WO1998048020A2 - Mammalian ubiquitin-isopeptidases that control cell proliferation - Google Patents

Abstract

The present invention relates to ubiquitin isopeptidases, to the polynucleotides encoding them, to methods for detecting such proteins and regulating their cellular activities.

Description

"Mammalian ubiquitin-isopeptidases that control cell proliferation"

The present invention relates to ubiquitin isopeptidases (UBPs) , to proteins interacting with them and to the encoding polinucleotides, to methods for detecting such proteins and regulating their cellular activities.

The ubiquitin pathway controls the degradation of a large number of intracellular proteins, many of which play key roles in controlling cell growth (Hochstrasser; Curr Op Cell Biol 7: 215-223, 1995; Pagano, Beer-Romero et al. Seventh Pezcoller Symposium, Cancer Genes: Functional Aspects, 1995; Hochstrasser. Cell 84: 813-815, 1996).

The intracellular levels of proteins like p53, the product of a gene which is inactivated in a large fraction of human tumors, or p27, an inhibitor of the cell division cycle, the transcription factor NFkB, whose activation is fundamental for the activation of inflammatory processes, are controlled by such system. Ubiquitin is a 76 a ino acid protein which is conjugated to protein substrates thus generating a signal for degradation. Ubiquitin conjugation is generated first through the formation of an isopeptide bond between glycine 76 of ubiquitin and an epsilon-amino group of a lysine within a protein substrate, and is then followed by the further conjugation of ubiquitin molecules one on the top of the other. The presence of ubiquitin trees on a given substrate is recognized by the proteasome, a high molecular weight multi protein complex, which is able to digest the target molecule, and to generate small peptides and free ubiquitin, ready to be re-utilized by the cell. The conjugation of ubiquitin to a given substrate is mediated by three enzyme types, which have been called El (ubiquitin- activating enzyme) , E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin-ligase) . The role of El appears to be limited to the generation of activated intermediaries of E2 and E3, which occur through the formation of thioesters between the active site cysteine of such enzymes and the C-terminus of ubiquitin. E2 and E3 of which many different forms exist within a single cell, are responsible for the specific recognition of a given substrate. Recently enzymatic activities have been identified which are able to hydrolyze the isopeptide bond generated by the conjugation of ubiquitin to a given protein substrate (Papa and Hochstrasser; Nature 366: 313-9, 1993) . Relatively little is known of these enzymes but it has been proposed that they could also be involved in actively regulating the degradation of intracellular proteins. The balance between the relative rates of ubiquitination and de-ubiquitination of a given protein could control its intracellular levels and as a consequence its function. De-ubiquitinating enzymes are also called ubiquitin-isopeptidases (UBPs) and have been identified in yeast, in the nematode C. elegans, in Drosophila and mammalian cells thus far. What specific defects have been identified so far in cells lacking or carrying an altered UBP? In Drosophila the product of the fat facets (Faf) gene encodes a UBP. Faf is required for Drosophila eye development and ovarian function (Fischer- Vize, et al., 1992. Development 116, 985-1000). It has been demonstrated that the Faf phenotype can be rescued by either expressing a fully functional Faf UBP, or by down regulating proteasome function (Huang, et al., 1995. Science 270, 1828- 1831) . This would argue that FAF protein antagonizes the proteasome because of its ability to remove ubiquitin from target substrates. In yeast several UBPs have been identified. The yeast UBP1, UBP2 and UBP3 proteins are all able to cleave Ub-fusion proteins and polyubiquitin chains. While a triple disruption of yeast UBP1, UBP2, UBP3 does not results in obvious growth defects (Baker, et al . , 1992. J Biol Chem 267, 23364-75), UBP3 protein has been found to interact with SIR4, a protein required for transcriptional silencing at the mating type locus. Yeast cells lacking UBP3 have in fact an improved ability to activate silencing, suggesting that UBP3 is an inhibitor of silencing (Moazed and Johnson, 1996. Cell 86, 667-77) . A Drosophila UBP has also been implicated in transcriptional silencing (Henchoz, et al., 1996. Mol Cell Biol 16, 5717-25). The yeast DOA4 gene, also called UBP4, was identified due to its ability to restore degradation of the MATa2 transcription factor (Papa end Hochstrasser. Nature 366: 313-9, 1993) . Yeast lacking DOA4 show slow growth, DNA repair defects, and overexpression of a dominant negative D0A4 Cys-to-Ser mutant causes a slowdown in cell growth. In mammalian cells several UBPs have been identified. The Tre213-ORF2 protein was discovered as the product of a genetic rearrangement in human cells transfected with DNA from Ewing sarcoma cells and was shown to share structural similarities with the yeast DOA4 protein (Huebner, et al., 1988. Oncogene 3, 449-55). The Tre 213-ORF2 protein has the hallmark signatures of a UBP, and it can hydrolyze ubiquitin fusion proteins (Papa and Hochstrasser, 1993. Nature 366, 313-9) (Nakamura, et al., 1988. Oncogene Res 2, 357-70, Nakamura, et al., 1992. Oncogene 7, 733-41). Given that a construct comprising a truncated, inactive form of Tre 213- ORF2 was able to transform cells in culture, the hypothesis has been made that Tre 213-ORF2, by removing ubiquitin from a growth suppressing protein, enhances its activity and that the mutant protein could counteract these effects thereby enhancing cell proliferation.

The DUB-1 UBP is the product of a cytokine inducible gene (Zhu, et al., 1996. Proc Natl Acad Sci U Ξ A 93, 3275-9, Zhu, et al., 1997. J Biol Chem 272, 51-7). Its mRNA and protein are induced very rapidly in response to IL-3 addition to a murine lymphocyte line, and then quickly down-regulated as cells progress into S-phase. The continuous expression of a wild-type DUB-1 protein, but not of a Cys-to-Ala mutant, causes cell cycle arrest in the Gl phase of the division cycle, suggesting that its down-regulation in Gl is essential for cell cycle progression. The same group has identified a second gene product, DUB-2, which is induced upon IL-2 addition to CTLL cells (Jaster, et al . , 1997. Mol Cell Biol 17, 3364-72) . The mouse UNP gene was identified because of its proximity to a retroviral insertion site (Gupta, et al., 1993. Oncogene 8, 2307-2310). Cells transfected with UNP cDNA cause tumors in athymic mice (Gupta, et al., 1994. Oncogene 9, 1729-31) . The human UNP mRNA was also found overexpressed in human small cell lung primary carcinomas and cell lines (Gray, et al., 1995. Oncogene 10, 2179-2183). Recently, a further human UBP has been identified due to its ability to interact with VmwllO, an immediate early protein of herpes simplex virus type I (Everett, et al., 1997. Embo J 16, 1519- 30) . A gene encoding a novel human UBP has also been found on the X chromosome within a region involved in X-linked retinal disorders and found to be highly expressed in retina (Swanson, et al., 1996. Hum Mol Genet 5, 533-8). Whether preferential substrates of UBPs exist is still a question. The existence of sixteen UBP family members in yeast would indicate that they either have distinct substrates or have distinct upstream regulatory mechanisms that control their activity selectively. In human cells, we have been able to detect expression of different members of this family in the same cell type. We also know that in primary tissues certain of these enzymes show a remarkable tissue specific expression and that they can be overexpressed in human tumors. Here we demonstrate that inhibition of a cellular UBP can have a striking effect on cell proliferation. UBPs are cysteine proteases and as such amenable to the development of specific chemical inhibitors. The selective inhibition of any member of this family which either showed a tissue and/or tumor specific expression could be considered as a strategy for the development of novel pharmaceutical agents.

Three novel ubiquitin isopeptidases have now been identified, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, and it has been found that such proteins are involved in controlling the cell division cycle and/or that alterations of the same proteins can be detected in human tumors.

Other two novel proteins having the a ino acid sequences reported as SEQ ID NO: 8, SEQ ID NO: 10, which interact with the above reported ubiquitin isopeptidases, have been identified.

Therefore the present invention refers to the proteins having the amino acid sequences reported as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and their functional derivatives.

A further aspect of the present invention are the polynucleotides, isolated or purified that operationally encode the above indicated SEQ ID NO: 2 protein, SEQ ID NO: 4 protein; SEQ ID NO: 6 protein, SEQ. ID NO.: 8 protein; SEQ ID NO: 10 protein. Specifically, such polynucleotides include the nucleotide sequence reported as SEQ ID NO: 1, which encodes the SEQ ID NO: 2 protein, the SEQ ID NO: 3 which encodes the SEQ ID NO: 4 protein, the SEQ ID NO: 5 which encodes the SEQ ID NO: 6 protein, the SEQ ID NO: 7 which encodes the SEQ ID NO: 8 protein; the SEQ ID NO: 9 which encodes the SEQ ID NO: 10 protein.

Another aspect of the present invention are the mRNAs which encode the amino acid sequences of the proteins part of the invention.

Specifically, object of the present invention are the mRNAs of the polynucleotide sequences SEQ ID NO:l, 3, 5, 7, 9.

In a preferred embodiment of the present invention are also the expression vectors needed for the production of the proteins of the invention in prokaryotic and eucaryotic cells and the cell lines containing such vectors for the expression of the proteins of the invention.

The term "functional derivative" is referred to a "fragment", a "variant", an "analogue" or a "chemical derivative" of the proteins of the present invention which maintains the same function of the native proteins described.

The term "fragment" is referred to any portion of the proteins of the present invention. The term "variant" is referred to molecules similar in the overall to the complete protein of the invention or to a fragment of such protein. For example, such variants include deletions, insertions, and / or substitutions of residues in the amino acid sequence. The term "analogue" is referred to a molecule which is not present in nature and is basically similar to the native protein or to a fragment of it.

The term "chemical derivative", as used herein, refers to proteins and peptides of the present invention containing chemical groups that do not normally belong to the protein.

Such "chemical derivatives" are obtained by chemical modification of specific amino acid residues with an organic derivatizing agent, known to the person skilled in the art, that is capable of reacting with selected side chains or terminal residues. Such modifications may improve the solubility, absorption, biological half life and the like, of the proteins and peptides of the invention. Such modifications are reported, for example , in Remington's Pharmaceutical Sciences 16th ed., Mack Publishing Co., Easton, PA (1980) .

Cysteinyl residues most commonly are reacted with alpha-haloacetates ( and corresponding amines ) , such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives . Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, alpha- bromo-beta- (5-imidazolyl) propionic acid, choroacetyl- phosphate, N-alkylmaleimides, 3-nitro-2pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l, 3-diazole. Histidyl residues are derivatized by reaction with diethylpirocarbonate at pH 5.5-7.0 because this agent is relativly specific for the histidyl side chain. Para- bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with this agent has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imido esters such as methyl picolynimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; 0- methylisourea; 2, 4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3- butanedione, 1, 2-cyclohexanedione, and ninhydrin. Deriva- tization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues per se has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazol and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R¹ -N-C-N-R' ) such as l-cyclohexyl-3- (2-morpholinyl- (4-ethyl) -carbodiimide or 1- ethyl-3-azonia-4 , 4-dimethylpentyl) carbo-diimide . Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues.

Alternatively, these residues are deaminated under mildly acidic conditions. Either form of these residues falls within the scope of this invention . Derivatization with bifunctional agents is useful for cross- linking the peptide to a water insoluble support matrix or to other macromolecular carriers. Commonly used cross-linking agents include, e.g. , 1, 1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, n-hy-droxysuccinimide esters , for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis (succinimi-dylpropionate) , and bifunctional maleimides such as bis-N-maleimido-1, 2-octane . Derivatizing agents such as methyl-3- ( (p-azidophenyl) dithio (propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide- activated carbohydrates and the reactive substrates described in U.S. -A-3, 969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alfa-amino groups of lysine, arginine, and histidine side chains (T . E. Creighton, Proteins : Structure and Molecule Properties, W.H. Freeman & Co., San Francisco pp.79.86 (1983)), acetylation of the N- terminal a ine and, in some istances, amidation of the C- terminal carboxyl groups.

The proteins of the present invention are referred to as isolated and / or purified, where the term "isolated" denotes that the material has been removed from its original environment and the term "purified" is intended relative to the material in its natural state an does not mean absolute purity .

With the term "protein" is intended a polypeptide with a molecular weight between about 5.000 and more than 150.000 Dalton. The concentration of proteins isolated and / or purified is preferably al least 1 μg/ml.

Another aspect of the invention consists in purified antibodies against the proteins of the invention, including both monoclonal and polyclonal antibodies. The invention further includes antisense RNA obtained from the polynucleotides of the invention.

In addition, the present invention includes homopurine and homopyrimidine sequences of the polynucleotides of the invention or of their fragments, and their use as triple helix probes.

Another aspect of the present invention is an assay for the enzymatic activity of ubiquitin isopeptidases which comprises the following steps: a) Preparation of a cell extract containing ubiquitinated proteins; b) Incubation of the cell extract obtained in a) . with the protein being tested. c) Quantitative measurement of ubiquitin release .

As an option, the cell extract could be treated with proteasome inhibitors or could be derived from cells treated with proteasome inhibitors .

Inhibitors of isopeptidase activity can be for example, N- ethyl maleimide, iodoacetamide or iodoacetate.

The methods to detect quantitative ubiquitin release can use, for example, immunoblotting with anti-ubiquitin antibodies or

ELISA methods with aiiti-ubiquitin antibodies.

Description of the Figures

Fig. 1: Identification of the UBP (SEQ ID NO: 2) protein in cell extracts (A, B) Immunoprecipitation and immunoblotting experiments.

(A) U20S (lanes 1-4) and WI-38 (lanes 5-8) cell extracts were prepared. 1 mg of lysate proteins were immunoprecipitated with either affinity purified antibodies to the SEQ ID NO: 2 protein (lanes 2 and 6) or the corresponding preimmune serum (lanes 1 and 5). 30 μg of a cell lysate (lanes 3 and 7) or of an immunoprecipitation supernatant (lanes 4 and 8), were subjected to SDS-PAGE (8%) , transferred to nitrocellulose and blotted with the immune serum. (B) The SEQ ID NO: 1 cDNA was transcribed and translated in rabbit reticulocyte lysate (RRL) . 5 μl of the programmed RRL (lane 4), or of a control (empty vector) mixture (lane 5), one half of an immunoprecipitation (500 μg of C33A lysate) with immune serum (lane 3) or preimmune serum (lane 2), 10 μg of lysate (lane 1), were subjected to SDS-PAGE (10 %) and processed as in A.

(C, D ) Immunoblotting upon transient transfection. C33A (C) and U20S (D) cells were transfected with the pCMV- neo-Bam control vector (lane 1) or with the same vector containing the SEQ ID NO: 1 cDNA in either sense (S) (lane 3) , or antisense (AS) orientation (lane 2) . 20 μg of each lysate were subjected to SDS-PAGE (8%) and processed as in A. (E) Immunoblotting from human cell lines. 20 μg of total lysate from the indicated cell lines were subjected to immunoblotting as in A. 1: SAOS; 2: U20S; 3: WI- 38; 4: VA13; 5: C33A; 6: HeLa; 7: U937.

Molecular size markers (kDa) are indicated. The position of the protein is indicated by arrowheads.

