENFEAFMKAI which functions in the GAGGGACACGACCAAGCAGCTCTCAGCAGGG polypeptides ability to bind Small hydrophobic molecules, CTMTGGGGATGATGAGGCTTGCAGCATG Such as lipids, Steroid hormones, and retinoids. Preferred GACACCAACTAA of SEO ID NO: 161. amino acids of SEO ID NO:277 and SEO ID NO:535 0598. The preferred polypeptides of the invention are include GKYOLOSOENFEAFMKAI, MSFS those which prevent or reverse ubiquitination of cellular GKYOLOSOENFEAF, and LOSOENFEAFMKAIGLPE. US 2006/0053498A1 Mar. 9, 2006 60

0604 Phosphorylation status modulates the activity of tion, treatment or attenuation of conditions in which lipo L-FABP. Preferred polypeptides of the invention include the philic compounds are elevated in the Serum of mammals, amino acids Sequence comprising the Sites of cAMP- and preferably humans. Such conditions are Selected from a cGMP-dependent protein kinase phosphorylation including group which include but are not limited to obesity, hyper residues of SEQ ID NO:277 and 535 comprising the lipidemia, hypercholesterolemia, hypertriglyceridemia, dia sequence KRIS. betes type I (IDDM) diabetes type II (NIDDM), atheroscle 0605 Further preferred polypeptides of the invention rosis, and hypertension. include the amino acid Sequence comprising the Sites of 0611 Mammary-derived growth inhibitor (MDGI) and Protein kinase C phosphorylation including residues at posi heart-fatty acid binding protein (FABP), which belong to the tions 4 to 6,94 to 96, and 124 to 126 of SEQ ID NO:277 and FABP family, specifically inhibit growth of normal mouse 535. Still further preferred polypeptides of SEQ ID NO:277 mammary epithelial cells (MEC) and promote morphologi and 535 include the amino acid Sequence comprising SGK, cal differentiation, Stimulates its own expression and pro TFK, and SKR. motes milk protein synthesis (U.S. Pat. No. 5,977.309, 24 Mar. 1995). In further preferred embodiments, polypeptides 0606 Further preferred are polypeptides of SEQ ID of the invention include those which locally signal growth NO:277 and 535 include the amino acid sequence compris cessation and Stimulate differentiation of the developing ing a Casein kinase II phosphorylation sites. Preferred epithelium. Further preferred polypeptides of the invention amino acids of SEQ ID NO:277 and 535 include positions 64 to 67,100 to 103, and 114 to 117. Further preferred amino SuppreSS the mitogenic effects of EGF family members, and acids comprise the sequences TVGE, SVTE, and TLGD. inhibit c-fos, c-myc and c-ras expression. 0607. A preferred polypeptide of SEQ ID NO:277 and 0.612) In a further aspect of the present invention, there is 535 is one in which the amino acid asparagine (ASn) is provided a method for producing Such polypeptide by located at residue 105, further referred to as the N-isoform. recombinant techniques comprising culturing recombinant Further preferred is the polypeptide of SEQ ID NO:277 and prokaryotic and/or eukaryotic host cells, containing a human 535 in which the amino acid aspartate (Asp) is located at fatty acid binding polypeptides or polynucleotides of the residue 105 further referred to as the D-isoform. The rat invention acid under conditions promoting expression of homologue of the human D-isoform of the present invention Said protein and Subsequent recovery of Said protein. was shown to have a greater affinity to lysophospholipids, 0613. In a further embodiment of the present invention, prostaglandins, retinoids, bilirubin and bile Salts compared there is provided a method for utilizing Such polypeptides, to the rathomologue of the human N-isoform of the present or polynucleotides of the invention for therapeutic purposes, invention by methods described by DiPietro and Santome, for example, as a cell growth inhibitor and as to cause Biochim Biophys Acta 1478:186-200 (2000) which disclo differentiation Stimulatory activity on various responsive Sure is hereby incorporated by reference in its entirety. The types of tissues and cells in vitro. Further preferred are rat homologues share only 82% identity with the of the methods for use of polypeptides, or polynucleotides of the human D- and N-isoforns, therefore it is not predictable to invention, in appropriate physiological form, for therapeutic find that the human D-isoform has equal or greater affinity purposes for to inhibit cell proliferation or to induce cell to lysophospholipids, prostaglandins, retinoids, bilirubin, differentiation in mammals, preferably humans. bile salts and fatty acid compared to the human N-isoform. 0.614. It will be appreciated that all characteristics and 0608 Further preferred polypeptides of the present uses of the polynucleotides of SEQ ID NO:108 and SEQ ID invention include the D-isoform polypeptide and fragments NO:421 and polypeptides of SEQ ID NO:277 and SEQ ID thereof which have an equal or at least 10%, 20%, 30%, NO:535, described throughout the present application also 40%, 50%, 60% or 75% greater affinity for fatty acids, and pertain to the human cDNA of clone 653966, and the lipophilic compounds Selected from a group including but polypeptides encoded thereby. not limited to lysophospholipids, prostaglandins, retinoids, bilirubin, bile Salts, Steroid hormones (Such as testosterone Proteins of SEQ ID NO:313 (Internal Designation Clone and estradiol), and cholesterol compared to the N-isoform. ID:633418). SEQ ID NO:314 (Internal Designation Clone ID:422878) and Related Protein of SEQ ID NO:552. 0609 Another embodiment of the invention includes polynucleotides or polypeptides of the invention or frag 0615. The polynucleotides of SEQ ID NO:144, 145 and ments thereof which bind lipophilic compounds Selected 438 and polypeptides of SEQ ID NO:313, 314and 552 from a group including but not limited to free fatty acids, encode a cleavage Stimulation factor important in mRNA ly Sophospholipids, proStaglandins, retinoids, bilirubin, bile processing and protein expression. Protein kinase C phos Salts, Steroid hormones (such as testosterone and estradiol), phorylation increases activity of Said polypeptides and pre and cholesterol in Serum or plasma. Further preferred are ferred amino acids include SEK and SGR. Further, sites of polypeptides of the invention which bind lipophilic com tyrosine kinase phosphorylation increase activity of Said pounds in Serum or plasma separated from whole blood in a polypeptides and preferred amino acids of SEQ ID NO:314, process of purifying Serum or plasma for use in Vitro or in 552 include KKLEENPY. vivo. Further preferred are polypeptides of the invention which bind lipophilic compounds in Serum or plasma in 0616) It will be appreciated that all characteristics and uses of the polynucleotides of SEQ ID NO:144, 145 and 438 WVO. and polypeptides of SEQID NO:313,314 and 552, described 0610. In a further embodiment, polynucleotides or throughout the present application also pertain to the human polypeptides of the invention or fragments thereof, in physi cDNA of clones 633418 and 422878, and the polypeptides ological appropriate formulations, are useful in the preven encoded thereby. US 2006/0053498A1 Mar. 9, 2006

0617) Proteins of SEQ ID NO:219 (Internal Designation be analyzed for relative expression analyses, etc. In addition, Clone ID:589848), SEQ ID NO:220 (Internal Designation such methods may be used to specifically remove RNA from Clone ID:211883). SEQ ID NO:221 (Internal Designation a Sample, for example during the purification of DNA. To Clone ID:642603). SEQ ID NO:222 (Internal Designation carry out any of these methods, the proteins of the invention Clone ID: 193316), and Related Protein of SEQID NO:497. or part thereof may be bound to a chromatographic Support, either alone or in combination with other RNA binding 0618) Polynucleotides of SEQ ID NO:50, 51, 52,53,380 proteins, to form an affinity chromatography column. A and polypeptides of SEQ ID NO:219, 220, 221 and 497 Sample containing a mixture of nucleic acids to purify is then encode RNA associated proteins with a ribosomal L34 run through the column. Immobilizing the proteins of the domain comprising the amino acid sequence NEYQPSNI invention or part thereof on a Support is particularly advan KRKNKHGWVRRLXTPAGXXXILRRMLKGRKSLSH tageous for embodiments in which the method is to be O NEYOPSNIKRKNKHGWVRRLX practiced on a commercial Scale. This immobilization facili TPAGVOVILRRMLKGRKSLSH. It will be appreciated tates the removal of RNAS from the batch of resin-coupled that all characteristics and uses of the polynucleotides of protein after binding, and allows Subsequent re-use of the SEQ ID NOS:50, 51, 52,53,380 and polypeptides of SEQ ID protein. Immobilization of the proteins of the invention or NOS:219, 220, 221 and 497, described throughout the part thereof can be accomplished, for example, by inserting present application also pertain to the human cDNA of any matrix binding domain in the protein according to clones 589848, 211883, 642603 and 1933 16, and the methods known to those skilled in the art. The resulting polypeptides encoded thereby. fusion product including the proteins of the invention or part Proteins of SEQ ID NO:302 (Internal Designation Clone thereof is then covalently, or by any other means, bound to ID:1000891255) and Related Protein of SEQ ID NO:543. a protein, carbohydrate or matrix (Such as gold, "Sephadex' 0619 Polynucleotides of SEQ ID NO:133 and 429 and particles, polymeric Surfaces). polypeptides of SEQ ID NO:302 and 543 encode human 0622 Another embodiment of the present invention ribosomal protein, hRIBPRT. An embodiment of the inven relates to methods and compositions using the proteins of tion includes the compositions of the polypeptides of SEQ the invention, or part thereof, to associate Specific mRNAS ID NO:302 and 543, comprising the amino acid sequence to the inner face of lipidic bilayers of liposomes in order to MVAAKKTKKSLESIKSRLOLVMKSGKYV further introduce these mRNAS into the cytoplasm of LGYKOTLKMIROGKAKLVILANNCPALRKSEI eukaryotic cells. Preferably, specific mRNAS are first asso EYYAMLAKTGVHHYSGNNIELG ciated with the protein of the invention and the RNA/protein TACGKYYRVCTLAIIDPXDSXIIRSMPEOTGEK, and complex formed in that way is then mixed with liposomes the polynucleotides of SEQ ID NO:133 and 429, respec according to methods known to those skilled in the art. tively, which encode human ribosomal protein, hRIBPRT. These lipoSomes are added to an in vitro culture of eukary The polypeptides of the invention contain the ribosomal otic cells. In Vivo, Such a method might treat and/or prevent protein L30e/L7Ae/S12e/Gadd4 signature (Koonin E V, J disorders linked to dysregulation of gene transcription Such Mol Med 75:236-238 (1997) and Nakanishi et al., Gene as cancer and other disorders relating to abnormal cellular 35:289-96 (1985)). differentiation, proliferation, or degeneration. 0620 Preferred polypeptides of the invention include the 0623) A decrease in ribosome function results in a sig amino acid sequence comprising KSLESIKSRLQLVMKS nificant inhibition of cell growth. Therefore, in another GKYVLGYKOTLKMIROGKAKLVILANNC embodiment, the present proteins and nucleic acids can be PALRKSEIEYYAMLAK TGVHHYSGNNIELG used to modulate the rate of cell growth in vitro or in vivo. TACGKYYRVCTLAIIDPXDSXIIR; Accordingly, compounds that inhibits the expression or KSLESIKSRLOLVMKSGKYVLGYKOTLK function of the proteins of the invention can be used to MIROGKAKLVILANNCPALRK; and SEIEYYAMLAKT inhibit the growth rate of cells, and can thus be used, e.g. in GVHHYSGNNIELGTACGKYYRVCTLAIDPXDSXIIR. the treatment or prevention of diseases or conditions asso Further preferred amino acids of the invention include sites ciated with excessive cell growth, Such as cancer or inflam of PKC phosphorylation, comprising the amino acid matory conditions. Such compounds include, but are not sequences of SEQ ID NO:302 and 543 including TKK limited to, antibodies, antisense molecules, dominant nega (positions 7-13); SIK (positions 13-15); SGK (positions tive forms of the proteins, and any heterologous compounds 2426); and TLK (positions 34-36). Further preferred amino that inhibit the expression or the activity of the proteins. acids of the invention include sites of Casein Kinase II 0624. It will be appreciated that all characteristics and phosphorylation, comprising the amino acid Sequences SEIE uses of the polynucleotides of SEQ ID NO:133 and 429 and (positions 58-61) and SMPE (positions 107-110). polypeptides of SEQ ID NO:302 and 543, described 0621. In another embodiment, the proteins of SEQ ID throughout the present application also pertain to the human NO:302 and 543 can be used to bind to nucleic acids, cDNA of clone 1000891255, and the polypeptides encoded preferably RNA, alone or in combination with other sub thereby. stances. For example, the proteins of the invention or part thereof can be added to a Sample containing RNAS in Proteins of SEQ ID NO:271 (Internal Designation Clone optimum conditions for binding, and allowed to bind to ID:493328), Related Clones 153261, 152042, 599054, and RNAS. In a preferred such embodiment, the proteins of the 650872 and Related Protein of SEO ID NO:533. invention or part thereof may be used to purify mRNAS, for 0625) The polynucleotides of SEQID Nos: 102, 103,104, example to Specifically isolate RNA, e.g. from a specific cell 105, and 106 and polypeptides of SEQ ID Nos:271, 272, type or from cells grown under particular conditions. Such 273, 274, 275 and 533 encode for the HUMAN GENSET RNAS could then be reverse transcribed and cloned, could BINDING PROTEIN or HGBP-1. Preferred polypeptides US 2006/0053498A1 Mar. 9, 2006 62 comprise the amino acid sequence MKVKIKCWNG 0631 Alternatively, the protein of the invention or part VATWLWVANDENCGICRMAFNGCCPDCK thereof may be bound to a chromatographic Support, either VPGDDCPLVWGOCSHCFHM HCILKWLHAOOVOOH alone or in combination with other DNA binding proteins, CPMCROEWKFKE. It will be appreciated that all using techniques well known in the art, to form an affinity characteristics and uses of the polynucleotides of SEQ ID chromatography column. A Sample containing nucleic acids Nos: 102, 103, 104, 105, and 106 and polypeptides of SEQ to purify is run through the column. Immobilizing the ID Nos:271, 272,273,274,275 and 533, described through protein of the invention or part thereof on a Support advan out the present application also pertain to the human cDNA tageous is particularly for those embodiments in which the of clones 493328, 153261, 152042, 599054, and 650872, method is to be practiced on a commercial Scale. This and the polypeptides encoded thereby. immobilization facilitates the removal of the protein from 0626. The protein of SEQ ID NO: 271 encoded by the the batch of product and Subsequent reuse of the protein. extended cDNA SEQ ID NO:102 is the same as a hepato Immobilization of the protein of the invention or part thereof cellular carcinoma associated ring finger protein (EMBL can be accomplished, for example, by inserting a cellulose AF247565) and Genset protein in WO0100806 (Genpep binding domain in the protein. One of skill in the art will accession AXO61622) with homology to an anaphase-pro understand that other methods of immobilization could also moting complex (APC) subunit from Drosophila (Embi be used and are described in the available literature. accession number AJ251510). In addition, HGBP-1 exhibits 0632. In another embodiment, the present invention the pfam PHD Zinc finger signature from positions 33 to 79. relates to compositions and methods using the protein of the 0627 Zinc binding domains which contain a CHC invention or part thereof, especially the Zinc binding Sequence motif are known as RING domains (Lovering, R. domain, to alter the expression of genes of interest in a target et al. (1993) Proc. Natl. Acad. Sci. USA90:2112-2116). Zinc cells. Such genes of interest may be disease related genes, finger domains are found in numerous Zinc binding proteins Such as oncogenes or exogenous genes from pathogens, Such which are involved in protein-protein and protein-nucleic as bacteria or viruses using any techniques known to those acid interactions. They are independently folded zinc-con skilled in the art including those described in U.S. Pat. Nos. taining mini-domains which are used in a modular repeating 5,861,495; 5,866,325 and 6,013,453. fashion to achieve Sequence-specific recognition of DNA 0633. In a further embodiment, the protein of the inven (Klug 1993 Gene 135, 83-92). Such zinc binding proteins tion or part thereof may be used to diagnose, treat and/or are commonly involved in the regulation of gene expression, prevent disorders linked to dysregulation of gene transcrip and usually Serve as transcription factors, either by directly tion Such as cancer and other disorders relating to abnormal affecting transcription or recruiting co-activators or co cellular differentiation, proliferation, or degeneration, repressors (see U.S. Pat. Nos. 5,866,325; 6,013,453 and including hyperaldosteronism, hypocortisolism (Addison's 5,861,495). PHD fingers are CHC zinc fingers spanning disease), hyperthyroidism (Grave's disease), hypothyroid approximately 50-80 residues and distinct from RING fin ism, colorectal polyps, gastritis, gastric and duodenal ulcers, gers or LIM domains. They are thought to be mostly DNA ulcerative colitis, and Crohn's disease. The invention relates or RNA binding domain but may also be involved in to methods of diagnosing, treating and/or preventing disor protein-protein interactions (for a review See Aasland et al., derS described herein, comprising delivering to a patient, or Trends Biochem Sci 20:56-59 (1995)). causing to be present therein, a Zinc finger polypeptide 0628 HGBP-1 or part thereof is a zinc binding protein, which inhibits the expression of a gene enabling the cells to which is able to bind nucleic acids, more preferably a divide. The target could be, for example an oncogene or a transcription factor. Preferred polypeptides of the invention normal gene, which is overexpressed in the cancer cells. are polypeptides comprising the amino acids of SEQID NO: ISPG Iron-Sulfur Cluster Protein (Clone ID:1000872335) 271 from positions 33 to 79. Other preferred polypeptides of the invention are fragments of SEQ ID NO: 271 having any 0634) The polynucleotides of SEQ ID NOs:43 and 374 of the biological activity described herein. The nucleic acid encodes the amino acids sequence of SEQ ID NOS:212 and binding activity of the protein of the invention or part thereof 491 respectively, an iron-Sulfur protein which mediates may be assayed using any of the assays known to those electron transfer in metabolic reactions, also referred to as ISPG. It will be appreciated that all characteristics and uses skilled in the art including those described in U.S. Pat. No. of the polynucleotides of SEQ ID NOS 43 and 374 and 6,013,453. polypeptides of SEQ ID NOS:212 and 491, described 0629. The invention relates to methods and compositions throughout the present application also pertain to the human using the protein of the invention or part thereof to bind to cDNA of clone 10008.72335, and the polypeptides encoded nucleic acids, preferably DNA, alone or in combination with thereby. other Substances. For example, the protein of the invention 0635 ISPG is an iron-sulfur protein that belongs to the or part thereof is added to a Sample containing nucleic acid broad family of the 2Fe-2S-type ferredoxins. The 2Fe-2S in conditions allowing binding, and allowed to bind to type ferredoxins are proteins or domains of around one nucleic acids. In a preferred embodiment, the protein of the hundred amino acid residues that bind a single 2Fe-2S invention or part thereof may be used to purify nucleic acids iron-Sulfur cluster and are found in plants, animals and Such as restriction fragments. bacteria. Iron-Sulfur cluster proteins are well known classes 0630. In another preferred embodiment, HGBP polypep of proteins and are recognized as ideal devices for accepting, tides or parts thereof may be used to visualize nucleic acids donating, Storing and shifting electrons. It will be appreci when the polypeptide is linked to an appropriate fusion ated by the skilled artisan that Stuctural aspect of iron-Sulfur partner, or is detected by probing with an antibody. Thus, proteins have been studied extensively (reviewed in Beinert HGBP polypeptides can be used to diagnose. et al, Nature 1 Aug. 1997, 277:653-659), allowing modifi US 2006/0053498A1 Mar. 9, 2006 cations of the ISPG proteins to fine tune its properties for 0640 Additionally, structural aspects of ISPG suggest desired uses while retaining its biological function in medi that it may be capable of mediating Steroid hormone Syn ating electron transfer and possible protein Stabilization and thesis, either in human or animals, or in engineered cell iron or sulfide storage functions. The ISPG protein of SEQ culture Systems for the large Scale production of hormones. ID NO 491 comprisese a glycosaminoglycan attachment Site ISPG may be used to act as an adrenal ferredoxin (known as at amino acid positios 34 (SGSG); protein kinase C phos adrenodoxin (ADX)), a vertebrate mitochondrial protein phorylation sites at amino acid positions 14 (SAR), 44 which transferS electrons from adrenodoxin reductase to (TTR), and 86 (SGR); N-myristoylation sites at amino acid cytochrome P450s.cc, which is involved in cholesterol side positions 11 (GGVSAR), 24 (GTXWNR), 31 (GGTSGS), chain cleavage. Its primary function as a Soluble electron 39 (GVALGT), 106 (GACEAS), a cytochrome c family carrier between the NADPH-dependent adrenodoxin reduc heme-binding Site at amino acid positions 114-119 and an tase and several cytochromes P450 makes it an irreplaceable iron-Sulfur binding region signature at amino acid positions component of the Steroid hormones biosynthesis in the 108-118. adrenal mitochondria of vertebrates. 0636. In view of its role in electron transfer reactions, ISPG is thought to be involved in a wide variety of meta Drug Metabolism bolic reactions and disorders, and may be useful in the 0641 Previous studies have revealed that cytochrome treatment of disorders of metabolism Such as obesity, in the P-450 isozymes are responsible for drug metabolism, and detection of toxic compounds, in prediction, diagnosis or oxidation by P-450 isozymes is a common aspect of the treatment of conditions or traits related to drug metabolism overall clearance of drugs. Further Studies have revealed that or in treatments related to the Synthesis of eg. Steroid genetic polymorphism of cytochrome P-450 isozymes hormones. underlies a wide Spectrum of Substrates Specificity in drug 0637. In a preferred example, overexpression or admin oxidation. In certain cases, genetic mutation and/or deletion istration of the ISPG protein may be used as a therapeutic of one critical isozyme gene results in a significant alteration treatment for obesity by accelerating the metabolic rate of a of a phenotype projected on Substrate specificity. It has been Subject in need of treatment. There is accumulating evidence reported that CYP2D6 oxidizes more than 30 drugs (for to Support the hypothesis that a low-energy-output pheno example, M. Eichelbaum et al., Pharmacol. Ther., Vol. 46, type is at high risk of weight gain and obesity, irrespective pp. 377-, 1990). Many anti-cancer drugs are known to be of whether this is owing to a low resting metabolic rate oxygenated by cytochrome P450 enzymes to yield metabo and/or physical inactivity. The low-energy-output phenotype lites that are cytotoxic or cytostatic toward tumor cells. is associated with impaired appetite control, which is These include Several commonly used cancer chemothera improved if energy output is increased, Serving as the peutic drugs, Such as cyclophosphamide (CPA), its isomer background for pharmacologic Stimulation of energy expen ifosfamide (IFA), dacarbazine, procarbazine, thio-TEPA, diture as a tool to improve the results of obesity manage etoposide, 2-aminoanthracene, 4-ipomeanol, and tamoxifen ment. The ISPG protein and agonists or stimulators thereof (LeBlanc, G. A. and Waxman, D. J., Drug Metab. Rev. may serve as a means to increase electron transfer and hence 20:395-439 (1989); Ng, S. F. and Waxman D. J., Intl. J. the metabolic rate of an individual in a similar goal as Oncology 2:731-738 (1993); Goeptar, A. R., et al., Cancer commonly cited targets Such as leptin receptors, the Sym Res. 54:2411-2418 (1994); van Maanen, J. M., et al., Cancer pathetic nervous System and its peripheral beta-adrenocep Res. 47:4658-4662 (1987); Dehal, S. S., et al., Cancer Res. tors, Selective thyroid hormone derivatives, and Stimulation 57:3402-3406 (1997); Rainov, N. G., et al., Human Gene of the mitochondrial uncoupling proteins. Therapy 9:1261-1273 (1998)). Bioreductive metabolism 0638. In addition, iron-sulfur proteins such as ISPG are that results in drug activation is also catalyzed by cyto generally recognized as being capable of Several functions chrome P450 enzymes for a variety of anti-cancer drugs. that are not of an oxidoreductive nature Such as the binding Examples of Such drugs include Adriamycin, mitomycin C, and activation of Substrates at the unique iron site (in the and tetramethylbenzoquinone (Goeptar, A. R., et al., Crit. catalytic function of aconitase and relates enzymes), and Rev. Toxicol. 25:25-65 (1995); Goeptar, A. R., et al., Mol. apparently Stabilizing radicals in reactions occurring by a Pharmacol. 44:1267-1277 (1993)). Those who have free-radical pathway. There is also evidence Suggesting that homozygous alteration in this recessive gene, are So-called Such iron-Sulfur clusters can function in coupling electron “poor metabolizers (PMs)” and may suffer from severe side transfer to proton transport. By binding CyS ligands from effects due to poor metabolism of drugs (for example, see M. different Subunits, iron-Sulfur clusters effect dimer forma Eichelbaum et al., Pharmacol. Ther., Vol. 46, pp. 377-, tion, as in the Fe protein of nitrogenase. Further, by Strad 1990). Such genetic alterations occur at rates of from I to dling protein Structural elements, iron-Sulfur clusters are 30% in different ethnic populations (for example, L. M. able to Stabilize Structures that are required for Specific Distlerath et al., J. Biol. Chem., Vol. 260, pp. 9057-1985). functions (eg. endonuclease III of E. coli). Proteins of 0642. The ISPG protein of the invention is thought to be ISPG's class have also been shown to protect proteins from capable of functioning as a Soluble electron carrier in the the attack of intracellular proteases. Finally, proteins of electron transport chain involving one or more of the Several ISPG's class are thought to be capable of Serving as Storage available cytochromes P450 enzymes. The ISPG protein devices for iron and possibly sulfide. may thus be useful in methods of killing neoplastic cells 0639 Thus, in a few examples, ISPG may be used involving P450 (and ISPG) gene transfer and the use of advantageously as an iron or metal biosensor, for the treat bioreductive drugs that are activated by cytochrome P450, ment and/or diagnosis of iron Overload disorders, or in and in methods for evaluating the Susceptibility of a Sample applications involving Stabilizing target proteins Such as for compound to metabolism with respect to a specific cyto protein production or for mediating protein interactions. chrome P450 isozyme system. US 2006/0053498A1 Mar. 9, 2006 64

0643. Thus, in a first aspect, a drug activation/gene invention comprises a method for killing neoplastic cells therapy Strategy has been developed based on a cytochrome comprising: (a) infecting the neoplastic cells with a vector P450 gene (“CYP” or “P450”) in combination with a cancer for gene delivery, the vector comprising an ISPG gene chemotherapeutic agent that is activated through a P450 capable of mediating enzymatic conversion of a chemo catalyzed monoxygenase reaction (Chen, L. and Waxman, therapeutic agent by a P450 enzyme; (b) optionally infecting D. J., Cancer Research 55:581-589 (1995); Wei, M. X., et the neoplastic cells with a vector for gene delivery, the al., Hum. Gene Ther. 5:969-978 (1994); U.S. Pat. No. vector comprising a cytochrome P450 gene and/or a gene 5,688,773, issued Nov. 18, 1997). Presently known drug encoding RED; (b) treating the neoplastic cells with a enzyme combinations can utilize established chemothera chemotherapeutic agent that is activated by the product of peutic drugs widely used in cancer therapy. Such methods to the cytochrome P450 gene; and (c) killing the neoplastic obtain enhanced chemoSensitivity have been demonstrated cells. both in Vitro and in Studies using a Subcutaneous rodent Solid 0646 The present invention also provides a reagent com tumor model and human breast tumor grown in nude mice position for use in evaluating drug metabolism by a specific in Vivo, and is Strikingly effective in Spite of the presence of cytochrome P450 isozyme, which comprises a liver a Substantial liver-associated capacity for drug activation in microsome lacking said specific P450, said specific P450 these animals (Chen, L., et al., Cancer Res. 55:581-589 isozyme and a carrier material. The liver microSome may be (1995); Chen, L., et al., Cancer Res. 56:1331-1340 (1996)). of human source lacking CYP2D6, CYP2C19, or CYP2A6. The P450-based approach also shows significant utility for The CYP2D6 isozyme, CYP2C19 isozyme, and CYP2A6 gene therapy applications in the treatment of brain tumors isozyme to be added may be a recombinant CYP2D6 (Wei, M. X., et al., Human Gene Ther. 5:969-978 (1994); expressing microSome, a recombinant CYP2C 19-express Manome, Y., et al., Gene Therapy 3:513-520 (1996); Chase, ing microSome, or a recombinant CYP2A6-expressing M., et al., Nature Biotechnol. 16:444-448 (1998)). microSome. The reagent composition may comprise more 0644 Although the P450/drug activation system has than one kind of PM microSomes. shown great promise against Several tumor types, further enhancement of the activity of this System is needed to 0647 According to the present invention, there can be achieve clinically effective, durable responses in cancer provided a reagent composition and a method for accurately patients. This requirement is necessitated by two character quantitating the contribution of certain P450 isozymes Such istics that are inherent to the P450 enzyme system: (1) P450 as CYP2D6, CYP2C19, and CYP2A6 in drug metabolism. enzymes metabolize drugs and other foreign chemicals, The present invention provides a method for evaluating the including cancer chemotherapeutic drugs, at low rates, with Susceptibility of a Sample compound to metabolism with a typical P450 turnover number (moles of metabolite respect to a Specific cytochrome P-450 isozyme, which formed/mole P450 enzyme) of only 10-30 per minute; and comprises contacting the Sample compound with a reagent (2) P450 enzymes metabolize many chemotherapeutic drugs composition prepared by adding Said specific cytochrome with high Km values, typically in the millimolar range. This P-450 isozyme and an ISPG protein to liver microsomes compares to plasma drug concentrations that are only in the lacking Said specific cytochrome P-450 isozyme in a carrier micromolar range for many chemotherapeutic drugs, includ material. ISPG would be useful in order to enhance effi ing drugs Such as CPA and IFA. Thus, current approaches to ciency of the P-450 isozyme in drug metabolism, thereby P450 gene therapy may result in intratumoral drug activation effectively amplifying the power of the assay to detect the at a low absolute rate and under conditions that are not contribution of a particular P-450 enzyme. In another Saturating with respect to drug Substrate. Furthermore, Since embodiment the contribution to drug metabolism of a par P450 is expressed at a very high level in liver tissue, only a ticular P-450 System can be assessed by focussing on a very Small fraction of the administered chemotherapeutic particular iron-Sulfur protein associated with Said specific drug is metabolized via the tumor cell P450 gene product P-450 System. In this aspect, the method comprises contact using the currently available methods for P450 gene therapy ing the Sample compound with a reagent composition pre (Chen, L. and Waxman, D. J., Cancer Res. 55:581-589 pared by adding said specific ISPG protein to liver (1995)). As described in U.S. Pat. No. 6,207,648, one microSomes lacking Said ISPG protein in a carrier material. enhancement involves introducing a P450 reductase (RED) The method may further comprise (a) incubating a mixture gene in combination with a cytochrome P450 gene (and thus of the Sample compound and the reagent composition; (b) a P450 gene product) into neoplastic cells, the enzymatic extraction of the reaction mixture obtained in Step (a); and conversion of a P450-activated chemotherapeutic drug to its (c) analyzing the reaction products isolated in Step (b). For therapeutically active metabolites is greatly enhanced within the purposes of quantitating the assay, a plurality of the the cellular and anatomic locale of the tumor, thereby reagent compositions having different amount of the Specific increasing both the selectivity and efficiency with which P-450 isozyme or ISPG protein may be subjected to Step (a) neoplastic cells are killed. to (c), respectively. For example, the specific P-450 isozyme 0645. In a preferred embodiment, further enhancements to be used in the method may be selected from CYP2D6, to known prodrug-enzyme Strategies may be achieved by CYP2C19, CYP2A6, CYPIA1 and CYP2E1. introducing an ISPG gene into neoplastic cells, either alone Iron Biosensor or in combination with a P450 gene and/or a P450 reductase gene. Suitable vectors for the introduction and expression of 0648 Iron-sulfur clusters have been found to serve as said ISTG and P450 genes are known to one of skill in the Sensors of iron, dioxygen, Superoxide ion and possibly nitric art. The introduction of the ISPG gene and subsequent oxide. Two main mechanisms of Sensing have been expression of the ISPG gene product may increase the described. In one example, the oxidation Fe°S'->Fe°S) enzymatic conversion of a P450-activated chemotherapeutic '" by dioxygen provides the signal for activation of a drug to its therapeutically active metabolites. Thus, the defense mechanism against Superoxide, as observed with the US 2006/0053498A1 Mar. 9, 2006

