AU1118301A - Tumour suppressor genes from chromosome 16 - Google Patents
Tumour suppressor genes from chromosome 16 Download PDFInfo
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- AU1118301A AU1118301A AU11183/01A AU1118301A AU1118301A AU 1118301 A AU1118301 A AU 1118301A AU 11183/01 A AU11183/01 A AU 11183/01A AU 1118301 A AU1118301 A AU 1118301A AU 1118301 A AU1118301 A AU 1118301A
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Description
WO 01/32861 PCT/AUOO/01329 TUMOUR SUPPRESSOR GENES FROM CHROMOSOME 16 Technical Field The present invention relates to a novel gene which has been identified at the distal tip of the long arm of 5 chromosome 16 at 16q24.3. The TSG16 gene encodes a polypeptide active in suppressing cellular proliferation. In particular, TSG16 functions as a tumour suppressor gene as well as having a role in immune/autoimmune/inflammatory disorders. 10 Background Art The development of human carcinomas has been shown to arise from the accumulation of genetic changes involving both positive regulators of cell function (oncogenes) and 15 negative regulators (tumour suppressor genes). For a normal somatic cell to evolve into a metastatic tumour it requires changes at the cellular level, such as immortalisation, loss of contact inhibition and invasive growth capacity, and changes at the tissue level, such as 20 evasion of host immune responses and growth restraints imposed by surrounding cells, and the formation of a blood supply for the growing tumour. Molecular genetic studies of colorectal carcinoma have provided substantial evidence that the generation of 25 malignancy requires the sequential accumulation of a WO 01/32861 PCT/AUOO/01329 2 number of genetic changes within the same epithelial stem cell of the colon. For a normal colonic epithelial cell to become a benign adenoma, progress to intermediate and late adenomas, and finally become a malignant cell, 5 inactivating mutations in tumour suppressor genes and activating mutations in proto-oncogenes are required (Fearon and Vogelstein,1990). Tumour suppressor genes were first identified in the childhood cancer retinoblastoma. Both inherited and 10 sporadic forms of this cancer exist, with the genetic form inherited as a highly penetrant autosomal dominant trait, which was mapped to chromosome 13q14 by genetic linkage analysis (Sparkes et al., 1983). From the observation that bilateral retinoblastoma was characteristic of the 15 inherited disease and occurred at an early age, whereas unilateral retinoblastoma was characteristic of the sporadic form and occurred at a later age, led to the hypothesis that the tumour arises from two mutational steps (Knudson, 1971). With this proposition, familial 20 cancers would result from an inherited germline. mutation of a gene suppressing the growth of cells (tumour suppressor gene), such that all cells would carry this mutation. A second mutation or "hit" in any cell therefore resulted in the manifestation of the recessive mutation 25 leading to cancer. The fact that only one more "hit" WO 01/32861 PCT/AUOO/01329 3 produces a cancerous cell meant that individuals with an inherited pre-disposition to the disease had an earlier age of onset and often bilateral tumours. In contrast, sporadic cases tended to be in one eye and later in onset 5 because two "hits" were needed to the genes in the same cell. This hypothesis was confirmed with the use of genetic markers mapping to 13q14 to type DNA isolated from blood and tumour samples taken from the same affected 10 individuals (Cavenee et al., 1983). In several cases the constitutional DNA from lymphocytes was heterozygous for some markers but the tumour cells appeared homozygous for the same markers. The apparent reduction to homozygosity (or loss of heterozygosity, LOH) through the loss of one 15 allele of these markers was suggested to be the second "hit" which was removing the remaining functional copy of the retinoblastoma gene in these individuals. The analysis of tumours in familial cases showed that the chromosome from the unaffected parent was in each instance the one 20 eliminated from the tumour. A number of mechanisms were proposed including mitotic recombination, mitotic non disjunction with loss of the wild-type allele or reduplication of the mutant allele, and gene conversion, deletion or mutation. 