Fig. 2: UBP (SEQ ID NO: 2) Deubiquitinating Activity. (A, B) The GST-UBP protein was expressed in bacteria, purified and its deubiquitinating activity tested. (A) In vitro ubiquitinated and NEM-inactivated RRL (Promega) was used as a substrate and incubated in the absence (lane 1) or in the presence of 0.5 μg (lane 2, indicated as +) , or 1 μg GST-UBP-SEQ ID NO: 2 (lane 3, indicated as ++) , or of 1 μg GST-UBP-SEQ ID NO: 2 pretreated with N-ethylmaleimide (lane 4). Reactions were stopped with Laemmli buffer and subjected to immunoblotting with anti-ubiquitin antiserum (Sigma) . (B) Ub6 was used as a substrate and incubated in the presence of 1 μg of GST-UBP (lane 2), or in the presence of 1 μg GST-UBP pretreated with N-ethylmaleimide (lane 1) . Reactions were stopped with Laemmli buffer and subjected to immunoblotting with monoclonal anti-Ha antibodies. (C, D) Testing UBP activity in immunoprecipitations. (C) Cell extracts from U20S wild type (lanes 1, 5,6) and from NIH-3T3 cells transiently transfected with pCMV-neo-Bam vector alone (lanes 2, 7), or with the same vector containing the UBP-SEQ ID NO: 1 cDNA encoding wild type (lanes 3, 8, 9) or Cys786Ala mutant protein (lanes 4, 10) were prepared and immunoprecipitations from 300 μg of each cell extract with affinity purified antibodies (lanes 5-10) were performed in the presence (lanes 6, 9) or the absence (lanes 5, 7, 10) of 10 mM NEM. 40 μg of cell extracts (lanes 1-4) and one half of the total immunoprecipitation (lanes 5-10) were subjected to anti-UBP-SEQ. ID NO. 2 immunoblotting.

(D) Ub6 was used as a substrate and incubated solely with protein A Sepharose beads (lane 1) or with UBP-SEQ ID NO: 2 immunoprecipitations (lanes 2-7). Reactions were stopped with Laemmli buffer and subjected to immunoblotting with anti Ha MAb to detect HA-tagged reaction products. Lanes 2-7 measure the activities of the samples indicated in panel C (lanes 5- 10 above) .

Fig. 3: UBP (SEQ. ID NO.:2) is essential for cell growth (A, B) Regulation of the UBP protein levels in response to changes in growth condition.

WI-38 human fibroblasts were arrested in G0/G1 phase by serum starvation for 72 hr (0.5 % serum), re-stimulated with 10% serum and further cultured for the indicated times. Cell extracts were prepared from exponentially (As) growing, starved, and re-stimulated cells. 20 μg of extracts were subjected to SDS-PAGE (8%), transferred to nitrocellulose membrane and blotted with antibodies against the indicated proteins. The different cell cycle phases were determined by propidium iodide flow-cytometry.

(C) Microinjection of antisense UBP-SEQ ID NO: 1 cDNA causes inhibition of S-phase entry. WI-38 human fibroblasts were made quiescent by serum starvation. Arrested cells (200-250 per point) were injected with the pCMV-neo-Bam empty vector or the same vector containing the UBP-SEQ ID NO: 1 cDNA in antisense orientation. Cells were re-stimulated with 10% serum and BrdU was added to the growth medium. Cells were fixed and stained 24 hours post-serum addition. The indicated values were calculated as the ratio of injected BrdU-positive cells to BrdU-positive surrounding cells multiplied by 100. The results are the mean (± s. e.) of three independent experiments.

Fig. 4: UBP (SEQ ID NO: 2) protein levels regulation by cell- cell adhesion

(A) WI-38 human fibroblasts (8 x 10⁵ cells/plate) and (B) human osteosarcoma U20S cells (5 x 10⁵ cells/plate) were plated and cultured until they reached confluence (day 4) and then grown for three further days (day 7), with medium changes every two days. Cell extracts were prepared from asynchronously growing (days 1 to 3) and from confluent cells (days 4 and 7) . 20 mg of each lysate were subjected to SDS- PAGE (12%), transferred to nitrocellulose membrane and blotted with antibodies against the indicated proteins.

Fig 5: UBP (SEQ. ID NO. 2) down-regulation causes accumulation of ubiquitinated proteins .

(A, B) U20S cells were cotransfected with 10 mg of HA-Ub plasmid and 10 μg of pCMV empty vector (V) , or of pCMV-UBP- SEQ ID NO: 1 antisense (AS), or 10 μg of pCMV-UBP-SEQ ID NO: 1 sense (S) , or 10 μg of pCMV-UBP-SEQ ID NO: 1 Cys786Ala sense (Sm) constructs.

(A) Lysates (30 μg) were subjected to SDS-PAGE (8%) and to anti-UBP-SEQ ID NO: 2 or to anti-HA immunoblotting. A control transfection (lane C) with 20 μg of pCMV empty vector without Ha-Ub plasmid was also performed.

(B) Lysates (30 μg) from a different experiment than in A were subjected to SDS-PAGE (15%) and to anti-HA immunoblotting to detect ubiquitinated proteins. In both the experiments each co-transfection was made in triplicate.

Fig. 6: Effect of altering UBP (SEQ ID NO: 2) expression on cell proliferation

(A) Dose-effect antisense experiment. U20S cells were cotransfected with 2 μg of GFP plasmid and 20 μg of the pCMV vector (control) , or 15 μg of the same empty vector and 5 μg of pCMV-UBP-SEQ ID NO: 1 antisense construct, or 10 μg of the same empty vector and 10 μg of pCMV-UBP-SEQ ID NO: 1 antisense construct, or 20 μg of pCMV-UBP-SEQ ID NO: 1 antisense construct. Propidium iodide staining of GFP- positive gated cells versus cell number is shown. Also indicated are the percentages of cells in the different phases of the division cycle. (B) Colony forming assay 8 x 10⁵ U20S cells were plated in a 10 cm dish and transfected with either 20 μg of control empty pCMV vector or of the same vector containing the UBP-SEQ ID NO: 1 cDNA in sense orientation. Drug resistant colonies (15 days after transfection) stained with crystal violet are shown.

Fig. 7: Two Hybrid System to test the binding specificity of G3BP (SEQ ID NO: 7 and 8) for UBP SEQ. ID. NO: 6. l.Full length UBP SEQ ID No: 6 (yeast strain L40 carrying the

UBP coding sequence linked to LexA-pLexA190FL) transformed with G3BP activator domain clone

2. Full length UBP SEQ ID NO: 2 (L40pLexA55) transformed with G3BP activator domain clone

3. Full length UBP UNPH (L40pLexAUNP) transformed with G3BP activator domain clone

4. Full length UBP SEQ ID NO: 6 (L40pLexA190FL) carrying a

Cys488Ala mutation in the catalytic site transformed with G3BP activator domain clone

5. Full length UBP SEQ ID NO: 6 (L40pLexA190FL) carrying a deletion in the carboxy terminal part (lacking both cysteine and histidine boxes) transformed with G3BP activator domain clone

Fig. 8: Immunoblotting with antibodies to UBP (SEQ. ID.

NO: 6)

1. 20 μg C33A lysate

2. 20 μg HL60 lysate 3. 20 μg U20S lysate

4. 1 μg GST190

5. 4 μl of in vitro translated product

Fig . 9 : Immunoprecipitaton with affinity purified antibodies to UBP (SEQ. ID. NO: 6)

1. 4 μl of in vitro transcribed/translated SEQ. ID. NO: 5 DNA.

2. 20 μg C33A cervical carcinoma cell lysate

3. Immunoprecipitation with preimmune serum (1 mg of C33A cervical carcinoma cell lysate)

4. Immunoprecipitation with preimmune serum (1 mg of C33A cervical carcinoma cell lysate) .

Arrow indicates the specific UBP band. Fig. 10: Characterization of the UBP (SEQ ID NO: 4) expression in human colon cancer cases by immunoblotting.

Matched normal and tumor samples derived from four different patients were run on SDS-PAGE and immunoblotted with a specific antibody to UBP-SEQ ID NO: 4. For comparison UBP-SEQ ID NO: 4 expression in normal fibroblasts (IMR90 strain) and a human breast cancer line (MCF7) is shown.

Fig.11: Characterization of the UBP (Seq. ID. NO: 4) expression in human colon tissue

Lysates from either normal or tumor colon epithelium were tested for UBP (SEQ ID NO: 4) expression by immunoblotting with a specific anti-C-terminal peptide antiserum. The figure shows that a band, indicated by the arrow, disappears upon treatment of the antiserum with the antigenic peptide.

Fig. 12: Tissue and tumor specific expression of human ubiquitin isopeptidases : Detection of UBP-SEQ ID NO: 6 in human epidermis by immunohistochemistry .

Detection of UBP-SEQ ID NO: 5 mRNA by in situ hybridization in a sample of colon mucosa showing features of normal, adenoma and adenocarcinoma tissue. : Detection of UBP-SEQ ID NO: 6 protein expression in normal

(C) , adenoma (D) and adenocarcinoma (E) colon tissue.

EXAMPLES

Example 1 : Ubiquitin isopeptidase expression

The UBP-SEQ ID NO: 1 cDNA clone was first identified in the human myeloblast cell line KG-1. The entire UBP-SEQ ID NO: 1 cDNA sequence (bases 1 to 4359) was submitted (13-APR-1994 ) to the DDBJ/EMBL/GenBank databases by Nomura, N. et Al . (Accession Number D29956) . The UBP-SEQ ID NO: 1 coding sequence extends between bases 318-3674 and is predicted to encode a 1118 amino acid protein. A 3539 bp fragment (bases 226 to 3765) was cloned in the pBscSK+ expression vector in Hind III site, then was cut out from this vector by Xhol-Clal digestion and subcloned in the LTR-2 expression vector in the same sites (Xhol-Clal). The 318-3674 bases fragment was amplified by PCR inserting 5 ' -BamHI and 3 ' -BamHl sites and subsequently cloned in pCMV-neo-Bam- cytomegalovirus (CMV) expression vector, in sense or antisense orientation. A cDNA fragment encoding amino acids 9-188 of UBP-SEQ ID NO: 2, whose sequence does not show significant similarities to other known UBPs was inserted into a pGEX expression vector and used to generate a GST-fusion protein in bacteria. The expressed GST-UBP-SEQ ID NO:2 9-188 protein was purified through chromatography on glutathione-Sepharose and used to generate rabbit polyclonal antisera. The obtained sera were affinity purified on Amino-Link conjugated GST-UBP-SEQ ID NO.:2 9-188 after removal of the anti-GST component by affinity chromatography onto immobilized GST. In Fig.l, we present the characterization of the UBP-SEQ ID NO: 2 protein in a panel of human cell lines. Human lung WI-38 fibroblasts, mouse NIH-3T3 fibroblasts, cervical carcinoma C33A and osteosarcoma U20S cell lines were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 2 mM glutamine, 100 U/ml of penicillin, 1 mg/ml of streptomycin and 10% fetal calf serum (FCS) for WI- 38 and NIH-3T3 cells, or 10% bovine calf serum (BCS) for C33A and U20S cells. For synchronization by serum starvation, WI- 38 fibroblasts (at 30-40 % density) were made quiescent by culturing them for 3 days in DMEM containing 0.5 % FCS. They were then given 10% FCS to induce synchronous entry into the cell cycle. Cell cycle phases were monitored by flow cytometry (see below) .

A protein doublet of Mr 130K was recognized specifically upon immunoblotting with anti-UBP-SEQ ID NO: 2 antibodies in lysates from both non-transformed human fibroblasts (strain WI-38) and the U20S human osteosarcoma cell line (Fig. 1A) . The antibodies were able to precipitate the protein from cell lysates and to quantitatively deplete them of UBP-SEQ ID NO: 1 (Fig. 1A) . In Fig. IB, a similar experiment performed in C33A cervical carcinoma cells shows that the lower band of the UBP-SEQ ID NO: 2 doublet co-migrated with in vitro translated UBP-SEQ ID NO: 2. To confirm the identity of the protein doublet we performed transfection experiments using both U20S and C33A cells. We generated a cDNA construct containing the UBP-SEQ ID NO: 1 open reading frame under the transcriptional control of a cytomegalovirus (CMV) promoter in sense or antisense orientation. Forty-eight hours after transfection cells were collected and lysates analyzed by immunoblotting with anti- UBP-SEQ ID NO: 2 antiserum. In lysates from cells transfected with the antisense vector we detected a decrease in the levels of both 130K bands as compared to lysates made from cells transfected with vector alone (Fig. 1 C, D) . Conversely, lysates from cells transfected with sense UBP-SEQ ID NO: 1 vector showed a substantial accumulation of both bands compared to control. It should be pointed out that the data presented are an under-estimate of the actual effects of sense/antisense transfections since the transfection efficiency was approximately thirty percent, and the immunoblots reflected protein expression in the total cell population. We can therefore estimate a nearly quantitative depletion of UBP-SEQ ID NO: 2 protein in cells transfected with antisense plasmid, and conversely, a substantial increase in UBP-SEQ ID NO: 2 levels in sense-transfected cells. Our data demonstrate that the anti-UBP-SEQ ID NO: 2 antibodies recognize a protein of the expected size in human cell lysates. The UBP-SEQ ID NO: 2 doublet, which in some cases even appeared as a triplet, could be resulting from a post-translational modification and it was detected in all human cell lines tested (Fig. IE). Indeed, we have evidence that UBP-SEQ ID NO: 2 is a phosphoprotein (Ciro Mercurio, SN and GFD, unpublished) .

Cell extracts were prepared as described (Pagano et al; Science 269, 682-685 ,1995). Briefly, 3-5 volumes of lysis buffer (50 mM Tris-HCl pH 7.4, 0.25 M NaCl, 0.5% NP40, 5 mM EDTA, 50 mM NaF, 1 mM Na3V04, 1 mM DTT) were added to pelleted cells. The following protease inhibitors were also added: 0.1 mM phenylmethyl sulfonylfluoride (PMSF), 1 mg/ml of leupeptin, 1 mg/ml of aprotinin, 2 mg/ml of pepstatin. After incubation on ice for 30 min, samples were centrifuged at 14,000 rpm in an Eppendorf microcentrifuge for 15 min. at 4 0C and the supernatant (NP40-total extract) was recovered. Aliquots were taken for protein quantification, using the method of Bradford (Bradford et al.; Anal . Biochem. 12, 248, 1976) . Immunoprecipitation and immunoblotting experiments were performed as described (Pagano et al., Science 269, 682- 685 ,1995). Typically, we employed 10-50 mg of lysate protein for direct immunoblotting and 0.3-2 mg of lysate protein for immunoprecipitation/immunoblotting experiments. Filters were subjected to immunoblotting using the ECL (Amersham) detection system according to the manufacturer's directions.

Example 2 : Assaying UBP enzymatic activity

To demonstrate that UBP-SEQ ID NO: 2 is a ubiquitin isopeptidase, we generated a pool of ubiquitinated proteins using rabbit reticulocyte lysate (RRL) and then incubated them with purified full-length recombinant GST-UBP-SEQ ID NO: 2.