SoxR protein of E. coli, and thus may serve a cytoprotective with profound iron deficiency syndrome. Detection of bio function (e.g. useful for treatment of ischemia, etc.). In an accessible iron is one of the most important measurements alternative mechanism, oxidative disassembly or reassembly that doctors can use for early detection if iron deficiency, of a cluster provides the controlling signal as in the FNR iron overload or other types of immunological disorders. To protein of E. coli. date iron is measured through a combination of blood tests that detect iron and iron binding capacity of transferrin, the 0649. In a further detailed example, the ISPG protein of protein that transports iron through the body. The current the invention maybe be used as an iron biosensor. An technology involves very Sophisticated instrumentation example of an iron biosensor is provided in U.S. Pat. No. which make this analysis prohibitively expensive and often 5,516,697 (Kruzel et al.). In summary, ISPG can be immo requires qualified perSonnel to analyze the Sample. There bilized in the vicinity of a device to measure the change in fore, there is a need for the direct assay of iron that combines pH, causing a detectable variation of the potential upon the simplicity and economics. ISPG may therefore be advanta binding of iron. geously used in development of a biosensor for detecting the 0650 In the process of sequestering iron, the sensing amount of iron in a Sample. element, the ISPG protein is expected to release a number of protons of hydrogen (H+) directly proportional to the atoms Steroid Biosynthesis of iron bound. The release of protons during the binding of 0653 As noted above, ISPG may be used as an adrenal iron by ISPG becomes the operative feature which is mea ferredoxin (known as adrenodoxin (ADX)), a vertebrate Sured by the biosensors. The release of protons causes a mitochondrial protein which transferS electrons from change in pH and is measured by an ion-Selective field effect adrenodoxin reductase to cytochrome P450s.cc, which is transistor or by pH Sensitive paper. A Sample containing iron involved in cholesterol side chain cleavage and is an irre is placed into a buffered Solution, usually water. The Sample placeable component of the Steroid hormones biosynthesis may be diluted one or more times. In one embodiment of the in the adrenal mitochondria. Sensors of the present invention, the release of protons is measured as the variation of the potential on the Surface of 0654) In therapeutic embodiments, ISPG may have par an ion-selective field effect transistor (an ISFET) (Reviewed ticular importance in treatment of disorders where it is in: Biosensor Technology, edited by Buck et al. and pub desired to increase the level of Steroid hormone Synthesis. AS lished by Marcel Decker, Inc., 1990, entitled “Solid State P450s.cc has a critical role in synthesis of the conversion of Potentiometric Sensors” by Jiri Janata, pp 17-34). In another cholesterol into pregnenolone, ISPG may be used as a embodiment of the present invention, the protons released limiter or enhancer of Steroid synthesis. In but one example, upon binding of iron by ISPG are detected by the change in evidence has been shown that cytochrome P450s.cc activity pH using pH Sensitive paper. Preferably, the iron Selective in the human placenta is limited by the Supply of electrons element (ISPG) is incorporated in close proximity or inte to the P450s.cc. Furthermore, Tuckey et al. Eur. J. Biochem. grated with the Signal transducer, to give a reagentleSS July 1999;263(2): 319-325 have shown that p450s.cc activity Sensing System for iron. Since the Signal can be amplified, can be increased considerably by adding adrenodoxin reduc only small quantities of ISPG are needed for detection of tase and adrenodoxin. Thus, ISPG may be useful in the iron. The ISFET is modified by immobilizing ISPG on the treatment of reproductive disorders by augmenting the elec surface of the ISFET or by a disposable membrane with tron supply to P450s.cc, and thus increasing the level of immobilized ISPG that is in close proximity to the ISFET by progesterone Synthesis. Accordingly, in another example, attaching the ISPG-modified membrane to the surface of the ISPG may be used to limit steroid synthesis, whether for ISFET. A Sample containing iron, for example a biological therapeutic or for research uses. Sample Such as body fluid from a mammal, particularly a 0655 ISPG may also be used in biological steroid syn human, is then contacted with the ISPG -modified ISFET. thesis processes for the production of Steroid hormones. For 0651). In order to produce an ISPG-modified ISFET as an example, Duport etal, Nat Biotechnol February 1998; 16(2): independent Sensor, an existing System which uses an ISFET 186-9 report a system for self-sufficient biosynthesis of designed to measure pH can be modified. Systems which pregnenolone and progesterone in engineered yeast wherein presently use an ISFET to measure pH are the Sentron 2001 the first two steps of the Steroidogenic pathway were repro pH System, manufactured by Integrated Sensor Technology, duced in Saccharomyces cerevisiae. Engineering of Sterol Federal Way, Wash.; the Corning 360i pH system, manu biosynthesis by disruption of the delta 22-desaturase gene factured by Corning Incorporated, Corning N.Y.; or Orion and introduction of the Arabidopsis thaliana delta 7-reduc 610 pH system, manufactured by Orion Analytical Technol tase activity and coexpression of bovine Side chain cleavage ogy, Inc., Boston, Mass. The modification required to mea cytochrome P450, adrenodoxin, and adrenodoxin reductase, Sure the amount of iron in a Sample is either to place an lead to pregnenolone biosynthesis from Simple carbon immobilized layer of ISPG on the ISFET of such a system Source. AS ISPG is thought to be capable of functioning as or, alternatively, to provide a ISPG-modified membrane, i.e. an adrenodoxin protein, ISPG may be used as a function a membrane coated with ISPG, which will be in close substitute in the system of Duport et al for adrenodoxin. proximity to the existing ISFETso as to detect the release of MTG (METALLOTHIONEIN) (Clone ID:654627) protons when the ISPG binds iron in a sample and records the change in potential. 0656 SEQ ID NOS 96 and 413 and clone FL 11:654627 182-5-3-0-F10-F encode the polypeptide of 0652 Iron sensitive biosensors (as well as treatments for SEQ ID NOS:265 and 527 respectively, a metallothionein iron overload disorders) are extremely valuable It is esti protein which binds heavy metal. Said polypeptide of the mated that 30,000,000 Americans suffer from different types invention is also referred herein as MTG. It will be appre of iron related disorders, including a Substantial proportion ciated that all characteristics and uses of the polynucleotides US 2006/0053498A1 Mar. 9, 2006 66 of SEQ ID NOS:96 and 413 and polypeptides of SEQ ID lothionein, a method of treating skin diseases and a method NO:265 and 527, described throughout the present applica of Screening ultraViolet rays, and further relates to cosmetic tion also pertain to the human cDNA of clone 654627, and compositions and UV Screening compositions. the polypeptides encoded thereby. 0663 Conventionally, steroids and Zinc oxide formula 0657 Metallothioneins (MT) 1.2.3 are small proteins tions have been topically used as medicines for treating skin which bind heavy metals. Such as Zinc, copper, cadmium, diseases Such as dermatitis, Sunburn, neurodermatitis, nickel, etc., through clusters of thiolate bonds. MT's occur eczema and anogenital pruritus. Steroids, however, have throughout the animal kingdom and are also found in higher been difficult to administer in large quantities for a pro plants, fungi and Some prokaryotes and are thought to play longed period due to their Strong adverse side effects. Zinc a role in metal detoxification or in the metabolism and oxide formulations, which have local astringent action, homeostasis of metals. On the basis of Structural relation involve problems with respect to the manufacture of phar ships MTs have been Subdivided into three classes. Class I maceuticals, Since they are insoluble in water and are not includes mammalian MT's as well as MTS from crustacean usually administered internally. and molluscs, but with clearly related primary Structure. 0664 Zinc, one of the indispensable trace metals in the Class II groups together MTS from various Species Such as living body, is known to participate in the development of Sea urchins, fungi, insects and cyanobacteria which display Sexual organs, promotion of wound healing and is also none or only very distant correspondence to class I MTS. known to be a component of a metalloenzyme, an accelera Class III MTS are atypical polypeptides containing gamma tor for dehydrogenase, and to have various functions Such as glutamylcysteinyl units. activating the immune System. Zinc is further known to be 0658) Vertebrate class I MT's such as the MTG protein of an inducing factor of metallothionein (MT), a metal-com the invention are proteins of typically 60 to 68 amino acid bining protein. It is reported that MT functions as a Scav residues, 20 of these residues are cysteines that bind to 7 enger of free radicals which are generated at the onset of bivalent metal ions. As a signature pattern we chose a region inflammations “Dermatologica”, Hanada, k., et al., 179 that SpanS 19 residues and which contains Seven of the (suppl. 1) 143 (1989)). metal-binding cysteines, this region is located in the N-ter 0665 As proposed in U.S. Pat. No. 5,582,817 (Otsu et minal Section of class-I MT's. A consensus pattern for class al), MTG may be useful in treatment of dermatological I Mrs is as follows: C-X-C-GSTAP-X(2-C-X-C-X(2)-C-X- inflammations caused by external irritative Stimulants, Such C-x(2)-C-X-K. as Sunburn or the like, where MTG could act to quench the 0659 The MTG protein of SEQ ID NO 527 has a free radicals released from leukocytes, especially granulo metallothionein domain (Prosite ref. PS00203) at amino acid cytes which gather at the inflamed region, and thereby positions 13-31; an N-glycosylation site at position 4 exhibit an anti-oxidation action to diminish cell damage, (NCSC), a protein kinase C phosphorylation site at amino especially to normal lymphocytes, to activate the immune acid positions 18 (SCK), 28 (SCK) and 55 (SQR); a casein System and further to prevent the accelerated aging of the kinase II phosphorylation site at amino acid position 41 skin. Formation of Sunburn cells (SBCs) could be Sup (TLVD); an N-myristoylation site at amino acid positions 10 pressed by administering zinc for inducing MTG to be (GVSCTC); and a prokaryotic membrane lipoprotein lipid present, or to increase MTG in the epidermal keratinous attachment site at amino acid position 3 (PNCSCAAGVSC). layer. Anti-oxidation action of MTG can also be useful in the treatment of skin problems resulting from radiation therapy Therapeutics by X rays, alpha rays, beta rays, gamma rays, neutron rays and accelerated electron rayS. 0660 Discovery of new proteins related to metallothio neins, and the polynucleotides that encode them, Satisfies a 0.666 Various zinc compounds have been studied by Otsu need in the art by providing new diagnostic or therapeutic et al (Supra) with respect to their pharmacological activities, compositions useful in diagnosing and treating heavy metal who reported that Zinc Salts or Zinc complexes of a certain compound have an excellent action of inducing metal toxicity, cancer, inflammatory disease and immune disor lothionen (MT) and suppressing Sunburn cell (SBC) pro derS. duction due to UV rays, and thereby useful as components 0661 Acute or chronic exposure to heavy metals such as of cosmetic compositions or medicineS for purposes of lead, arsenic, mercury or cadmium leads to a variety of ameliorating Sunburn, preventing Sunburn, ameliorating Suf diseases and disorders involving neuromuscular, CNS, car ferings from skin diseases and ameliorating other radiation diovascular, and gastrointestinal effects. MTS may play a induced disorders, leading to completion of the invention. role in the prevention or alleviation of these conditions. In addition, MTS are transcriptionally regulated by glucocorti 0667 There are two different types of dermatological coids, which Suggests that MTS have a direct role in the reactions caused by Sunlight, one is an acute inflammatory effects of glucocorticoids to treat inflammatory disease, change in the skin called Sunburn, and the other is a immune disorders, and cancer. It is therefore thought that Subsequent melanin pigmentation called Suntan. The light MTG may have important applications in the treatment of having a wave length in the range of 320 nm or less, called inflammatory disease, immune disorders, and cancer as well UVB, induces Sunburn and is responsible for erythematous as in cytoprotection in a variety of therapeutic applications. change. The erythemic reaction caused by UV rays, as opposed to a burn injury, does not occur immediately after 0662. In one preferred example, the MTG nucleic acids the exposure to the Sunlight, but rather occurs after a latent and protein may be used for Suppressing the production of period of several hours. When Sunburned skin is histopatho Sunburn cells which is applicable in various manners with logically examined, various degrees of inflammatory minimal adverse Side effects, a method of inducing metal changes are recognized in the epidermis and dermis depend US 2006/0053498A1 Mar. 9, 2006 67 ing on the dose of radiation. Among Such changes, a notable Chem. 263,5989-5992, 1988). This instability is inherent to one is the generation of so-called Sunburn cells (SBC) in the the presently available expression systems of CHO dhfr epidermis. A histologically stained tissue Sample presents Sup.- cells. For many years, Several promoters have been Strongly and acidophilically Stained cells which have used to drive the expression of the target genes Such as the pyknotic nuclei. This phenomenon indicates the necrosis of SV40 early promoter, the CMV early promoter and the epidermal cells (“Fragrance Journal”, 9, 15-20 (1991). In SR.alpha. promoter. The CMV and SR.alpha. promoters are order to prevent Sunburn, para-aminobenzoic acid deriva claimed to be the strongest (Wenger et al, Anal. Biochem. tives, cinnamic acid derivatives or the like UV absorbers 221, 416-418, 1994). mentioned above are used, but their UV absorbing effects are not necessarily Satisfactory. What is more, they raise 0671 In one report, the beta-interferon promoter has problems of cumberSome handling upon use, poor Stability, also been used to drive the expression of the beta.-interferon low compatibility with other components of the composi gene in the mutant CHO dhfr.Sup.- cells (U.S. Pat. No. tion, and also involve unsolved problems in water-resistance 5,376,567). In this system, however, the selected CHO dhfr and oil-resistance. cells had to be superinduced by the method of Tan et al (Tan et al., PNAS U.S.A. 67, 464471, 1970; Tan et al., U.S. Pat. 0668. In the field of medicines for the treatment of skin No. 3,773,924) to effect a higher level of beta-interferon diseases, development of medicineS which have minimal production. In this System a significant percentage of the adverse side effects, and which have novel functions obtain Superinduced beta.-interferon produced by the CHO dhfr able by both external and internal administrations has been cells was not glycosylated. The mouse metallothionein gene desired. Also, in the field of the therapy and prevention of (mMT1) promoter has also been used for the expression of radiation disorders, medicineS which can Suppress and cure beta-interferon genes in CHO cells, BHK and LTK.Sup.- the disorders caused by oxidative reactions have been mouse cells (Reiser et al 1987 Drug Res. 37, 4, 482485). desired. Lastly, in the field of the manufacture of cosmetics, However, the expression of beta-interferon with this pro cosmetics which overcome the above-mentioned problems moter was not as good as the SV40 early promoter in CHO Such as handling upon use and Stability of the composition cells. Further, beta.-interferon expression from these cells have been desired. Accordingly, the present invention mediated by the mMT1 promoter was inducible by heavy encompasses providing therapeutic agents for treating skin metals. Heavy metals are however extremely toxic to the diseases having the above-mentioned characteristics, cells and this System was therefore abandoned. Instead, wherein Said agents are capable of inducing MTG for Reiser et al used the CHO dhfr- expression system in Suppressing the formation of Sunburn cells, and for use in conjunction with the SV40 early promoter (Reiser et al., cosmetic compositions. Also encompassed are methods of Drug Res. 37.4, 482485 (1987) and EP-A-0529300) to Screening for therapeutic agents for treating skin diseases produce beta-interferon in CHO dhfr-cells as derived by comprising bringing a test compound into contact with a the method of Urlaub et al (1980). cell, tissue or animal model of disease, and detecting induc tion of MTG expression or function. 0672. As described in U.S. Pat. No. 6,207,146 (Tan et al) beta.-interferon was expressed in wild-type CHO cells using Gene Expression Systems a metallothionein based system. MTG may thus be used in Similar applications So as to provide a System for expression 0669 The MTG nucleic acid and proteins of the inven of recombinant proteins. Tan et al demonstrates wild-type tion may also be advantageously used in the production of CHO cells transfected with a vector comprising a beta.- recombinant proteins as biopharmaceutical products at com interferon gene under the control of a mouse Sarcoma viral mercial Scale. enhancer and mouse metallothionein promoter (MSV 0670 Previously, genes have been extensively expressed mMT1), a neogene under the control of promoter capable in mammalian cell lines, particularly in mutant Chinese of driving expression of the neogene in both E. coli and Hamster Ovary (CHO) cells deficient in the dihydrofolate mammalian cells and a human metallothionein gene having reductase gene (dhfr) as devised by the method of Urlaub et its own promoter. Transfected cells capable of expressing al, PNAS U.S.A. 77, 42164220, 1980. A variety of expres ..beta.-interferon were Selected by first exposing cells to Sion Systems have been used. Many vectors for the expres geneticin (antiobiotic G418) and thus eliminating cells lack Sion of genes in Such cells are therefore available. Typically, ing the neogene and then exposing the Surviving cells to the Selection procedures used to isolate cells transformed increasing concentrations of a heavy metal ion. with the expression vectors rely on using methotrexate to 0673. The heavy metal ion enhanced the MSV-mMT1 select for transformants in which both the dhfr and the target promoter for the beta-interferon gene, thus increasing genes are coamplified. The dhfr gene, which enables cells to ..beta.-interferon expression. The heavy metal ion also withstand methotrexate, is usually incorporated in the vector induced the human metallothionein gene promoter, causing with the gene whose expression is desired. Selection of cells expression of human metallothionein. The human metal under increasing concentrations of methotrexate is then lothionein protected the cells against the toxic effect of the performed. This leads to amplification of the number of dhfr heavy metal ion. The presence of the heavy metal ion genes present in each cell of the population, as cells with ensured that there was continual Selection of cells which had higher copy numbers withstand greater concentrations of the transfecting vector, or at least the beta.-interferon gene methotrexate. AS the dhfr gene is amplified, the copy num and the human metallothionein gene and their respective ber of the gene of interest increases concomitantly with the promoters, integrated into their genome. copy number of the dhfr gene, So that increased expression of the gene of interest is achieved. Unfortunately, these 0674) The selected cells that had been successfully trans amplified genes have been reported to be variably unstable fected expressed beta.-interferon. Expression was Surpris in the absence of continued selection (Schimke, J. Biol. ingly improved when the cells were cultured in the presence US 2006/0053498A1 Mar. 9, 2006 68 of Zn". The beta-interferon had improved properties, in application where a temporally controlled Solubilization of a particular a higher bioavailability, than prior beta.-interfer protein of interest is desired. For example, a fragment OS. comprising the CDPG GPI-anchor domain can be used in the production of Soluble molecules by replacing the trans 0675. These findings have general applicability and Sug membrane domains of the cDNA of a protein of interest with gest that the MTG gene of the present invention may be used a Sequence comprising the CDPG glycosylphosphatidyl accordingly in expression Systems. Accordingly, the present inositol (GPI) linkage. These chimeric cDNAS are then invention provides a nucleic acid vector comprising: transferred into an expression vector containing a Strong 0676 (i) a coding sequence which encodes a protein of promoter and a mutant (e.g. DHFR) gene allowing high interest and which is operably linked to a promoter capable levels of transcriptional expression and amplification of the of directing expression of the coding Sequence in a mam gene. These chimeric genes are then cotransfected into a malian cell in the presence of a heavy metal ion; (ii) a first Selected cell type, preferably lacking the endogenous protein Selectable marker Sequence which comprises an MTG gene of interest, and transfectants are Selected and can also be of the invention and which is operably linked to a promoter screened with antibodies for the protein of interest. These capable of directing expression of the MTG gene in a GPI linked proteins of interest can then be solubilized by mammalian cell in the presence of a heavy metal ion; and cleavage with the enzyme phosphatidyl inositol Specific optionally (iii) a second Selectable marker Sequence which phospholipase C (PI-PLC) and purified/concentrated from comprises a neogene and which is operably linked to a the Supernatant (e.g. by passage over a protein of interest promoter capable of directing expression of the neogene in reactive antibody affinity column). a mammalian cell; Therapeutics CDPG (Glycosyl Phosphatidylinositol-Linked Glycopro 0681 CDPG or inhibitors of CDPG may be used in the tein) (Clone ID:1000902917) treatment of any disorder where it is desired to regulate 0677 SEQ ID NOS 3 and 341 and clone B-cell proliferation or differentiation. CDPG or inhibitors FL11:1000902917 223-524-0-G3-F encode the polypep thereof may be useful in the treatment of B-cell neoplasms, tide of SEQ ID NOS 172 and 458 respectively, a glycosyl a heterogeneous group of diseases characterized by different phosphatidylinositol-linked glycoprotein protein which is maturation states of the B-cell, which are related to the thought to be a signal transducing polypeptide expressed in aggressiveness of the disorder. Chronic lymphocytic leuke lymphoid, myeloid, and erythroid cells. Said polypeptide mia (CLL) is characterized by proliferation and accumula comprises a CD24 signal transducing domain as well as a tion of B-lymphocytic leukemia (BLL) is characterized by GPI-anchor of the invention is also referred herein as CDPG. proliferation and accumulation of B-lymphocytes that CDPG is believed to be highly glycosylated, and it is appear morphologically mature but are biologically imma expected that CDPG molecular weight will vary among cell ture. This disorder accounts for 30% of leukemias in West types and cell developmental Stage due to differences in ern countries. The disorder is characterized by proliferation glycosylation patterns, providing further specificity in its use of biologically immature lymphocytes, unable to produce as a therapeutic target. It will be appreciated that all char immunoglobulins, which cause lymph node enlargement. AS acteristics and uses of the polynucleotides of SEQ ID NOS: a regulator of B-cell proliferation and differentiation, CDPG 3 and 341 and polypeptides of SEQ ID NO: 172 and 458, and/or inhibitors of CDPG may be useful for inhibiting described throughout the present application also pertain to proliferation of leukemic B-cells in CLL patients. the human cDNA of clone 1000902917, and the polypep 0682 CDPG may also be useful in the modulation of cell tides encoded thereby. growth in the CNS. CD24 is known to be highly expressed 0678. It is suggested that CDPG may have a specific role in neurons and has been demonstrated as capable of inhib to play in early thymocyte development. The CDPG protein iting neurite outgrowth of dorsal root ganglion neurons is thought to be extensively O-glycosylated may be capable while promoting neurite outgrowth of cerebellar neurons via of modulating b-cell activation responses. AS a Signaling interaction with an L1 protein. transducer, the CDPG polypeptide's signal transducing function may in Some embodiments be triggered by the Selectable Cell Markers binding of a lectin-like ligand to the CD24 domain carbo 0.683. In one aspect, the CDPG polypeptide may be used hydrates, and the release of Second messengerS allowing as a Selectable cell marker and to a method of using the Signaling. The CDPG polypeptide is thought to have impor Selectable marker to identify a cell. Viruses Such as recom tant functions in regulating the differentiation and/or growth binant retroviruses have been used as a vehicle for gene of lymphoid, myeloid, and erythroid cells, including Spe transfer based on their potential for highly efficient infection cifically promoting antigen dependent proliferation of and non-toxic integration of their genome into a wide range b-cells, and preventing their terminal differentiation into of cell types. The transfer of exogenous genes into mam antibody-forming cells. malian cells may be used, for example in gene therapy to correct an inherited or acquired disorder through the Syn 0679. Additionally, based on a growing body of evidence thesis of missing or defective gene products in Vivo. The characterizing the CD24 domain function, it is proposed that expression of exogenous genes in cells may be useful in CDPG may have a role as a potent stimulator of neurite Somatic gene therapy, to correct hereditable disorders at the outgrowth, and thus may be useful in the treatment of central level of the gene. Hemopoietic Stem cells are particularly nervous System disorders. Suited to Somatic gene therapy as regenerative bone marrow 0680 Fragments of CDPG may also be useful, eg. GPI cells may be readily isolated, modified by gene transfer and anchor domain for example in the development of Soluble transplanted into an immunocompromised host to reconsti T-cell receptors (U.S. Pat. No. 6,080,840) or any suitable tute the hosts hemopoietic System. US 2006/0053498A1 Mar. 9, 2006 69