25 In addition to retinoblastoma, studies of other WO 01/32861 PCT/AUOO/01329 4 cancers have supported the model that LOH is a specific event in the pathogenesis of cancer. In Von Hippel-Lindau (VHL) syndrome both sporadic and inherited cases of the syndrome show LOH for the short arm of chromosome 3. 5 Somatic translocations involving 3p in sporadic tumours, and genetic linkage to the same region in affected families has also been observed. Similarly, in colorectal carcinoma, inherited forms of the disease have been mapped to the long arm of chromosome 5 while LOH at 5q has been 10 reported in both the familial and sporadic versions of the disease and the APC gene, mapping to this region, has been shown to be involved (Groden et al., 1991). Other examples include the TP53 and NF2 genes, which firmly establishes the fact that -a general mechanism in human cancer is the 15 inactivation of tumour suppressor genes by LOH. Breast cancer is the most common malignancy seen in women affecting approximately 10% of females in the Western world. The route to breast cancer is not as well mapped as that of colon cancer due to the histological 20 stages of breast cancer development being largely. unknown. It is known however, that breast cancer is derived from the epithelial lining of terminal mammary ducts or lobuli. Hormonal influences, such as those exerted by oestrogen, are believed to be important because of the marked 25 increase in breast cancer incidence in post-menopausal WO 01/32861 PCT/AUOO/01329 5 women, but the initial steps in breast cancer development probably occur before the onset of menopause. As with colon carcinoma, it is believed that a number of genes need to become involved in a stepwise progression during 5 breast tumourigenesis. Certain women appear to be at an increased risk of developing breast cancer. Genetic linkage analysis has shown that 5 to 10% of all breast cancers are due to at least two autosomal dominant susceptibility genes. 10 Generally, women carrying a mutation in a susceptibility gene develop breast cancer at a younger age compared to the general population, often have bilateral breast tumours, and are at an increased risk of developing cancers in other organs, particularly carcinoma of the 15 ovary. Genetic linkage analysis on families where the onset of breast cancer occurred before the age of 46 was successful in mapping the first susceptibility gene, BRCA1, to chromosome 17q21 (Hall et al., 1990). Subsequent 20 to this, the BRCA2 gene was mapped to chromosome 13q12-q13 (Wooster et al., 1994) with this gene conferring a higher incidence of male breast cancer and a lower incidence of ovarian cancer when compared to BRCA1. Additional inherited breast cancer syndromes exist, however they are 25 rare. Inherited mutations in the TP53 gene have been WO 01/32861 PCT/AUOO/01329 6 identified in individuals with Li-Fraumeni syndrome, a familial cancer resulting in epithelial neoplasms occurring at multiple sites including the breast. Similarly, germline mutations in the MMAC1/PTEN gene 5 involved in Cowden's disease and the ataxia telangiectasia (AT) gene have been shown to confer an increased risk of developing breast cancer, among other clinical manifestations, but together account for only a small percentage of families with an inherited predisposition to 10 breast cancer. In 1994, the BRCA1 gene was positionally cloned and shown to encode a protein consisting of 1,863 amino acids (Miki et al., 1994). Numerous mutations in this gene have been identified in susceptible individuals without 15 evidence of mutation clustering, with the most common mutations leading to truncation of the protein product. BRCA1 protein co-localises, co-immunoprecipitates, and forms a complex with the HsRad5l protein (Scully et al., 1997), suggesting that it may participate in Rad5l 20 functions. These