The GST-UBP-SEQ ID NO: 2 full-length and the GST-UBP-SEQ ID NO: 2 9-188 fusion proteins were obtained by recombinant PCR of the appropriate fragment from the UBP-SEQ ID NO: 1-cDNA followed by cloning in the expression vectors pGEX-KG (BamHI- ECORI sites) for GST-UBP-SEQ ID NO: 2 FL, and in pGEX-KT (Xba-Xho sites) for GST-UBP-SEQ ID NO: 2 9-188, in frame with the GST moiety. The GST-UBP-SEQ ID NO: 2 FL protein contains the full length UBP-SEQ ID NO: 2 protein without the initiator methionine (aa 2 to 1118). The GST-UBP-SEQ ID NO: 2 9-188 protein contains the N-terminal portion of the UBP-SEQ ID NO:l-cDNA between amino acids 9 and 188. Purification of the recombinant fusion proteins from bacterial supernatants was performed on glutathione-Sepharose according to the manufacturer's directions (Pharmacia).

To block endogenous deubiquitinating or ubiquitin-conjugating activities, prior to the addition of GST-UBP-SEQ ID NO: 2 the RRL was treated with 20 mM N-ethyl maleimide (NEM) and then the NEM removed by centrifugation and washing through Centricon filters (see Methods) . In Fig. 2A we show the result of an experiment in which after the incubation with or without GST-UBP-SEQ ID NO: 2, samples were analyzed by SDS- PAGE and immunoblotting with anti-ubiquitin antibodies. Incubation with UBP-SEQ ID NO: 2 resulted in the disappearance of most of the ubiquitin immunoreactivity (the bands detected at 60-90 K in the presence of UBP-SEQ ID NO: 2 were caused by antibody cross-reactivity with the GST-UBP-SEQ ID NO: 2 preparation) . Under the same conditions, a GST-UBP- SEQ ID NO: 2 protein treated with NEM prior to incubation with the ubiquitinated substrates failed to generate de- ubiquitination. The disappearance of ubiquitinated bands from the blot was not paralleled by a disappearance of proteins as detected by Ponceau red staining, demonstrating that the UBP- SEQ ID NO: 2 preparation was free of contaminating proteolytic activities; furthermore no effect was seen upon incubation of the sole GST moiety with the RRL proteins (data not shown) . The above results show that UBP-SEQ ID NO: 2 can hydrolyze ubiquitin-isopeptide bonds.

UBP-SEQ ID NO: 2 could also cleave purified linear ubiquitin chains. In Fig. 2B, the results of an experiment in which GST-UBP-SEQ ID NO: 2 was incubated with Ha-tagged Ub-hexamer (Ub6, see Methods) are shown. Furthermore, as is the case for other UBPs, UBP-SEQ ID NO: 2 was able to cleave Ub-b- galactosidase fusion proteins upon expression in E. coli (data not shown) . To study the biological role of human isopeptidases it is essential to be able to measure the deubiquitinating activity of UBPs directly isolated from cell extracts rather than from recombinant sources. To achieve this we set up an assay to measure the deubiquitinating activity in anti-UBP-SEQ ID NO: 2 immunoprecipitations, by testing the cleavage of Ub6. We employed lysates from either U20S cells which express UBP-SEQ ID NO: 2 or from NIH-3T3 cells in which our antibodies do not detect UBP-SEQ ID NO. 2 protein. NIH3T3 cells were transfected with various UBP-SEQ ID NO: 1 constructs. As shown in Fig. 2C, upon transfection with a sense cDNA construct NIH3T3 cells expressed the protein to amounts comparable to the endogenous levels detected in U20S cells. Both a wild type protein as well as a Cys786Ala mutant could be expressed in NIH3T3 and i munoprecipitated using UBP-SEQ ID NO: 2 antibodies.

The Cys786Ala mutation was introduced into UBP-SEQ ID NO: 1 by a two-step PCR-based approach. Oligodeoxynucleotides 5'- GGAAATACTGCTTATATGAAC-3' and 5 ' GTTCATATAAGCAGTATTTCC-3 ' were used to mutate the Cys786 codon. Oligodeoxynucleotides 5'- CAACACTGTTCATATGTACC-3' and 5'-

CCGGGGATCCTTATGTGGCTACATCAGTTA-3' served as flanking primers. PCMV-neo-Bam UBP-SEQ ID NO: 1 was used as a template. The second set of PCRs used the flanking primers and the two initial PCR reaction products. The resulting DNA fragments • were digested with Ndel and BamHI (the Ndel restriction site was constitutively present in UBP-SEQ ID NO: 1 sequence, while the BamHI one was introduced into the second flanking primer) and gel purified. The 5'NdeI-BamHI 3' mutated DNA fragment was utilized to replace the corresponding wild type fragment and cloned as a three piece ligation into the PCMV- UBP-SEQ ID NO: 1 plasmid. The mutant construct was verified by dideoxy nucleotide DNA sequencing. In Fig. 2D, we show that a detectable NEM-sensitive deubiquitinating activity could be revealed in immunoprecipitations from U20S cell extracts and that immunoprecipitations from NIH-3T3 cells transfected with wild-type, but not a Cys786 mutant UBP-SEQ ID NO: 1 construct, expressed a detectable NEM-sensitive deubiquitinating activity. These experiments demonstrate that a. endogenous UBP-SEQ ID NO: 2 is an active deubiquitinating enzyme; b. the UBP-SEQ ID NO: 2 expressed upon transfection is also active; c. as predicted, a mutation of Cys786 inactivates the enzyme. These results also imply that by upregulating UBP-SEQ ID NO: 2 levels, an increase in its cellular enzymatic activity can be generated (see also below) . Protein ubiquitination was generated as described (Pagano et al., Science 269, 682-685, 1995) with some modifications.

Rabbit reticulocyte lysate (Promega) was used both as source of ubiquitinating enzymes and of substrate proteins, and was incubated at 370C for 5-10 min. in 300 ml of ubiquitination mix [final concentration 33% (v/v) RRL, 50 mM Tris-HCl pH 8.3, 5 mM MgC12, 2 mM DTT, 20 mg of added ubiquitin (Sigma), 1 mM ATPgS, 50 mM LLnL] . Reaction was stopped by the addition of N-ethyl-maleimide (NEM 20 mM final concentration) . Sample was then centrifuged and washed several times, at 3,200 rpm for 20 min in a Centricon-3 concentrator (Amicon) to remove NEM. Aliquots of the ubiquitinated lysate (40 mg of proteins) were used as substrate for deubiquitinating activity: 0.5-1 mg of GST-UBP-SEQ ID NO: 2 FL were incubated at 37 °C for 5 to 20 min in 20 ml of deubiquitination mix (40 mg of in vitro ubiquitinated proteins, 50 mM Tris-HCl pH 8.3, 5 mM MgC12, 2 mM DTT) . Where indicated GST-UBP-SEQ ID NO: 2 FL was preincubated with NEM to block its active site cysteine. Reactions were stopped with Laemmli buffer, boiled, resolved by SDS-PAGE (8%), and transferred to nitrocellulose. Filters were subjected to immunoblotting with anti ubiquitin antiserum (Sigma) diluted (1:100) in 5% dry fat-free milk in TBS/0.05% Tween-20, and incubated at 4°C over/night. UBP-SEQ ID NO: 2 deubiquitinating activity was also tested using a linear hexamer of HA-tagged ubiquitin (Ub6) as a substrate. The cDNA sequence encoding HA-tagged ubiquitin was excised from the parental plasmid pMT123 (Treier et al.,Cell 18, 787-798, 1994), and inserted into pET23 bacterial expression vector to transform BL21 bacteria. Methods for expressing and purifying polyubiquitin chains in bacteria have been described previously (Jonnalagadda et al.,J Biol Chem; 262, 17750-6, 1987). Purification of the recombinant Ub6 from the bacterial supernatant was performed onto S200 16/60 column, the cleanest fractions were pooled, concentrated and used as substrate for UBP-SEQ ID NO: 2 deubiquitinating activity. To test this activity, UBP-SEQ ID NO: 2 immunoprecipitations from cell extracts were washed in lysis buffer (see above) with or without 10 mM NEM and further washed twice in the reaction buffer (50 mM Tris-HCl pH 8.3, 5 mM MgC12, 2 mM DTT ) again with or without 10 mM NEM and finally incubated at 37°C for 10 to 30 min in 90 ml of the same reaction buffer containing 20 ng/ml Ub6. At each time point, after spinning to pellet the beads, aliquots of the reactions were mixed with Laemmli buffer, boiled, resolved by SDS-PAGE (12%), and transferred to nitrocellulose. Filters were subjected to immunoblotting with monoclonal anti HA antibodies to detect reaction products using the ECL (Amersham) system.

A new method that we are using to measure the enzymatic activities of protein isopeptidases involves the use of any cell extract derived from cells treated with proteasome inhibitors, using which an accumulation of ubiquitinated proteins is obtained. The ubiquitinated proteins which accumulate in these cell extracts can be utilized as substrates of enzymes having isopeptidase activity. In the example shown in Fig. 2 a cell extract incubated in the presence or the absence of UBP-SEQ ID NO: 2, or in the presence of N-ethyl-maleimide inactivated UBP-SEQ ID NO: 2, was run on SDS-PAGE gel electrophoresis and then transferred to nitrocellulose and incubated with anti-ubiquitin antibodies to monitor the reaction by immunoblotting. Other means of testing de-ubiquitination could make use for example, "ELISA" methods with anti-ubiquitin antibodies.

Example 3 : Blocking UBP f nction affects cell proliferation

We have studied the regulation of UBP-SEQ ID NO: 2 levels in response to changes in cell growth conditions. We analyzed the expression of the protein upon removal of growth factors. WI-38 human fibroblasts were deprived of serum for 72 hours to induce GO entry and then re-stimulated by the addition of medium containing 10% fetal bovine serum. At different times after serum stimulation, samples were analyzed for cell cycle status by propidium iodide flow citometry and for UBP-SEQ ID NO: 2 and cell cycle marker expression by immunoblotting. UBP-SEQ ID NO: 2 expression was undetectable in serum- deprived cells and reappeared as cells progressed through the Gl phase of the cycle (Fig. 3A, B) .

Based on the above results we asked whether the up-regulation of UBP-SEQ ID NO: 2 levels observed in response to serum stimulation was an event necessary for cell growth. To answer this question we employed microinjection of UBP-SEQ ID NO: 1 antisense cDNA constructs and measured the effects of preventing its appearance on cell cycle re-entry. Fig. 3C shows that compared to controls, in three separate experiments, cells microinjected with antisense constructs failed to enter S-phase as shown by the reduced incorporation of bromodeoxyuridine (BrdU) added together with serum for 24 hours. This result indicates that UBP-SEQ ID NO: 2 plays a critical function for cell proliferation, which appears to be non redundant since at least two more enzymes of the UBP family are expressed in the same cells (not shown) .

Cell monolayers growing on glass coverslips (at about 60% density) were microinjected with an automated microinjection system (AIS, Zeiss) connected with an Eppendorf injector. All microinjection experiments were carried out in 3.5 cm petri dishes containing 5 ml of freshly added growth medium. Cells were injected at a pressure between 50-150 hecta-Pascal (hPa) . The computer settings for injection were angle "45", speed "10", and time of injection "0.1 sec". Using this system the percentage of successfully microinjected cells was >50 %. pCMV -neo-Bam empty vector or the same vector containing UBP-SEQ ID NO: 1 full length cDNA in 3 '-5' antisense orientation were injected at the concentration of 50 ng/ml in the presence of a microinjector marker (GFP- plasmid 20 ng/ml ) . Immediately after injection fresh growth medium was added and supplemented with BrdU ( Sigma ) at a final concentration of 100 mM. Cells were fixed 24 hr after serum re-addition. GFP (green fluorescence protein) detection by fluorescence microscope to identify microinjected cells, and BrdU incorporation (see above) were utilized to determine the effects of plasmid microinjection.

Cells grown on glass coverslips were washed twice with PBS, fixed for 10 min at room temperature with 4% paraformaldehyde in PBS, washed once in PBS and permeabilized with 0.25 %

Triton X-100 in PBS for 5 min at room temperature. Cells were then incubated for 20 sec at room temperature in 50 mM NaOH. After three washes with PBS cells were incubated for 30 min with monoclonal anti-BrdU (Becton Dickinson) . After three further washes, cells were incubated for 30 min with cy3- conjugated anti-mouse antibodies (Jackson Immunoresearch Laboratories), and then washed three times; counterstaining for DNA was performed by adding DAPI (1 mg/ml) into the final PBS ^"wash. The coverslips were then washed once in distilled water and dried. The dried coverslips were mounted on slides with Mowiol and analyzed with a fluorescence microscope (Leitz Aristoplan) . A JVC KYF55BE three-color digital video camera was used to obtain digitized images, which were then analyzed with the Image Grabber 24 1.2 software (Neotech) . We also maintained cells in culture until they had reached confluence (Fig. 4A) . In non-immortalized human fibroblasts, which are inhibited by cell cell contact, we observed a down- regulation of the protein coincident with the cells becoming arrested, as monitored by the disappearance of cyclin A and the accumulation of the p27 Cdk-inhibitory protein (Guadagno et al., Science, 262 , 1572-1575 1993; Sherr and Roberts, Genes & dev.;3, 1149-1163, 1995). Interestingly, in the transformed osteosarcoma line U20S, which does not show contact inhibition, such down regulation of the UBP-SEQ ID NO: 2 protein levels was not observed, despite the fact that cells became confluent as monitored by the increase in p27 levels (Fig. 4B) . Similar results were obtained in other transformed cell lines, including normal fibroblasts made to express SV40 large T antigen (not shown) . Altogether the above experiments identify UBP-SEQ ID NO: 2 as a growth regulated protein that plays a critical role for cell proliferation. Example 4 : Altered expression causes dramatic changes in overall protein ubiquitination