0684 Gene therapy involving hone marrow transplant sequence into the cell. Preferably, the CDPG nucleotide with recombinant primary hemopoietic Stem cells requires Sequence is operatively linked to one or more regulatory efficient gene transfer into the Stem cells. As a very Small elements. The recombinant viral vector of the invention may number of primary Stem cells can reconstitute the entire host be used as a marker for an exogenous gene to be expressed hemopoietic System it is important that the transferred gene in a host cell. The invention further provides a method of be efficiently expressed in the recombinant Stem cells trans identifying a cell and progeny thereof comprising: providing ferred. The transfer of foreign genes into a reconstituted host a cell; infecting the cell with a recombinant viral vector of hemopoietic system has been limited by the availability of a the invention under Suitable conditions to allow expression Selectable marker which permits the rapid and non-toxic of the cell surface protein CDPG on the cell; and, identifying Selection of cells which are efficiently expressing the trans the cell and progeny thereof by detecting expression of the ferred gene. Currently available Selection markers may not cell Surface protein CDPG on the cell or progeny thereof. be Suitable for primary hemopoietic Stem cells Since they Cells infected with a recombinant viral vector of the inven may alter the proliferative ability or biological characteris tion and expressing the cell Surface protein may be trans tics of the cells. The transfer of foreign genes into a planted into a host, and the cell and progeny thereof may be reconstituted host hemopoietic System has also been limited identified after transplantation by removing biological by the availability of a viral vector capable of expression in Samples from the host, and assaying for cells expressing the hemopoietic Stem cells, especially where more than one cell Surface protein. A recombinant viral vector of the transcriptional unit is present in the vector (Botrell, D. R. L. invention may be directly introduced into a host. et al., 1987, Mol. Biol. Med. 4:229). 0685 U.S. Pat. No. 5,804,177 (Humphries et al) has Enriching Stem Cell Compositions demonstrated that the cell surface protein CD24 (also 0689. As it is proposed that CDPG is involved in early M1/69-J11d heat stable antigen) can be used as a dominant thymocyte development and is found on the cell Surface due marker in a recombinant Viral vector. A nucleotide Sequence to its GPI-anchor, CDPG nucleic acids and polypeptides of encoding the cell Surface protein CD24 in a recombinant the present invention may also be used to obtain novel Viral vector was used to infect hematopoietic Stem cells and antibody compositions useful for preparing cell preparations cells infected with the recombinant viral vector were rapidly containing human hematopoietic cells. and non-toxically Selected for in vitro using fluorescence activated cell Sorting (FACS), demonstrating a good corre 0690. There is a continued interest in developing stem lation between proviral copy number and expression of cell purification techniques. Pure populations of stem cells Selectable marker. will facilitate Studies of hematopoiesis. Transplantation of hematopoietic cells from peripheral blood and/or bone mar 0686 CD24 is a signal transducing molecule found on row is also increasingly used in combination with high-dose the Surface of most human B cells that can modulate their chemo- and/or radiotherapy for the treatment of a variety of responses to activation signals, and is structurally closely disorders including malignant, nonmalignant and genetic related to CDPG. The CD24 cDNA (approximately 300 bps) disorders. Very few cells in Such transplants are capable of has been cloned (Kay, R. et al., 1991, J. Immunol. 147: 1412) long-term hematopoietic reconstitution, and thus there is a and encodes a mature peptide of only 31 to 35 amino acids Strong Stimulus to develop techniques for purification of that is extensively glycosylated and attached to the outer hematopoietic Stem cells. Furthermore, Serious complica Surface of the plasma membrane by a glycosyl phosphati tions and indeed the Success of a transplant procedure is to dylinositol lipid anchor. M1/69-J11d heat stable antigen is a a large degree dependent on the effectiveness of the proce genetically similar homologous murine peptide widely dures that are used for the removal of cells in the transplant expressed on a variety of hemopoietic cell types (Kay, R. et that pose a risk to the transplant recipient. Such cells include al., 1990, J. Immunol. 145:1952). T lymphocytes that are responsible for graft verSuS host 0687. It is thus proposed that a recombinant viral vector disease (GVHD) in allogenic grafts, and tumour cells in can be used to successfully transfer and express the CDPG autologous transplants that may cause recurrence of the gene in primitive hemopoietic Stem cells Such that they are malignant growth. able to repopulate lethally irradiated recipients. Preferably 0691 Hematopoietic cells have been separated on the foreign CDPG antigen expression in repopulated animals basis of physical characteristics Such as density and on the persists post transplantation Such that the biological function basis of Susceptibility to certain pharmacological agents of the repopulated hemopoietic cells is not affected by the which kill cycling cells. The advent of monoclonal antibod expression of the CDPG antigen. CDPG may subsequently ies against cell Surface antigens has greatly expanded the be found to be expressed in any or all of hemopoietic potential to distinguish and Separate distinct cell types. lineages including granulocytes, macrophages, pro-erythro There are two basic approaches to Separating cell popula cytes, erythrocytes and T and B lymphocytes. Therefore, the tions from bone marrow and peripheral blood using mono cell surface protein CDPG may be particularly useful as a clonal antibodies. They differ in whether it is the desired or marker for hematopoietic Stem cells capable of repopulation undesired cells which are distinguished/labeled with the in Vivo and as a Selectable marker in gene therapy. The antibody(s). In positive Selection techniques the desired cells recombinant Viral vectorS also have the advantage that the are labeled with antibodies and removed from the remaining nucleotide Sequence encoding the marker is very Small, unlabeled/unwanted cells. In negative Selection, the leaving a large amount of Space for the insertion of addi unwanted cells are labeled and removed. Antibody/comple tional genes of interest Such as those coding for exogenous ment treatment and the use of immunotoxins are negative geneS. Selection techniques, but FACS Sorting and most batch wise 0688. In a preferred embodiment of the invention a immunoadsorption techniques can be adapted to both posi recombinant viral vector is used to introduce the nucleotide tive and negative Selection. In immunoadsorption techniques US 2006/0053498A1 Mar. 9, 2006 70 cells are Selected with monoclonal antibodies and preferen comprises a tyrosine at amino acid position 40. The HSTG tially bound to a surface which can be removed from the protein also comprises a Pattern-DE: Protein kinase C remainder of the cells e.g. column of beads, flasks, magnetic phosphorylation site at amino acid position 51 (SSK). It will particles. Immunoadsorption techniques have won favor be appreciated that all characteristics and uses of the poly clinically and in research because they maintain the high nucleotides of SEQID NOS: 22 and 358 and polypeptides of Specificity of targeting cells with monoclonal antibodies, but SEQ ID NO: 191 and 475, described throughout the present unlike FACSorting, they can be Scaled up to deal directly application also pertain to the human cDNA of clone with the large numbers of cells in a clinical harvest and they 1000839315, and the polypeptides encoded thereby. avoid the dangers of using cytotoxic reagents Such as 0697 SEQ ID NOS 8 and 345 and clone FL11:338112 immunotoxins, and complement. 174-1-1-0-A11-F encode the polypeptide of SEQ ID NOS 0692 Current positive selection techniques for the puri 177 and 462 respectively, a proline-rich protein referred to fication of hematopoietic Stem cells target and isolate cells herein as PRSG believed to be a component of saliva and a which express CD34. However, positive selection proce calcium binding protein also possessing potent antimicrobial dures Suffer from many disadvantages including the pres properties. Preferably, the amino acid sequence of PRSG ence of materials. Such as antibodies and/or magnetic beads (SEQ ID NO 462) comprises a proline residue at amino acid on the CD34" cells, and damage to the cells resulting from position 96, an arginine residue at amino acid position 100, the removal of these materials. a glutamine residue at amino acid positoin 102, and/or a glycine residue at amino acid position 103. The PRSG 0693 Negative selection has been used to remove minor protein also comprises a casein kinase II phosphorylation populations of cells from clinical grafts. These cells are site at amino acid positions 15 (SAOD), 24 (SQED), and 59 either T-cells or tumour cells that pose a risk to the transplant (SAGD) and an N-myristoylation site at amino acid position recipient. The efficiency of these purges varies with the 52 (GGOOSQ). It will be appreciated that all characteristics technique and depends on the type and number of antibodies and uses of the polynucleotides of SEQ ID NOS:8 and 345 used. Typically, the end product is very similar to the Start and polypeptides of SEQ ID NO: 177 and 462, described Suspension, missing only the tumor cells or T-cells. throughout the present application also pertain to the human 0694. As described in U.S. Pat. No. 5,877.299, Thomas et cDNA of clone 338112, and the polypeptides encoded al developed a negative Selection technique that uses an thereby. antibody composition containing antibodies specific for gly 0.698) A study of saliva and its tooth-protective compo cophorin A, CD3, CD24, CD16, CD14 and optionally nents reveals at least four important functions of saliva: (1) CD45RA, CD36, CD2, CD19, CD56, CD66a, and CD66b, buffering ability, (2) a cleansing effect, (3) antibacterial which reportedly gave a cell preparation highly enriched for action, and (4) maintenance of a saliva SuperSaturated in human hematopoietic and progenitor cells. Maximum calcium phosphate. Several Salivary constituents Serve one enrichment of early progenitor and stem cells (CD34", or more of these functions. Research has yielded important CD38 cells) was observed when anti-CD45R and anti information about organic and inorganic Secretory products. CD36 were included in the antibody composition. However, It is also clear that Saliva as a unique biologic fluid has to be as CDPG is proposed as acting in early thymocyte devel considered in its entirety to account fully for its effects on opment, CDPG may be used advantageuously to develop teeth. Saliva is greater then the Sum of its parts. One reason more effective antibody compositions for Selecting hemato for this is that Salivary components display redundancy of poietic Stem cells. Accordingly, the invention encompasses function, each often having more than one function. This antibodies specific for CDPG polypeptides of the invention redundancy, however, does not imply that proteins that share and antibody compositions comprising, consisting of or functional roles all contribute to the same degree. For consisting essentially of antibodies Specific for CDPG, gly instance, when comparing proteins that inhibit calcium cophorin A, CD3, CD24, CD16, CD14 and optionally phosphate precipitation, Statherin and acidic proline-rich CD45RA, CD36, CD2, CD19, CD56, CD66a, and CD66b. proteins are most potent, whereas histatins, cyStatins, and mucins appear to play lesser roles. The complex interaction 0695) Use of the antibody composition comprising between proteins is another major factor contributing to CDPG in a negative Selection technique to prepare a cell Saliva's function. In this regard, heterotypic complexes of preparation which is enriched for hematopoietic Stem cells various proteins have been shown to form on hydroxyapa and progenitor cells may offer Significant advantages over tite. Mucin binding to other Salivary proteins, including conventional techniques. The antibody composition is proline-rich proteins, histatins, cyStatins, and Statherin, is applied in one Step to a Sample of peripheral blood, bone well documented. The complexes, whether adsorbed to the marrow, cord blood or frozen bone marrow, preferably tooth Surface or in Saliva, have important implications for without additional enrichments steps which could result in bacterial clearance, Selective bacterial aggregation on the loSS of, or damage to, progenitor and Stem cells. tooth Surface, and control of mineralization and demineral ization. Finally, proteolytic activity of Saliva generates PRSG (Proline-Rich Calcium-Binding Protein) and HSTG numerous products whose biologic activities are often dif (Basic Histidine Rich Salivary Gland Peptide) (Clone ferent from their parent compounds. The ability of saliva to ID:338112 and 1000839315 Respectively) deliver fluoride to the tooth Surface constantly makes Sali 0696 SEQ ID NOS 22 and 358 and clone FLI 1: vary fluoride an important player in caries protection largely 1000839315 220-26-1-0-F3-F encode the polypeptide of by promoting remineralization and reducing demineraliza SEQ ID NOS 191 and 475 respectively, a basic histidine rich tion. Saliva is well adapted to protection against dental salivary gland peptide referred to herein as HSTG and caries. Saliva's buffering capability; the ability of the saliva expected to have potent antimicrobial properties. Preferably, to wash the tooth Surface, to clear bacteria, and to control the amino acid sequence of HSTG (SEQ ID NO 475) demineralization and mineralization; Saliva's antibacterial US 2006/0053498A1 Mar. 9, 2006 activities, and perhaps other mechanisms all contribute to its 0701. In further embodiments, the skilled artisan will essential role in the health of teeth. The fact that the appreciate that fragments and analogues of PRSG and protective function of saliva can be overwhelmed by bac HSTG may readily be generated and selected. Selection of terial action indicates the importance of prevention and preferred fragments and analogies may be carried out by therapy as in other infectious diseases. With knowledge of assaying for a desired antimicrobial activity. For example, Salivary components and their interactions, the use of modi Synthetic histatin analogues and methods for obtaining Such fied oral molecules as therapeutic agents may become a analogies with broad-spectrum antimicrobial activity are important contributor to oral health. described in Helmerhorst EJ et al, Biochem J 1997 Aug. 15:326 (Pt 1):3945, where histatin analogies inhibited the 0699 Proline-rich proteins are major components of growth of the Second most common yeast found in clinical parotid and Submandibular Saliva in humans as well as other isolates, Torulopsis glabrata, of oral- and non-oral patho animals. They can be divided into acidic, basic and glyco gens Such as PrevOtella intermedia and StreptococcuS Sylated proteins. The proline-rich proteins are apparently mutants, and of a methicillin-resistant StaphylococcuS Synthesized the acinar cells of the Salivary glands and their LifeS. phenotypic expression is under complex genetic control. The acidic proline-rich proteins will bind calcium with a 0702) Thus, in preferred embodiments, the PRSG and/or Strength which indicates that they may be important in HSTG polypeptides or fragments thereof may be used in maintaining the concentration of ionic calcium in Saliva. oral, injectable, topical or edible compositions for the treat Moreover they can inhibit formation of hydroxyapatite, ment of infection PRSG and/or HSTG polypeptides may whereby growth of hydroxyapatite crystals on the tooth also be used as antimicrobiavantifungal compositions for surface in vivo may be avoided. Both of these activities as disinfection of Surfaces (e.g. in industrial Settings). well as the binding site for hydroxyapatite are located in the 0703. In a preferred example further discussed below, the N-terminal proline-poor part of the protein. PRSG and/or HSTG polypeptides or fragments thereof are used in oral, topical (e.g. mouthwash) or edible composi 0700 Basic histidine rich salivary gland peptides such as tions optionally containing additional Salivary proteins to the peptide of SEQ ID NO. 191 and 475, also referred to as provide an anticaries effect. While there is an interest in histatins are a group of electrophoretically distinct histidine developing and marketing products which reduce caries rich polypeptides with microbicidal activity found in human without reliance on a high level of fluoride ions (such as in parotid and Submandibular gland Secretions. Histatins 1, 3, fluoridated water and fluoride toothpastes), there have not and 5 are homologous proteins that consist of 38, 32, and 24 been many reports of Such approaches meeting with Success. amino acid residues, respectively, that have been shown to While certain cysteine-rich proteins have been proposed kill the pathogenic yeast, Candida albicans. More recently useful in the treatment of dental caries (U.S. Pat. No. histatins 2, 4, 6, and 7-12 were isolated and characterized 5,688,766, Revisetal), the present invention provides PRSG Troxier RF et al., J Dent Res January 1990;69(1):2-6. Hista and HSTG polypeptides which may provide higher potency, tin 2 was found to be identical to the carboxyl terminal 26 residues of histatin histatin 4 was found to be identical to the efficacy and range of disinfection and protection. carboxyl terminal 20 residues of histatin 3; and histatin 6 0704 PRSG and HSTG polypeptide compositions can be was found to be identical to histatin 5, but contained an administered in a formulation comprising a carrier. A pre additional carboxyl terminal arginine residue. The amino ferred carrier composition for the active(s) of this invention acid Sequences of histatins 7-12 formally corresponded to are oral compositions. Such compositions include tooth residues 12-24, 13-24, 12-25, 13-25, 5-11, and 5-12, respec pastes, mouthrinses, liquid dentifrices, lozenges, chewing tively, of histatin 3, but could also arise proteolytically f gums or other vehicle Suitable for use in the oral cavity. histatin 5 or 6. Troxler et al provides further guidance on the Toothpastes and mouthrinses are the preferred Systems. The Structural elements and relationship of histatins to one abrasive polishing material contemplated for use in the another in the context of their genetic origin, biosynthesis toothpaste compositions of the present invention can be any and Secretion into the oral cavity, and potential as reagents material which does not excessively abrade dentin. These in anti-candidal studies. The HSTG polypeptide and frag include, for example, Silicas including gels and precipitates, ments thereof are therefore expected to have valuable prop calcium carbonate, dicalcium orthophosphate dihydrate, cal erties and uses in antimicrobial applications, particularly in cium pyrophosphate, tricalcium phosphate, calcium poly antifungal applications. Supporting Such uses is a consider metaphosphate, insoluble Sodium polymetaphosphate, able body of evidence, including MacKay B J et al., Infect hydrated alumina, and resinous abrasive materials. Such as Immun. June 1984;44(3):695-701, Growth-inhibitory and particulate condensation products of urea and formaldehyde, bactericidal effects of human parotid Salivary histidine-rich and others such as disclosed by Cooley et al. in U.S. Pat. No. polypeptides on StreptococcuS mutants, MacKay B J et al., 3,070,510, Dec. 25, 1962, incorporated herein by reference. Infect Immun. June 1984;44(3):688-94, Isolation of milli Mixtures of abrasives may also be used. Silica dental gram quantities of a group of histidine-rich polypeptides abrasives, of various types, can provide the unique benefits from human parotid saliva; Pollock J J et al., Infect Immun. of exceptional dental cleaning and polishing performance June 1984;44(3):702-7, Fungistatic and fungicidal activity without unduly abrading tooth enamel or dentin. For these of human parotid Salivary histidine-rich polypeptides on reasons, they are preferred for use herein. Flavoring agents Candida albicans, and Xu T et al., Infect Immun. August can also be added to toothpaste compositions. Suitable 1991;59(8):2549-54, Anticandidal activity of major human flavoring agents include oil of wintergreen, oil of pepper salivary histatins. Furthermore, tissue distribution of RNAS mint, oil of Spearmint, oil of Sassafras, and oil of clove. for cyStatins, histatins, Statherin, and proline-rich Salivary Sweetening agents which can be used include aspartame, proteins in humans and macaques is further discussed in aceSulfame, Saccharin, dextrose, levulose and Sodium cycla Sabatini et al., J Dent Res July 1989;68(7): 113845. mate. Flavoring and Sweetening agents are generally used in US 2006/0053498A1 Mar. 9, 2006 72 toothpastes at levels of from about 0.005% to about 2% by 223 and 498 respectively, a hydroxysteroid dehydrogenase weight. Toothpaste compositions can also contain emulsi referred to herein as HSDG. As the HSDG polypeptide is fying agents. Suitable emulsifying agents are those which implicated in Steroid hormone regulation, preferably gluco are reasonably Stable and foam throughout a wide pH range, corticoid metabolism, HSDG may be useful in any applica including non-Soap anionic, nonionic, cationic, Zwittefionic tions where Steroid hormones levels are to be increased or and amphoteric organic Synthetic Surfactants. Water is also inhibited. HSDG may be useful in the treatment of disease present in the toothpastes of this invention. Water employed treatable by steroid hormones. HSDG inhibitors may also be in the preparation of commercially Suitable toothpastes useful in systems for self-sufficient biosynthesis of steroid should preferably be deionized and free of organic impuri hormones Such as glucocorticoids Such as in engineered ties. In preparing toothpastes, it is necessary to add Some cells comprising elements of the synthesis pathway. HSDG thickening material to provide a desirable consistency. Pre inhibitors of endogenous HSDG activity may allow the ferred thickening agents are carboxyvinyl polymers, carra recovery of higher amounts of glucocorticoids and/or other geenan, hydroxyethyl cellulose and water Soluble Salts of Synthesized Steroid hormones from these cell Systems. cellulose etherS Such as Sodium carboxymethyl cellulose and 0708. It will be appreciated that all characteristics and Sodium carboxymethyl hydroxyethyl cellulose. Natural uses of the polynucleotides of SEQID NOS: 54 and 381 and gums. Such as gum karaya, Xanthan gum, gum arabic, and polypeptides of SEQ ID NO: 223 and 498, described gum tragacanth can also be used. Colloidal magnesium throughout the present application also pertain to the human aluminum Silicate or finely divided Silica can be used as part cDNA of clone 495917, and the polypeptides encoded of the thickening agent to further improve texture. Thick thereby. ening agents in ane amount from 0.2% to 5.0% by weight of the total composition can be used. It is also desirable to 0709) The HSDG polypeptides of the invention comprise include Some humectant material in a toothpaste to keep it leucine zipper pattern (Prosite ref. PS00029) at amino acid from hardening. Suitable humectants include glycerin, Sor positions 58-79 as well as a N-myristoylation site at amino bitol, and other edible polyhydric alcohols at a level of from acid position 36 (GANAGV) of SEQ ID NO 498. In one about 15% to about 70%. example, HSDG polypeptides may be used in the production or therapeutic modulation of glucocorticoids. The skilled 0705) Another preferred embodiment of the present artisan will recognize that any suitable HSDG polypeptides invention is a mouthwash composition. Conventional or variants or fragments thereof capable of metabolizing mouthwash composition components can comprise the car glucocorticoids can readily be used. rier for the agents of the present invention. Mouthwashes generally comprise from about 20:1 to about 2:1 of a 0710. In one embodiment HSDG activity can be deter water/ethyl alcohol Solution or be alcohol free and prefer mined by detecting levels of glucocorticoids or metabolites ably other ingredients Such as flavor, Sweeteners, humec or glucocorticoids. Moreover, Structural aspects of 11beta tants and Sudsing agents Such as those mentioned above for hydroxysteroid dehydrogenase have been documented in the dentifrices. The humectants, Such as glycerin and Sorbitol art and may serve as a guidelines for developing Suitable give a moist feel to the mouth. Generally, on a weight basis HSDG variants and fragments. The HSDG polypeptides of the mouthwashes of the invention comprise 0% to 60% the invention may allow modulation of glucocorticoid activ (preferably 5% to 20%) ethyl alcohol, 0% to 20% (prefer ity or identification (e.g. drug Screening) of compounds ably 5% to 20%) of a humectant, 0% to 2% (preferably capable of Specifically modulating glucocorticoid levels or 0.01% to 1.0%) emulsifying agents, 0% to 0.5% (preferably activity. HSDG may be useful in allowing the modulation of 0.005% to 0.06%) Sweetening agent such as saccharin or Steroid hormone Synthesis, or glucocorticoid Synthesis to be natural Sweeteners such as stevroside 0% to 0.3% (prefer carried in a tissue Specific manner, thereby offering ably 0.03% to 0.3%) flavoring agent, and the balance water. improved methods for treating disease with decreased risk of side effects. 0706 The pH of the present compositions and/or the pH in the mouth can be any pH which is safe for the mouth's 0711 Corticosteroids, also referred to as glucocorticoids hard and soft tissues. Such pHs are generally from about 3 are Steroid hormones, the most common form of which is to about 10, preferably from about 4 to about 8. Other cortisol. Modulation of glucocorticoid activity is important acceptable oral carders include gums, lozenges, as well as in regulating physiological processes in a wide range of other forms. Such Suitable forms are disclosed in U.S. Pat. tissues and organs. Glucocorticoids act within the gonads to No. 4,083,955, Apr. 11, 1978 to Grabenstetter et al. incor directly Suppress testosterone production (Monder, C et al., porated herein in its entirety by reference. Edible composi (1994) Steroids 59, 69-73). High levels of glucocorticoids tions are also Suitable for use as the carrier compositions may also result in excessive Salt and water retention by the herein. Edible compositions include many types of Solid as kidneys, producing high blood pressure. well as liquid compositions. Such compositions include, for 0712) Glucocorticoid action is mediated via binding of example, Soft drinks, citrus drinks, cookies, cakes, breads the molecule to a receptor, defined hereinafter as either a among many others. Such compositions may contain Sugar mineralocorticoid receptor (MR) or a glucocorticoid recep or another Sweetener, water, flour, shortening, other fibers tor (GR). Krozowski, Z. S. et al., (1983) Proc. Natl. Acad. Such as wheat, corn, barley, rye, oats, psyllium and mixtures Sci. USA 80, 6056-6060) and Beaumont, K. et al., (1983) thereof. Endocrinology 113, 2043-2049) showed that MR of adrena HSDG (Hydroxysteroid Dehydrogenase) (Clone lectomised rats have an equal affinity for the mineralocor ticoid aldosterone and glucocorticoids, for example corti ID:495917) costerone and cortisol. Confirmatory evidence has been 0707 SEQ ID NOS 54 and 381 and clone FL11:495917 found for human MR (Arriza, J. Let al., (1988) Neuron I, 160-22-4-0-D8-F encode the polypeptide of SEQ ID NOS 887-900). In patients suffering from the congenital syn US 2006/0053498A1 Mar. 9, 2006 73 drome of Apparent Mineralocorticoid Excess (AME), cor the S100 calcium binding protein referred to herein as tisol levels are reportedly elevated and bind to and activate S100G. SEQ ID NOS 301 and 542 provide the amino acid MRS normally occupied by aldosterone, the steroid that Sequence corresponding to the nucleic acid Sequences of regulates Salt and water balance in the body. Salt and water SEQ ID NOS 132 and 428, respectively. It will be appre are retained in AME patients causing Severe hypertension. ciated that all characteristics and uses of the polynucleotides 0713. Like HSDG, the enzyme 113-hydroxysteroid dehy of SEQ ID NOs: 132 and 428 and polypeptides of SEQ ID drogenase (11BHSD), also discussed in U.S. Pat. No. 5,965, NO: 301 and 542, described throughout the present appli 372 (Funder et al) may be involved in converting glucocor cation also pertain to the human cDNA of clone200895, and ticoids into metabolites that are unable to bind to MRS the polypeptides encoded thereby. (Edwards et al., (1988) Lancet. 2:986-9; Funder et al., (1988) Science 242, 583,585), present in mineralocorticoid target Background tissues, for example kidney, pancreas, Small intestine, colon, 0717) In nearly all eukaryotic cells, calcium (Ca") func as well as the hippocampus, placenta and gonads. For tions as an intracellular signaling molecule in diverse cel example, in aldosterone target tissues 11 BHSD inactivates lular processes including cell proliferation and differentia glucocorticoid molecules, allowing the much lower circu tion, neurotransmitter Secretion, glycogen metabolism, and lating levels of aldosterone to maintain renal homeostasis. skeletal muscle contraction. Within a resting cell, the con When the 11 BHSD enzyme is inactivated, for example in centration of Ca" in the cytosol is extremely low, <107 M. AME patients or following administration of glycyrrhetinic However, when the cell is stimulated by an external Signal, acid, a component of licorice, Severe hypertension results. Such as a neural impulse or a growth factor, the cytosolic Further, placental 11 BHSD activity may protect the foetus concentration of Ca" increases by about 50-fold. This influx from high circulating levels of glucocorticoid which may of Ca" is caused by the opening of plasma membrane Cat" predispose to hypertension in later life (Edwards et al., channels and the release of Ca" from intracellular stores 1993). Biochemical characterisation of activity has indicated the presence of at least two 11 BHSD isoenzymes (11 BHSD1 Such as the endoplasmic reticulum. Ca' directly activates and 11 BHSD2) with different cofactor requirements and regulatory enzymes, Such as protein kinase C, which trigger substrate affinities. The 11 BHSD1 enzyme is a low affinity Signal transduction pathways. enzyme that prefers NADP+ as a cofactor (Agarwal et al., 0718 The protein of SEQ ID NOS 301 and 542 is a 1989). The 11.f3HSD2 enzyme is a high affinity enzyme (Km calcium binding S100 protein typically found in heart and for glucocorticoid=10 nM), requiring NAD+, not NADP+ as muscle. S100 proteins are low-molecular weight calcium the preferred cofactor, belonging to a class of glucocorticoid binding proteins that are believed to play an important role dehydrogenase enzymes hereinafter referred to as “NAD+ in various cellular processes Such as cytoskeletaVmembrane dependent glucocorticoid dehydrogenase' enzymes. interactions, cell division and differentiation. The expression 0714 Inverse correlation between 113HSD enzyme of S100 proteins has been evaluated in a variety of disorders. activity in human granulosa-lutein cells and the Success of For example, S100 protein have been evaluated as markers IVF has further been shown, suggesting that activity of this of inflammatory disease, including ulcerative colitits, enzyme might be related to the Success of embryo attach Crohn's disease, and as Serum markers for Subjects with ment and implantation following IVF. The measurement of infections diseases including AIDS and malaria and for ovarian 11BHSD enzyme activity as a prognostic indicator Subjects with hematological disease. Evidence has accumu for the outcome of assisted conception in all Species, is the lated that indicates that S100 proteins can alter cellular subject of UK Patent Application No 9305984. However, the invasion and metastatic spead of cancer. For example, S100 disclosure of Michael et al. (1993) Lancet 342, 711-712), protein is expressed in dendritic cells in human transitional and corresponding UK Patent Application No 93.05984 do cell carcinoma of the bladder and the invasive potential of not identify, or even Suggest which isoenzyme in the ovary these tumor has been found to correlate with the presence of might be a predictive indicator of IVF embryo transfer, or a S100 protein expressing cells. means of distinguishing isoenzymes of 11 BHSD in the prediction of IVF embryo transfer outcomes. In fact, the Therapeutics and Diagnostics enzyme assay procedure might detect all isoenzymes of 0719. The S100G protein of SEQ ID NOS 301 and 542 11 BHSD activity in the cell, some of which may be hitherto disclosed herein provides new calcium binding S100 protein uncharacterised. compositions useful in the diagnosis, prevention, and treat 0715 Thus, the human HSDG hydroxysteroid dehydro ment of cancer, reproductive disorders, immune disorders, genase enzyme and the nucleic acids encoding it providing neuronal disorders, Vesicle trafficking disorders and devel novel means for the development of gene therapies and opmental disorders. identification of HSDG activators and inhibitors which alter the endogenous activity of this hydroxysteroid dehydroge 0720 CBPs are implicated in a variety of disorders and nase enzyme in a cell. The present invention also permits the several CBPs have proven to be effective therapeutic targets Screening, through genetic or immunological means, levels for which small molecule inhibitors could be developed. of expression of genes encoding the NAD+ dependent However, while several CBPs are targets for widely-used glucocorticoid dehydrogenase enzyme in various tissue or therapeutic treatments, it would be advantageous to provide organ types, including for example, Skin, colon, kidney, further CBPs allowing more selective therapeutic treatments placenta, and gonads, amongst others. for disease to be developed. In one example, calcineurin is found in the cells of all eukaryotes ranging from yeast to S100G Calcium Binding Protein (Clone ID:200895) mammals. Calcineurin is a target for inhibition by the 0716) The polynucleotides of clone FL11:200895 116 immunosuppressive agents cycloSporin A and FK506 O55-1-0-H11-F and SEO ID NOS 132 and 428 encode for emphasizing its importance in immune disorders (Kissinger, US 2006/0053498A1 Mar. 9, 2006 74