functions primarily concern the double strand-break repair pathway, with the Saccharomyces cerevisiae homologue, ScRad5l, shown to be essential for mitotic and meiotic recombination (Rockmill et al., 1995). The BRCA2 gene was cloned in 1995 (Wooster et al., 1995) 25 and has also been shown to bind the Rad5l gene (Sharan et WO 01/32861 PCT/AUOO/01329 7 al., 1997). Brca2 knockout mice have also shown early embryonic lethality similar to that seen in Rad5l and Brcal knockout mice. Also, hypersensitivity to irradiation has been observed in Brca2 mutated cancer cells 5 suggesting, as with BRCA1, this gene may be involved in the repair of DNA breaks, thereby controlling cell cycle progression. Somatic mutations in the TP53 gene have been shown to occur in a high percentage of individuals with sporadic 10 breast cancer. However, although LOH has been observed at the BRCA1 and BRCA2 loci at a frequency of 30 to 40% in sporadic cases (Cleton-Jansen et al., 1995; Saito et al., 1993), there is virtually no sign of somatic mutations in the retained allele of these two genes in sporadic cancers 15 (Futreal et al., 1994; Miki et al., 1996). The use of both RFLP and STRP markers has identified numerous regions of allelic imbalance in breast cancer suggesting the presence of additional tumour suppressor genes, which may be implicated in breast cancer. Data compiled from more than 20 30 studies reveals the loss of DNA from at -least 11 chromosome arms at a frequency of more than 25%, with regions such as 16q and 17p affected in more than 50% of tumours (Devilee and Cornelisse, 1994; Brenner and Aldaz, 1995). Furthermore, some of these regions are known to 25 harbour tumour suppressor genes shown to be mutated in WO 01/32861 PCT/AUOO/01329 8 individuals with both sporadic (TP53 and RB genes) and familial (TP53, RB, BRCA1, and BRCA2 genes) forms of breast cancer. Cytogenetic studies have implicated loss of the long 5 arm of chromosome 16 as an early event in breast carcinogenesis since it is found in tumours with few or no other cytogenetic abnormalities. Alterations in chromosome 1 and 16 have also been seen in several cases of ductal carcinoma in situ (DCIS), the preinvasive stage of ductal 10 breast carcinoma. In addition, LOH studies on DCIS samples identified loss of 16q markers in 29 to 89% of the cases tested (Chen et al., 1996; Radford et al., 1995). Together, these findings suggest the presence of a tumour suppressor gene mapping to the long arm of chromosome 16 15 that is critically involved in the early development of a large proportion of breast cancers, but to date no such gene has been identified. Disclosure of the Invention 20 The present invention provides an isolated DNA molecule comprising the nucleotide sequence set forth in SEQ ID NO:1. It also provides an isolated DNA molecule comprising the nucleotide sequence set forth in SEQ ID NO:1, or a 25 fragment thereof, which encodes a polypeptide active in WO 01/32861 PCT/AUOO/01329 9 suppressing cellular proliferation. It also provides an isolated DNA molecule comprising the nucleotide sequence set forth in SEQ ID NO:1, or a fragment thereof, which encodes a polypeptide active in 5 suppressing cellular functions. The invention also encompasses an isolated DNA molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:1 and which encodes a polypeptide active in suppressing 10 cellular proliferation. It also encompasses an isolated DNA molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:1 and which encodes a polypeptide active in suppressing cellular 15 functions. Such variants will have preferably at least about 85%, and most preferably at least about 95% sequence identity to the nucleotide sequence encoding TSG16. A particular aspect of the invention encompasses a variant 20 of SEQ ID NO:1 which has at least about 70%, more preferably at least about 85%, and most preferably at least about 95% sequence identity to SEQ ID NO:1. Any one of the polynucleotide variants described above can encode an amino acid sequence, which contains at least one 25 functional or structural characteristic of TSG16.