To identify biochemical changes related to altered UBP-SEQ ID NO: 2 expression, we analyzed the overall protein ubiquitination status in extracts from cells transiently transfected with various UBP-SEQ ID NO: 1 constructs along with Ha-Ub plasmids (Fig. 5) . It has been demonstrated that epitope tagged ubiquitin can be correctly conjugated in vivo and in vitro to cellular proteins which then become target of proteolytic cleavage by the proteasome (Ellison and Hochstrasser; J Biol Chem, 266, 21150-7, 1991; Treier et al.; Cell 18, 787-798, 1994; Hateboer et al.; Genes Dev, 10, 2960- 70, 1996; Diehl et al . ; Genes Dev, 21,957-72, 1997). The Ha- Ub allowed the quantitative detection of protein ubiquitination by immunoblotting. To our surprise we consistently found that by either inhibiting UBP-SEQ ID NO: 2 accumulation with an antisense constructs (AS in Fig. 5) , or by inducing the expression of a Cys786 mutant of the protein (Sm in Fig. 5) a substantial increase in total cell protein ubiquitination occurred. Conversely, upon overexpression of the wild-type protein (S in Fig. 5) a decrease in protein ubiquitination was found. The results of a separate experiment (Fig. 5B) in which samples were run on a higher density gel show that even lower Mr ubiquitinated species accumulated in antisense or mutant transfected cells (AS or Sm) . The observed biochemical changes were not generated in vitro since NEM was added at the time of cell lysis. These experiments indicate that UBP-SEQ ID NO: 2 is essential for the execution of a key general step in ubiquitin processing. Cells (8 x 105/plate) were split and were co-transfected 20 hours later with calcium-phosphate precipitates of 22 μm of plasmid DNA (ratio 10:1 between plasmid of interest and GFP- plasmid) for each 100 mm dish. After 24 hours, the cells were washed twice with phosphate-buffered saline (PBS) , and incubated with fresh 10% serum containing medium. 24-48 hours after removal of DNA precipitates, cells were washed twice with PBS, rinsed off the plates with 0.05% trypsin, 0.2% EDTA in PBS, collected in 10% serum-containing medium and pelleted. Cell pellets were washed twice with PBS, divided into two aliquots and processed for immunoblotting and cell cycle analysis. For cell cycle analysis the washed pellets were fixed in cold 70% methanol in PBS, pelleted, washed in 1% BSA in PBS. Fixed and washed cells were pelleted, and stained in a solution of 50 mg/ml of propidium iodide and 100 mg/ml of ribonuclease (RNAse) A for 1-2 hours at 37°C. Flow cytometry analysis was performed on a Becton-Dickinson FACScan, and data from 50,000-100,000 cells per sample were analyzed with the ModiFIT Cell Cycle Analysis software. A gate was set to select GFP-positive cells with a fluorescence at least 50 fold stronger than that in the negative non transfected cells. The propidium iodide signal was used as a measure of DNA content and hence cell cycle stage. The DNA histograms referring to GFP-positive cells each contain data from 10,000 to 20,000 cells. On the basis of the results obtained with the transfection experiments, we decided to evaluate the biological consequences of altering UBP-SEQ ID NO: 2 function. We show (Fig. 3C) that UBP-SEQ ID NO: 2 antisense microinjection in starved fibroblasts, which are depleted of the protein, results in an inhibition of S-phase entry. In U20S cells also, altering UBP-SEQ ID NO: 2 expression caused dramatic changes. Examples of our findings are depicted in Fig.: 6. Down-regulation of UBP-SEQ ID NO: 2 levels in asynchronously growing U20S cells by antisense plasmid transfection determined an accumulation of cells in the S-phase of the cell cycle. To measure this, we used a modification of the technique described by van den Heuvel and Harlow (Science, 252,2050-4,1993) which makes use of a marker gating flow citometry procedure to identify transfected cells. Using co- transfection of the UBP-SEQ ID NO: 1 antisense plasmid and of a plasmid encoding a modified green fluorescent protein we were able to assess changes in cell cycle distribution resulting from UBP-SEQ ID NO: 2 down regulation. By increasing the dosage of UBP-SEQ ID NO: 1-antisense plasmid, we could observe a progressive increase in S-phase cell accumulation (Fig. 6A) . The magnitude of these effects is similar to, if not greater than, the results obtained upon acute overexpression of the E2F-1 transcription factor which induces an accumulation of cells in S-phase (Mueller et al.,Moll Cell Biol; in press).

A different experiment is shown in Fig. 6B. Transfection of a UBP-SEQ ID NO: 1 sense plasmid accompanied by selection of the transfected clones with a neomycin resistance marker resulted in a severe inhibition of cell growth compared to controls, likely as a result of the dramatic changes in protein ubiquitination shown above (Fig. 5). Similar effects were seen upon transfection of a mutated (Cys786Ala) UBP-SEQ ID NO: 1 construct, which also subverted protein ubiquitination (data not shown) .

For colony forming assay, 8 x 105 U20S cells were plated in a 10 cm dish and transfected by calcium phosphate precipitation either with 20 μg of control empty pCMV vector or of the same vector containing the UBP-SEQ ID NO: 1 sequence in sense orientation. After 24 hours, the cells were washed twice with phosphate-buffered saline (PBS), and incubated with fresh 10% containing serum medium. 24 hours after the removal of DNA precipitates, cells were washed twice with PBS, rinsed off the plates with 0.05% trypsin, 0.2% EDTA in PBS, collected in 10% serum containing medium and then split at different dilutions and subcoltured into medium containing 500 mg/ml G418. Drug resistant colonies appeared 10 to 15 days later and were either isolated for further analysis or counted after crystal violet staining.

In conclusion, UBP-SEQ ID NO: 2 appears to play a critical role in the maintenance of the overall protein ubiquitination status. Acute alterations of its function can result in discrete effects on cell proliferation that reflect the particular time in the cell division cycle at which they occur. To date no other studies have demonstrated the effects of down-regulating a ubiquitin isopeptidase in mammalian cells. We have been able to inhibit UBP-SEQ ID NO: 2 accumulation using an antisense cDNA vector and could demonstrate that G0- arrested cells were prevented from entering S-phase after serum stimulation. Surprisingly we found that de-regulation of UBP-SEQ ID NO: 2 accumulation generates a substantial derangement of the overall cell protein ubiquitination. Our data indicate that UBP-SEQ ID NO: 2 plays a general function in the ubiquitin pathway. One possible way for exerting this function could be through the recycling of Ub-tree peptide remnants which are generated after ubiquitinated proteins are cleaved by the proteasome. The consequences of inhibiting cleavage of these remnants by decreasing the cellular levels of UBP-SEQ ID NO: 2 or by expressing a Cys786 mutant which we know can avidly bind poly-ubiquitinated species (SN and GFD, unpublished) are consistent with our findings of an increase in the level of ubiquitinated species, likely due to the clogging of the proteasome by the Ub-remnants. A similar biochemical function has been proposed for the yeast Doa4 UBP (Papa and Hochstrasser; Nature, 366, 313-9, 1993) . UBP-SEQ ID NO: 2 could exert a similar function, or could indirectly affect this function by controlling the ubiquitination state of one or more proteins which themselves control the activity of the proteasome.

As far as UBP-SEQ ID NO: 2 is concerned, our data indicate that this protein plays an essential regulatory function which appears to be non redundant with other UBPs we know to be expressed in the cell lines under study. As it happens for other critical cellular components, the acute subversion of their function can result in specific phenotypes. We show that microinjection of antisense UBP-SEQ. ID NO. 1 cDNA in quiescent human cells will prevent S-phase entry. A similar experiment performed in growing osteosarcoma cells will instead determine an accumulation of cells in S-phase. These apparently opposing effects can be explained by the fact that the ubiquitin system plays a role in many biochemical cascades required for cell division (see Pagano; Faseb J, 11 , 1067-75, 1997, for review) and that at any given time an alteration of proteasome function can have discrete effects that will depend on the cell cycle phase at which the alteration occurs. One other likely explanation for these phenomena is that, compared to normal cells, the lack of Gl checkpoint function in transformed cells makes them less sensitive to UBP-SEQ. ID NO. 2 down regulation early in the cell cycle. Further investigation will be worth of this issue. Example 5 : Cloning of full length ubiquitin isopeptidase cDNA.

The SEQ ID NO: 5 partial cDNA clone was first identified in the human myeloblast cell line KG-1. The SEQ ID NO: 5 cDNA sequence (bases 1 to 3280 ) was submitted ( 12-Dec-1995) to the DDBJ/EMBL/GenBank databases by Nomura N. et Al. (Accession Number D80012) . The UBP-SEQ ID NO: 5 coding sequence extends from base 2 to base 2443 and is predicted to encode a 814 Kd protein. By screening a ML1 Lamda ZipII human cDNA library, we found a complete cDNA (see SEQ ID NO: 5) having a coding sequence of 2590bp and coding for a protein of 862Kd.

The full lenght SEQ ID NO: 5 was cloned in the pBscKS cloning vector using Hindi and NotI as cloning sites. Then it was cut out from this vector by EcoRI-NotI digestion and subcloned in the pGEX4-Tl expression vector to generate a fusion protein that was then purified and injected into rabbits to obtain specific polyclonal antibodies. A mutant Cys488Ala was generated by PCR. The coding region of SEQ ID NO: 6 was subcloned in the pBMN-GFP mammalian expression vector , using Xho/Not or Not/Sail as cloning sites, in sense or antisense orientation; this was similarly done with the insert encoding the mutated Cys488Ala protein. Example 6 : Identification of ubiquitin isopeptidase interacting clones .

The yeast tow hybrid system was used to identify proteins that interact with SEQ ID NO: 6. The first tools we used were developed by S .M. H. R. Sternglanz and H. Weintraub (unpublished data) (Vojtek et.al. 1993, Cell, 74, 205-214). The bait was made as a fusion between LexA DNA binding domain (aa 1-211) and KIA190FL (SEQ ID NO: 6) (aa 14-862). The second hybrid is a fusion between a nuclear localized VP16 acidic activation domain and random cDNA fragments derived from 9.5 and 10.5 days mouse embryos. Approximately 10.10⁷ yeast transforms were screened, 30 clones were found positive on Xgal/Hist- plates in the presence of 5mM 3-aminotriazole . The recovered plasmids were subjected to restriction and sequence analysis and from this screen 10 clones were identified that correspond to the same cDNA. This fragment encoded the murine protein G3BP which has a human homologous .( Parker et al.,1996 Mol Cell Biol 16,2561-9) (SEQ ID NO:8). Other 4 positives were identified to be the mouse xl6 cDNA, homologous to the human SRP20 gene (SEQ ID NO: 10) (Jumaa and Nielsen, 1997, Embo J

16, 5077-85, Jumaa et al . , 1997, Moll Cell Biol 17, 3116-24). 5xl0^δ indipendent yest trasformants were screened from a cDNA library from human lymphocytes. Of the 12 positives isolated, one clone turned out to be the human G3BP (SEQ ID NO: 10) . We tested the ability of the isolated clone to bind to a panel of known ubiquitin isopeptidases linked to LexA baits using yeast interaction assays. We found that the activator domain-tagged G3BP fragment strongly interacted with the ubiquitin isopeptidase bait (SEQ ID NO: 6) , with an N- terminal truncation of it and with a catalytically inactive mutant, but not with other ubiquitin isopeptidases, like the UNPH and the SEQ ID NO: 2 ones (Fig. 7) . The exclusive binding of our isopeptidase to G3BP suggests a possible role for it in regulating progression through the Gl phase of the cell division cycle.

Example 7 : Deregulation of UBP expression in tumors

To identify UBP-SEQ ID NO: 4 we immunized rabbits with a peptide derived from its carboxy-terminal sequence (the last nine amino acids) with the addition of a cysteine residue on its amino-terminus (CDYEKYSMLQ) , which was used to conjugated it to ovalbumin. The obtained antiserum recognized one or multiple bands of approx. relative molecular mass 110,000 which were specifically removed when antigenic peptide was added to the reaction mixture, demonstrating that specific epitopes were recognized by the antiserum (Fig. 11). The protein was detected in all cell lines tested and its distribution has been then in primary normal and tumoral

(adenocarcinoma) human colon tissue. In the example shown in Fig. 10, it can be seen that the protein is present at significantly higher levels in the tumor tissues compared to normal samples. As seen in cell lines, the band identified as the product of UBP-SEQ ID NO: 4 and found to be expressed at high levels in tumor tissues, was eliminated by incubation of the antiserum with its antigenic peptide. Our data demonstrate that it will be possible to use measurements of UBP-SEQ ID NO: 4 protein expression to diagnose colon tumors, and that specific inhibitors of UBP-SEQ ID NO: 4 expression might be able to specifically affect the function of tumor but not normal cells.

Polyclonal antibodies specific for the UBP-SEQ ID NO: 6 gene product were generated by immunizing rabbit with a GST fusion protein. Affinity-purified antibodies were obtained from total serum by affinity chromatography on the fusion protein bound to Sepharose.

Five μM formalin-fixed, paraffin-embedded tissue sections were used for immunohistochemical staining with a polyclonal antibodies against the UBP indicated as SEQ ID NO: 4 and SEQ ID NO: 6. Antigen-antibody reactions were revealed utilizing the avidin biotin complex (ABC) method using diaminobenzidine (DAB) as substrate. The specificity of the polyclonal antibodies had been previously assessed by immunoblotting. Digoxigenin-labeled probes were utilized for in situ hybridization. Riboprobes were generated with T3 and T7 RNA polymerase for 1 hour at 37 °C in IX transcription buffer (Promega, Madison, WI . ) , 10 mM dithiothreitol (DTT), 40 U of RNAse inhibitor, 1 mM each of ATP, CTP and GTP, as well as a mixture of cold UTP and digoxigenin-UTP (6.5 and 3.5 mM respectively) (Boehringer Mannheim, Indianapolis, IN) (Galaktionov, et al., 1995, Science 269, 1575-1577). 5 μM formalin-fixed paraffin-embedded sections were dewaxed, rehydrated, washed in phosphate buffered saline (PBS). Sections were digested with proteinase K (50 μg/ml) in 1 M Tris-EDTA buffer (pH 8) for 18 min at 37°C, fixed in 4% paraformaldehyde in PBS for 5 min at 4°C, then washed in PBS. Prior to prehybridization, sections are acetylated for 10 min at RT . Pre-hybridization is carried out at 37° for 15 min in 50% formamide and 2X SSC. Hybridization was performed at 42°C overnight applying 10 pM of digoxigenin-labeled riboprobe in 50 μl of hybridization buffer (50% deionized formamide, 2XSSC, 10% dextran sulphate, 1% sodium dodecyl sulphate [SDS] , 10 mg/ml denatured herring sperm DNA) per section under a coverslip. The highest stringency of post- hybridization washes is 50°C in 0. IX SSC for 15 min. Anti- digoxigenin antibody (1:500) is applied for 30 min at 37°C. Detection is accomplished with nitro-blue tetrazolium/ 5- bromo 4-chloro 3-indolyl phosphate (NBT/BCIP) for 30-90 minutes for all sections.