C. R. et al. (1995) Nature 378:641-644). Calcineurin also 0721. However, despite the many uses and therapeutics plays a critical role in transcriptional regulation and growth based on S100 proteins and other-CBPs, it would be advan control in T-lymphocytes (Wang, M. G. et al. (1996) Cyto tageous to selectively target CBPs which are involved in a genet. Cell Genet. 72:236-241). However, inhibition of disorder and which are found Specifically in the targeted calcineurin phosphatase activity has been implicated both in cells or tissues. Thus, in a first embodiment, the S100G the mechanism of immunosuppression and in the observed protein of the invention can be used for the development of toxic Side effects of FKS06 in nonlymphoid cells, Suggesting Selective inhibitors of calcium signaling, e.g. preferably that identification of a new (FK binding proteins (FKBPs) inhibitors of transcriptional regulation and cell growth con that can mediate calcineurin inhibition and are restricted in trol. its expression to T cells could provide new immunosuppres Sive drugs may be identified that, by Virtue of their specific 0722. In one embodiment, an antagonist of S100G may interaction with the FKBP, would be targeted in their site of be administered to a Subject to prevent or treat a neuronal action (Baughman G, et al Mol Cell Biol August disorder. Such disorders may include, but are not limited to, 1995;15(8):4395.402). In another CBP example, levels of akathesia, Alzheimer's disease, amnesia, amyotrophic lat CaM are increased several-fold in tumors and tumor-derived eral Sclerosis, bipolar disorder, catatonia, cerebral neo cell lines for various types of cancer (Rasmussen, C. D. and plasms, dementia, depression, Down's Syndrome, tardive Means, A. R. (1989) Trends in Neuroscience 12:433438). dyskinesia, dystonias, epilepsy, Huntington's disease, mul Calcium binding S100B is another example of a CBP tiple Sclerosis, neurofibromatosis, Parkinson's disease, para involved in a variety of disorders. Like the S100G protein of noid psychoses, Schizophrenia, and Tourette's disorder. In the invention, S100B contains an EF-hand motif. S100B is one aspect, an antibody which specifically binds S100G may abundantly expressed in the nervous system. S100B levels be used directly as an antagonist or indirectly as a targeting are increased in the blood and cerebroSpinal fluid of patients or delivery mechanism for bringing a pharmaceutical agent with neurological injury resulting from cerebral infarction, to cells or tissue which express S100G. transient ischemic attacks, hemorrhagia, head trauma, and 0723. In one embodiment, an antagonist of S100G may Down's syndrome. Furthermore, S100B and other neural be administered to a Subject to prevent or treat a vesicle Specific CBPS may also protect against neurodegenerative trafficking disorder. Such disorders may include, but are not disorders, Such as Alzheimer's, Parkinson's, and Hunting limited to, cystic fibrosis, glucose-galactose malabsorption ton's diseases. S100B is produced and Secreted by glial cells Syndrome, hypercholesterolemia, diabetes mellitus, diabetes in the central and peripheral nervous Systems (Allore, R. J. insipidus, hyper- and hypoglycemia, Grave's disease, goiter, et al. (1990) J. Biol. Chem. 265:15537-15543). The accu Cushing's disease, and Addison's disease, gastrointestinal mulation of S100B in mature glial cells is associated with the disorders including ulcerative colitis, gastric and duodenal microtubule network. S100B promotes neuronal differentia ulcers; other conditions associated with abnormal vesicle tion and Survival but may be detrimental to cells if overex trafficking including AIDS; allergies including hay fever, pressed. The Selective overproduction has been implicated asthma, and urticaria (hives), autoimmune hemolytic ane in the progression of the neuropathological changes in mia; proliferative glomerulonephritis, inflammatory bowel Alzheimer's disease which may involve mitotic protein disease; multiple Sclerosis, myasthenia gravis, rheumatoid kinases (Marshak, D. R. and Pena, L. A. (1992) Prog. Clin. and Osteoarthritis, Scleroderma; Chediak-Higashi and Biol. Res. 379:289-307). Adult T-cell leukaemia (ATL) is a Sjogren's Syndromes, Systemic lupus erythematosus, toxic mature T-cell malignancy which is caused by human T Shock Syndrome; traumatic tissue damage; and Viral, bacte lymphotrophic virus type-I. Diminished Surface expression rial, fungal, helminth, and protozoal infections. In one of the T-cell receptor alpha beta (TCRO. B+) complex is a aspect, an antibody which specifically binds S100G may be specific feature of ATL cells. S100B is not detectable in used directly as an antagonist or indirectly as a targeting or CD4+, TCRC. B+ ATL cells, but is expressed in CD4-, CD8-, delivery mechanism for bringing a pharmaceutical agent to TCRO.f3+ leukaemic cells from four ATL patients. This cells or tissue which express S100G. Suggested that increased levels of S100B may be associated with the diminished surface expression of the TCRCfB+ 0724. In one embodiment, an antagonist of S100G may complex in ATL (Suzushima, H. et al. (1994) Leuk. Lym be administered to a Subject to prevent or treat an immuno phoma 13:257-262). Elevated serum levels of S100B are logical disorder. Such disorders may include, but are not asSociated with disseminated malignant melanoma limited to, AIDS, Addison's disease, adult respiratory dis metastases, Suggesting that Serum S100B may be of value as treSS Syndrome, allergies, anemia, asthma, atherosclerosis, a clinical marker for progression of metastatic melanoma bronchitis, cholecystitis, Crohn's disease, ulcerative colitis, (Henze, G. et al. (1997) Dermatology 194:208-212). In yet atopic dermatitis, dermatomyositis, diabetes mellitus, another example, messenger RNA levels encoding human emphysema, erythema nodosum, atrophic gastritis, glom calgizZarin (an S100-like protein), as well as those encoding erulonephritis, gout, Graves disease, hypereosinophilia, phospholipase A, are elevated in colorectal cancers com irritable bowel Syndrome, lupus erythematosus, multiple pared with those of normal colorectal mucosa (Tanaka, M. Sclerosis, myasthenia gravis, myocardial or pericardial et al. (1995) Cancer Lett. 89:195-200). Finally, an intracel inflammation, osteoarthritis, osteoporosis, pancreatitis, lular S100 calcium-binding protein has been isolated from polymyositis, rheumatoid arthritis, Scleroderma, Sjogren's rat peritoneum. This protein, MRP14, is one of two migra Syndrome, Werner Syndrome, and autoimmune thyroiditis, tion inhibitory factor-related proteins that are expressed in complications of cancer, hemodialysis, and extracorporeal peritoneal macrophages in the arthritis-Susceptible Lewis/N circulation; viral, bacterial, fungal, parasitic, protozoal, and rat (Imamichi, T. et al. (1993) Biochem. Biophys. Res. helminthic infections, and trauma. In one aspect, an anti Comm. 194:819-825). body which specifically binds S100P may be used directly as US 2006/0053498A1 Mar. 9, 2006 75 an antagonist or indirectly as a targeting or delivery mecha according to conventional pharmaceutical principles. The nism for bringing a pharmaceutical agent to cells or tissue combination of therapeutic agents may act Synergistically to which express S100G. effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to 0725. In one embodiment, an antagonist of S100G may achieve therapeutic efficacy with lower dosages of each be administered to a Subject to prevent or treat a neoplastic agent, thus reducing the potential for adverse side effects. disorder. Such disorders may include, but are not limited to, adenocarcinoma, leukemia, lymphoma, melanoma, 0729 Expression of S100 proteins have been evaluated myeloma, Sarcoma, teratocarcinoma, and, in particular, can as Serum markers for melanoma (Henze et al, Dermatology cers of the adrenal gland, bladder, bone, bone marrow, brain, 194:208-212; Buer et al., 1997 Brit. J. Cancer 75: 1373-1376; breast, cervix, gall bladder, ganglia, gastrointestinal tract, Sherbert et al. 1998; Anticancer Res. 18:2415-2422) and heart, kidney, liver, lung, muscle, Ovary, pancreas, parathy more recently as Serum markers for cancer in general roid, penis, prostate, Salivary glands, skin, Spleen, testis, (particularly breast, colon and lung cancers) and that their thymus, thyroid, and uterus. In one aspect, an antibody detectability in Serum can have prognostic and/or therapeu which specifically binds S100P may be used directly as an tic significance in cancer. Furthermore auto-antibodies to antagonist or indirectly as a targeting or delivery mechanism S100 proteins were found in cancer patients. (International for bringing a pharmaceutical agent to cells or tissue which Patent Publication No. WO 00/26668). The S100G protein express S100G. of the invention may thus be used in the diagnosis and prevention of cancer, for identification of Subjects predis 0726. In addition, the S100G nucleic acids and polypep posed to cancer, for monitoring patients undergoing treat tides of the present invention can be used to identify ment for cancer based on the increased level of S100G compounds for the treatment of a Subject experiencing protein(s) in biological fluid Samples of Subjects. negative side effects from the administration of other phar maceuticals, Such as those drugs that disrupt the body's 0730 Methods for diagnosis and prognosis of cancer in a calcium homeostasis. Co-administration of Said compounds Subject may comprise would be useful to counter-effect iatrogenically caused dyS 0731) a) detecting a S100G protein in a biological fluid function of calcium metabolism. Sample obtained from a Subject, and 0727. An antagonist of S100G may be produced using 0732 b) comparing the level of protein detected in the methods which are generally known in the art. In particular, Subject's Sample to the level of protein detected in a control purified S100G may be used to produce antibodies or to Sample, Screen libraries of pharmaceutical agents to identify those which specifically bind S100G. In one example, a positive 0733 wherein an increase in the level of S100G protein Screening for drugs that Specifically inhibit the Ca2+-Sig detected in the Subject's Sample as compared to control naling activity was carried out on the basis of the growth Samples is an indicator of a Subject with cancer of at promoting effect on a yeast mutant with a peculiar pheno increased risk for cancer. type (Shitamukai A et al, Biosci Biotechnol Biochem Sep 0734 The invention also comprises methods for diagno tember 2000;64(9): 1942-6). An inappropriate activation of a sis and prognosis of a Subject with cancer comprising: Signaling pathway in yeast often has a deleterious physi ological effect and causes various defects, including growth 0735) a) contacting a serum sample derived from a sub defects. In a certain genetic background (deltazds1) of ject with a Sample containing S100G protein antigens under Saccharomyces cerevisiae, the cell-cycle progression in G2 conditions Such that a specific antigen-antibody complex is specifically blocked in the medium with CaCl2 by the binding can occur; and hyperactivation of the Ca2+-signaling pathways. Shitamu 0736 b) detecting the presence of immunospecific bind kai et al provide an example of a drug Screening procedure ing of autoantibodies present in the Suject's Serum Samples designed to detect the active compounds that Specifically to the S100G protein; attenuate the Ca2+-signaling activity on the basis of the ability to abrogate the growth defect of the cells Suffering 0737 wherein the presence of immunospecific binding of from the hyperactivated Ca2+Signal. Screening conditions autoantibodies indicates the presence of cancer. were established for the drugs that SuppreSS the Ca2+- 0738 Assays for detection of S100G protein in a sample induced growth inhibition using known calcineurin inhibi can be accomplished by any Suitable method, including tors as model compounds, and an indicator Strain with an immunoassays where in S100G proteins are detected by increased drug Sensitivity was constructed with a Syr1/erg3 their interaction with an S100G specific antibody. In addi null mutation. tion, reagents other than antibodies Such as for example 0728. In another embodiment, a vector expressing the polypeptides that specifically bind S100G may be used. complement of the polynucleotide encoding S100P may be 0739. In yet further embodiments, the S100G protein of administered to a Subject to treat or prevent a neuronal the invention may be useful for the development of specific disorder, immunological disorder, neoplastic disorder or anti-S100 antibodies which are specific for S100 proteins vesicle trafficking disorder including, but not limited to, other than S100G. As several S100 proteins have been those described above. In other embodiments, any of the implicated in disease, it may be advantageous to develop a proteins, antagonists, antibodies, agonists, complementary panel of S 100 specific antibodies to characterize disease, e.g. Sequences or vectors of the invention may be administered cancers. Thus, S100G proteins and antibodies thereto may in combination with other appropriate therapeutic agents. be advantageous to distinguish cancer types. S100G proteins Selection of the appropriate agents for use in combination and antibodies may also be useful in the screening of S100 therapy may be made by one of ordinary skill in the art, Specific antibodies by determining the Selectivity of a given US 2006/0053498A1 Mar. 9, 2006 76 anti-S100 antibody for its target, and eliminating antibodies oped by using any Suitable means known in the art to detect which are cross-reactive with S100G proteins. a conformational change induced by the binding of calcium to the EF-hand domains of the S100G polypeptides. Pref Stuctural Aspects of S100 Proteins of the Invention erably, the conformational change is detected by detecting a change in the ability of the S100G protein to bind a selected 0740 The S100 proteins are a group of low molecular Secondary effector protein mass (approximately 10-12 kDa) acidic Caf'-binding pro teins, so named after the solubility of the first isolated 0744. As the distribution of particular S100 proteins is protein in 100% saturated ammonium sulfate. The most dependent on Specific cell types, the S100 proteins may be Striking conserved feature of these proteins is the presence involved in transducing the Signal of an increase in intrac of an EF-hand. The S100 proteins have two Ca"-binding ellular calcium in a cell type-specific fashion (Wu, T. et al. domains. One of these domains is a basic helix-loop-helix (1997) J. Biol. Chem. 272:17145-17153). domain, the other domain is an acidic helix-loop-helix EF-hand (Kligman, D. and Hilt, D. C. (1988) Trends CaMLP (Calcium Binding Protein) (Clone ID:500742698) Biochem. Sci. 13:437442). The EF-hand domain also 0745 Calcium is one of the “second messengers” which encompasses a part of a region within S100 proteins which relayS chemical and electrical signals within a cell. This specifically identifies members of the S100 family of pro Signal transduction and, hence the regulation of biological teins which have a low affinity for Ca" ions (S100/ICaBP; processes, involves interaction of calcium ion with high PROSITE PS00303, SWISSPROT, PFAM PF01023). The affinity calcium-binding proteins (CBPs). Disclosed herein EF-hand is characterized by a twelve amino acid residue in SEQ ID NOS 184 and 469 is one such protein, encoded containing loop, flanked by two alpha-helices, orientated by the nucleic acid sequences of SEQ ID NOS 15 and 352, approximately 90 degrees with respect to one another. respectively, and the clone FLI 1:500742698 204-614-0- Aspartate (D) and glutamate (E) residues are usually found B2-F, and further referred to herein as CAMLP, which is bordering the twelve amino acid loop. In addition, a con thought to act as a Ca" sensing and binding protein Served glycine residue in the central portion of the loop is involved in diverse aspects of cell proliferation (Such as for found in most Ca"-binding EF-hand domains. Oxygen example of hepatocytes, melanoma cells, leukemic lympho ligands within this domain coordinate the Cat" ion cytes, and HUVEC (human umbilical vein endothelial (Kretsinger, R. H. and Nockolds, C. E. (1973) J. Biol. Chem. cells)) and differentiation. It will be appreciated that all 248:3313-3326). It will also be appreciated by the skilled characteristics and uses of the polynucleotides of SEQ ID artisan that modifications of the S100G polypeptide may NOS:15 and 352 and polypeptides of SEQ ID NO:184 and readily be made based on extensive knowledge of CBP 469, described throughout the present application also per Stucture and Ca(2+) binding mechanisms, Such as Sastry M tain to the human cDNA of clone 500742698, and the et al., (Structure 1998;6:223-23 1), describing the three polypeptides encoded thereby. Notably, the CaMLP dimensional structure of Ca(2+)bound calcyclin and its polypeptide contains EF hand calcium-binding domains implications for Ca(2+}signal transduction by S100. (PROSITE PS00018) at amino acid positions 81-93 and at 0741. Recently, a protein designated S100A13 has been position 129-141 of SEQ ID NO 469. CaMLP discovered and characterized which shared significant pri 0746) The cellular processes in which Ca" functions as mary structure similarity with the S100G polypeptide of the an intracellular Signaling molecule are diverse, including invention. (Wicki et al., (1996) BBRC 227:594-599). cell proliferation and differentiation, neurotransmitter Secre 0742 The binding to calcium induces a conformational tion, glycogen metabolism, and Skeletal muscle contraction. change in the S100 proteins, and this may then affect the Within a resting cell, the concentration of Ca" in the cytosol Secondary effector proteins. This mode of protein-protein is extremely low, <10. Sup.-7 M. However, when the cell is Stimulated by an external Signal, Such as a neural impulse or interaction and modulation of the activity of the Secondary a growth factor, the cytosolic concentration of Ca" effector protein is Similar to that seen with calmodulin, also increases by about 50-fold. This influx of Ca" is caused by containing the EF-hands. The S100G protein of the inven the opening of plasma membrane Ca" channels and the tion comprises an ICaBP type calcium binding domain release of Ca" from intracellular stores such as the endo domain at amino acid positions 9 to 52 and casein kinase II plasmic reticulum. Ca' directly activates regulatory phosphorylation site patterns at amino acid position 7 enzymes, Such as protein kinase C, which trigger Signal (TELE); 34 (SVNE); and 55 (SLDE) of SEQ ID NO 542. transduction pathways. Ca" also binds to specific Ca"- 0743. The S100G polypeptides of the invention may thus binding proteins (CBPs) such as calbindins, troponin C, also be used in any situation in Vivo or in vitro where it is calmodulin, and S-100 proteins which then activate multiple desired to modulate, preferably decrease the level of free target proteins including enzymes, membrane transport calcium, or in applications involving Sensing a change in pumps, and ion channels. Calmodulin (CaM) is the most calcium concentration. In one aspect, S100G nucleic acids widely distributed and the most common mediator of cal and polypeptides may be used to develop a calcium biosen cium effects and appears to be the primary sensor of Ca" Sor. Calcium biosensors may have particular utility in detect changes in eukaryotic cells. The binding of Ca" to CaM ing abnormalities in calcium transport that result in uncom induces marked conformational changes in the protein per pensated influx into, or efflux from, the extracellular fluid, mitting interaction with, and regulation of over 100 different will result in hypercalcaemia or hypocalcaemia, respec proteins. CBP interactions are involved in a multitude of tively. Such abnormalities in Serum calcium concentration cellular processes including, but not limited to, gene regu may have profound effects on neurological, gastrointestinal, lation, DNA 30 Synthesis, cell cycle progression, mitosis, and renal function (Bushinsky DA et al, Lancet 1998 Jul. cytokinesis, cytoskeletal organization, muscle contraction, 25:352 (9124):306-11). Calcium biosensors may be devel Signal transduction, ion homeostasis, exocytosis, and meta US 2006/0053498A1 Mar. 9, 2006 77 bolic regulation (Celio, M. R. et al. (1996) Guidebook to invention may be used in drug Screening processes to Calcium-binding Proteins, Oxford University Press, Oxford, identify selective modulators of both calmodulin and UK, pp. 15-20). CaMLP, thereby identifying compounds having increased potency. Therapeutics 0751. Upon calcium binding, CaMLP may interact with a 0747 The CaMLP protein of SEQ ID NOS 184 and 469 number of protein targets in a calcium dependent manner, disclosed herein provides new calcium binding protein com thereby altering a number of complex biochemical pathways positions useful in the diagnosis, prevention, and treatment that can affect the overall behavior of cells. The calcium of cancer, reproductive disorders, immune disorders, neu calmodulin complex for example controls the biological ronal disorders and developmental disorders. activity of more than thirty different proteins including Several enzymes, ion transporters, receptors, motor proteins, 0748 Calcium binding proteins (CBPs) are implicated in transcription factors, and cytoskeletal components in a variety of disorders and several CBPs have proven to be eukaryotic cells. effective therapeutic targets for which Small molecule inhibitors could be developed. However, while several CBPs 0752. As described in U.S. Pat. No. 5,840,697, Blondelle are targets for widely-used therapeutic treatments, it would et all have peptide inhibitors of calmodulin. A number of be advantageous to provide further CBPs allowing more other calmodulin targeted compounds are known and used Selective therapeutic treatments for disease to be developed. for a variety of therapeutic applications. For instance, chlo Evidence has accumulated for a large number of CBPs rpromazine (Thorazine. RTM.) and related phenothiazine Suggesting involvement in cell proliferative disorders. It is derivatives, disclosed, for example, in U.S. Pat. No. 2,645, proposed that CaMLP may be useful as a tissue specific 640, are calmodulin antagonists useful as tranquilizers and calmodulinhomologue allowing the development of Specific Sedatives. Naphthalenen-Sulfonamides, also calmodulin inhibitors and activators having increased Selectivity and antagonists, are known to inhibit cell proliferation, as dis Safety (decreased side effect profile). To date, calmodulin closed, for example, in Hidaka et al. (1981), PNAS, antagonists are reportedly useful for the treatment of Some 78:43544357) and are useful as antitumor agents. In addi malignant tumors, particularly those of the central nervous tion, the cyclic peptide cyclosporin A (Sandimmune(E), System, as well as lung tumors. The antitumor activity of disclosed in U.S. Pat. No. 4,117,118, is as an immunosup calmodulin antagonists, as well as Successful chemotherapy pressive agent which is thought to work by inhibiting using the same, has been described, for example, in Sculler calmodulin mediated responses in lymphoid cells. et al. Cancer Res., 50:1645-1649 (1990) and Hait et al. 0753. Many existing calmodulin inhibitors have undesir Cancer Res.,50:6636-6640 (1990). U.S. Pat. No. 5,340,565, able biological effects when administered at concentrations additionally describes the use of calmodulin antagonists or sufficient to block calmodulin. These undesirable biological inhibitors as agents which enhance the effectiveness of a effects include non-specific binding to other proteins or chemotherapeutic agent or radiation treatment. Specifically, receptors, as described, for example, in Polak et al., (1991), described therein is a method of inhibiting or killing a tumor J. Neurosci. 11:534-542.) In addition, negative side effects or cancer cell in a human patient undergoing radiation Such as toxicity can occur. A specific example is the toxic therapy or chemotherapy, for example with Such chemo Side effects from cycloSporin A. Therefore, a need exists for therapeutic agents as cisplatin (Platinole), by additionally calmodulin targeted agents, and in particular calmodulin administering a calmodulin binding agent which inhibits antagonists which inhibit calmodulin without having addi calmodulin activity. tional, undesirable biological or Side effects. In particular 0749 Calmodulin is also believed to play a pathogenic there is a need for inhibitors which are specific to calmodulin role in the tissue damage caused by burns and frostbite and which do not have toxic side effects. (Beitner et al., Gen. Pharmac. 20: 641-646, 1989), as well as 0754) In addition, the CaMLP nucleic acids and polypep in dermatitis and other conditions involving keratinocyte tides of the present invention can be used to identify hyperproliferation. The methods of the present invention compounds for the treatment of a Subject experiencing may be applied to the treatment of these and other conditions negative side effects from the administration of other phar wherein antagonism of calmodulin activity is desirable. maceuticals, Such as those drugs that disrupt the body's calcium homeostasis. Co-administration of Said compounds 0750 Thus, as discussed above, CaMLP protein of the would be useful to counter-effect iatrogenically caused dyS invention shares Structural Similarity with the ubiquitous function of calcium metabolism. Such disorders include, but intracellular receptor protein calmodulin Suggesting that are not limited to, organ damage, autoimmune disorders, CaMLP may be useful in the development of selective psychotic disorders, tumors and drug induced dysfunction, CaMLP inhibitors. The nucleic acids and polypeptides of the Such as negative side effects Subsequent to administration of invention thus provide a novel therapeutic target in particu pharmaceuticals. For example, organ or tissue transplanta lar for cell proliferative disorders. In one aspect, Said nucleic tion can result in autoimmune disorders, Such as tissue graft acids and protein may be used in drug Screening processes (allograft) rejections. to develop Selective calmodulin and other calcium binding protein antagonists which do not inhibit the polypeptide of 0755. It is well known that calmodulin-targeted com the invention. In another aspect, the nucleic acids and pounds which are antagonists can be used as immunoSup polypeptides of the invention may be used in drug Screening pressive agents. In addition, also as described above, Such processes to identify selective modulators of the CaMLP compounds are widely used as Sedative or anti-psychotic without inhibiting calmodulin, thereby identifying com agents. Furthermore, there is evidence that calmodulin (and pounds less likely to cause unwanted Side effects. In yet hence CaMLP) antagonists are useful for the treatment of another aspect, the nucleic acids and polypeptides of the Some malignant tumors, particularly those of the central US 2006/0053498A1 Mar. 9, 2006 78 nervous System, as well as lung tumors. The antitumor those skilled in the art including those described in Gold and activity of calmodulin antagonists, as well as Successful Schweiger, M. Methods in Enzymology, Vol. XX, Part C pp. chemotherapy using the Same, has been described, for 537-542, Ed. Moldave, Academic Press, New York and Lon example, in Sculler et al. Cancer Res., 50:1645-1649 (1990) don, 1971 and in the U.S. Pat. No. 4,255,517, which and Hait et al. Cancer Res., 50:6636-6640 (1990), both of disclosures are hereby incorporated by reference in their which are incorporated herein by reference. U.S. Pat. No. entireties. 