WO 01/32861 PCT/AUOO/01329 10 Typically sequence identity is calculated using the BLASTN algorithm with the BLOSSUM62 default matrix. The invention also encompasses an isolated DNA molecule that encodes a polypeptide active in suppressing 5 cellular proliferation, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:1. It also provides an isolated DNA molecule that encodes a polypeptide active in suppressing cellular 10 functions, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:l. Under stringent conditions, hybridization will most preferably occur at 42 0 C in 750 mM NaCl, 75 mM trisodium 15 citrate, 2% SDS, 50% formamide, 1X Denhart's, 10% (w/v) dextran sulphate and 100 pg/ml denatured salmon sperm DNA. Useful variations on these conditions will be readily apparent to those skilled in the art. The washing steps which follow hybridization most preferably occur at 65 0 C in 20 15 mM NaCl, 1.5 mM trisodium citrate, and _1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art The invention also provides an isolated DNA molecule which encodes a polypeptide having the amino acid sequence 25 set forth in SEQ ID NO:2.
WO 01/32861 PCT/AUOO/01329 11 Still further, the invention encompasses an isolated DNA molecule wherein the amino acid sequence has at least 70%, preferably 85%, and most preferably 95%, sequence identity to the sequence set forth in SEQ ID NO:2. 5 Preferably, sequence identity is determined using the BLASTP algorithm with the BLOSSUM62 default matrix. In a further aspect the invention provides an isolated gene comprising the nucleotide sequence set forth in SEQ ID NO:1 and TSG16 control elements. 10 Preferably, the TSG16 control elements are those which mediate expression in breast tissue. The nucleotide sequences of the present invention can be engineered using methods accepted in the art so as to alter TSG16-encoding sequences for a variety of purposes. 15 These include, but are not limited to, modification of the cloning, processing, and/or expression of the gene product. PCR reassembly of gene fragments and the use of synthetic oligonucleotides allow the engineering of TSG16 nucleotide sequences. For example, oligonucleotide 20 mediated site-directed mutagenesis can introduce mutations that create new restriction sites, alter glycosylation patterns and produce splice variants etc. As a result of the degeneracy of the genetic code, a number of polynucleotide sequences encoding TSG16, some 25 that may have minimal similarity to the polynucleotide WO 01/32861 PCT/AUOO/01329 12 sequences of any known and naturally occurring gene, may be produced. Thus, the invention includes each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon 5 choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring TSG16, and all such variations are to be considered as being specifically disclosed. 10 The polynucleotides of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified, as will be appreciated by those skilled in the art, and the DNA molecules of this 15 invention may be in any one of the forms listed. In some instances it may be advantageous to produce nucleotide sequences encoding TSG16 or its derivatives possessing a substantially different codon usage than that of the naturally occurring TSG16. For example, codons may be 20 selected to increase the rate of expression of the peptide in a particular prokaryotic or eukaryotic host corresponding with the frequency that particular codons are utilized by the host. Other reasons to alter the nucleotide sequence encoding TSG16 and its derivatives 25 without altering the encoded amino acid sequences include WO 01/32861 PCT/AUOO/01329 13 the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence. The invention also encompasses production of DNA 5 sequences, which encode TSG16 and its derivatives, or fragments thereof, entirely by synthetic chemistry. Synthetic sequences may be inserted into expression vectors and cell systems that contain the necessary elements for transcriptional and translational control of 10 the inserted coding sequence in a suitable host. These elements may include regulatory sequences, promoters, 5' and 3' untranslated regions and specific initiation signals (such as an ATG initiation codon and Kozak consensus sequence) which allow more efficient translation 15 of sequences encoding TSG16. In cases where the complete TSG16 coding sequence including its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, additional control signals may not be needed. However, in cases where only coding 20 sequence, or a fragment thereof, is inserted, exogenous translational control signals as described above should be provided by the vector. Such signals may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers 25 appropriate for the particular host cell system used WO 01/32861 PCT/AUOO/01329 14 (Scharf et al., 1994). The present invention allows for the preparation of purified TSG16 polypeptide or protein, or variants thereof. In order to do this, host cells may be 5 transfected with an expression vector comprising a DNA molecule according to the invention. A variety of expression vector/host systems may be utilized to contain and express sequences encoding TSG16. These include, but are not limited to, microorganisms such as bacteria 10 transformed with plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); or mouse or other animal or human tissue cell systems. Mammalian cells can also be used to express 15 the TSG16 protein using a vaccinia virus expression system. The invention is not limited by the host cell employed. TSG16 can be stably expressed in cell lines to allow long term production of recombinant proteins in mammalian 20 systems. Sequences encoding TSG16 can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. The selectable marker confers resistance 25 to a selective agent, and its presence allows growth and WO 01/32861 PCT/AUOO/01329 15 recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type. 5 The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode TSG16 may be designed to 10 contain signal sequences which direct secretion of TSG16 through a prokaryotic or eukaryotic cell membrane. In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired 15 fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, glycosylation, phosphorylation, and acylation. Post-translational cleavage of a "prepro" form of the protein may also be used to specify protein targeting, folding, and/or 20 activity. Different host cells having specific cellular machinery and characteristic mechanisms for post translational activities (e.g., CHO or HeLa cells), are available from the American Type Culture Collection (ATCC) and may be chosen to ensure the correct modification and 25 processing of the foreign protein.