The obtained results show that deregulated expression of the ubiquitin isopeptidase indicated as SEQ ID NO: 4 and 6 is a frequent event in human tumors. The data also show that the expression of these proteins can be used to monitor oncogenic transformation. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Istituto Europeo di Oncologia

(B) STREET: Via Filodrammatici, 8 (C) CITY: Milan

(E) COUNTRY: Italy

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(ii) TITLE OF INVENTION: Mammalian ubiquitin-isopeptidases that control cell proliferation

(iii) NUMBER OF SEQUENCES: 10

(iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3357 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ATGCCTGCTG TGGCTTCAGT TCCTAAAGAA CTCTACCTCA GTTCTTCACT AAAAGACCTT 60

AATAAGAAGA CAGAAGTTAA ACCAGAGAAA ATAAGCACTA AGAGTTATGT GCACAGTGCC 120 CTGAAGATCT TTAAGACAGC AGAAGAATGC AGATTAGATC GTGATGAGGA AAGGGCCTAT 180

GTACTATATA TGAAATACGT GACTGTTTAT AATCTTATCA AAAAAAGACC TGATTTCAAG 240

CAACAGCAGG ATTATTTCCA TTCAATACTT GGACCTGGAA ACATCAAAAA AGCTGTCGAA 300

GAAGCTGAAA GACTCTCTGA AAGCCTTAAA TTAAGATATG AAGAAGCTGA AGTCCGGAAA 360

AAACTTGAGG AAAAAGACAG GCAGGAGGAA GCACAGCGGC TACAACAAAA AAGGCAGGAA 420 ACAGGAAGAG AGGATGGTGG CACATTGGCT AAAGGCTCTT TGGAGAATGT TTTGGATTCC 480

AAAGACAAAA CCCAAAAGAG CAATGGTGAA AAGAATGAAA AATGTGAGAC CAAAGAGAAA 540

GGAGCAATCA CAGCAAAGGA ACTATACACA ATGATGACGG ATAAAAACAT CAGCTTGATT 600 ATAATGGATG CTCGAAGAAT GCAGGATTAT CAGGATTCCT GTATTTTACA TTCTCTCAGT 660

GTTCCTGAAG AAGCCATCAG TCCAGGAGTC ACTGCTAGTT GGATTGAAGC ACACCTGCCA 720

GATGATTCTA AAGACACATG GAAGAAGAGG GGGAATGTGG AGTATGTGGT ACTTCTTGAC 780

TGGTTTAGTT CTGCCAAAGA TTTACAGATT GGAACAACTC TCCGGAGTCT GAAAGATGCA 840 CTTTTCAAGT GGGAAAGTAA AACTGTCCTG CGCAATGAGC CTTTGGTTTT AGAGGGAGGC 900

TATGAAAACT GGCTCCTTTG TTATCCCCAG TATACAACAA ATGCTAAGGT CACTCCACCC 960

CCACGACGCC AGAATGAAGA GGTGTCTATC TCATTGGATT TTACTTATCC CTCATTGGAA 1020

GAATCAATTC CTTCTAAACC TGCTGCCCAG ACGCCACCTG CATCTATAGA AGTAGATGAA 1080

AATATAGAAT TGATAAGTGG TCAAAATGAG AGAATGGGAC CACTGAATAT ATCAACTCCA 1140 GTTGAACCAG TTGCTGCTTC TAAATCTGAT GTTTCACCCA TAATTCAGCC AGTGCCTAGT 1200

ATAAAGAATG TTCCACAGAT TGATCGTACT AAAAAACCAG CAGTCAAATT GCCTGAAGAG 1260

CATAGAATAA AATCTGAAAG TACAAACCAT GAGCAACAAT CTCCTCAGAG TGGAAAAGTT 1320

ATTCCTGATC GTTCCACCAA GCCAGTAGTT TTTTCTCCAA CTCTCATGTT AACAGATGAA 1380

GAAAAGGCTC GTATTCATGC AGAAACTGCT CTTCTAATGG AAAAAAACAA ACAAGAAAAA 1440 GAACTTCGGG AAAGGCAGCA AGAGGAACAG AAAGAGAAAC TGAGGAAGGA AGAACAAGAA 1500

CAAAAAGCCA AAAAGAAACA AGAAGCTGAA GAAAATGAAA TTACAGAGAA GCAACAAAAA 1560

GCAAAAGAAG AAATGGAGAA GAAAGAAAGT GAACAGGCCA AGAAAGAAGA TAAAGAAACC 1620

TCAGCAAAGA GGGGCAAAGA AATAACAGGA GTAAAAAGAC AAAGTAAAAG TGAACATGAA 1680

ACTTCTGATG CCAAGAAATC TGTAGAAGAT AGGGGGAAAA GGTGTCCAAC CCCAGAAATA 1740 CAGAAAAAGT CAACAGGAGA TGTGCCCCAT ACATCTGTGA CAGGGGATTC AGGTTCAGGC 1800

AAGCCATTTA AGATTAAAGG ACAACCAGAA AGTGGAATTC TAAGGACAGG AACTTTTAGA 1860

GAGGATACAG ACGATACCGA AAGAAATAAA GCTCAACGAG AACCTTTGAC AAGAGCACGA 1920

AGTGAAGAAA TGGGGAGGAT CGTACCAGGA CTGCCTTCAG GCTGGGCCAA GTTTCTTGAC 1980

CCAATCACTG GAACCTTTCG TTATTATCAT TCACCCACCA ACACTGTTCA TATGTACCCA 2040 CCGGAAATGG CTCCTTCATC TGCACCTCCT TCCACCCCTC CAACTCATAA AGCCAAGCCA 2100

CAGATTCCTG CTGAGCGGGA TAGGGAACCT TCCAAACTGA AGCGCTCCTA CTCCTCCCCA 2160

GATATAACCC AGGCTATTCA AGAGGAAGAG AAGAGGAAGC CAACAGTAAC TCCAACAGTT 2220

AATCGGGAAA ACAAGCCAAC ATGTTATCCT AAAGCTGAGA TCTCAAGGCT TTCTGCTTCT 2280

CAGATTCGGA ACCTCAATCC TGTTTTTGGA GGTTCTGGAC CAGCTCTTAC TGGACTTCGT 2340 AACTTAGGAA ATACTTGTTA TATGAACTCA ATATTGCAGT GCCTATGTAA CGCTCCACAT 2400 TTGGCTGATT ATTTCAACCG AAACTGTTAT CAGGATGATA TTAACAGGTC AAATTTGTTG 2460

GGGCATAAAG GTGAAGTGGC AGAAGAATTT GGTATAATCA TGAAAGCCCT GTGGACAGGA 2520

CAGTATAGAT ATATCAGTCC AAAGGACTTT AAAATCACCA TTGGGAAGAT CAATGACCAG 2580

TTTGCAGGAT ACAGTCAGCA AGATTCACAA GAATTGCTTC TGTTCCTAAT GGATGGTCTC 2640 CATGAAGATC TAAATAAAGC TGATAATCGG AAGAGATATA AAGAAGAAAA TAATGATCAT 2700

CTCGATGACT TTAAAGCTGC AGAACATGCC TGGCAGAAAC ACAAGCAGCT CAATGAGTCT 2760

ATTATTGTTG CACTTTTTCA GGGTCAATTC AAATCTACAG TACAGTGCCT CACATGTCAC 2820

AAAAAGTCTA GGACATTTGA GGCCTTCATG TATTTGTCTC TACCACTAGC ATCCACAAGT 2880

AAATGTACAT TACAGGATTG CCTTAGATTA TTTTCCAAAG AAGAAAAACT CACAGATAAC 2940 AACAGATTTT ACTGCAGTCA TTGCAGAGCT CGACGGGATT CTCTAAAAAA GATAGAAATC 3000

TGGAAGTTAC CACCTGTGCT TTTAGTGCAT CTGAAACGTT TTTCCTACGA TGGCAGGTGG 3060

AAACAAAAAT TACAGACATC TGTGGACTTC CCGTTAGAAA ATCTTGACTT GTCACAGTAT 3120

GTTATTGGTC CAAAGAACAA TTTGAAGAAA TATAATTTGT TTTCTGTTTC AAATCACTAC 3180

GGTGGGCTGG ATGGAGGCCA CTACACAGCC TATTGTAAAA ATGCAGCAAG ACAACGGTGG 3240 TTTAAGTTTG ATGATCATGA AGTTTCTGAT ATCTCCGTTT CTTCTGTGAA ATCTTCAGCA 3300

GCTTATATCC TCTTTTATAC TTCATTGGGA CCACGAGTAA CTGATGTAGC CACATAA 3357

(2) INFORMATION FOR SEQ ID NO: 2:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1118 amino acids

(B) TYPE: amino ac d

(C) STRANDEDNESS: (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Pro Ala Val Ala Ser Val Pro Lys Glu Leu Tyr Leu Ser Ser Ser 1 5 10 15

Leu Lys Asp Leu Asn Lys Lys Thr Glu Val Lys Pro Glu Lys lie Ser 20 25 30

Thr Lys Ser Tyr Val His Ser Ala Leu Lys lie Phe Lys Thr Ala Glu 35 40 45

Glu Cys Arg Leu Asp Arg Asp Glu Glu Arg Ala Tyr Val Leu Tyr Met 50 55 60 Lys Tyr Val Thr Val Tyr Asn Leu lie Lys Lys Arg Pro Asp Phe Lys 65 70 75 80

Gin Gin Gin Asp Tyr Phe His Ser lie Leu Gly Pro Gly Asn lie Lys 85 90 95

Lys Ala Val Glu Glu Ala Glu Arg Leu Ser Glu Ser Leu Lys Leu Arg 100 105 110 Tyr Glu Glu Ala Glu Val Arg Lys Lys Leu Glu Glu Lys Asp Arg Gin 115 120 125

Glu Glu Ala Gin Arg Leu Gin Gin Lys Arg Gin Glu Thr Gly Arg Glu 130 135 140

Asp Gly Gly Thr Leu Ala Lys Gly Ser Leu Glu Asn Val Leu Asp Ser 145 150 155 160

Lys Asp Lys Thr Gin Lys Ser Asn Gly Glu Lys Asn Glu Lys Cys Glu 165 170 175

Thr Lys Glu Lys Gly Ala He Thr Ala Lys Glu Leu Tyr Thr Met Met 180 185 190

Thr Asp Lys Asn He Ser Leu He He Met Asp Ala Arg Arg Met Gin 195 200 205

Asp Tyr Gin Asp Ser Cys He Leu His Ser Leu Ser Val Pro Glu Glu 210 215 220

Ala He Ser Pro Gly Val Thr Ala Ser Trp He Glu Ala His Leu Pro 225 230 235 240

Asp Asp Ser Lys Asp Thr Trp Lys Lys Arg Gly Asn Val Glu Tyr Val 245 250 255

Val Leu Leu Asp Trp Phe Ser Ser Ala Lys Asp Leu Gin He Gly Thr 260 265 270 Thr Leu Arg Ser Leu Lys Asp Ala Leu Phe Lys Trp Glu Ser Lys Thr 275 280 285

Val Leu Arg Asn Glu Pro Leu Val Leu Glu Gly Gly Tyr Glu Asn Trp 290 295 300

Leu Leu Cys Tyr Pro Gin Tyr Thr Thr Asn Ala Lys Val Thr Pro Pro 305 310 315 320

Pro Arg Arg Gin Asn Glu Glu Val Ser He Ser Leu Asp Phe Thr Tyr 325 330 335

Pro Ser Leu Glu Glu Ser He Pro Ser Lys Pro Ala Ala Gin Thr Pro 340 345 350 Pro Ala Ser He Glu Val Asp Glu Asn He Glu Leu He Ser Gly Gin 355 360 365

Asn Glu Arg Met Gly Pro Leu Asn He Ser Thr Pro Val Glu Pro Val 370 375 380 Ala Ala Ser Lys Ser Asp Val Ser Pro He He Gin Pro Val Pro Ser 385 390 395 400

He Lys Asn Val Pro Gin He Asp Arg Thr Lys Lys Pro Ala Val Lys 405 410 415

Leu Pro Glu Glu His Arg He Lys Ser Glu Ser Thr Asn His Glu Gin 420 425 430

Gin Ser Pro Gin Ser Gly Lys Val He Pro Asp Arg Ser Thr Lys Pro 435 440 445

Val Val Phe Ser Pro Thr Leu Met Leu Thr Asp Glu Glu Lys Ala Arg 450 455 460

He His Ala Glu Thr Ala Leu Leu Met Glu Lys Asn Lys Gin Glu Lys 465 470 475 480

Glu Leu Arg Glu Arg Gin Gin Glu Glu Gin Lys Glu Lys Leu Arg Lys 485 490 495

Glu Glu Gin Glu Gin Lys Ala Lys Lys Lys Gin Glu Ala Glu Glu Asn 500 505 510 Glu He Thr Glu Lys Gin Gin Lys Ala Lys Glu Glu Met Glu Lys Lys 515 520 525

Glu Ser Glu Gin Ala Lys Lys Glu Asp Lys Glu Thr Ser Ala Lys Arg 530 535 540

Gly Lys Glu He Thr Gly Val Lys Arg Gin Ser Lys Ser Glu His Glu 545 550 555 560

Thr Ser Asp Ala Lys Lys Ser Val Glu Asp Arg Gly Lys Arg Cys Pro 565 570 575

Thr Pro Glu He Gin Lys Lys Ser Thr Gly Asp Val Pro His Thr Ser 580 585 590 Val Thr Gly Asp Ser Gly Ser Gly Lys Pro Phe Lys He Lys Gly Gin 595 600 605

Pro Glu Ser Gly He Leu Arg Thr Gly Thr Phe Arg Glu Asp Thr Asp 610 615 620

Asp Thr Glu Arg Asn Lys Ala Gin Arg Glu Pro Leu Thr Arg Ala Arg 625 630 635 640

Ser Glu Glu Met Gly Arg He Val Pro Gly Leu Pro Ser Gly Trp Ala 645 650 655

Lys Phe Leu Asp Pro He Thr Gly Thr Phe Arg Tyr Tyr His Ser Pro 660 665 670 Thr Asn Thr Val His Met Tyr Pro Pro Glu Met Ala Pro Ser Ser Ala 675 680 685

Pro Pro Ser Thr Pro Pro Thr His Lys Ala Lys Pro Gin He Pro Ala 690 695 700 Glu Arg Asp Arg Glu Pro Ser Lys Leu Lys Arg Ser Tyr Ser Ser Pro 705 710 715 720

Asp He Thr Gin Ala He Gin Glu Glu Glu Lys Arg Lys Pro Thr Val 725 730 735

Thr Pro Thr Val Asn Arg Glu Asn Lys Pro Thr Cys Tyr Pro Lys Ala 740 745 750

Glu He Ser Arg Leu Ser Ala Ser Gin He Arg Asn Leu Asn Pro Val 755 760 765

Phe Gly Gly Ser Gly Pro Ala Leu Thr Gly Leu Arg Asn Leu Gly Asn 770 775 780

Thr Cys Tyr Met Asn Ser He Leu Gin Cys Leu Cys Asn Ala Pro His 785 790 795 800

Leu Ala Asp Tyr Phe Asn Arg Asn Cys Tyr Gin Asp Asp He Asn Arg 805 810 815

Ser Asn Leu Leu Gly His Lys Gly Glu Val Ala Glu Glu Phe Gly He 820 825 830

He Met Lys Ala Leu Trp Thr Gly Gin Tyr Arg Tyr He Ser Pro Lys 835 840 845

Asp Phe Lys He Thr He Gly Lys He Asn Asp Gin Phe Ala Gly Tyr 850 855 860

Ser Gin Gin Asp Ser Gin Glu Leu Leu Leu Phe Leu Met Asp Gly Leu 865 870 875 880

His Glu Asp Leu Asn Lys Ala Asp Asn Arg Lys Arg Tyr Lys Glu Glu 885 890 895

Asn Asn Asp His Leu Asp Asp Phe Lys Ala Ala Glu His Ala Trp Gin 900 905 910 Lys His Lys Gin Leu Asn Glu Ser He He Val Ala Leu Phe Gin Gly 915 920 925

Gin Phe Lys Ser Thr Val Gin Cys Leu Thr Cys His Lys Lys Ser Arg 930 935 940

Thr Phe Glu Ala Phe Met Tyr Leu Ser Leu Pro Leu Ala Ser Thr Ser 945 950 955 960

Lys Cys Thr Leu Gin Asp Cys Leu Arg Leu Phe Ser Lys Glu Glu Lys 965 970 975

Leu Thr Asp Asn Asn Arg Phe Tyr Cys Ser His Cys Arg Ala Arg Arg 980 985 990 Asp Ser Leu Lys Lys He Glu He Trp Lys Leu Pro Pro Val Leu Leu 995 1000 1005