5,340,565, which is incorporated herein by reference, addi 0758 Lysozymes, which are ubiquitous proteins found in tionally describes the use of calmodulin antagonists or most body Secretions, are defined as 1,4-beta-N-acetylmu inhibitors as agents which enhance the effectiveness of a ramidase which cleave the glycoside bond between the C-1 chemotherapeutic agent or radiation treatment. Specifically, of N-acetyl-muramic acid and the C-4 of N-acetylglu described therein is a method of inhibiting or killing a tumor cosamine in the peptidoglycan of bacteria. They have vari or cancer cell in a human patient undergoing radiation ous therapeutic properties, Such as antiviral, antibacterial, therapy or chemotherapy, for example with Such chemo anti-inflammatory and antihistaminic effects. The activity of therapeutic agents as cisplatin (Platinol), by additionally lysozymes as an anti-bacterial agent appears to be based on administering a calmodulin binding agent which inhibits both its direct bacteriolytic activity and also on Stimulatory calmodulin activity. effects in connection with phagocytosis of polymorpho 0756. It has also been found that extracellular calmodulin nuclear leucocytes and macrophages (Biggar and Sturgess, inhibits TNF release and facilitates elastase release, provid J. M. Infect Immunol. 16: 974-982 (1977); Thacore and ing further suggestion that CaMLP, CaMLP analogues and Willet, Am. Rev. Resp. Dis. 93: 786-790 (1966); Klockars CaMLP receptor agonists are useful agents for regulating the and Roberts, P. Acta Haematol 55: 289-292 (1976)), which inflammatory process. CaMLP antagonists, which include disclosures are hereby incorporated by reference in their CaMLP receptor antagonists and CaMLP -binding mol entireties. Lysozymes have proven to be not only a Selective ecules, may be used to block the interaction of CaMLP with factor but also an effective factor against microorganisms of a receptor, thus providing the opposite effect from CaMLP, the mouth (Iacono et al., J. J. Infect. Immunol. 29: 623-632 its analogues and receptor agonists. CaMLP may serve as a (1980)), which disclosure is hereby incorporated by refer potent modulator of Self-directed inflammation by assisting ence in its entirety. Lysozymes can also kill pathogens by in the recognition of Self vs. non-Self as prokaryotes (e.g., acting Synergistically with other proteins Such as comple bacterial pathogens) do not contain CaMLP. In Some situa ment or antibody to lyse pathogenic cells. Lysozymes, also tions Such as in tumor necrosis, release of extracellular inhibit chemotaxis of polymorphonuclear leukocytes and CaMLP may lead to an inappropriate host response and limit the production of oxygen free radicals following an failure of the immune/inflammatory Systems to eradicate infection. This limits the degree of inflammation, while at tumor cells. Further, a diagnostic test has been developed the same time enhances phagocytosis by these cells. Other which can discern patient variabilities in TNF inhibition by postulated functions of lysozymes include immune Stimu calmodulin and other Substances. This test can be utilized in lation (Jolles, P. Biomedicine 25: 275-276 (1976) Osser monitoring individual patients for determining effective mann, E. F. Adv. Pathobiol 4: 98-102 (1976)) and immuno therapies, and for predicting efficacy of therapy with extra logical and non-immunological monitoring of host cellular CAMLP, CaMLP analogues or CaMLP receptor membranes for any neoplastic transformation (Jolles, P. agonists on the one hand and CaMLP antagonists on the Biomedicine 25: 275-276 (1976); Ossermann, E. F. Adv. other. A diagnostic test for elastase has also been developed Pathobiol 4: 98-102 (1976)), which disclosures are hereby with similar utility. incorporated by reference in their entireties. Lysozymes may thus be used in a wide spectrum of applications (see U.S. Lysozyme C Protein of SEQ ID NO: 196 (Internal Desig Pat. No. 5,618,712, which disclosure is hereby incorporated nation 482.181) and Related Protein of SEQ ID NO:479 by reference in its entirety). Determination of the lysozymes 0757. The polypeptides of SEQ ID NO: 196 and SEQ ID from Serum and/or urine is used to diagnose various diseases NO:479encoded by the cDNA of SEQ ID NO:27 and 362, or as an indicator for their development. In acute lympho respectively, belong to the widely conserved family of blastic leukaemia the lysozyme Serum level is significantly lysozyme C precursors (Prager and Jollés, Lysozymes: reduced, whereas in chronic myelotic leukaemia and in acute model enzymes in biochemistry and biology, ed. Jolles, monoblastic and myelomonocytic leukaemia the lysozyme 9-321 (1996), Qasba and Kumar, Crit. Rev. Biochem. Mol. concentration in the Serum is greatly increased. The thera Biol. 32:255-306 (1997)), which disclosures are hereby peutically effective use of lysozyme is possible in the incorporated by reference in their entireties. The protein of treatment of various bacterial and virus infections (Zona, SEQ ID NOs: 196 and 479 or part thereof plays a role in Herpes Zoster), in colitis, various types of pain, in allergies, glycoprotein and/or peptidoglycan metabolism, probably as inflammation and in pediatrics (the conversion of cows milk a glycosyl hydrolase of family 22. Thus, the protein of the into a form suitable for infants by the addition of lysozyme). invention or part thereof is involved in immune and inflam 0759. The invention relates to methods and compositions matory responses and has antiviral, antibacterial, anti-in using the protein of the invention or part thereof to hydro flammatory and/or anti-histaminic functions. Preferred lyze one or Several Substrates, alone or in combination with polypeptides of the invention are polypeptides comprising other Substances, preferably antiviral, antifungal and/or anti the amino acids of SEQ ID NO:196 from positions 19 to bacterial Substances including but not limited to immuno 100, or from positions 1 to 100. Other preferred polypep globulins, lactoferrin, betalysin, fibronectin, and comple tides of the invention are fragments of SEQ ID NO: 196 ment components. Such Substrates are glycosylated having any of the biological activities described herein. The compounds, preferably containing beta-1-4-glycoside glycolytic activity of the protein of the invention or part bonds, more preferably containing beta-1-4-glycoside bonds thereof may be assayed using any of the assays known to between n-acetylomuraminic acid and n-acetyloglu US 2006/0053498A1 Mar. 9, 2006 79 cosamine. For example, the protein of the invention or part with other hydrolytic enzymes, using techniques well known thereof is added to a sample containing the Substrate(s) in in the art, to form an affinity chromatography column. A conditions allowing hydrolysis, and allowed to catalyze the Sample containing the undesirable Substrate is run through hydrolysis of the Substrate(s). In a preferred embodiment, the column to remove the Substrate. Immobilizing the pro the hydrolysis is carried out using a Standard assay Such as tein of the invention or part thereof on a Support advanta those described by Gold and Schweiger, Supra, and U.S. Pat. geous is particularly for those embodiments in which the Nos. 5,871,477 and 4,255,517, which disclosures are hereby method is to be practiced on a commercial Scale. This incorporated by reference in their entireties. In a preferred immobilization facilitates the removal of the enzyme from embodiment, the protein of the invention or part thereof may the batch of product and Subsequent reuse of the enzyme. be used to lyze recombinant bacteria in order to recover the Immobilization of the protein of the invention or part thereof recombinant DNA, the recombinant protein of interest, or can be accomplished, for example, by inserting a cellulose both using, for example, any of the assays described in binding domain in the protein. One of skill in the art will Sambrook, et al., Molecular Cloning: A Laboratory Manual, understand that other methods of immobilization could also Second Edition, Cold Spring Harbor Laboratory Press be used and are described in the available literature. Alter (1989), which disclosure is hereby incorporated by reference natively, the same methods may be used to identify new in its entirety. Substrates. 0760. In an embodiment, the protein of the invention or 0761. In addition, the protein of the invention or part part thereof is used to hydrolyze contaminating Substrates, thereof may be useful to identify or quantify the amount of preferably exogenous Substrates from bacterial, fungal or a given Substrate in biological fluids, foods, water, air, Viral origins, in an aqueous Sample or onto a material, Solutions and the like. In a preferred embodiment, the preferably glassware and plasticware. In particular, the pro protein of the invention or part thereof is used in assays and tein of the invention or part thereof may be used as a diagnostic kits for the identification and quantification of disinfectant in dental rinse, in protection of aqueous Systems exogenous Substrates in bodily fluids including blood, or in preparing material for medical applications using any lymph, Saliva or other tissue Samples, in addition to bacte of the methods and compositions described in U.S. Pat. Nos. rial, fungal, plant, yeast, Viral or mammalian cell cultures. In 5,069,717, 4,355,022 and 5,001,062, which disclosures are a preferred embodiment, the protein of the invention or part hereby incorporated by reference in their entireties. In a thereof is used to detect, identify, and or quantify eubacteria preferred embodiment, the protein of the invention is used as using reagents and assays described in U.S. Pat. No. 5,935, a host resistance factor in infants formulas to convert cow's 804, which disclosure is hereby incorporated by reference in milk into a form more Suitable for infants as described in its entirety. Briefly, the protein of the invention of part U.S. Pat. No. 6,020,015, which disclosure is hereby incor thereof is catalytically inactived, i.e. capable of binding but porated by reference in its entirety. In another preferred not cleaving a peptidoglycan comprising NAc-muramic acid embodiment, the protein of the invention or part thereof may in the eubacteria, using any of the methods known to those be used as a food preservative (see Hayashi et al., Agric. skilled in the art including those which produce a mutant Biol. Chem. (European Edition of Japanese Journal of enzyme, a recombinant-enzyme, or a chemically inactivated Agriculture, Biochemistry and Chemistry), Vol. 53, pp. enzyme. The catalytically inactive protein of the invention is 3173-3177, 1989), which disclosure is hereby incorporated then incubated with an aliquot of a biological Sample under by reference in its entirety. In addition, the protein of the conditions Suitable for binding of the inactive enzyme to the invention or part thereof may be used to clarify Xanthan gum peptidoglycan Substrate. Then, the bound enzyme is detected fermented broth for applications in food and in cosmetic to assess the presence or amount of the eubacteria in the industries using the method described in U.S. Pat. No. biological Sample. 5,994,107, which disclosure is hereby incorporated by ref 0762. In another embodiment, the nucleic acid of the erence in its entirety. In another preferred embodiment, invention or part thereof may be used to increase disease compositions comprising the protein of the present invention resistance of plants to bacterial, fungal and/or viral infec or part thereof are added to Samples or materials as a tions. A polynucleotide containing the nucleic acid of the “cocktail” with other antimicrobial substances, preferably invention or part thereof is introduced into the plant genome antibiotics or hydrolytic enzymes Such as those described in in conditions allowing correct expression of the transgenic U.S. Pat. Nos. 5,458.876 and 5,041,326, which disclosures are hereby incorporated by reference in their entireties, to protein using any methods known to those skilled in the art decontaminate the Samples. For example, the protein of the including those disclosed in U.S. Pat. Nos. 5,349,122 and invention or part thereof may be used in place or in com 5,850,025, which disclosures are hereby incorporated by bination with antibiotics in cell cultures. The advantage of reference in their entireties. using a cocktail of hydrolytic enzymes is that one is able to 0763. In another preferred embodiment, the protein of the hydrolyze a wide range of Substrates without knowing the invention or part thereof may be useful to treat and/or Specificity of any of the enzymes. Using a cocktail of prevent bacterial, fungal and Viral infections in humans or in hydrolytic enzymes also protects a Sample or material from animals caused by various agents including but not limited a wide range of future unknown contaminants from a vast to Streptococcus, Veillonella alcalescens, Actinomyces, Her number of Sources. For example, the protein of the invention pes simplex, Candida albicans, MicrococcuS lySOdeikticuS or part thereof is added to Samples where contaminating and HIV by hydrolyzing the glycosylated compounds con Substrates, preferably exogenous Substrates from bacterial, tained in Such micro-organisms. In Still a preferred embodi fungal or viral origins, is undesirable in an amount Sufficient ment, the protein of the invention or part thereof is used to to promote hydrolysis of Said Substrates. Alternatively, the prevent and/or treat bacterial, fungal and Viral infections in protein of the invention or part thereof may be bound to a immunocompromised individuals who lack fully functional chromatographic Support, either alone or in combination immune Systems, Such as neonates or geriatric patients or US 2006/0053498A1 Mar. 9, 2006

HIV-infected individuals, or who suffer from a disease atic ribonucleases are pyrimidic-specific ribonucleases affecting the respiratory tract Such as cystic fibrosis or the present in high quantity in the pancreas of a number of gastrointestinal tract Such as ulcerative colitis or Sprue. mammalia taxa and of a few reptiles. In addition to their 0764. In still another embodiment, the protein of the function in hydrolysis of RNA, ribonucleases have evolved invention or part thereof may be used as a growth factor for to Support a variety of other physiological activities. Such in vitro cell culture, preferably for T cells and T cell lines, activities include anti-parasite, anti-bacterium, anti-Virus, using techniques and methods taught in U.S. Pat. No. anti-neoplastic activities, neurotoxicity, and angiogenesis. 5,468,635, which disclosure is hereby incorporated by ref For example, bovine Seminal ribonuclease is anti-neoplastic erence in its entirety. (Laceetti, et al. (1992) Cancer Res. 52: 4582-4586, which disclosure is hereby incorporated by reference in its 0765. In addition, the protein of the invention or part entirety). Some frog ribonucleases display both anti-viral thereof may be used to identify inhibitors for mechanistic and anti-neoplastic activity (Youle, et al. (1994) Proc. Natl. and clinical applications. Such inhibitors may then be used Acad. Sci. USA 91: 6012-6016; Mikulski, et al. (1990) J. to identify or quantify the protein of the invention in a Natl. Cancer Inst.82: 151-152; and Wu, et al. (1993) J. Biol. Sample, and to diagnose, treat or prevent any of the disorders Chem. 268: 10686-10693), which disclosures are hereby where the protein's hydrolytic, immunostimulatory and/or incorporated by reference in their entireties. Eosinophil inflammatory activities is/are undesirable and/or deleterious derived neurotoxin (EDN) and eosinophil cationic protein including but not limited to amyloidosis, colitis, lySOSomal (ECP) are related ribonucleases which possess neurotoxicity diseases, inflammatory and immune disorders including (Beintema, et al. (1988) Biochemistry 27:45304538; Ack allergies and leukaemia. The protein of the invention may erman, (1993) In Makino, and Fukuda, Eosinophils: Bio also be used to monitor host cell membranes for neoplastic logical and Clinical Aspects. CRC Press, Boca Raton, Fla., transformation. pp 33-74), which disclosures are hereby incorporated by 0766. It will be appreciated that all characteristics and reference in their entireties. In addition, ECP exhibits cyto uses of the polynucleotides of SEQ ID NOS:27 and 362 and toxic, anti-parasitic, and anti-bacterial activities. A EDN polypeptides of SEQ ID NO:196 and 479, described related ribonuclease, named RNase k6, is shown to express throughout the present application also pertain to the human in normal human monocytes and neutrophils, Suggesting a cDNA of clone 482181, and the polypeptides encoded role for this ribonuclease in host defense (Rosenberg, and thereby. Dyer, (1996) Nuc. Acid. Res. 24: 3507-3513), which dis Angiogenin Protein of SEQ ID NO: 176 (Internal Designa closure is hereby incorporated by reference in its entirety. tion 114180) and Related Protein of SEQ ID NO:461 0769 Angiogenin is a tRNA-specific ribonuclease which 0767 The polypeptides of SEQ ID NO:176 and SEQ ID binds protein partners on the Surface of endothelial cells for NO:461 encoded by the extended cDNASEQ ID NO:7 and endocytosis. Potential partners of angiogenin include hep SEQ ID NO:344, respectively, are ribonucleases that arin, plasminogen, elastase, angiostatin, actin, and a 170 belongs to the pancreatic ribonuclease family (see reviews kDa receptor on the surface of endothelial cells Strydom, from Beintema, (1998) Cell. Mol. Life Sci. 54:763-5; (1998) Cell. Mol. Life Sci. 54,811-824, which disclosure is Beintema and Kleineidam, (1998) Cell. Mol. Life Sci. hereby incorporated by reference in its entirety . Endocy 54:825-32, which disclosures are hereby incorporated by tosed angiogenin is translocated to the nucleus where it reference in their entireties). It will be appreciated that all promotes endothelial invasiveness required for blood vessel characteristics and uses of the polynucleotides of SEQ ID formation (Moroianu, and Riordan, (1994) Proc. Natl. Acad. NO:7 and SEQ ID NO:344 and polypeptides of SEQ ID Sci. USA 91: 1217-1221, which disclosure is hereby incor NO:176 and SEQ ID NO:461, described throughout the porated by reference in its entirety). present application also pertain to the human cDNA of clone 114180, and the polypeptides encoded thereby. In addition, 0770 Although originally isolated from medium condi the protein of the invention plays a role in angiogenesis as tioned by human colon cancer cells (Fett et al. (1985), an angiogenin variant protein (see review from Badet, Supra), and Subsequently shown to be produced by several (1999) Pathol. Biol. 74:345-51, which disclosure is hereby other histological types of human tumors Rybak, et al. incorporated by reference in its entirety). Preferred polypep (1987) Biochem. Biophys. Res, Commun. 146, 1240-1248; tides of the invention are polypeptides comprising the amino Olson, et al., (1995) Proc. Natl. Acad. Sci. U.S.A. 92, acids of SEQ ID NO: 176 from positions 19 to 75, from 442446, which disclosures are hereby incorporated by ref positions 26 to 75, or from positions 63 to 69. Other erence in their entireties, angiogenin also is a constituent of preferred polypeptides of the invention are fragments of human plasma and normally circulates at a concentration of SEQ ID NO: 176 having any of the biological activity 250-360 ng/ml Shimoyama, et al. (1996) Cancer Res. 56, described herein. The ribonuclease activity of the protein of 2703-2706; Blaser, et al. (1993) Eur. J. Clin. Chem. Clin. the invention or part thereof may be assayed using any of the Biochem. 31, 513-516, which disclosures are hereby incor assays known to those skilled in the art including those porated by reference in their entireties. It has also been described in U.S. Pat. No. 5,866,119, which disclosure is shown that recurrent gastric cancer patients had a much hereby incorporated by reference in its entirety. The angio higher Serum concentration of angiogenin than primary genic activity of the protein of the invention or part thereof gastric cancer patients Shimoyama, and Kaminishi, (2000) may be assayed using any of the assays known to those J. Cancer Res. Clin. Oncol. 126, 468474, which disclosure skilled in the art including those described by Fett et al. is hereby incorporated by reference in its entirety. (1985) Biochem. 24, 5480-5486, which disclosure is hereby 0771 Angiogenin is a potent inducer of angiogenesis incorporated by reference in its entirety. Fett, et al. Supra). Angiogenesis is a complex process of 0768 Ribonucleases are proteins which catalyze the blood vessel formation comprising of Several Separate but hydrolysis of phosphodiester bonds in RNA chains. Pancre interconnected Steps at the cellular and biochemical level US 2006/0053498A1 Mar. 9, 2006 including: (i) activation of endothelial cells by the action of the substrate. Immobilizing the protein of the invention or an angiogenic stimulus, (ii) adhesion and invasion of acti part thereof on a Support is particularly advantageous for Vated endothelial cells into the Surrounding tissues and those embodiments in which the method is to be practiced on migration toward the Source of the angiogenic Stimulus, and a commercial Scale. This immobilization facilitates the (iii) proliferation and differentiation of endothelial cells to removal of the enzyme from the batch of product and form a new microvasculature Folkman, and Shing, (1992) Subsequent reuse of the enzyme. Immobilization of the J. Biol. Chem. 267, 10931-10934; Moscatelli, and Rifkin, protein of the invention or part thereof can be accomplished, (1988) Biochim. Biophys. Acta 948, 67-85, which disclo for example, by inserting a cellulose-binding domain in the Sures are hereby incorporated by reference in their entire protein. One of skill in the art will understand that other ties. While angiogenesis is a tightly-controlled process methods of immobilization could also be used and are under usual physiological conditions, abnormal angiogen described in the available literature. Alternatively, the same esis can have devastating consequences in pathological methods may be used to identify new Substrates. conditions Such as arthritis, diabetic retinopathy and tumor growth. It is now well-established that the growth of virtu 0774. In another embodiment, the protein of the inven ally all Solid tumors is angiogenesis dependent Folkman, tion or part thereof may be used to decontaminate or (1989) J. Natl. Cancer Inst. 82, 4-6, which disclosure is disinfect Samples infected by undesirable parasite, bacteria hereby incorporated by reference in its entirety. Angiogen and/or viruses using any of the methods known to those esis is also a prerequisite for the development of metastasis, skilled in the art including those described in Youle et al., Since it provides the means whereby tumor cells disseminate (1994), supra; Mikulski et al (1990) supra, Wu et al (1993) from the original primary tumor and establish at distant Sites Supra. Mahadevan, and Hart, (1990) Rev. Oncol. 3, 97-103; 0775. In another embodiment, the present invention Blood, and Zetter (1990) Biochim. Biophys. Acta 1032, relates to compositions and methods using the protein of the 89-118, which disclosures are hereby incorporated by ref invention or part thereof to selectively kill cells. The protein erence in their entireties. Therefore, interference with the of the invention or part thereof is linked to a recognition process of tumor-induced angiogenesis can be an effective moiety capable of binding to a chosen cell, Such as lectins, therapy for both primary and metastatic cancers. Indeed, receptors or antibodies thus generating cytotoxic reagents Several anti-angiogenic agents have been produced and are using methods and techniques described in U.S. Pat. No. currently in the clinical trial Stage. 5,955,073, which disclosure is hereby incorporated by ref 0772 The invention relates to methods and compositions erence in its entirety. using the protein of the invention or part thereof to hydro 0776. In still another embodiment, the invention relates lyze one or Several Substrates, preferably nucleic acids, more to compositions and methods using the protein of the preferably RNA, alone or in combination with other sub invention or part thereof to stimulate cell proliferation both stances. For example, the protein of the invention or part in vitro and in Vivo, especially endothelial cell growth. For thereof is added to a sample containing the Substrate(s) in example, Soluble forms of the protein of the invention or part conditions allowing hydrolysis, and allowed to catalyze the thereof may be added to cell culture medium in an amount hydrolysis of the substrate(s). Hydrolysis conditions as effective to stimulate cell proliferation. described in the U.S. Pat. No. 5,866,119 may be used, which disclosure is hereby incorporated by reference in its entirety. 0777. In still another embodiment, the protein of the invention or part thereof may be used in the diagnosis, 0773) In a preferred embodiment, the protein of the prevention and/or treatment of disorders associated with invention or part thereof may be used to remove contami excessive angiogenesis Such as tumor growth, arthritis or nating RNA in a biological Sample, alone or in combination diabetic retinopathy. with other nucleases. In a more preferred embodiment, the protein of the invention or part thereof may be used to purify 0778 In a preferred embodiment, the protein of the DNA preparations from contaminating RNA, to remove invention may be used as a diagnostic marker to evaluate the RNA templates prior to Second Strand Synthesis and prior to risk of a given individual to develop a tumor, to evaluate the analysis of in vitro translation products. Compositions com risk of recurrence of tumors or to evaluate the degree of prising the protein of the present invention or part thereofare cancer aggressiveness based on the facts that the level of added to biological Samples as a "cocktail” with other circulating angiogenin is lower in normal individuals than in nucleases. The advantage of using a cocktail of hydrolytic patients bearing tumors, and is lower in patients with pri enzymes is that one is able to hydrolyze a wide range of mary cancers compared to patients with reoccurent tumors, Substrates without knowing the Specificity of any of the as Stated above. Thus, quantitative immunoassays can be enzymes. Such cocktails of nucleases are commonly used in used for the detection of abnormal levels of either the protein molecular biology assays, for example to remove unbound of SEQ ID NO: 176 or the mRNA encoding such protein as RNA in RNASe protection assays. Using a cocktail of the polynucleotide of SEQ ID No.:7, thereby identifying hydrolytic enzymes also protects a Sample from a wide those individuals at risk for the development of tumors or the range of future unknown RNA contaminants from a vast recurrence of tumors. Detection of abnormal levels of the number of Sources. For example, the protein of the invention protein of the invention may be performed using any tech or part thereof is added to Samples where contaminating niques known to those skilled in the art including those Substrates is undesirable. Alternatively, the protein of the described elsewhere in the application. For example, anti invention or part thereof may be bound to a chromatographic bodies binding specifically to the protein of the invention, or Support, either alone or in combination with other hydrolytic fragments thereof, may be used in routine immunoassays to enzymes, using techniques well known in the art, to form an Screen for the presence or absence of the protein of the affinity chromatography column. A Sample containing the invention, or fragments thereof. Alternatively, the nucleic undesirable Substrate is run through the column to remove acids which encode the protein of the invention, or frag US 2006/0053498A1 Mar. 9, 2006 82 ments thereof, may be used in hybridization assays to detect digestion. For example, the absence or insufficiency of a and/or quantity the expression of Said protein. protease can result in a pathological condition that can be 0779) Another aspect of the invention provides for mol treated by replacement or augmentation therapy. Such thera ecules which inhibit, or reduce, the biological activity or pies include the treatment of hemophilia with clotting fac expression of SEQ ID NO: 276. Such molecules may be be tors VIII, IX, and VIIa. In another application, the pro administered to patients to prevent vascularization, espe teolytic enzyme tissue plasminogen activator (t-PA) is used cially tumor vascularization, thereby limiting tumor growth. to activate the body's clot lysing mechanism, thereby reduc Such antagonists and/or inhibitors may be antibodies Spe ing morbitity resulting from myocardial infarction. The cific for the protein of the invention that can be used directly protease thrombin is used to initiate the clotting of fibrino as an antagonist, or indirectly as a targeting or delivery gen-based tissue adhesives during Surgery. Neutrophils pro mechanism for bringing a pharmaceutical agent to cells or duce Several antibacterial Serine proteases (Gabay, Ciba tissue which express the protein of the invention. Neutral Found. Symp. 186:237-247, 1994; Scocchi et al., Eur. J. izing antibodies, (i.e., those which inhibit protein-protein Biochem. 209:589-595, 1992, which disclosures are hereby interactions) are especially preferred for therapeutic use. incorporated by reference in their entireties). Proteases also Alternatively, Such molecules may be mutated forms of the regulate cellular processes through receptor-mediated path protein of the invention or truncated forms which will be ways by proteolytic activation of the cognate receptor (Vu et able to bind to the partners of the protein of the invention and al., Cell 64:1057-1068, 1991; Blackhartet al., J. Biol. Chem. compete with if for partners but without eliciting any of its 271:16466-16471, 1996, which disclosures are hereby biological activities. Other methods to inhibit the expression incorporated by reference in their entireties). of the protein of the invention include antisense and triple 0783. Overproduction or lack of regulation of proteases helix Strategies as described herein. Other antagonists or can also have pathological consequences. Elastase, released inhibitors of the protein of the invention may be produced within the lung in response to the presence of foreign using methods which are generally known in the art, includ particles, can damage lung tissue if its activity is not tightly ing the Screening of libraries of pharmaceutical agents to regulated. Emphysema in SmokerS is believed to arise from identify those which specifically bind the protein of the an imbalance between elastase and its inhibitor, alpha-1- invention. The protein of the invention, or part thereof, antitrypsin. This balance may be restored by administration preferably its functional or immunogenic fragments, or of exogenous alpha-1-antitrypsin. oligopeptides related thereto, can be used for Screening libraries of compounds in any of a variety of drug Screening 0784. In addition, protease inhibitors have been shown to techniques including those described herein. inhibit the growth of microorganisms including human pathogenic bacteria Such as Strains of group A Streptococci, Protease Inhibitor Protein of SEQ ID NO: 181 (Internal including antibiotic-resistant Strains (Merigan, T. et al Designation 1000771934) and Related Protein of SEQ ID (1996) Ann Intern Med 124:1039-1050; Stoka, V. (1995) NO:466 FEBS. Lett 370:101-104; Vonderfecht, S. etal (1988) J Clin 0780. The protein of SEQ ID NOs: 181 and 466 encoded Invest 82:2011-2016; Collins, A. et al (1991) Antimicrob by the extended cDNA SEQ ID NO:12 and 349, respec Agents Chemother 35:2444-2446, which disclosures are tively, is a protease inhibitor belonging to the WAP-type hereby incorporated by reference in their entireties). disulfide core family. It will be appreciated that all charac teristics and uses of the polynucleotides of SEQ ID NOS:12 0785. In view of the growing use of proteases in industry, and 349 and polypeptides of SEQ ID NOs: 181 and 466, research, and medicine, there is an ongoing need in the art described throughout the present application also pertain to for new enzymes and new enzyme inhibitors. The present the human cDNA of clone 1000771934, and the polypep invention addresses these needs. tides encoded thereby. Preferred polypeptides of the inven 0786 The invention relates to compositions and methods tion are polypeptides comprising the amino acid fragments using the protein of the invention or part thereof to inhibit of SEQID NO: 181 from positions 32 to 73, 49 to 62, 76 to proteases, both in vitro or in Vivo. Since proteases play an 122,97 to 110 or any combination thereof. Other preferred important role in the regulation of many biological processes polypeptides of the invention are fragments of SEQ ID in Virtually all living organisms as well as a major role in NO:181 having any of the biological activity described diseases, inhibitors of proteases are useful in a wide variety herein. The protease inhibitor activity of the protein of the of applications. invention or part thereof may be assessed using any tech niques known to those skilled in the art. Possible substrates 0787. In one embodiment, the protein of the invention or for the protein of the invention include are not limited to part thereof may be useful to quantify the amount of a given trypsin, chymotrypsin, leukoproteinase, elastase, Subtilisin, protease in a biological Sample, and thus used in assays and type IV collagenase and other Serine proteases. diagnostic kits for the quantification of proteases in bodily 0781 Proteases, which cleave proteins, are largely used fluids or other tissue Samples, in addition to bacterial, fungal, in industry including in food processing, brewing, and plant, yeast, Viral or mammalian cell cultures. In a preferred alcohol production. Proteases are important components of embodiment, the Sample is assayed using a Standard pro laundry detergents and other products. Within biological tease Substrate. A known concentration of protease inhibitor research, proteases are used in purification processes to is added, and allowed to bind to a particular protease present. degrade unwanted proteins. It is often desirable to employ The protease assay is then rerun, and the loSS of activity is proteases of low Specificity or mixtures of more specific correlated to the protease inhibitor activity using techniques proteases to obtain the necessary degree of degradation. well known to those skilled in the art. 0782 Proteases are also key components of a broad range 0788. In addition, the protein of the invention or part of biological pathways, including blood coagulation and thereof may be used to remove, identify or inhibit contami US 2006/0053498A1 Mar. 9, 2006 nating proteases in a Sample. Compositions comprising the Serpin Protein of SEQ ID NO: 179 (Internal Designation polypeptides of the present invention may be added to 784.093) and Related Protein of SEQ ID NO:464 biological Samples as a “cocktail” with other protease inhibi tors to prevent degradation of protein Samples. The advan 0793. The protein of SEQ ID NOs: 179 and 464 and tage of using a cocktail of protease inhibitors is that one is encoded by the extended cDNA SEQ ID NOS:10 and 347, able to inhibit a wide range of proteases without knowing the respectively, is a Serine protease inhibitor. It will be appre Specificity of any of the proteases. Using a cocktail of ciated that all characteristics and uses of the polynucleotides protease inhibitors also protects a protein Sample from a of SEQ ID NOS:10 and 347 and polypeptides of SEQ ID wide range of future unknown proteases which may con NOs: 179 and 464, described throughout the present appli taminate a protein Sample from a vast number of Sources. cation also pertain to the human cDNA of clone 784.093, and For example, the protein of the invention or part thereof are the polypeptides encoded thereby. Preferred polypeptides of added to Samples where proteolytic degradation by contami the invention are polypeptides comprising the amino acid nating proteases is undesirable. Such protease inhibitor fragments of SEQ ID NO:179 from positions 47 to 139. cocktails (see for example the ready to use cocktails Sold by Other preferred polypeptides of the invention are fragments Sigma) are widely used in research laboratory assays to of SEQ ID NO:179 having any of the biological activity inhibit proteases Susceptible of degrading a protein of inter described herein. The protease inhibitor activity of the est for which the assay is to be performed. Alternatively, the protein of the invention or part thereof may be assessed protein of the invention or part thereof may be bound to a using any techniques known to those skilled in the art chromatographic Support, either alone or in combination including those disclosed in the U.S. Pat. No. 5,955,284. with other protease inhibitor, using techniques well known Possible substrates for the protein of the invention include in the art, to form an affinity chromatography column. A are not limited to Serine proteaseS Such as elastase, trypsin, Sample containing the undesirable protease is run through chymotrypsin, thrombin III, plasmin, heparin, complement the column to remove the protease. Alternatively, the same II, plasminogen activator, protein C, interleukin-IB covert methods may be used to identify new proteases. ing enzyme, preferably trypsin, elastase, and chymotrypsin. 0789. In a preferred embodiment, the protein of the 0794. Proteases are key components of a broad range of invention or part thereof may be used to inhibit proteases biological pathways, including blood coagulation and diges implicated in a number of diseases where cellular proteoly tion. For example, the absence or insufficiency of a protease sis occur Such as diseases characterized by tissue degrada can result in a pathological condition that can be treated by tion including but not limited to arthritis, muscular dystro replacement or augmentation therapy. Such therapies phy, inflammation, tumor invasion, glomerulonephritis, include the treatment of hemophilia with clotting factors parasite-borne infections, Alzheimer's disease, periodontal VIII, IX, and VIIa. In another application, the proteolytic enzyme tissue plasminogen activator (t-PA) is used to acti disease, and cancer metastasis. Vate the body's clot lysing mechanism, thereby reducing 0790. In another preferred embodiment, the protein of the morbitity resulting from myocardial infarction. The protease invention or part thereof may be useful to inhibit exogenous thrombin is used to initiate the clotting of fibrinogen-based proteases, both in Vivo and in vitro, implicated in a number tissue adhesives during Surgery. Neutrophils produce Several of infectious diseases including but not limited to gingivitis, antibacterial Serine proteases (Gabay, Ciba Found. Symp. malaria, leishmaniasis, filariasis, osteoporosis and Osteoar 186:237-247, 1994; Scocchi et al., Eur. J. Biochem. thritis, and other bacterial, and parasite-borne or viral infec 209:589-595, 1992, which disclosures are hereby incorpo tions. In particular, the protein of the invention or part rated by reference in their entireties). Proteases also regulate thereof may offer applications in Viral diseases where the cellular processes through receptor-mediated pathways by proteolysis of primary polypeptide precursors is essential to proteolytic activation of the cognate receptor (Vu et al., Cell the replication of the virus, as for HIV and HCV. 64:1057-1068, 1991; Blackhart et al., J. Biol. Chem. 271:16466-16471, 1996, which disclosures are hereby 0791. In another embodiment, the protease inhibitors of incorporated by reference in their entireties). the present invention may be used as antibacterial agents to retard or inhibit the growth of certain bacteria either in vitro 0795 Overproduction or lack of regulation of proteases or in Vivo. Particularly, the polypeptides of the present can also have pathological consequences. Elastase, released invention may be used to inhibit the growth of group A within the lung in response to the presence of foreign Streptococci on non-living matter Such as Surgical instru particles, can damage lung tissue if its activity is not tightly ments, laboratory glassware and plasticware, and in culture regulated. Emphysema in SmokerS is believed to arise from of living plant, fungi, and animal cells. an imbalance between elastase and its inhibitor, alpha-1- antitrypsin. This balance may be restored by administration 0792 Furthermore, the protease inhibitors of the present of exogenous alpha-1-antitrypsin. invention find use in drug potentiation applications. For example, therapeutic agents Such as antibiotics or antitumor 0796) The serine proteases (SP) are a large family of drugs can be inactivated through proteolysis by endogenous proteolytic enzymes that include the digestive enzymes, proteases, thus rendering the administered drug less effec trypsin and chymotrypsin, components of the complement tive or inactive. Accordingly, the protease inhibitors of the cascade and of the blood-clotting cascade, and enzymes that invention may be administered to a patient in conjunction control the degradation and turnover of macromolecules of with a therapeutic agent in order to potentiate or increase the the extracellular matrix. SP are so named because of the activity of the drug. This co-administration may be by presence of a Serine residue in the active catalytic Site for Simultaneous administration, Such as a mixture of the pro protein cleavage. They are characterized by a catalytic triad tease inhibitor and the drug, or by Separate Simultaneous or of Serine, histidine, and aspartic acid residues. SP have a Sequential administration. wide range of Substrate Specificities and can be Subdivided US 2006/0053498A1 Mar. 9, 2006 84 into Subfamilies on the basis of these Specificities. The main able to inhibit a wide range of proteases without knowing the Sub-families are trypases (cleavage after arginine or lysine), Specificity of any of the proteases. Using a cocktail of aspases (cleavage after aspartate), chymases (cleavage after protease inhibitors also protects a protein Sample from a phenylalanine or leucine), metases (cleavage after methion wide range of future unknown proteases which may con ine), and Serases (cleavage after Serine). taminate a protein Sample from a vast number of Sources. 0797 Serine proteases are used for a variety of industrial For example, the protein of the invention or part thereof are purposes. For example, the Serine protease Subtilisin is used added to Samples where proteolytic degradation by contami in laundry detergents to aid in the removal of proteinaceous nating proteases is undesirable. Such protease inhibitor stains (e.g., Crabb, ACS Symposium Series 460:82-94, cocktails (see for example the ready to use cocktails Sold by 1991, which disclosure is hereby incorporated by reference Sigma) are widely used in research laboratory assays to in its entirety). In the food processing industry, Serine inhibit proteases Susceptible of degrading a protein of inter proteases are used to produce protein-rich concentrates from est for which the assay is to be performed. Alternatively, the fish and livestock, and in the preparation of dairy products protein of the invention or part thereof may be bound to a (Kida et al., Journal of Fermentation and Bioengineering chromatographic Support, either alone or in combination 80:478-484, 1995; Haard and Simpson, in Martin, A. M., with other protease inhibitor, using techniques well known ed., Fisheries Processing: Biotechnological Applications, in the art, to form an affinity chromatography column. A Chapman and Hall, London, 1994, 132-154; Bos et al., Sample containing the undesirable protease is run through European Patent Office Publication 494 149 A1, which the column to remove the protease. Alternatively, the same disclosures are hereby incorporated by reference in their methods may be used to identify new proteases. entireties). 0802. In a preferred embodiment, the protein of the 0798 Serpins are irreversible serine protease inhibitors invention or part thereof may be used to inhibit proteases which are principally located extracellularly. Proteins which implicated in a number of diseases where cellular proteoly have been assigned to the Serpin family include the follow sis occur Such as diseases characterized by tissue degrada ing: alpha.-1 protease inhibitor, alpha.-1-antichymotrypsin, tion including but not limited to arthritis, muscular dystro antithrombin III, alpha.-2-antiplasmin, heparin cofactor II, phy, inflammation, tumor invasion, glomerulonephritis, complement C1 inhibitor, plasminogen activator inhibitorS 1 parasite-borne infections, Alzheimer's disease, periodontal and 2, glia derived nexin, protein C inhibitor, rat hepatocyte disease, and cancer metastasis. In a more preferred embodi inhibitors, crma (a viral serpin which inhibits interleukin ment, the invention relates to compositions and methods to 1-beta. cleavage enzyme), human Squamous cell carcinoma use the protein of the invention or part thereof in diseases antigen which may modulate the host immune response characterized by an abnormally elevated levels of trypsin, against tumor cells, human maspin which Seems to function chymotrypsin or elastase, including but not limited to as a tumor Suppressor, lepidopteran protease inhibitor, leu chronic emphysema of the lungs, cirrhosis, liver failure, kocyte elastase inhibitor (the only known intracellular Ser cystic fibrosis, alpha1-antitrypsin deficiency associated dis pin), and products from three orthopoxviruses (these prod orderS Such as aneurysm or toxic shock. For prevention ucts may be involved in the regulation of the blood clotting and/or treatment purposes, the protein of the invention may cascade and/or of the complement cascade in the mamma be used using any of the gene therapy methods described lian host). herein or known to those skilled in the art. 0799. In view of the growing use of proteases in industry, 0803. In another preferred embodiment, the protein of the research, and medicine, there is an ongoing need in the art invention or part thereof may be useful to inhibit exogenous for new enzymes and new enzyme inhibitors. The present proteases, both in Vivo and in vitro, implicated in a number invention addresses these needs. of infectious diseases including but not limited to gingivitis, malaria, leishmaniasis, filariasis, osteoporosis and Osteoar 0800 In one embodiment, the protein of the invention or thritis, and other bacterial, and parasite-borne or viral infec part thereof may be useful to quantify the amount of a given tions. In particular, the protein of the invention or part protease in a biological Sample, and thus used in assays and thereof may offer applications in Viral diseases where the diagnostic kits for the quantification of proteases in bodily proteolysis of primary polypeptide precursors is essential to fluids or other tissue Samples, in addition to bacterial, fungal, the replication of the virus, as for HIV and HCV. plant, yeast, Viral or mammalian cell cultures. In a preferred embodiment, the Sample is assayed using a Standard pro 0804. In another embodiment, the protease inhibitors of tease Substrate. A known concentration of protease inhibitor the present invention may be used as antibacterial agents to is added, and allowed to bind to a particular protease present. retard or inhibit the growth of certain bacteria either in vitro The protease assay is then rerun, and the loSS of activity is or in Vivo. Particularly, an amount of the polypeptides of the correlated to the protease inhibitor activity using techniques present invention effective to inhibit proliferation may be well known to those skilled in the art. Preferred proteases in used to inhibit the growth of group A Streptococci on this embodiment are Serine protease, more preferably non-living matter Such as Surgical instruments, laboratory elastase, trypsin and chymotrypsin. glassware and plasticware, and in culture of living plant, fungi, and animal cells. 0801. In addition, the protein of the invention or part thereof may be used to remove, identify or inhibit contami 0805. Furthermore, the protease inhibitors of the present nating proteases in a Sample. Compositions comprising the invention find use in drug potentiation applications. For polypeptides of the present invention may be added to example, therapeutic agents Such as antibiotics or antitumor biological Samples as a “cocktail” with other protease inhibi drugs can be inactivated through proteolysis by endogenous tors to prevent degradation of protein Samples. The advan proteases, thus rendering the administered drug less effec tage of using a cocktail of protease inhibitors is that one is tive or inactive. Accordingly, the protease inhibitors of the US 2006/0053498A1 Mar. 9, 2006 invention may be administered to a patient in conjunction types and in chloroplast and bacterial membranes. This with a therapeutic agent in order to potentiate or increase the universality indicates the central importance of this enzyme activity of the drug. This co-administration may be by to ATP metabolism. Transcriptional regulation of these Simultaneous administration, Such as a mixture of the pro nuclear encoded genes appears to be the predominant means tease inhibitor and the drug, or by Separate Simultaneous or for controlling the biogenesis of ATP synthase. Multiple Sequential administration. mitochondrial pathologies exist because of the essential role of mitochondrial oxidative phosphorylation in cellular ATPKf Protein Sequence of SEQ ID No.253 (Internal Des energy production, in the generation of reactive oxygen ignation 1000867870) Species and in the initation of apoptosis (Wallace, Science, 0806) The protein of SEQ ID NO:253 encoded by the 283: 1482-1488, 1999, which disclosure is hereby incorpo extended cDNA SEQ ID NO:84 and relate polynucleotides rated by reference in its entirety). It is now clear that of SEQ ID NO:404 is a variant of the human mitochondrial mitochondrial diseases encompass an assemblage of clinical ATP synthase f subunit or ATPK (E.C. 3.6.1.34) and, as problems commonly involving tissues that have high energy Such, plays a role in cellular respiration. Preferred polypep requirements Such as heart, muscle and the renal and endo tides of the invention are are polypeptides comprising the crine Systems. Over the past 11 years, a considerable body amino acids of SEQ ID NO: 253 from positions 5 to 88. of evidence has accumulated implicating defects in the Other preferred polypeptides of the invention are fragments mitochondrial energy-generating pathway, oxidative phos of SEQ ID NO: 253 having any of the biological activity phorylation, in a wide variety of degenerative diseases described herein. It will be appreciated that all characteris including myopathy and cardiomyopathy. Most classes of tics and uses of the polynucleotides of SEQ ID NOS:84 and pathogenic mitochondrial DNA mutations affect the heart, in 404 and polypeptides of SEQ ID NO: 253 described asSociation with a variety of other clinical manifestations throughout the present application also pertain to the human that can include skeletal muscle, the central nervous System cDNA of clone 1000867870, and the polypeptides encoded (including eye), the endocrine System, and the renal System. thereby. Nuclear mutations causing mitochondrial disorders have been described. They are often found in highly conserved 0807. The mitochondrial electron transport (or respira Subunits. Mitochondrial disorders with nuclear mutations tory) chain is a Series of enzyme complexes in the mito include: myopathies (PEO, MNGIE, congenital muscular chondrial membrane that is responsible for the transport of dystrophy, carnitine disorders), encephalopathies (Leigh, electrons from NADH to oxygen and the coupling of this oxidation to the synthesis of ATP (oxidative phosphoryla Infantile, Wilson's disease, Deafness-Dystonia syndrome), tion). ATP then provides the primary source of energy for other Systemic disorders and cardiomyopathies. driving a cell's many energy-requiring reactions. ATP Syn 0808 The discovery of a new ATP synthase subunit, and thase (F0 F1 ATPase) is the enzyme complex at the terminus polynucleotides encoding it Satisfy a need in the art by of this chain and Serves as a reversible coupling device that providing new compositions which are useful for the diag interconverts the energies of an electrochemical proton nosis, prevention, and treatment of cancer, myopathies, gradient acroSS the mitochondrial membrane into either the immune disorders, and neurological disorders. synthesis or hydrolysis of ATP. This gradient is produced by 0809. An object of the present invention relates to com other enzymes of the respiratory chain in the course of positions and methods of targeting heterologous com electron transport from NADH to oxygen. When the cell's pounds, either polypeptides or polynucleotides to mitochon energy demands are high, electron transport from NADH to dria by recombinantly or chemically fusing a fragment of the oxygen generates an electrochemical gradient acroSS the protein of the invention to an heterologous polypeptide or mitochondrial membrane. Proton translocation from the polynucleotide. Preferred fragments are signal peptide, outer to the inner side of the membrane drives the synthesis amphiphilic alpha helices and/or any other fragments of the of ATP Under conditions of low energy requirements and protein of the invention, or part thereof, that may contain when there is an excess of ATP present, this electrochemical targeting Signals for mitochondria including but not limited gradient is reversed and ATP synthase hydrolyzes ATP. The to matrix targeting Signals as defined in Herrman and energy of hydrolysis is used to pump protons out of the Neupert, Curr. Opinion Microbiol. 3:210-4 (2000); Bhagwat mitochondrial matrix. ATP synthase is, therefore, a dual et al. J. Biol. Chem. 274:24014-22 (1999), Murphy Trends complex, the FO portion of which is a transmembrane proton Biotechnol. 15:326-30 (1997); Glaser et al. Plant Mol Biol carrier or pump, and the F1 portion of which is catalytic and 38:311-38 (1998); Ciminale et al. Oncogene 18:4505-14 synthesizes or hydrolyzes ATP Mammalian ATP synthase (1999), which disclosures are hereby incorporated by refer complex consists of sixteen different polypeptides (Walker, ence in their entireties. Such heterologous compounds may J. E. and Collinson, T. R. (1994) FEBS Lett.346: 39-43, which disclosure is hereby incorporated by reference in its be used to modulate mitochondria's activities. For example, entirety). Six of these polypeptides (subunits alpha, beta, they may be used to induce and/or prevent mitochondrial gamma, delta, epsilon, and an ATPase inhibitor protein IFI) induced apoptosis or necrosis. In addition, heterologous comprise the globular catalytic F1 ATPase portion of the polynucleotides may be used for mitochondrial gene therapy complex, which lies outside of the mitochondrial membrane. to replace a defective mitochondrial gene and/or to inhibit The remaining ten polypeptides (Subunits a, b, c, d, e, f, g, the deleterious expression of a mitochondrial gene. F6, OSCP, and A6L) comprise the proton-translocating, 0810) The invention further relates to methods and com membrane Spanning FO portion of the complex. Like other positions using the protein of the invention or part thereof to members of the respiratory chain, all but two of the polypep diagnose, prevent and/or treat Several disorders in which tide Subunits of ATP synthase are nuclear gene products that mitochondrial respiratory electron transport chain is are imported into the mitochondria. Enzyme complexes impaired, including but not limited to mitochondriocytopa similar to mammalian ATP synthase are found in all cell thies, necrosis, aging, myopathies, cancer and neurodegen US 2006/0053498A1 Mar. 9, 2006 86 erative diseaseS Such as Alzheimer's disease, Huntington's their entireties. Because of their usefulness in biotechnology, disease, Parkinson's disease, epilepsy, Down's Syndrome, it is thus highly interesting to isolate new multimerization dementia, multiple Sclerosis, and amyotrophic lateral Scle domains. rosis. For diagnostic purposes, the expression of the protein of the invention could be investigated using any of the 0816. The multimerization activity of the protein of the Northern blotting, RT-PCR or immunoblotting methods invention or part thereof may be assayed using any of the described herein and compared to the expression in control assays known to those skilled in the art including circular individuals. For prevention and/or treatment purposes, the dichroism Spectrum, gel filtration chromatography and ther protein of the invention may be used to enhance electron mal melting analyses. transport and increase energy delivery using any of the gene 0817. In one embodiment, the invention relates to com therapy methods described herein or known to those skilled positions and methods of using the protein of the invention in the art. or part thereof for preparing Soluble multimeric proteins, 0811. In another embodiment, the invention further which consist in multimers of fusion proteins containing a relates to methods and compositions using the protein of the multimerization domain fused to a protein of interest, using invention or part thereof to diagnose, prevent and/or treat any technique known to those skilled in the art including Several disorders in which mitochondrial respiratory elec those described in international patent WO9410308, which tron transport chain needs to be impaired, including but not disclosure is hereby incorporated by reference in its entirety. limited to Sjogren's Syndrome, Addison's disease, bronchi 0818. In another embodiment, the protein of the inven tis, dermatomyositis, polymyositis, glomerulonephritis, dia tion or part thereof or derivative thereof is used for detection betes mellitus, emphysema, Graves disease, atrophic gas and determination of an analyte in a biological liquid using tritis, lupus erythematosus, myasthenia gravis, multiple the teachings of U.S. Pat. No. 5,643,731, which disclosure Sclerosis, autoimmune thyroiditis, ulcerative colitis, anemia, is hereby incorporated by reference in its entirety. Briefly, a pancreatitis, Scleroderma, rheumatoid and Osteoarthritis, first multimerization domain is immobilized on a Solid asthma, allergic rhinitis, atopic dermatitis, dermatomyositis, Support and the Second multimerization domain is coupled polymyositis, and gout, using any techniques known to those to a specific binding partner for an analyte in a biological skilled in the art including the antisense or triple helices fluid. The two peptides are then brought into contact thereby Strategies described herein. immobilizing the binding partner on the Solid phase. The 0812 Moreover, antibodies to the protein of the invention biological Sample is then contacted with the immobilized or part thereof may be used for detection of mitochondria binding partner and the amount of analyte in the sample organelles and/or mitochondrial membranes using any tech bound to the binding partner determined. niques known to those skilled in the art. 0819. In still another embodiment, the protein of the Oligomerization Protein Sequence of SEQ ID No. 310 invention or part thereof may be used to construct multim erization devices comprising hybrid molecules with a func (Internal Designation D150568) tional domain fused to a multimerization domain in order to 0813 The protein of SEQ ID NO: 310 encoded by the yield multimeric complexes with improved pharmacokinetic cDNA of SEQ ID NO: 141 and 435, is able to form and pharmacological properties as described in homo-oligomers. Preferred polypeptides of the invention are WO0102440, which disclosure is hereby incorporated by polypeptides comprising the amino acids of SEQID NO:310 reference in its entirety. In a preferred embodiment, the from positions 1 to 109. Other preferred polypeptides of the protein of the invention or part thereof may be used to invention are fragments of SEQ ID NO: 310 having any of construct different fusion proteins with different functional the biological activities described herein. domains Such as enzyme moieties or cytotoxic moieties. Vectors encoding these different proteins may then be trans 0814 Multivalency is a prerequisite for a variety of fected in the same host cell in conditions allowing for macromolecular interactions Such as binding of antibodies multimerization, thus yielding multimeric multifunctional or lectins to Specific targets, ligand recognition, activation or complexes. inhibition of receptors and cell adhesion. Dimerization and oligomerization of proteins are thus general biological con Chaperone Protein of SEQ ID NO:303 (Internal Designa trol mechanisms that contribute to the activation of cell tion D637548) membrane receptors, transcription factors, Vesicle fusion 0820) The protein of SEQ ID NO:303 encoded by the proteins, and other classes of intra- and extracellular pro cDNA of SEQ ID NO:134 is a chaperonin. Accordingly, the teins. protein of SEQ ID NO:303 plays a role in protein synthesis/ 08.15 Multimerization domains have been shown to be folding, cellular trafficking, and the cellular StreSS response. useful tools in Several areas of biotechnology, especially in In addition, the protein of SEQ ID No. 303 has immunosu protein engineering. For example, TSO et all have used preSSant and growth factor properties. It is able to depress leucine ZipperS for producing bispecific antibody het delayed type hyperSensitivity reactions. It is a product of erodimers (U.S. Pat. No. 5,932,448)/Methods of preparing primary and neoplastic cell proliferation and under these Soluble oligomeric proteins using leucine ZipperS have been conditions acts as a growth factor. It is also a product of described by Conrad et al (U.S. Pat. No. 5,965,712), Cia platelet activation and may play a part in wound healing and rdelli et al (U.S. Pat. No. 5,837.816), Spriggs et al skin repair. Preferred polypeptides of the invention are (WO9410308)/Leucine zipper forming sequences have been polypeptides comprising the amino acids of SEQID NO:303 used by Pelletier et al in protein fragment complementation from positions 9 to 33, or from positions 7 to 101. Other assays to detect biomolecular interactions (WO9834120), preferred polypeptides of the invention are fragments of which disclosures are hereby incorporated by reference in SEQ ID NO: 303 having any of the biological activities US 2006/0053498A1 Mar. 9, 2006 87 described herein. The different activities of the protein of the 0825. In still another embodiment, the protein of the invention or part thereof may be assayed using any of the invention or part thereof may be used to treat and/or prevent assays described in U.S. Pat. No. 6,117,421 or any of the infertility and miscarriage using the Simple administration of assays referred into U.S. Pat. No. 6117,421, which disclo the protein of the invention or part thereof or using any of Sures are hereby incorporated by reference in their entireties. the gene therapy methods described elsewhere in the appli 0821 Chaperonins belong to a wider class of molecular cation and the teaching of the U.S. Pat. No. 6,117,421. chaperones, molecules involved in post-translational fold 0826. In another embodiment, the present invention pro ing, targeting and assembly of other proteins, but which do vide methods of using the present proteins to identify not themselves form part of the final assembled Structure as Specific cell types in vitro and in Vivo. For example, as discussed by Ellis et al., 1991, Annu. Rev. Biochem. 