Val His Leu Lys Arg Phe Ser Tyr Asp Gly Arg Trp Lys Gin Lys Leu 1010 1015 1020 Gin Thr Ser Val Asp Phe Pro Leu Glu Asn Leu Asp Leu Ser Gin Tyr 1025 1030 1035 1040

Val He Gly Pro Lys Asn Asn Leu Lys Lys Tyr Asn Leu Phe Ser Val 1045 1050 1055

Ser Asn His Tyr Gly Gly Leu Asp Gly Gly His Tyr Thr Ala Tyr Cys 1060 1065 1070

Lys Asn Ala Ala Arg Gin Arg Trp Phe Lys Phe Asp Asp His Glu Val 1075 1080 1085

Ser Asp He Ser Val Ser Ser Val Lys Ser Ser Ala Ala Tyr He Leu 1090 1095 1100

Phe Tyr Thr Ser Leu Gly Pro Arg Val Thr Asp Val Ala Thr 1105 1110 1115

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3270 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

ATGCCCCAAC GGCTTCCCCA TGCCAGGCAG CACACACCCC TCCCTCTGGG ATCAGCAGAC 60

TACAGGCGTG TCGTCAGTGT CAGACCACAG GGGCCACACA GAGACCCCAA GGACTCCAGA 120

GATGCAGCCA AACGCGAGCA AGGGTCCTTG GCACCCAGGC CTGTGCCGGC TTCACGTGGT 180 GGGAAGACCC TCTGCAAGGG GTATAGGCAG GCCCCTCCAG GCCCACCAGC CCAGTTCCAG 240

CGGCCCATTT GCTCAGCTTC CCCGCCATGG GCATCTCGTT TTTCCACGCC CTGTCCTGGT 300

GGGGCTGTCC GGGAAGACAC GTACCCTGTG GGCACTCAGG GTGTGCCCAG CCTGGCCCTG 360

GCTCAGGGAG GACCTCAGGG TTCCTGGAGA TTCCTGGAGT GGAAGTCAAT GCCCCGGCTC 420

CCAACGGACC TGGATATAGG GGGCCCTTGG TTCCCCCATT ATGATTTTGA ACGGAGCTGC 480 TGGGTCCGTG CCATATCCCA GGAGGACCAG CTGGCCACCT GCTGGCAGGC TGAACACTGC 540

GGAGAGGTTC ACAACAAAGA TATGAGTTGG CCTGAGGAGA TGTCTTTTAC AGCAAATAGT 600

AGTAAAATAG ATAGACAAAA GGTTCCCACA GAAAAGGGAG CCACAGGTCT AAGCAACCTG 660

GGAAACACAT GCTTCATGAA CTCAAGCATC CAGTGCGTTA GTAACACACA GCCACTGACA 720

CAGTATTTTA TCTCAGGGAG ACATCTTTAT GAACTCAACA GGACAAATCC CATTGGTATG 780 AAGGGGCATA TGGCTAAATG CTATGGTGAT TTAGTGCAGG AACTCTGGAG TGGAACTCAG 840 AAGAGTGTTG CCCCATTAAA GCTTCGGCGG ACCATAGCAA AATATGCTCC CAAGTTTGAT 900

GGGTTTCAGC AACAAGACTC CCAAGAACTT CTGGCTTTTC TCTTGGATGG TCTTCATGAA 960

GATCTCAACC GAGTCCATGA AAAGCCATAT GTGGAACTGA AGGACAGTGA TGGCCGACCA 1020

GACTGGGAAG TAGCTGCAGA GGCCTGGGAC AACCATCTAA GAAGAAATAG ATCAATTATT 1080

GTGGATTTGT TCCATGGGCA GCTAAGATCT CAAGTCAAAT GCAAGACATG TGGGCATATA 1140

AGTGTCCGAT TTGACCCTTT CAATTTTTTG TCTTTGCCAC TACCAATGGA CAGTTACATG 1200

GACTTAGAAA TAACAGTGAT TAAGTTAGAT GGTACTACCC CTGTACGGTA TGGACTAAGA 1260

CTGAATATGG ATGAAAAGTA CACAGGTTTA AAAAAACAGC TGAGGGATCT CTGTGGACTT 1320

AATTCAGAAC AAATCCTACT AGCAGAAGTA CATGATTCCA ACATAAAGAA CTTTCCTCAG 1380 GATAACCAAA AAGTACAACT CTCAGTGAGC GGATTTTTGT GTGCATTTGA AATTCCTGTC 1440

CCTTCATCTC CAATTTCAGC TTCTAGTCCA ACACAAATAG ATTTCTCCTC TTCACCATCT 1500

ACAAATGGAA TGTTCACCCT AACTACCAAT GGGGACCTAC CCAAACCAAT ATTCATCCCC 1560

AATGGAATGC CAAACACTGT TGTGCCATGT GGAACTGAGA AGAACTTCAC AAATGGAATG 1620

GTTAATGGTC ACATGCCATC TCTTCCTGAC AGCCCCTTTA CAGGTTACAT CATTGCAGTC 1680 CACCGAAAAA TGATGAGGAC AGAACTGTAT TTCCTGTCAC CTCAGGAGAA TCGCCCCAGC 1740

CTCTTTGGAA TGCCATTGAT TGTTCCATGC ACTGTGCATA CCCAGAAGAA AGACCTATAT 1800

GATGCGGTTT GGATTCAAGT ATCCTGGTTA GCAAGACCAC TCCCACCTCA GGAAGCTAGT 1860

ATTCATGCCC AGGATCGTGA TAACTGTATG GGCTATCAAT ATCCATTCAC TCTACGAGTT 1920

GTGCAGAAAG ATGGGATCTC CTGTGCTTGG TGCCCACAGT ATAGATTTTG CAGAGGCTGT 1980 AAAATTGATT GTGGGGAAGA CAGAGCTTTC ATTGGAAATG CCTATATTGC TGTGGATTGG 2040

CACCCCACAG CCCTTCACCT TCGCTATCAA ACATCCCAGG AAAGGGTTGT AGATAAGCAT 2100

GAGAGTGTGG AGCAGAGTCG GCGAGCGCAA GCCGAGCCCA TCAACCTGGA CAGCTGTCTC 2160

CGTGCTTTCA CCAGTGAGGA AGAGCTAGGG GAAAGTGAGA TGTACTACTG TTCCAAGTGT 2220

AAGACCCACT GCTTAGCAAC AAAGAAGCTG GATCTCTGGA GGCTTCCACC CTTCCTGATT 2280 ATTCACCTTA AGCGATTTCA ATTTGTAAAT GATCAGTGGA TAAAATCACA GAAAATTGTC 2340

AGATTTCTTC GGGAAAGTTT TGATCCGAGT GCTTTTTTGG TACCACGAGA CCCGGCCCTC 2400

TGCCAGCATA AACCACTCAC ACCCCAGGGG GATGAGCTCT CCAAGCCCAG GATTCTGGCA 2460

AGAGAGGTGA AGAAAGTGGA TGCGCAGAGT TCGGCTGGAA AAGAGGACAT GCTCCTAAGC 2520

AAAAGCCCAT CTTCACTCAG CGCTAACATC AGCAGCAGCC CAAAAGGTTC TCCTTCTTCA 2580 TCAAGAAAAA GTGGAACCAG CTGTCCCTCC AGCAAAAACA GCAGCCCTAA TAGCAGCCCA 2640 CGGACTTTGG GGAGGAGCAA AGGGAGGCTC CGGCTGCCCC AGATTGGCAG CAAAAATAAG 2700

CCGTCAAGTA GTAAGAAGAA CTTGGATGCC AGCAAAGAGA ATGGGGCTGG GCAGATCTGT 2760

GAGCTGGCTG ACGCCTTGAG CCGAGGGCAT ATGCGGGGGG GCAGCCAACC AGAGCTGGTC 2820

ACTCCTCAGG ACCATGAGGT AGCTTTGGCC AATGGATTCC TTTATGAGCA TGAAGCATGT 2880

GGCAATGGCT GTGGCGATGG CTACAGCAAT GGTCAGCTTG GAAACCACAG TGAAGAAGAC 2940

AGCACTGATG ACCAAAGAGA AGACACTCAT ATTAAGCCTA TTTATAATCT ATATGCAATT 3000

TCATGCCATT CAGGAATTCT GAGTGGGGGC CATTACATCA CTTATGCCAA AAACCCAAAC 3060

TGCAAGTGGT ACTGTTATAA TGACAGCAGC TGTGAGGAAC TTCACCCTGA TGAAATTGAC 3120

ACCGACTCTG CCTACATTCT TTTCTATGAG CAGCAGGGGA TAGACTACGC ACAATTTCTG 3180 CCAAAGATTG ATGGCAAAAA GATGGCAGAC ACAAGCAGTA CGGATGAAGA CTCTGAGTCT 3240

GATTACGAAA AGTACTCTAT GTTACAGTAA 3270

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1089 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Pro Gin Arg Leu Pro His Ala Arg Gin His Thr Pro Leu Pro Leu 1 5 10 15

Gly Ser Ala Asp Tyr Arg Arg Val Val Ser Val Arg Pro Gin Gly Pro 20 25 30

His Arg Asp Pro Lys Asp Ser Arg Asp Ala Ala Lys Arg Glu Gin Gly 35 40 45

Ser Leu Ala Pro Arg Pro Val Pro Ala Ser Arg Gly Gly Lys Thr Leu 50 55 60

Cys Lys Gly Tyr Arg Gin Ala Pro Pro Gly Pro Pro Ala Gin Phe Gin 65 70 75 80

Arg Pro He Cys Ser Ala Ser Pro Pro Trp Ala Ser Arg Phe Ser Thr 85 90 95

Pro Cys Pro Gly Gly Ala Val Arg Glu Asp Thr Tyr Pro Val Gly Thr

100 105 110 Gin Gly Val Pro Ser Leu Ala Leu Ala Gin Gly Gly Pro Gin Gly Ser 115 120 125

Trp Arg Phe Leu Glu Trp Lys Ser Met Pro Arg Leu Pro Thr Asp Leu 130 135 140

Asp He Gly Gly Pro Trp Phe Pro His Tyr Asp Phe Glu Arg Ser Cys 145 150 155 160

Trp Val Arg Ala He Ser Gin Glu Asp Gin Leu Ala Thr Cys Trp Gin 165 170 175

Ala Glu His Cys Gly Glu Val His Asn Lys Asp Met Ser Trp Pro Glu 180 185 190

Glu Met Ser Phe Thr Ala Asn Ser Ser Lys He Asp Arg Gin Lys Val 195 200 205

Pro Thr Glu Lys Gly Ala Thr Gly Leu Ser Asn Leu Gly Asn Thr Cys 210 215 220

Phe Met Asn Ser Ser He Gin Cys Val Ser Asn Thr Gin Pro Leu Thr 225 230 235 240

Gin Tyr Phe He Ser Gly Arg His Leu Tyr Glu Leu Asn Arg Thr Asn 245 250 255

Pro He Gly Met Lys Gly His Met Ala Lys Cys Tyr Gly Asp Leu Val 260 265 270

Gin Glu Leu Trp Ser Gly Thr Gin Lys Ser Val Ala Pro Leu Lys Leu 275 280 285 Arg Arg Thr He Ala Lys Tyr Ala Pro Lys Phe Asp Gly Phe Gin Gin 290 295 300

Gin Asp Ser Gin Glu Leu Leu Ala Phe Leu Leu Asp Gly Leu His Glu 305 310 315 320

Asp Leu Asn Arg Val His Glu Lys Pro Tyr Val Glu Leu Lys Asp Ser 325 330 335

Asp Gly Arg Pro Asp Trp Glu Val Ala Ala Glu Ala Trp Asp Asn His 340 345 350

Leu Arg Arg Asn Arg Ser He He Val Asp Leu Phe His Gly Gin Leu 355 360 365 Arg Ser Gin Val Lys Cys Lys Thr Cys Gly His He Ser Val Arg Phe 370 375 380

Asp Pro Phe Asn Phe Leu Ser Leu Pro Leu Pro Met Asp Ser Tyr Met 385 390 395 400

Asp Leu Glu He Thr Val He Lys Leu Asp Gly Thr Thr Pro Val Arg 405 410 415

Tyr Gly Leu Arg Leu Asn Met Asp Glu Lys Tyr Thr Gly Leu Lys Lys 420 425 430 Gin Leu Arg Asp Leu Cys Gly Leu Asn Ser Glu Gin He Leu Leu Ala

435 440 445

Glu Val His Asp Ser Asn He Lys Asn Phe Pro Gin Asp Asn Gin Lys

450 455 460

Val Gin Leu Ser Val Ser Gly Phe Leu Cys Ala Phe Glu He Pro Val

465 470 475 480

Pro Ser Ser Pro He Ser Ala Ser Ser Pro Thr Gin He Asp Phe Ser 485 490 495

Ser Ser Pro Ser Thr Asn Gly Met Phe Thr Leu Thr Thr Asn Gly Asp 500 505 510

Leu Pro Lys Pro He Phe He Pro Asn Gly Met Pro Asn Thr Val Val 515 520 525

Pro Cys Gly Thr Glu Lys Asn Phe Thr Asn Gly Met Val Asn Gly His 530 535 540

Met Pro Ser Leu Pro Asp Ser Pro Phe Thr Gly Tyr He He Ala Val 545 550 555 560

His Arg Lys Met Met Arg Thr Glu Leu Tyr Phe Leu Ser Pro Gin Glu 565 570 575

Asn Arg Pro Ser Leu Phe Gly Met Pro Leu He Val Pro Cys Thr Val 580 585 590

His Thr Gin Lys Lys Asp Leu Tyr Asp Ala Val Trp He Gin Val Ser 595 600 605 Trp Leu Ala Arg Pro Leu Pro Pro Gin Glu Ala Ser He His Ala Gin 610 615 620

Asp Arg Asp Asn Cys Met Gly Tyr Gin Tyr Pro Phe Thr Leu Arg Val 625 630 635 640

Val Gin Lys Asp Gly He Ser Cys Ala Trp Cys Pro Gin Tyr Arg Phe 645 650 655

Cys Arg Gly Cys Lys He Asp Cys Gly Glu Asp Arg Ala Phe He Gly 660 665 670

Asn Ala Tyr He Ala Val Asp Trp His Pro Thr Ala Leu His Leu Arg 675 680 685 Tyr Gin Thr Ser Gin Glu Arg Val Val Asp Lys His Glu Ser Val Glu 690 695 700

Gin Ser Arg Arg Ala Gin Ala Glu Pro He Asn Leu Asp Ser Cys Leu 705 710 715 720

Arg Ala Phe Thr Ser Glu Glu Glu Leu Gly Glu Ser Glu Met Tyr Tyr 725 730 735

Cys Ser Lys Cys Lys Thr His Cys Leu Ala Thr Lys Lys Leu Asp Leu 740 745 750 Trp Arg Leu Pro Pro Phe Leu He He His Leu Lys Arg Phe Gin Phe 755 760 765