60 chaperone proteins are often upregulated in response to 321-347, which disclosures are hereby incorporated by cellular StreSS, the detection of cells expressing elevated reference in their entireties. Most molecular chaperones are levels of the proteins provides a tool for detecting cells under “heat shock” or “stress” proteins (hsp); i.e. their production StreSS. AS cellular StreSS has been implicated in a number of is induced or increased by a variety of cellular insults (Such disorders, Such as cardiovascular disorders, neurodegenera as metabolic disruption, oxygen radicals, inflammation, tive disorders, and cancer, the ability to detect Such StreSS infection and transformation), heat being only one of the thus provides a diagnostic or Screening tool for Such con better studies stresses as reviewed by Lindquist et al., 1988, ditions. Annu. Rev. Genet. 22 631-677, which disclosure is hereby incorporated by reference in its entirety. AS well as these 0827. In addition, the present polypeptides and poly quantitative changes in Specific protein levels, StreSS can nucleotides can be used to develop diagnostic and Screening induce the movement of constitutively produced StreSS pro assays for diseases characterized by an abnormal level or teins to different cellular compartments as referred to in the activity of the protein of SEQID NO:303 Such as malignant Lindquist reference mentioned above. The heat shock disorders of various types, and autoimmune diseases includ response is one of the most highly conserved genetic System ing type I diabetes, rheumatoid arthritis, Systemic lupus known and the various heat shock protein families are erythematosus, Sjogren Syndrome, Graves disease, multiple among the most evolutionarily Stable proteins in existence. Sclerosis, and mixed connective tissue disease. Such assays The major StreSS proteins accumulate to very high levels in can be performed using any biological Sample, Such as Stressed cells but occur at low to moderate levels in cells that Serum or plasma. have not been stressed. As well as enabling cells to cope 0828. In another embodiment, various disorders can be under adverse conditions, members of these families per treated, attenuated and/or prevented by a protein of SEQ ID form essential functions in normal cells. NO: 303, or part thereof, or any other compound that can 0822 Chaperones are also involved in a number of affect the level or activity of the proteins Such as nucleic disorders, especially autoimmune diseaseS Such as type 1 acids, antibodies, or chemical Substances. In a preferred diabetes, rheumatoid arthritis, Systemic lupus erythemato embodiment, proteins or other compounds directed to the Sus, Sjogren Syndrome, and mixed connective tissue disease proteins of the invention can be used to treat or prevent (Feige et al. EXS 1996; 77:359-73; Feili-Hariri et al. J disorders in which the activity or level of the protein of SEQ Autoimmun 2000; 14:133-42, which disclosures are hereby ID NO:303 is unbalanced. Such diseases include, but are not incorporated by reference in their entireties). Chaperones are limited to, infectious diseases, neurogenerative disorders as also involved in various disorders including tuberculosis and Alzheimer and Parkinson diseases, Schizophrenia, alopecia, leprosy (Zugel et al. Clin Microbiol Rev 1999; 12:19-39), aging, atherosclerosis, malignant disorders of various types, neurogenerative disorderS Such as Alzheimer and Parkinson and autoimmune diseases including type I diabetes, rheu diseases (Yoo et al. J Neural Transm Suppl 1999; 57.315 matoid arthritis, Systemic lupus erythematosus, Sjogren Syn 22), and malignant disorders (Csermely et al. Pharmacol drome, mixed connective tissue disease, malignant disor Ther 1998; 7.9:129-68), which disclosures are hereby incor ders, autoimmune and any other neurodegenerative disorder. porated by reference in their entireties. In another embodiment, the proteins of SEQ ID NO:303 or part thereof can be used as vaccines for various disorders 0823. In one embodiment, the protein of the invention or including, but not limited, to cancer (Wang et al. Immunol part thereof may be used to detect a potential pregnancy, Invest 2000:29:131-7), tuberculosis (Silva et al. Microbes preferably within 6-24 hours of fertilization using the teach Infect 1999; 1:429-35), diabetes (Int Immunol 1999; 11:957 ing of Morton et al., 1976, Proc. R. Soc. B. 193 413-41 and 66), and atherosclerosis (Xu et al. Arterioscler Thromb U.S. Pat. No. 6,117,421, which disclosures are hereby 1992; 12:789-99), which disclosures are hereby incorpo incorporated by reference in their entireties. Detection of the rated by reference in their entireties. expression or activity of the protein of the invention may be performed using any techniques known to those skilled in 0829. One embodiment of the present invention relates to the art including those described elsewhere in the applica methods and compositions using the protein of SEQ ID tion. NO:303 or fragments thereof as a stabilizing adjuvant to Slow down protein degradation, boost the yields of recom 0824. In another embodiment, molecules able to block binant proteins, prevent the aggregation of proteins or regen the expression or activity of the protein of the invention, erate denatured proteins. In a preferred embodiment, the Such as antibodies, antisense or triple helix oligonucleotides, protein of SEQ ID NO:303 of fragment thereof is mixed dominant negative forms of the protein, polypeptides or with a composition comprising the protein for which it is small molecule inhibitors of the expression or activity of the desired to Slow down degradation, boost yield, or regenerate proteins, may be used to induce abortion as described in U.S. denatured proteins under conditions which facilitate the Pat. No. 6,117,421. desired result. For example, numerous commercial assay US 2006/0053498A1 Mar. 9, 2006 88 kits commonly used by those skilled in the arts of molecular by reference in its entirety. The advantage of using a cocktail biology and biochemistry depend on the biological proper of chaperone proteins is to accommodate differences in ties of proteins (mostly enzymes) which can be very short binding specificity of the Hsp different families and the lived in vitro due to the low stability of those proteins. An different members within each family. example is described in Eur. Patent DE4124286, the disclo Sure of which is incorporated herein by reference in its 0832. In another embodiment of the present invention, entirety, wherein the low intrinsic stability of test solutions the protein of SEQ ID NO:303 may be used to promote used in optical tests is increased by addition of chaperone tissue repair and/or increase cell Survival in StreSS conditions proteins, thus making the test more Sensitive. Another Such as hypoxy, oxidative StreSS, genotoxic agents and more example is given in U.S. Pat. No. 6,013,488, which disclo generally harmful conditions leading to programmed cell Sure is hereby incorporated by reference in its entirety, death. Those conditions include but are not limited to wherein a heat-labile reverse transcriptase is able to perform infarction, heart Surgery, Stroke, neurodegenerative diseases, cDNASynthesis at high temperature levels in the presence of epilepsy, trauma, atherOSclerosis, restenosis after angio a chaperone. plasty, and nerve damage. 0830) The protein of SEQ ID NO:303 may also be used 0833. In addition, the invention relates to compositions to increase the yield or activity of recombinant proteins, and methods for promoting cell growth both in vitro and in preferably secreted proteins. In recombinant DNA technol Vivo using any of the techniques known to those skilled in ogy, a major unsolved problem is the Solubility and biologi the art including those described in the U.S. Pat. No. cal activity of the recombinantly overexpressed protein in a 6,117,421. For example, soluble forms of the protein of the host, especially a bacterial or yeast host. Many eukaryotic invention or part thereof may be added to cell culture proteins, especially the Secreted ones, require for correct medium in an amount effective to Stimulate cell prolifera folding a Specific cellular machinery which is lacking in tion. Alternatively, any of the gene therapy methods bacterial hosts Such as E. coli or becomes insufficient in described herein may be used to overexpress the protein of mammalian/yeast cells due to high expression of the protein. the invention or part thereof in vivo. Alternatively, the The ability of the protein of SEQ ID NO:303 or fragments protein of the invention or part thereof may be directly thereof to ensure proper folding of recombinant proteins administered in an amount effective to promoting cell may be utilized as follows. The protein of SEQ ID NO:303, growth in Said Subject. These applications are particularly or fragment thereof, may be coexpressed with the recombi important in individuals Suffering from wounds or tissue nant protein in bacterial or eukaryotic hosts to cause the damage to enhance tissue repair, in individuals to which hosts to express the heterologous proteins or polypeptides in organ or skin grafts have been applied, in individuals a form having increased Solubility and/or biological activity. Suffering from an inflammatory condition or an allergic For example, the protein of SEQ ID NO:303 or fragments disease. thereof may be used in the methods described in U.S. Pat. 0834. In addition, the invention relates to compositions No. 5,773,245, the disclosure of which is incorporated and methods for promoting immunoSupression in a Subject herein by reference in its entirety. Therefore the invention using any of the techniques known to those skilled in the art relates to a method for the correct folding, deaggregation or including those described in the U.S. Pat. No. 6,117,421. For prevention of aggregation of a monomeric protein in vivo example, the protein of the invention or part thereof may be comprising: (a) constructing a host cell transformed with (i) directly administered in an amount effective to achieve a first DNA encoding a polypeptide having the amino acid immunoSupression in Said Subject. Alternatively, any of the Sequence of a bioactive protein or a precursor thereof, gene therapy methods described herein may be used to wherein Said polypeptide or precursor can aggregate within overexpress the protein of the invention or part thereof in the cell to result in a multimeric, non-bioactive protein or Vivo. These applications are particularly important in cases precursor thereof and (ii) a second DNA which enable the in which immunoSupression is desired Such as in individuals cell to co-express the protein of the invention or part thereof Suffering from autoimmune disease including any of the with the said polypeptide or precursor, (b) growing said host diseases cited above and in individuals that have received an cell for Sufficient time under conditions wherein said first heterologous graft they could reject. DNA and said second DNA express said bioactive protein and Said protein of the invention, respectively; and (c) Ion Transport Protein of SEQ ID NO: 276 (Internal Desig obtaining monomeric protein that is a bioactive protein. nation D538694) Alternatively the protein of SEQ ID NO:303 or fragments 0835. The protein of SEQ ID NO: 276 encoded by the thereof may be exogeneously added to the cell cultures as cDNA of SEQ ID NO:107 belongs to the FXDY family of described in PCT application WO 00/08135, the disclosure Small ion transport regulators or channels (Sweadner and of which is incorporated herein by reference in its entirety. Rael (2000) Genomics 68:41-56, which disclosure is hereby 0831. The protein of SEQ ID NO:303 or fragments incorporated by reference in its entirety). The protein of thereof may further be used to regenerate denatured proteins. SEQ ID NO: 276 or part thereof plays a role in the control Recombinantly expressed proteins with poor biological of ion transport. Preferred polypeptides of the invention are activity are routinely denatured with a potent denaturing polypeptides comprising the amino acids of SEQID NO:276 agent, Such as guanidine hydrochloride, followed by refold from positions 9 to 63, or from positions 16 to 29. Other ing by dilution with a large amount of a diluent to reduce the preferred polypeptides of the invention are fragments of concentration of the denaturing agent. However, this method SEQ ID NO: 276 having any of the biological activities often results in a poor refolding rate which may be signifi described herein. The activity of the protein of the invention cantly increased by addition of a cocktail of chaperone or part thereof may be assayed using any of the assays proteins in a fashion similar to that described in Eur. Patent known to those skilled in the art including those described EP0650975, the disclosure of which is incorporated herein . US 2006/0053498A1 Mar. 9, 2006 89

0836. Osmoregulation occurs in all organisms, though invention may be used to enhance ion transport and prevent the mechanisms differ according to the organism's environ or treat oSmoregulatory disorders using any of the gene ment. Fresh water inhabitants need to retain Salts, whereas therapy methods described herein. ocean inhabitants need to retain water. Terrestrial inhabitants need to conserve both water and Salts. Organisms must Uses of Antibodies balance these needs with a requirement to eliminate meta 0840 Antibodies of the present invention have uses that bolic waste, Such as nitrogenous waste, and generate include, but are not limited to, methods known in the art to Secreted body fluids, Such as Saliva for digestion and Sweat purify, detect, and target the polypeptides of the present for thermoregulation. invention including both in vitro and in Vivo diagnostic and 0837. In mammals, Sweat glands, salivary glands, and the therapeutic methods. An example of Such use using immu kidney all produce a primary Secretion that is essentially noaffinity chromatography is given below. The antibodies of isosmotic with blood and extracellular fluids. Modification the present invention may be used either alone or in com of this primary Secretion then occurs as much of the Sodium bination with other compositions. For example, the antibod chloride and water are reabsorbed as they pass through the ies have use in immunoassays for qualitatively and quanti excretory ducts of the glands and kidney, whereas potassium tatively measuring levels of antigen-bearing Substances, and bicarbonate ions are Secreted. This modification of the including the polypeptides of the present invention, in primary Secretion is important in the Sweat glands to con biological Samples (See, e.g., Harlow et al., 1988). (Incor Serve Sodium chloride in hot environments, and in the porated by reference in the entirety). The antibodies may Salivary glands to conserve Sodium chloride when excessive also be used in therapeutic compositions for killing cells quantities of Saliva are lost. This modification is critical in expressing the protein or reducing the levels of the protein the kidney to maintain proper Sodium and water balance in in the body. the extracellular fluids, a balance which also regulates 0841. The invention further relates to antibodies that act arterial pressure. LOSS of this modification activity by the as agonists or antagonists of the polypeptides of the present duct cells causes a large loSS of Sodium and water, resulting invention. For example, the present invention includes anti in severe dehydration and low blood volume, and ultimately bodies that disrupt the receptor/ligand interactions with the to circulatory collapse. polypeptides of the invention either partially or fully. 0838 Sodium absorption by the intestines, especially in Included are both receptor-specific antibodies and ligand the colon, is necessary to prevent loss of Sodium in the Specific antibodies. Included are receptor-specific antibod Stools. The loSS of Sodium absorption produces a failure to ies, which do not prevent ligand binding but prevent receptor absorb anions and water as well. The unabsorbed sodium activation. Receptor activation (i.e., Signaling) may be deter chloride and water then lead to diarrhea, with further loss of mined by techniques described herein or otherwise known in Sodium chloride from the body. Other body fluids may be the art. Also include are receptor-specific antibodies which under regulation Similar to that Seen in the Systems described both prevent ligand binding and receptor activation. Like above. For example, cerebroSpinal fluid is produced by wise, included are neutralizing antibodies that bind the active Sodium ion transport from the capillaries acroSS the ligand and prevent binding of the ligand to the receptor, as epithelium of the choroid plexus, which in turn attracts well as antibodies that bind the ligand, thereby preventing chloride ions and water. A counter flow of potassium and receptor activation, but do not prevent the ligand from bicarbonate ions move out of the cerebrospinal fluid into the binding the receptor. Further included are antibodies that capillaries. A dysfunction in Osmoregulation is associated activate the receptor. These antibodies may act as agonists with Several disease States, including hyponatremia, renal for either all or less than all of the biological activities failure, and hypernatremia. (Strange, K. (1992) J Am. Soc. affected by ligand-mediated receptor activation. The anti Nephrol. 3:12-27, which disclosure is hereby incorporated bodies may be specified as agonists or antagonists for by reference in its entirety). biological activities comprising Specific activities disclosed herein. The above antibody agonists can be made using 0839. In one embodiment, the protein of the invention may be useful in the diagnosis, prevention and/or treatment methods known in the art. See e.g., WO 96/40281; U.S. Pat. of Osmoregulatory disorders including but not limited to No. 5,811,097; Deng et al. (1998); Chen et al. (1998); diabetes insipidus, diarrhea, peritonitis, chronic renal fail Harrop et al. (1998); Zhu et al. (1998); Yoon et al. (1998); ure, Addison's disease, Syndrome of inappropriate antidi Prat et al. (1998); Pitard et al. (1997); Liautard et al. (1997); uretic hormone (SIADH), hypoaldosteronism, hyponatre Carlson et al. (1997); Taryman et al. (1995); Muller et al. mia, adrenal insufficiency, hypothyroidism, hypernatremia, (1998); Bartunek et al. (1996) (said references incorporated hypokalemia, Barter's Syndrome, Cushing's Syndrome, by reference in their entireties). metabolic acidosis, metabolic alkalosis, encephalopathy, 0842) As discussed above, antibodies of the polypeptides edema, hypotension, and hypertension. For example, any of of the invention can, in turn, be utilized to generate anti the gene therapy methods described herein may be used to idiotypic antibodies that “mimic' polypeptides of the inven overexpress the protein of the invention or part thereof in tion using techniques well known to those skilled in the art Vivo. Alternatively, the protein of the invention or part (See, e.g. Greenspan and Bona (1989) and Nissinoff (1991), thereof may be directly administered in an amount effective which disclosures are hereby incorporated by reference in to promoting cell growth in Said Subject. For diagnostic their entireties). For example, antibodies which bind to and purposes, the expression of the protein of the invention competitively inhibit polypeptide multimerization or bind could be investigated using any of the Northern blotting, ing of a polypeptide of the invention to ligand can be used RT-PCR or immunoblotting methods described herein and to generate anti-idiotypes that "mimic' the polypeptide compared to the expression in control individuals. For multimerization or binding domain and, as a consequence, prevention and/or treatment purposes, the protein of the bind to and neutralize polypeptide or its ligand. Such neu US 2006/0053498A1 Mar. 9, 2006 90 tralization anti-idiotypic antibodies can be used to bind a 5'ESTs (i.e. cDNA fragments) expressed in a particular polypeptide of the invention or to bind its ligands/receptors, tissue referred to by its name is indicated in parentheses and thereby block its biological activity. (second column). In addition, the bias in the Spatial distri bution of the polynucleotide Sequences of the present inven Immunoaffinity Chromatography tion was examined by comparing the relative proportions of 0843 Antibodies prepared as described herein are the biological polynucleotides of a given tissue using the coupled to a Support. Preferably, the antibodies are mono following Statistical analysis. The under- or over-represen clonal antibodies, but polyclonal antibodies may also be tation of a polynucleotide of a given cluster in a given tissue used. The Support may be any of those typically employed was performed using the normal approximation of the in immunoaffinity chromatography, including Sepharose binomial distribution. When the observed proportion of a CL-4B (Pharmacia, Piscataway, N.J.), Sepharose CL-2B polynucleotide of a given tissue in a given consensus had (Pharmacia, Piscataway, N.J.), Affi-gel 10 (Biorad, Rich less than 1% chance to occur randomly according to the chi2 mond, Calif.), or glass beads. test, the frequency bias was reported as "preferred”. The results are given in Table V as follows. For each polynucle 0844. The antibodies may be coupled to the support using otide showing a bias in tissue distribution as referred to by any of the coupling reagents typically used in immunoaf its Sequence identification number in the first column, the list finity chromatography, including cyanogen bromide. After of tissues where the polynucleotides are under-represented is coupling the antibody to the Support, the Support is contacted given in the Second column entitled “low expression' and with a Sample which contains a target polypeptide whose the list of tissues where the polynucleotides are over isolation, purification or enrichment is desired. The target represented is given in the third column entitled “high polypeptide may be a polypeptide Selected from the group expression'. consisting of sequences of SEQ ID NOs: 170-338, 456-560, 785-918 and polypeptides encoded by the clone inserts of Evaluation of Expression Levels and Patterns of GENSET the deposited clone pool, Variants and fragments thereof, or Polypeptide-Encoding mRNAS a fusion protein comprising Said Selected polypeptide or a fragment thereof. 0849. The spatial and temporal expression patterns of GENSET polypeptide-encoding mRNAS, as well as their 0845 Preferably, the sample is placed in contact with the expression levels, may also be further determined as fol Support for a Sufficient amount of time and under appropriate lows. conditions to allow at least 50% of the target polypeptide to Specifically bind to the antibody coupled to the Support. 0850. Expression levels and patterns of GENSET polypeptide-encoding mRNAS may be analyzed by Solution 0846. Thereafter, the support is washed with an appro hybridization with long probes as described in International priate wash Solution to remove polypeptides which have Patent Application No. WO97/05277, the entire contents of non-specifically adhered to the Support. The wash Solution which are hereby incorporated by reference. Briefly, a may be any of those typically employed in immunoaffinity GENSET polynucleotide, or fragment thereof, correspond chromatography, including PBS, Tris-lithium chloride buffer ing to the gene encoding the mRNA to be characterized is (0.1M lysine base and 0.5M lithium chloride, pH 8.0), inserted at a cloning site immediately downstream of a Tris-hydrochloride buffer (0.05M Tris-hydrochloride, pH bacteriophage (T3, T7 or SP6) RNA polymerase promoter to 8.0), or Tris/Triton/NaCl buffer (50 mM Tris.cl, pH 8.0 or produce antisense RNA. Preferably, the GENSET poly 9.0, 0.1% Triton X-100, and 0.5 MNaCl). nucleotide is at least a 100 nucleotides in length. The 0847. After washing, the specifically bound target plasmid is linearized and transcribed in the presence of polypeptide is eluted from the Support using the high pH or ribonucleotides comprising modified ribonucleotides (i.e. low pH elution Solutions typically employed in immunoaf biotin-UTP and DIG-UTP). An excess of this doubly labeled finity chromatography. In particular, the elution Solutions RNA is hybridized in solution with mRNA isolated from may contain an eluant Such as triethanolamine, diethy cells or tissues of interest. The hybridizations are performed lamine, calcium chloride, Sodium thiocyanate, potasSSium under standard stringent conditions (40-50° C. for 16 hours bromide, acetic acid, or glycine. In Some embodiments, the in an 80% formamide, 0.4 M NaCl buffer, pH 7-8). The elution Solution may also contain a detergent Such as Triton unhybridized probe is removed by digestion with ribonu X-100 or octyl-beta-D-glucoside. cleases specific for single-stranded RNA (i.e. RNases CL3, T1, Phy M, U2 or A). The presence of the biotin-UTP modification enables capture of the hybrid on a microtitra Expression of Genset Gene Products tion plate coated with streptavidin. The presence of the DIG Spatial Expression of the GENSET Genes of the Invention modification enables the hybrid to be detected and quantified by ELISA using an anti-DIG antibody coupled to alkaline 0848 Tissue expression of the cDNAs of the present phosphatase. invention was examined. Tables III and IV lists the number of hits for the cDNAS in Genset's libraries of tissues and cell 0851. The GENSET polypeptide-encoding cDNAs, or types as well as in public databases. The tissues and cell fragments thereof, may also be tagged with nucleotide types examined for polynucleotide expression were, for Sequences for the Serial analysis of gene expression (SAGE) Table III: Brain; Fetal brain; Fetal kidney; Fetal liver; as disclosed in UK Patent Application No. 2305 241 A, the Pituitary gland; Liver; Placenta; Prostate; Salivary gland; entire contents of which are incorporated by reference. In Stomach/Intestine; and Testis. For each cDNA referred to by this method, cDNAS are prepared from a cell, tissue, organ its corresponding Sequence identification number from the ism or other Source of nucleic acid for which it is desired to priority application (see Table I for corresponding SEQ ID determine gene expression patterns. The resulting cDNAS NO in present application), the number of proprietary are separated into two pools. The cDNAS in each pool are US 2006/0053498A1 Mar. 9, 2006

cleaved with a first restriction endonuclease, called an with radioactive nucleotides. After hybridization and wash "anchoring enzyme, having a recognition site which is ing in controlled conditions, the hybridized mRNAS are likely to be present at least once in most cDNAs. The detected by phospho-imaging or autoradiography. Duplicate fragments which contain the 5' or 3' most region of the experiments are performed and a quantitative analysis of cleaved cDNA are isolated by binding to a capture medium differentially expressed mRNAS is then performed. Such as Streptavidin coated beads. A first oligonucleotide linker having a first Sequence for hybridization of an ampli 0854. Alternatively, expression analysis of GENSET fication primer and an internal restriction Site for a "tagging genes can be done through high density nucleotide arrays as endonuclease” is ligated to the digested cDNAS in the first described by Lockhart et al. (1996) and Sosnowski et al. pool. Digestion with the Second endonuclease produces (1997), which disclosures are hereby incorporated by refer short “tag” fragments from the cDNAs. A second oligo ence in their entireties. Oligonucleotides of 15-50 nucle nucleotide having a Second Sequence for hybridization of an otides corresponding to Sequences of a GENSET polynucle amplification primer and an internal restriction site is ligated otide or fragments thereof are Synthesized directly on the to the digested cDNAS in the second pool. The cDNA chip (Lockhart et al., Supra) or synthesized and then fragments in the Second pool are also digested with the addressed to the chip (Sosnowski et al., Supra). Preferably, "tagging endonuclease' to generate short “tag” fragments the oligonucleotides are about 20 nucleotides in length. derived from the cDNAs in the second pool. The “tags' cDNA probes labeled with an appropriate compound, Such resulting from digestion of the first and Second pools with as biotin, digoxigenin or fluorescent dye, are Synthesized the anchoring enzyme and the tagging endonuclease are from the appropriate mRNA population and then randomly ligated to one another to produce “ditags.” In Some embodi fragmented to an average size of 50 to 100 nucleotides. The ments, the ditags are concatamerized to produce ligation Said probes are then hybridized to the chip. After washing as products containing from 2 to 200 ditags. The tag Sequences described in Lockhart et al., (Supra) and application of are then determined and compared to the Sequences of the different electric fields (Sosnowsky et al., Supra), the dyes or GENSET polypeptide-encoding cDNAS to determine which labeling compounds are detected and quantified. Duplicate genes are expressed in the cell, tissue, organism, or other hybridizations are performed. Comparative analysis of the Source of nucleic acids from which the tags were derived. In intensity of the Signal originating from cDNA probes on the this way, the expression pattern of a GENSET polypeptide Same target oligonucleotide in different cDNA Samples encoding gene in the cell, tissue, organism, or other Source indicates a differential expression of the GENSET polypep of nucleic acids is obtained. tide-encoding mRNA. 0852 Quantitative analysis of GENSET gene expression Uses of GENSET Gene Expression Data may also be performed using arrayS. For example, quanti 0855 Once the expression levels and patterns of a tative analysis of gene expression may be performed with GENSET polypeptide-encoding mRNA has been deter GENSET polynucleotides, or fragments thereof in a comple mined using any technique known to those skilled in the art, mentary DNA microarray as described by Schena et al. in particular those described in the section entitled “Evalu (1995 and 1996) which disclosures are hereby incorporated ation of Expression Levels and Patterns of GENSET by reference in their entireties. GENSET polypeptide-en polypeptide-encoding mRNAS', or using the instant disclo coding cDNAS or fragments thereof are amplified by PCR Sure, these information may be used to design GENSET and arrayed from 96-well microtiter plates onto silylated gene Specific markers for detection, identification, Screening microScope Slides using high-speed robotics. Printed arrayS and diagnosis purposes as well as to design DNA constructs are incubated in a humid chamber to allow rehydration of the with an expression pattern similar to a GENSET gene array elements and rinsed, once in 0.2% SDS for 1 min, expression pattern. twice in water for 1 min and once for 5 min in Sodium borohydride Solution. The arrays are Submerged in water for Detection of GENSET Polypeptide Expression and/or Bio 2 min at 95 C., transferred into 0.2% SDS for 1 min, rinsed logical Activity twice with water, air dried and stored in the dark at 25 C. 0856. The invention further relates to methods of detec Cell or tissue mRNA is isolated or commercially obtained tion of GENSET polypeptide expression and/or biological and probes are prepared by a single round of reverse activity in a biological Sample using the polynucleotide and transcription. Probes are hybridized to 1 cm microarrays polypeptide Sequences described herein. Such method Scan under a 14x14 mm glass coverslip for 6-12 hours at 60° C. be used, for example, as a Screen for normal or abnormal Arrays are washed for 5 min at 25 C. in low stringency GENSET polypeptide expression and/or biological activity wash buffer (1xSSC/0.2% SDS), then for 10 min at room and, thus, can be used diagnostically. The biological Sample temperature in high stringency wash buffer (0.1xSSC/0.2% for use in the methods of the present invention includes a SDS). Arrays are scanned in 0.1xSSC using a fluorescence Suitable Sample from, for example, a mammal, particularly laser Scanning device fitted with a custom filter Set. Accurate a human. For example, the Sample can be issued from tissues differential expression measurements are obtained by taking or cell lines having the same origin as tissueS or cell lines in the average of the ratios of two independent hybridizations. which the polypeptide is known to be expressed, e.g. using 0853 Quantitative analysis of the expression of genes data from Tables III, IV, or V. may also be performed with GENSET polypeptide-encoding cDNAS or fragments thereof in complementary DNA arrays Detection of GENSET Polypeptides as described by Pietu et al. (1996), which disclosure is 0857. The invention further relates to methods of detec hereby incorporated by reference in its entirety. The tion of GENSET polypeptide or encoding polynucleotides in GENSET polynucleotides of the invention or fragments a Sample using the Sequences described herein and any thereof are PCR amplified and spotted on membranes. Then, techniques known to those skilled in the art. For example, a mRNAS originating from various tissueS or cells are labeled labeled polynucleotide probe having all or a functional US 2006/0053498A1 Mar. 9, 2006 92 portion of the nucleotide sequence of a GENSET polypep amplification primer has a Sequence comprised in Said tide-encoding polynucleotide can be used in a method to Selected Sequence or in the Sequence complementary to Said detect a GENSET polypeptide-encoding polynucleotide in a Selected Sequence. Sample. In one embodiment, the Sample is treated to render the polynucleotides in the sample available for hybridization 0867 Alternatively, a method of detecting GENSET to a polynucleotide probe, which can be DNA or RNA. The polypeptide expression in a test Sample can be accomplished resulting treated Sample is combined with a labeled poly using any product which binds to a GENSET olypeptide of nucleotide probe having all or a portion of the nucleotide the present invention or a portion of a GENSET polypeptide. sequence of the GENSET polypeptide-encoding cDNA or Such products may be antibodies, binding fragments of genomic Sequence, under conditions appropriate for hybrid antibodies, polypeptides able to bind Specifically to ization of complementary Sequences to occur. Detection of GENSET polypeptides or fragments thereof, including hybridization of polynucleotides from the sample with the GENSET polypeptide agonists and antagonists. Detection of labeled nucleic probe indicates the presence of GENSET Specific binding to the antibody indicates the presence of a polypeptide-encoding polynucleotides in a Sample. The GENSET polypeptide in the sample (e.g., ELISA). presence of GENSET polypeptide-encoding mRNA is 0868 Consequently, the invention is also directed to a indicative of GENSET polypeptide-encoding gene expres method for detecting specifically the presence of a GENSET SO. polypeptide according to the invention in a biological 0858 Consequently, the invention comprises methods for Sample, Said method comprising the Steps of: detecting the presence of a polynucleotide comprising a 0869 a) bringing into contact said biological sample with nucleotide Sequence Selected from a group consisting of the a product able to bind to a polypeptide of the invention or sequences of SEQ ID NOS:1-169,339-455, 561-784, the fragments thereof; Sequences of clone inserts of the deposited clone pool, 0870 b) allowing said product to bind to said polypeptide Sequences fully complementary thereto, fragments and Vari to form a complex, and ants thereof in a Sample. In a first embodiment, Said method comprises the following Steps of: 0871 b) detecting said complex. 0859 a) bringing into contact said sample and a nucleic 0872. In a preferred embodiment of the above detection acid probe or a plurality of nucleic acid probes which method, the product is an antibody. In a more preferred hybridize to said Selected nucleotide Sequence; and embodiment, said antibody is labeled with a detectable molecule. In another more preferred embodiment of the 0860 b) detecting the hybrid complex formed between above detection method, said antibody has been immobi Said probe or Said plurality of probes and Said polynucle lized on a Substrate. otide. 0873. In addition, the invention also relates to methods of 0861. In a preferred embodiment of the above detection determining whether a GENSET gene product (e.g. a poly method, Said nucleic acid probe or Said plurality of nucleic nucleotide or polypeptide) is present or absent in a biologi acid probes is labeled with a detectable molecule. In another cal Sample, Said methods comprising the Steps of preferred embodiment of the above detection method, said nucleic acid probe or Said plurality of nucleic acid probes 0874) a) obtaining said biological sample from a human has been immobilized on a Substrate. In Still another pre or non-human animal, preferably a mammal; ferred embodiment, Said nucleic acid probe or said plurality 0875 b) contacting said biological sample with a product of nucleic acid probes has a Sequence comprised in a able to bind to a GENSET polypeptide or encoding poly Sequence complementary to Said Selected Sequence. nucleotide of the invention; and 0862 In a second embodiment, said method comprises 0876 c) determining the presence or absence of said the Steps of: GENSET polypeptide-encoding gene product in Said bio logical Sample. 0863 a) contacting said sample with amplification reac tion reagents comprising a pair of amplification primers 0877. The present invention also relates to kits that can be located on either side of the region of Said nucleotide used in the detection of GENSET polypeptide-encoding Sequence to be amplified; gene expression products. The kit can comprise a compound that specifically binds a GENSET polypeptide (e.g. binding 0864 b) performing an amplification reaction to synthe proteins, antibodies or binding fragments thereof (e.g. Size amplification products containing Said region of Said F(ab')2 fragments) or a GENSET polypeptide-encoding Selected nucleotide Sequence; and mRNA (e.g. a complementary probe or primer), for example, disposed within a container means. The kit can O865) c) detecting said amplification products. further comprise ancillary reagents, including buffers and 0866. In a preferred embodiment of the above detection the like. method, when the polynucleotide to be amplified is a RNA molecule, preliminary reverse transcription and Synthesis of Detection of GENSET Polypeptide Biological Activity a second cDNA strand are necessary to provide a DNA 0878 The invention further includes methods of detect template to be amplified. In another preferred embodiment ing specifically a GENSET polypeptide biological activity, of the above detection method, the amplification product is and to identify compounds capable of modulating the activ detected by hybridization with a labeled probe having a ity of a GENSET polypeptide. Assessing the GENSET Sequence which is complementary to the amplified region. polypeptide biological activity may be performed by the In Still another preferred embodiment, at least one of Said detection of a change in any cellular property associated US 2006/0053498A1 Mar. 9, 2006 with the GENSET polypeptide, using a variety of tech nochemistry, ELISA). Examples of Such techniques are niques, including those described herein. To identify modu described in more detail below. Therefore, the invention lators of the polypeptides, a control is preferably used. For encompasses uses of the polynucleotides and polypeptides example, a control Sample includes all of the same reagents of the invention as tissue markers. In a preferred embodi but lacks the compound or agent being assessed; it is treated ment, polynucleotides preferentially expressed in given tis in the same manner as the test Sample. A number of SueS as indicated in Tables III-V and polypeptides encoded potentially assayable biological activities for many of the by Such polynucleotides are used for this purpose. The herein-described proteins are described Supra, under the invention also encompasses uses of polypeptides of the heading, “Uses of polypeptides of the invention.” invention as organelle markers. 0879 The present invention also relates to kits that can be 0882 Consequently, the present invention encompasses used in the detection of GENSET polypeptide biological methods of identification of a tissue/cell type/subcellular activity. The kit can comprise, e.g. Substrates for GENSET compartment, wherein Said method includes the Steps of polypeptides, GENSET-binding compounds, antibodies to 0883 a) contacting a biological sample which identity is GENSET polypeptides, etc., for example, disposed within a to be assayed with a product able to bind a GENSET gene container means. The kit can further comprise ancillary product; and reagents, including buffers and the like. 0884 b) determining whether a GENSET gene product is Identification of a Specific Context of GENSET Polypep expressed in Said biological Sample. tide-Encoding Gene Expression 0885 Products that are able to bind specifically to a 0880. When the expression pattern of a GENSET GENSET gene product, namely a GENSET polypeptide or polypeptide-encoding mRNA shows that a GENSET a GENSET polypeptide-encoding mRNA, include GENSET polypeptide-encoding gene is Specifically expressed in a polypeptide binding proteins, antibodies or binding frag given context, probes and primerS Specific for this gene as ments thereof (e.g. F(ab')2 fragments), as well as GENSET well as antibodies binding to the GENSET polypeptide polynucleotide complementary probes and primers. encoding polynucleotide may then be used as markers for the Specific context. Examples of Specific contexts are: 0886 Step b) may be performed using any detection Specific expression in a given tissue/cell or tissue/cell type method known to those skilled in the art including those (See, e.g., Tables III-V), expression at a given stage of disclosed herein, especially in the Section entitled "Detec development of a proceSS Such as embryo development or tion of GENSET polypeptide expression and/or biological disease development, or specific expression in a given activity”. organelle. Such primers, probes, and antibodies are useful Identification of Tissue Types or Cell Species by Means of commercially to identify tissues/cells/organelles of Labeled Tissue Specific Antibodies unknown origin, for example, forensic Samples, differenti ated tumor tissue that has metastasized to foreign bodily 0887. Identification of specific tissues is accomplished by Sites, or to differentiate different tissue types in a tissue the Visualization of tissue Specific antigens by means of croSS-Section using any technique known to those skilled in antibody preparations which are conjugated, directly (e.g., the art including in situ PCR or immunochemistry for green fluorescent protein) or indirectly to a detectable example. marker. Selected labeled antibody species bind to their Specific antigen binding partner in tissue Sections, cell 0881. For example, the cDNAS and proteins of the Suspensions, or in extracts of Soluble proteins from a tissue Sequence listing and fragments thereof, may be used to Sample to provide a pattern for qualitative or Semi-qualita distinguish human tissues/cells from non-human tissues/ tive interpretation. cells and to distinguish between human tissues/cells/or 0888 Antisera for these procedures must have a potency ganelles that do and do not express the polynucleotides exceeding that of the native preparation, and for that reason, comprising the cDNAS. By knowing the expression pattern antibodies are concentrated to a mg/ml level by isolation of of a given GENSET polypeptide, either through routine the gamma globulin fraction, for example, by ion-exchange experimentation or by using the instant disclosure, the chromatography or by ammonium Sulfate fractionation. polynucleotides and polypeptides of the present invention Also, to provide the most specific antisera, unwanted anti may be used in methods of determining the identity of an bodies, for example to common proteins, must be removed unknown tissue/cell Sample/organelle. AS part of determin from the gamma globulin fraction, for example by means of ing the identity of an unknown tissue/cell Sample/organelle, insoluble immunoabsorbents, before the antibodies are the polynucleotides and polypeptides of the present inven labeled with the marker. Either monoclonal or heterologous tion may be used to determine what the unknown tissue/cell Sample is and what the unknown Sample is not. For example, antisera is Suitable for either procedure. if a cDNA is expressed in a particular tissue/cell type/ A. Immunohistochemical Techniques organelle, and the unknown tissue/cell Sample/organelle does not express the cDNA, it may be inferred that the 0889 Purified, high-titer antibodies, prepared as unknown tissue/cells are either not human or not the same described above, are conjugated to a detectable marker, as human tissue/cell type/organelle as that which expresses the described, for example, by Fudenberg, (1980) or Rose et al., cDNA. These methods of determining tissue/cell/organelle (1980), which disclosures are hereby incorporated by refer identity are based on methods which detect the presence or ence in their entireties. absence of the mRNA (or corresponding cDNA) in a tissue/ 0890. A fluorescent marker, either fluorescein or cell Sample using methods well know in the art (e.g., rhodamine, is preferred, but antibodies can also be labeled hybridization, PCR based methods, immunoassays, immu with an enzyme that Supports a color producing reaction US 2006/0053498A1 Mar. 9, 2006 94 with a Substrate, Such as horseradish peroxidase. Markers proteins is transferred by blotting to a nitrocellulose filter can be added to tissue-bound antibody in a Second Step, as paper, a process that maintains the pattern of resolution. described below. Alternatively, the Specific anti-tissue anti Multiple copies are prepared. The procedure, known as bodies can be labeled with ferritin or other electron dense Western Blot Analysis, is well described in Davis et al., particles, and localization of the ferritin coupled antigen (1986) Section 19-3. One set of nitrocellulose blots is antibody complexes achieved by means of an electron stained with Coomassie Blue dye to visualize the entire set microScope. In yet another approach, the antibodies are of proteins for comparison with the antibody bound proteins. radiolabeled, with, for example ‘I, and detected by over The remaining nitrocellulose filters are then incubated with laying the antibody treated preparation with photographic a Solution of one or more specific antisera to tissue specific emulsion. Preparations to carry out the procedures can proteins prepared as described herein. In this procedure, as comprise monoclonal or polyclonal antibodies to a single in procedure A above, appropriate positive and negative protein or peptide identified as Specific to a tissue type, for Sample and reagent controls are run. example, brain tissue, or antibody preparations to Several 0892. In either procedure A or B, a detectable label can be antigenically distinct tissue specific antigens can be used in attached to the primary tissue antigen-primary antibody panels, independently or in mixtures, as required. Tissue complex according to various Strategies and permutations Sections and cell Suspensions are prepared for immunohis thereof. In a Straightforward approach, the primary Specific tochemical examination according to common histological antibody can be labeled; alternatively, the unlabeled com techniques. Multiple cryostat Sections (about 4 um, unfixed) plex can be bound by a labeled Secondary anti-IgG antibody. of the unknown tissue and known control, are mounted and In other approaches, either the primary or Secondary anti each slide covered with different dilutions of the antibody body is conjugated to a biotin molecule, which can, in a preparation. Sections of known and unknown tissues should Subsequent Step, bind an avidin conjugated marker. Accord also be treated with preparations to provide a positive ing to yet another Strategy, enzyme labeled or radioactive control, a negative control, for example, pre-immune Sera, protein A, which has the property of binding to any IgG, is and a control for non-specific Staining, for example, buffer. bound in a final Step to either the primary or Secondary Treated sections are incubated in a humid chamber for 30 antibody. The Visualization of tissue specific antigen binding min at room temperature, rinsed, then washed in buffer for at levels above those Seen in control tissues to one or more 30–45 min. Excess fluid is blotted away, and the marker tissue specific antibodies, prepared from the gene Sequences developed. If the tissue specific antibody was not labeled in identified from cDNA sequences, can identify tissues of the first incubation, it can be labeled at this time in a Second antibody-antibody reaction, for example, by adding fluores unknown origin, for example, forensic samples, or differ cein- or enzyme-conjugated antibody against the immuno entiated tumor tissue that has metastasized to foreign bodily globulin class of the antiserum-producing Species, for Sites. example, fluorescein labeled antibody to mouse IgG. Such Screening and Diagnosis of Abnormal GENSET Polypep labeled Sera are commercially available. The antigen found tide Expression and/or Biological Activity in the tissues by the above procedure can be quantified by 0893 Moreover, antibodies and/or primers specific for measuring the intensity of color or fluorescence on the tissue GENSET polypeptide expression may also be used to iden Section, and calibrating that Signal using appropriate Stan tify abnormal GENSET polypeptide expression and/or bio dards. logical activity, and Subsequently to Screen and/or diagnose B. Identification of Tissue Specific Soluble Proteins disorders associated with abnormal GENSET polypeptide 0891. The visualization of tissue specific proteins and expression. For example, a particular disease may result identification of unknown tissues from that procedure is from lack of expression, Over expression, or under expres carried out using the labeled antibody reagents and detection sion of a GENSET polypeptide-encoding mRNA. By com Strategy as described for immunohistochemistry; however paring mRNA expression patterns and quantities in Samples the Sample is prepared according to an electrophoretic taken from healthy individuals with those from individuals technique to distribute the proteins extracted from the tissue Suffering from a particular disorder, genes responsible for in an orderly array on the basis of molecular weight for this disorder may be identified. Primers, probes and anti detection. A tissue Sample is homogenized using a Virtis bodies specific for this GENSET polypeptide may then be apparatus, cell Suspensions are disrupted by Dounce homog used to elaborate kits of Screening and diagnosis for a enization or osmotic lysis, using detergents in either case as disorder in which the gene of interest is specifically required to disrupt cell membranes, as is the practice in the expressed or in which its expression is Specifically dysregu art. Insoluble cell components Such as nuclei, microSomes, lated, i.e. underexpressed or overexpressed. and membrane fragments are removed by ultracentrifuga Screening for Specific Disorders tion, and the Soluble protein-containing fraction concen 0894. The present invention also relates to methods and trated if necessary and reserved for analysis. A Sample of the uses of GENSET polypeptides for identifying individuals Soluble protein Solution is resolved into individual protein having elevated or reduced levels of GENSET polypeptides, Species by conventional SDS polyacrylamide electrophore which individuals are likely to benefit from therapies to sis as described, for example, by Davis et al., Section 19-2 SuppreSS or enhance GENSET polypeptide-encoding gene (1986), using a range of amounts of polyacrylamide in a set expression, respectively. One example of Such methods and of gels to resolve the entire molecular weight range of uses comprises the Steps of: proteins to be detected in the Sample. A size marker is run in parallel for purposes of estimating molecular weights of 0895) a) obtaining from a mammal a biological Sample; the constituent proteins. Sample size for analysis is a con 0896 b) detecting the presence in said sample of a venient volume of from 5 to 55 ul, and containing from GENSET polypeptide-encoding gene product (mRNA or about 1 to 100 ug protein. An aliquot of each of the resolved protein); US 2006/0053498A1 Mar. 9, 2006

0897 c) comparing the amount of said GENSET product. In accordance with this method, the presence in the polypeptide-encoding gene product present in Said Sample Sample of altered (e.g. increased or decreased) levels of the with that of a control Sample, and GENSET product indicates that the subject is predisposed to the disease or condition. Biological Samples Suitable for use 0898 d) determing whether said human or non-human in this method include biological fluids including, but not mammal has a reduced or elevated level of GENSET gene limited to, blood, Saliva, milk, and urine. Tissue Samples expression compared to the control Sample. (e.g. biopsies) can also be used in the method of the 0899. A biological sample from a subject affected by, or invention, including Samples derived from any of the tissues at risk of developing, any disease or condition associated listed in Tables III-V. Cell cultures or cell extracts derived, with a GENSET polypeptide can be screened for the pres for example, from tissue biopsies can also be used. ence of increased or decreased levels of GENSET gene product, relative to a normal population (standard or con 0907. The diagnostic methodologies described herein are trol), with an increased or decreased level of the GENSET applicable to both humans and non-human mammals. polypeptide relative to the normal population being indica Detection of GENSET Gene Mutations tive of predisposition to or a present indication of the disease 0908. The invention also encompasses methods and uses or condition, or any Sympton associated with the disease or of GENSET polynucleotides to detect mutations in condition. Such individuals would be candidates for thera GENSET polynucleotides of the invention. Such methods pies, e.g., treatment with pharmaceutical compositions com may advantageously be used to detect mutations occurring prising the GENSET polypeptide, a polynucleotide encod in GENSET genes and preferably in their regulatory regions. ing the GENSET polypeptide, or any other compound that When the mutation was proven to be associated with a affects the expression or activity of the GENSET polypep disease, the detection of Such mutations may be used for tide. Generally, the identification of elevated levels of the Screening and diagnosis purposes. GENSET polypeptide in a patient would be indicative of an individual that would benefit from treatment with agents that 0909. In one embodiment of the oligonucleotide arrays of SuppreSS GENSET polypeptide expression or activity, and the invention, an oligonucleotide probe matrix may advan the identification of low levels of the GENSET polypeptide tageously be used to detect mutations occurring in GENSET in a patient would be indicative of an individual that would genes and preferably in their regulatory regions. For this benefit from agents that induce GENSET expression or particular purpose, probes are specifically designed to have activity. a nucleotide Sequence allowing their hybridization to the genes that carry known mutations (either by deletion, inser 0900 Biological samples suitable for use in this method tion or substitution of one or several nucleotides). By known include any biological fluids, including, but not limited to, mutations, it is meant, mutations on the GENSET genes that blood, Saliva, milk, and urine. Tissue samples (e.g. biopsies) have been identified according, for example to the technique can also be used in the method of the invention, including used by Huang et al. (1996) or Samson et al. (1996), which samples derived from any tissue associated with GENSET disclosures are hereby incorporated by reference in their gene expression (See, e.g. Tables III-V). Cell cultures or cell entireties. extracts derived, for example, from tissue biopsies can also 0910 Another technique that is used to detect mutations be used. The detection Step of the present method can be in GENSET genes is the use of a high-density DNA array. performed using Standard protocols for protein/mRNA Each oligonucleotide probe constituting a unit element of detection. Examples of suitable protocols include Northern the high density DNA array is designed to match a specific blot analysis, immunoassays (e.g. RIA, Western blots, Subsequence of a GENSET genomic DNA or cDNA. Thus, immunohistochemical analyses), and PCR. an array consisting of oligonucleotides complementary to 0901 Thus, the present invention further relates to meth Subsequences of the target gene Sequence is used to deter ods and uses of GENSET polypeptides for identifying mine the identity of the target Sequence with the wild gene individuals or non-human animals at increased risk for Sequence, measure its amount, and detect differences developing, or present State of having, certain diseases/ between the target Sequence and the reference wild gene disorders associated with abnormal GENSET polypeptide Sequence of the GENSET gene. In one Such design, termed expression or biological activity. One example of Such 4 L tiled array, is implemented a set of four probes (A, C, G, methods comprises the Steps of T), preferably 15-nucleotide oligomers. In each set of four probes, the perfect complement will hybridize more Strongly 0902 a) obtaining from a human or non-human mammal than mismatched probes. Consequently, a nucleic acid target a biological Sample, of length L is Scanned for mutations with a tiled array 0903 b) detecting the presence in said sample of a containing 4 L probes, the whole probe Set containing all the GENSET gene product (mRNA or protein); possible mutations in the known wild reference Sequence. The hybridization signals of the 15-mer probe set tiled array 0904 c) comparing the amount of said GENSET gene are perturbed by a Single base change in the target Sequence. product present in Said Sample with that of a control Sample; AS a consequence, there is a characteristic loSS of Signal or and a "footprint” for the probes flanking a mutation position. 0905 d) determing whether said human or non-human This technique was described by Chee et al. in 1996, which mammal is at increased risk for developing, or present State disclosure is hereby incorporated by reference in its entirety. of having, a diseases or disorder. Construction of DNA Constructs with a GENSET Gene 0906. In preferred embodiments, the biological sample is Expression Pattern taken from animals presenting any Symptom asSociated with 0911. In addition, characterization of the spatial and any disease or condition associated with a GENSET gene temporal expression patterns and expression levels of