Val Asn Asp Gin Trp He Lys Ser Gin Lys He Val Arg Phe Leu Arg 770 775 780

Glu Ser Phe Asp Pro Ser Ala Phe Leu Val Pro Arg Asp Pro Ala Leu 785 790 795 800

Cys Gin His Lys Pro Leu Thr Pro Gin Gly Asp Glu Leu Ser Lys Pro 805 810 815

Arg He Leu Ala Arg Glu Val Lys Lys Val Asp Ala Gin Ser Ser Ala 820 825 830

Gly Lys Glu Asp Met Leu Leu Ser Lys Ser Pro Ser Ser Leu Ser Ala 835 840 845

Asn He Ser Ser Ser Pro Lys Gly Ser Pro Ser Ser Ser Arg Lys Ser 850 855 860

Gly Thr Ser Cys Pro Ser Ser Lys Asn Ser Ser Pro Asn Ser Ser Pro 865 870 875 880

Arg Thr Leu Gly Arg Ser Lys Gly Arg Leu Arg Leu Pro Gin He Gly 885 890 895

Ser Lys Asn Lys Pro Ser Ser Ser Lys Lys Asn Leu Asp Ala Ser Lys 900 905 910

Glu Asn Gly Ala Gly Gin He Cys Glu Leu Ala Asp Ala Leu Ser Arg 915 920 925

Gly His Met Arg Gly Gly Ser Gin Pro Glu Leu Val Thr Pro Gin Asp 930 935 940

His Glu Val Ala Leu Ala Asn Gly Phe Leu Tyr Glu His Glu Ala Cys 945 950 955 960

Gly Asn Gly Cys Gly Asp Gly Tyr Ser Asn Gly Gin Leu Gly Asn His 965 970 975

Ser Glu Glu Asp Ser Thr Asp Asp Gin Arg Glu Asp Thr His He Lys 980 985 990

Pro He Tyr Asn Leu Tyr Ala He Ser Cys His Ser Gly He Leu Ser 995 1000 1005 Gly Gly His Tyr He Thr Tyr Ala Lys Asn Pro Asn Cys Lys Trp Tyr 1010 1015 1020

Cys Tyr Asn Asp Ser Ser Cys Glu Glu Leu His Pro Asp Glu He Asp 1025 1030 1035 1040

Thr Asp Ser Ala Tyr He Leu Phe Tyr Glu Gin Gin Gly He Asp Tyr 1045 1050 1055

Ala Gin Phe Leu Pro Lys He Asp Gly Lys Lys Met Ala Asp Thr Ser 1060 1065 1070 Ser Thr Asp Glu Asp Ser Glu Ser Asp Tyr Glu Lys Tyr Ser Met Leu 1075 1080 1085

Gin

(2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3426 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

ATTTTACCGG GAAGGCCGAG ATGGGAGGAC CGTTTTCCTA TGTGTAGTAA GGACACTGTT 60 CTCTCTGTGT GTGCTCTCTA CTGGAGGAAA GGGATTCAAA GTCATACTCC ATTGATTGGG 120

GCCTGGTCCT GCCGGCGGGG GAAGCAGCGT GAGCAGCCGG -AGGATCGCGG AGTCCCAATG 180

AAACGGGCAG CCATGGCCCT CCACAGCCCG CAGTATATTT TTGGAGATTT TAGCCCTGAT 240

GAATTCAATC AATTCTTTGT GACTCCTCGA TCTTCAGTTG AGCTTCCTCC ATACAGTGGA 300

ACAGTTCTGT GTGGCACACA GGCTGTGGAT AAACTACCTG ATGGACAAGA ATATCAGAGA 360 ATTGAGTTTG GTGTCGATGA AGTCATTGAA CCCAGTGACA CTTTGCCGAG AACCCCCAGC 420

TACAGTATTT CAAGCACACT GAACCCTCAG GCCCCTGAAT TTATTCTCGG TTGTACAGCT 480

TCCAAAATAA CCCCTGATGG TATCACTAAA GAAGCAAGCT ATGGCTCCAT CGACTGCCAG 540

TACCCAGGCT CTGCCCTCGC TTTGGATGGA AGTTCTAATG TGGAGGCGGA AGTTTTGGAA 600

AATGATGGTG TCTCAGGTGG TCTTGGACAA AGGGAGCGTA AAAAGAAGAA AAAGCGGCCA 660 CCTGGATATT ACAGCTATTT GAAAGATGGT GGCGATGATA GTATCTCCAC AGAAGCCCTG 720

GTCAATGGCC ATGCCAATTC AGCAGTCCCG AACAGTGTCA GTGCAGAGGA TGCAGAATTT 780

ATGGGTGACA TGCCCCCGTC AGTTACGCCC AGGACTTGTA ACAGCCCCCA GAACTCCACA 840

GACTCTGTCA GTGACATTGT GCCTGACAGT CCTTTCCCCG GAGCACTCGG CAGTGACACC 900

AGGACTGCAG GGCAGCCAGA GGGGGGCCCC GGGGCTGATT TTGGTCAGTC CTGCTTCCCT 960 GCAGAGGCTG GCAGAGACAC CCTGTCAAGG ACAGCTGGGG CTCAGCCCTG CGTTGGTACC 1020

GATACTACTG AAAACCTTGG AGTTGCTAAT GGACAAATAC TTGAATCCTC GGGTGAGGGC 1080

ACAGCTACCA ACGGGGTGGA GTTGCACACC ACGGAAAGCA TAGACTTGGA CCCAACCAAA 1140 CCCGAGAGTG CATCACCTCC TGCTGACGGC ACGGGCTCTG CATCAGGCAC CCTTCCTGTC 1200

AGCCAGCCCA AGTCCTGGGC CAGCCTCTTT CATGATTCTA AGCCCTCTTC CTCCTCGCCG 1260 GTGGCCTATG TGGAAACTAA GTATTCCCCT CCCGCCATAT CTCCCCTGGT TTCTGAAAAG 1320

CAGGTTGAAG TCAAAGAAGG GCTTGTTCCG GTTTCAGAGG ATCCTGTAGC CATAAAGATT 1380

GCAGAGTTGC TGGAGAATGT AACCCTAATC CATAAACCAG TGTCGTTGCA ACCCCGTGGG 1440

CTGATCAATA AAGGGAACTG GTGCTACATT AATGCTACAC TGCAGGCATT GGTTGCTTGC 1500

CCGCCGATGT ACCACCTGAT GAAGTTCATT CCTCTGTATT CCAAAGTGCA AAGGCCTTGT 1560 ACGTCAACAC CCATGATAGA CAGCTTTGTT CGGCTAATGA ATGAGTTCAC TAATATGCCA 1620

GTACCTCCAA AACCCCGACA AGCTCTTGGA GATAAAATCG TGAGGGATAT TCGCCCTGGA 1680

GCTGCCTTTG AGCCCACATA TATTTACAGA CTCCTGACAG TTAACAAGTC AAGCCTGTCT 1740

GAAAAGGGTC GACAAGAAGA TGCTGAGGAA TACTTAGGCT TCATTCTAAA TGGACTTCAT 1800

GAGGAAATGT TGAACCTAAA GAAGCTTCTC TCACCAAGTA ATGAAAAACT TACGATTTCC 1860 AACGGCCCCA AAAACCACTC GGTCAATGAA GAAGAGCAGG AAGAACAAGG TGAAGGAAGC 1920

GAGGATGAAT GGGAACAAGT GGGCCCCCGG AACAAGACTT CCGTCACCCG CCAGGCGGAT 1980

TTTGTTCAGA CTCCAATCAC CGGCATTTTT GGTGGACACA TCAGGTCTGT GGTTTACCAG 2040

CAGAGTTCAA AAGAATCTGC CACTTTGCAG CCATTTTTCA CGTTGCAGTT GGATATCCAG 2100

TCAGACAAGA TACGCACAGT CCAGGATGCA CTGGAGAGCT TGGTGGCAAG AGAATCTGTC 2160 CAAGGTTATA CCACAAAAAC CAAACAAGAG GTTGAGATAA GTCGAAGAGT GACTCTGGAA 2220

AAACTCCCTC CTGTCCTCGT GCTGCACCTG AAACGATTCG TTTATGAGAA GACTGGTGGG 2280

TGCCAGAAGC TTATCAAAAA TATTGAATAT CCTGTGGACT TGGAAATTAG TAAAGAACTG 2340

CTTTCTCCAG GGGTTAAAAA TAAGAATTTT AAATGCCACC GAACCTATCG GCTCTTTGCA 2400

GTGGTCTACC ATCACGGCAA CAGTGCGACG GGCGGCCATT ACACTACAGA CGTCTTCCAG 2460 ATCGGTCTGA ATGGCTGGCT GCGCATCGAT GACCAGACAG TCAAGGTGAT CAACCAGTAC 2520

CAGGTGGTGA AACCAACTGC TGAACGCACA GCCTACCTCC TGTATTACCG CCGAGTGGAC 2580

CTGCTGTAAA CCCTGTGTGC GCTGTGTGTG CGCCCAGTGC CCGCTTCGTA GGACACCACC 2640

TCACACTCAC TTCCCGCCTC TCTTTAGTGG CTCTTTAGAG AGAAACTCTT TCTCCCTTTG 2700

CAAAAATGGG CTAGAATGAA AAGGAGATGC CTTGGGGTTC GTGCACAACA CAGCTTCTGT 2760 TGACTCTAAC TTCCAAATCA AAATCATTTG GTTGAAACAG ACTGTTGCTT GATTTTAGAA 2820

AATACACAAA AACCCATATT TCTGAAATAA TGCTGATTCC TGAGATAAGA AAGTGGATTT 2880

GATCCCCAGT CTCATTGCTT AGTAGAATAA ATCCTGCACC AGCAACAACA CTTGTAAATT 2940 TGTGAAAATG AATTTTATCT TTCCTTAAAA AAGAAATTTT TTAATCCATC ACACTTTTCT 3000

TCCCTACCCT TTAGTTTTTG ATAAATGATA AAAATGAGCC AGTTATCAAA GAAGAACTAG 3060

TTCTTACTTC AAAAGAAAAA TAAACATAAA AAATAAGTTG CTGGTTCCTA ACAGGAAAAA 3120

TTTTAATAAT TGTACTGAGA GAAACTGCTT ACGTACACAT TGCAGATCAA ATATTTGGAG 3180

TTAAAATGTT AGTCTACATA GATGGGTGAT TGTAACTTTA TTGCCATTAA AAGATTTCAA 3240

ATTGCATTCA TGCTTCTGTG TACACATAAT GAAAAATGGG CAAATAATGA AGATCTCTCC 3300

TTCAGTCTGC TCTGTTTAAT TCTGCTGTCT GCTCTTCTCT AATGCTGCGT CCCTAATTGT 3360 ACACAGTTTA GTGATATCTA GGAGTATAAA GTTGTCGCCC ATCAATAAAA ATCACAAAGT 3420

TGGTTT 3426

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 849 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

^" Met Cys Ser Lys Asp Thr Val Leu Ser Val Cys Ala Leu Tyr Trp Arg 1 5 10 15

Lys Gly He Gin Ser His Thr Pro Leu He Gly Ala Trp Ser Cys Arg 20 25 30

Arg Gly Lys Gin Arg Glu Gin Pro Glu Asp Arg Gly Val Pro Met Lys 35 40 45

Arg Ala Ala Met Ala Leu His Ser Pro Gin Tyr He Phe Gly Asp Phe 50 55 60

Ser Pro Asp Glu Phe Asn Gin Phe Phe Val Thr Pro Arg Ser Ser Val 65 70 75 80

Glu Leu Pro Pro Tyr Ser Gly Thr Val Leu Cys Gly Thr Gin Ala Val 85 90 95

Asp Lys Leu Pro Asp Gly Gin Glu Tyr Gin Arg He Glu Phe Gly Val 100 105 110 Asp Glu Val He Glu Pro Ser Asp Thr Leu Pro Arg Thr Pro Ser Tyr 115 120 125

Ser He Ser Ser Thr Leu Asn Pro Gin Ala Pro Glu Phe He Leu Gly 130 135 140 Cys Thr Ala Ser Lys He Thr Pro Asp Gly He Thr Lys Glu Ala Ser 145 150 155 160

Tyr Gly Ser He Asp Cys Gin Tyr Pro Gly Ser Ala Leu Ala Leu Asp 165 170 175

Gly Ser Ser Asn Val Glu Ala Glu Val Leu Glu Asn Asp Gly Val Ser 180 185 190

Gly Gly Leu Gly Gin Arg Glu Arg Lys Lys Lys Lys Lys Arg Pro Pro 195 200 205

Gly Tyr Tyr Ser Tyr Leu Lys Asp Gly Gly Asp Asp Ser He Ser Thr 210 215 220

Glu Ala Leu Val Asn Gly His Ala Asn Ser Ala Val Pro Asn Ser Val 225 230 235 240

Ser Ala Glu Asp Ala Glu Phe Met Gly Asp Met Pro Pro Ser Val Thr 245 250 255

Pro Arg Thr Cys Asn Ser Pro Gin Asn Ser Thr Asp Ser Val Ser Asp 260 265 270

He Val Pro Asp Ser Pro Phe Pro Gly Ala Leu Gly Ser Asp Thr Arg 275 280 285

Thr Ala Gly Gin Pro Glu Gly Gly Pro Gly Ala Asp Phe Gly Gin Ser 290 295 300

Cys Phe Pro Ala Glu Ala Gly Arg Asp Thr Leu Ser Arg Thr Ala Gly 305 310 315 320

Ala Gin Pro Cys Val Gly Thr Asp Thr Thr Glu Asn Leu Gly Val Ala 325 330 335

Asn Gly Gin He Leu Glu Ser Ser Gly Glu Gly Thr Ala Thr Asn Gly 340 345 350 Val Glu Leu His Thr Thr Glu Ser He Asp Leu Asp Pro Thr Lys Pro 355 360 365

Glu Ser Ala Ser Pro Pro Ala Asp Gly Thr Gly Ser Ala Ser Gly Thr 370 375 380

Leu Pro Val Ser Gin Pro Lys Ser Trp Ala Ser Leu Phe His Asp Ser 385 390 395 400

Lys Pro Ser Ser Ser Ser Pro Val Ala Tyr Val Glu Thr Lys Tyr Ser 405 410 415

Pro Pro Ala He Ser Pro Leu Val Ser Glu Lys Gin Val Glu Val Lys 420 425^' 430 Glu Gly Leu Val Pro Val Ser Glu Asp Pro Val Ala He Lys He Ala 435 440 445

Glu Leu Leu Glu Asn Val Thr Leu He His Lys Pro Val Ser Leu Gin 450 455 460 Pro Arg Gly Leu He Asn Lys Gly Asn Trp Cys Tyr He Asn Ala Thr 465 470 475 480

Leu Gin Ala Leu Val Ala Cys Pro Pro Met Tyr His Leu Met Lys Phe 485 490 495

He Pro Leu Tyr Ser Lys Val Gin Arg Pro Cys Thr Ser Thr Pro Met 500 505 510

He Asp Ser Phe Val Arg Leu Met Asn Glu Phe Thr Asn Met Pro Val 515 520 525

Pro Pro Lys Pro Arg Gin Ala Leu Gly Asp Lys He Val Arg Asp He 530 535 540

Arg Pro Gly Ala Ala Phe Glu Pro Thr Tyr He Tyr Arg Leu Leu Thr 545 550 555 560

Val Asn Lys Ser Ser Leu Ser Glu Lys Gly Arg Gin Glu Asp Ala Glu 565 570 575

Glu Tyr Leu Gly Phe He Leu Asn Gly Leu His Glu Glu Met Leu Asn 580 585 590

Leu Lys Lys Leu Leu Ser Pro Ser Asn Glu Lys Leu Thr He Ser Asn 595 600 605

Gly Pro Lys Asn His Ser Val Asn Glu Glu Glu Gin Glu Glu Gin Gly 610 615 620

Glu Gly Ser Glu Asp Glu Trp Glu Gin Val Gly Pro Arg Asn Lys Thr 625 630 635 640

Ser Val Thr Arg Gin Ala Asp Phe Val Gin Thr Pro He Thr Gly He 645 650 655

Phe Gly Gly His He Arg Ser Val Val Tyr Gin Gin Ser Ser Lys Glu 660 665 670 Ser Ala Thr Leu Gin Pro Phe Phe Thr Leu Gin Leu Asp He Gin Ser 675 680 685

Asp Lys He Arg Thr Val Gin Asp Ala Leu Glu Ser Leu Val Ala Arg 690 695 700

Glu Ser Val Gin Gly Tyr Thr Thr Lys Thr Lys Gin Glu Val Glu He 705 710 715 720

Ser Arg Arg Val Thr Leu Glu Lys Leu Pro Pro Val Leu Val Leu His 725 730 735

Leu Lys Arg Phe Val Tyr Glu Lys Thr Gly Gly Cys Gin Lys Leu He 740 745 750 Lys Asn He Glu Tyr Pro Val Asp Leu Glu He Ser Lys Glu Leu Leu 755 760 765

Ser Pro Gly Val Lys Asn Lys Asn Phe Lys Cys His Arg Thr Tyr Arg 770 775 780 Leu Phe Ala Val Val Tyr His His Gly Asn Ser Ala Thr Gly Gly His 785 790 795 800

Tyr Thr Thr Asp Val Phe Gin He Gly Leu Asn Gly Trp Leu Arg He 805 810 815

Asp Asp Gin Thr Val Lys Val He Asn Gin Tyr Gin Val Val Lys Pro 820 825 830

Thr Ala Glu Arg Thr Ala Tyr Leu Leu Tyr Tyr Arg Arg Val Asp Leu 835 840 845

Leu

(2) INFORMATION FOR SEQ ID NO: 7:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1769 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(11) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

GAATTCGGGC GGGGTTTGTA CTATCCTCGG TGCTGTGGTG CAGAGCTAGT TCCTCTCCAG 60

CTCAGCCGCG TAGGTTTGGA CATATTTACT CTTTTCCCCC CAGGTTGAAT TGACCAAAGC 120 AATGGTGATG GAGAAGCCTA GTCCCCTGCT GGTCGGGCGG GAATTTGTGA GACAGTATTA 180

CACACTGCTG AACCAGGCCC CAGACATGCT GCATAGATTT TATGGAAAGA ACTCTTCTTA 240

TGTCCATGGG GGATTGGATT CAAATGGAAA GCCAGCAGAT GCAGTCTACG GACAGAAAGA 300

AATCCACAGG AAAGTGATGT CACAAAACTT CACCAACTGC CACACCAAGA TTCGCCATGT 360

TGATGCTCAT GCCACGCTAA ATGATGGTGT GGTAGTCCAG GTGATGGGGC TTCTCTCTAA 420 CAACAACCAG GCTTTGAGGA GATTCATGCA AACGTTTGTC CTTGCTCCTG AGGGGTCTGT 480

TGCAAATAAA TTCTATGTTC ACAATGATAT CTTCAGATAC CAAGATGAGG TCTTTGGTGG 540

GTTTGTCACT GAGCCTCAGG AGGAGTCTGA AGAAGAAGTA GAGGAACCTG AAGAAAGACA 600

GCAAACACCT GAGGTGGTAC CTGATGATTC TGGAACTTTC TATGATCAGG CAGTTGTCAG 660

TAATGACATG GAAGAACATT TAGAGGAGCC TGTTGCTGAA CCAGAGCCTG ATCCTGAACC 720 AGAACCAGAA CAAGAACCTG TATCTGAAAT CCAAGAGGAA AAGCCTGAGC CAGTATTAGA 780

AGAAACTGCC CCTGAGGATG CTCAGAAGAG TTCTTCTCCA GCACCTGCAG ACATAGCTCA 840

GACAGTACAG GAAGACTTGA GGACATTTTC TTGGGCATCT GTGACCAGTA AGAATCTTCC 900 ACCCAGTGGA GCTGTTCCAG TTACTGGGAT ACCACCTCAT GTTGTTAAAG TACCAGCTTC 960

ACAGCCCCGT CCAGAGTCTA AGCCTGAATC TCAGATTCCA CCACAAAGAC CTCAGCGGGA 1020

TCAAAGAGTG CGAGAACAAC GAATAAATAT TCCTCCCCAA AGGGGACCCA GACCAATCCG 1080

TGAGGCTGGT GAGCAAGGTG ACATTGAACC CCGAAGAATG GTGAGACACC CTGACAGTCA 1140

CCAACTCTTC ATTGGCAACC TGCCTCATGA AGTGGACAAA TCAGAGCTTA AAGATTTCTT 1200

TCAAAGTTAT GGAAACGTGG TGGAGTTGCG CATTAACAGT GGTGGGAAAT TACCCAATTT 1260

TGGTTTTGTT GTGTTTGATG ATTCTGAGCC TGTTCAGAAA GTCCTTAGCA ACAGGCCCAT 1320

CATGTTCAGA GGTGAGGTCC GTCTGAATGT CGAAGAGAAG AAGACTCGAG CTGCCAGGGA 1380

AGGCGACCGA CGAGATAATC GCCTTCGGGG ACCTGGAGGC CCTCGAGGTG GGCTGGGTGG 1440

TGGAATGAGA GGCCCTCCCC GTGGAGGCAT GGTGCAGAAA CCAGGATTTG GAGTGGGAAG 1500

GGGGCTTGCG CCACGGCAGT AATCTTCATG GATCTTCATG CAGCCATACA AACCCTGGTT 1560

CCAACAGAAT GGTGAATTTT CGACAGCCTT TGGTATCTTG GAGTATGACC CCAGTCTGTT 1620 ATAAACTGCT TAAGTTTGTA TAATTTTACT TTTTTTGTGT GTTAATGGTG TGTGCTCCCT 1680

CTCCCTCTCT TCCCTTTCCT GACCTTTAGT CTTTCACTTC CAATTTTGTG GAATGATATT 1740

TTAGGAATAA CGGACTTTTA CCCGAATTC 1769 (2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 466 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Met Val Met Glu Lys Pro Ser Pro Leu Leu Val Gly Arg Glu Phe Val 1 5 10 15

Arg Gin Tyr Tyr Thr Leu Leu Asn Gin Ala Pro Asp Met Leu His Arg 20 25 30

Phe Tyr Gly Lys Asn Ser Ser Tyr Val His Gly Gly Leu Asp Ser Asn 35 40 45

Gly Lys Pro Ala Asp Ala Val Tyr Gly Gin Lys Glu He His Arg Lys 50 55 60

Val Met Ser Gin Asn Phe Thr Asn Cys His Thr Lys He Arg His Val 65 70 75 80 Asp Ala His Ala Thr Leu Asn Asp Gly Val Val Val Gin Val Met Gly 85 90 95

Leu Leu Ser Asn Asn Asn Gin Ala Leu Arg Arg Phe Met Gin Thr Phe 100 105 110

Val Leu Ala Pro Glu Gly Ser Val Ala Asn Lys Phe Tyr Val His Asn 115 120 125 Asp He Phe Arg Tyr Gin Asp Glu Val Phe Gly Gly Phe Val Thr Glu 130 135 140

Pro Gin Glu Glu Ser Glu Glu Glu Val Glu Glu Pro Glu Glu Arg Gin 145 150 155 160

Gin Thr Pro Glu Val Val Pro Asp Asp Ser Gly Thr Phe Tyr Asp Gin 165 170 175

Ala Val Val Ser Asn Asp Met Glu Glu His Leu Glu Glu Pro Val Ala 180 185 190

Glu Pro Glu Pro Asp Pro Glu Pro Glu Pro Glu Gin Glu Pro Val Ser 195 200 205 Glu He Gin Glu Glu Lys Pro Glu Pro Val Leu Glu Glu Thr Ala Pro 210 215 220

Glu Asp Ala Gin Lys Ser Ser Ser Pro Ala Pro Ala Asp He Ala Gin 225 230 235 240

Thr Val Gin Glu Asp Leu Arg Thr Phe Ser Trp Ala Ser Val Thr Ser 245 250 255

Lys Asn Leu Pro Pro Ser Gly Ala Val Pro Val Thr Gly He Pro Pro 260 265 270

His Val Val Lys Val Pro Ala Ser Gin Pro Arg Pro Glu Ser Lys Pro 275 280 285

Glu Ser Gin He Pro Pro Gin Arg Pro Gin Arg Asp Gin Arg Val Arg 290 295 300

Glu Gin Arg He Asn He Pro Pro Gin Arg Gly Pro Arg Pro He Arg 305 310 315 320

Glu Ala Gly Glu Gin Gly Asp He Glu Pro Arg Arg Met Val Arg His 325 330 335

Pro Asp Ser His Gin Leu Phe He Gly Asn Leu Pro His Glu Val Asp 340 345 350

Lys Ser Glu Leu Lys Asp Phe Phe Gin Ser Tyr Gly Asn Val Val Glu 355 360 365 Leu Arg He Asn Ser Gly Gly Lys Leu Pro Asn Phe Gly Phe Val Val 370 375 380

Phe Asp Asp Ser Glu Pro Val Gin Lys Val Leu Ser Asn Arg Pro He 385 390 395 400 Met Phe Arg Gly Glu Val Arg Leu Asn Val Glu Glu Lys Lys Thr Arg 405 410 415

Ala Ala Arg Glu Gly Asp Arg Arg Asp Asn Arg Leu Arg Gly Pro Gly 420 425 430

Gly Pro Arg Gly Gly Leu Gly Gly Gly Met Arg Gly Pro Pro Arg Gly 435 440 445

Gly Met Val Gin Lys Pro Gly Phe Gly Val Gly Arg Gly Leu Ala Pro 450 455 460

Arg Gin 465

(2) INFORMATION FOR SEQ ID NO: 9:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 495 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

ATGCATCGTG ATTCCTGTCC ATTGGACTGT AAGGTTTATG TAGGCAATCT TGGAAACAAT 60

GGCAACAAGA CGGAATTGGA ACGGGCTTTT GGCTACTATG GACCACTCCG AAGTGTGTGG 120 GTTGCTAGAA ACCCACCCGG CTTTGCTTTT GTTGAATTTG AAGATCCCCG AGATGCAGCT 180

GATGCAGTCC GAGAGCTAGA TGGAAGAACA CTATGTGGCT GCCGTGTAAG AGTGGAACTG 240

TCGAATGGTG AAAAAAGAAG TAGAAATCGT GGCCCACCTC CCTCTTGGGG TCGTCGCCCT 300

CGAGATGATT ATCGTAGGAG GAGTCCTCCA CCTCGTCGCA GATCTCCAAG AAGGAGAAGC 360

TTCTCTCGCA GCCGGAGCAG GTCCCTTTCT AGAGATAGGA GAAGAGAGAG ATCGCTGTCT 420 CGGGAGAGAA ATCACAAGCC GTCCCGATCC TTCTCTAGGT CTCGTAGTCG ATCTAGGTCA 480

AATGAAAGGA AATAG 495

(2) INFORMATION FOR SEQ ID NO: 10:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 164 ammo acids

(B) TYPE: amino acid

(C) STRANDEDNESS: (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Met His Arg Asp Ser Cys Pro Leu Asp Cys Lys Val Tyr Val Gly Asn 1 5 10 15

Leu Gly Asn Asn Gly Asn Lys Thr Glu Leu Glu Arg Ala Phe Gly Tyr

20 25 30 Tyr Gly Pro Leu Arg Ser Val Trp Val Ala Arg Asn Pro Pro Gly Phe

35 40 45

Ala Phe Val Glu Phe Glu Asp Pro Arg Asp Ala Ala Asp Ala Val Arg 50 55 60

Glu Leu Asp Gly Arg Thr Leu Cys Gly Cys Arg Val Arg Val Glu Leu 65 70 75 80

Ser Asn Gly Glu Lys Arg Ser Arg Asn Arg Gly Pro Pro Pro Ser Trp 85 90 95

Gly Arg Arg Pro Arg Asp Asp Tyr Arg Arg Arg Ser Pro Pro Pro Arg 100 105 110

Arg Arg Ser Pro Arg Arg Arg Ser Phe Ser Arg Ser Arg Ser Arg Ser 115 120 125

Leu Ser Arg Asp Arg Arg Arg Glu Arg Ser Leu Ser Arg Glu Arg Asn 130 135 140

His Lys Pro Ser Arg Ser Phe Ser Arg Ser Arg Ser Arg Ser Arg Ser 145 150 155 160

Asn Glu Arg Lys

Claims

l.A protein selected from the group consisting of the proteins having the sequences reported as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 SEQ ID NO: 10 or a functional derivative theareof

2. A polynucleotide operationally encoding a protein according to claim 1 or a functional derivative thereof.

3. A polynucleotide operationally encoding a protein according to claim 1, comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO : 5 , SEQ ID NO:7 or SEQ ID NO : 9 .

4. A polynucleotide according to claim 2, werein said polynucleotide is a mRNA.

5. A recombinant vector comprising a polynucleotide^" according to claims 2 or 3.

6. A host cell transfected with the recombinant vector according to claim 5.

7. A purified antibody against a protein of claim 1

8. The antibody of claim 7 , wherein the antibody is conjugated to a cytotoxic agent.

9. The antibody of claim 7 , wherein the antibody is bound to a detectable group

10. The antibody of claim 7, wherein the antibody is bound to a solid support.

11. Use of an antibody of claims 7 to 10 for the diagnosis of a tumor or another proliferative desease.

12. A mRNA or DNA antisense or a chemical derivative thereof, complementary to a mRNA encoding a protein of claim 1.

13. A mRNA or DNA antisense or a chemical derivative thereof according to claim 12 for therapeutic or diagnostic use .

14 Homopurine or homopiridine sequences of the polynucleotide sequences reported as SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID N0:7 or SEQ ID NO : 9 .

15. Use of the homopurine or homopiridine sequences of claim 14 as triple helix probe.

16. A method for assaying the enzymatic activity of ubiquitin isopeptidases which comprises the following steps: a) Preparation of a cell extract containing ubiquitinated proteins, optionally treated with proteasoma inhibitors or derived from cell treated with proteasoma inhibitors; b) Incubation of the cell extract obtained in step a) . with the protein being tested. c) Quantitative measurement of ubiquitin release .