WO 01/32861 PCT/AUOO/01329 16 When large quantities of TSG16 are needed such as for antibody production, vectors which direct high levels of expression of TSG16 may be used such as those containing the T5 or T7 inducible bacteriophage promoter. The present 5 invention also includes the use of the expression systems described above in generating and isolating fusion proteins which contain important functional domains of the protein. These fusion proteins are used for binding, structural and functional studies as well as for the 10 generation of appropriate antibodies. In order to express and purify the protein as a fusion protein, the appropriate TSG16 cDNA sequence is inserted into a vector which contains a nucleotide sequence encoding another peptide (for example, 15 glutathionine succinyl transferase) . The fusion protein is expressed and recovered from prokaryotic or eukaryotic cells. The fusion protein can then be purified by affinity chromatography based upon the fusion vector sequence and the TSG16 protein obtained by enzymatic cleavage of the 20 fusion protein. Fragments of TSG16 may also be produced by direct peptide synthesis using solid-phase techniques. Automated synthesis may be achieved by using the ABI 431A Peptide Synthesizer (Perkin-Elmer). Various fragments of TSG16 may 25 be synthesized separately and then combined to produce the WO 01/32861 PCT/AUOO/01329 17 full length molecule. According to the invention there is provided an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2. 5 According to a still further aspect of the invention there is provided an isolated polypeptide, comprising the amino acid sequence set forth in SEQ ID NO:2, or a fragment thereof, active in suppressing cellular proliferation. 10 Still further, there is provided an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, or a fragment thereof, active in suppressing cellular functions. The invention also encompasses an isolated 15 polypeptide active in suppressing cellular proliferation and having at least 70%, preferably 85%, and more preferably 95%, identity with the amino acid sequence set forth in SEQ ID NO:2. It also encompasses an isolated polypeptide active in 20 suppressing cellular functions and having at least 70% identity with amino acid sequence set forth in SEQ ID NO:2. Preferably, sequence identity is determined using the BLASTP algorithm with the BLOSSUM62 default matrix. 25 In a further aspect of the invention there is WO 01/32861 PCT/AUOO/01329 18 provided a method of preparing a polypeptide as described above, comprising the steps of: (1) culturing the host cells under conditions effective for production of the polypeptide; and 5 (2) harvesting the polypeptide. Substantially purified TSG16 protein or fragments thereof can then be used in further biochemical analyses to establish secondary and tertiary structure for example by x-ray crystallography of crystal of TSG16 protein or by 10 NMR. Determination of structure allows for the rational design of pharmaceuticals to interact with the protein, alter protein charge configuration or charge interaction with other proteins, or to alter its function in the cell. Chemical and structural similarity in the context of 15 sequences and motifs, exists between regions of TSG16 and ankyrin repeat containing family of proteins including BARD1 and IKB. TSG16 also interacts with members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family, which are 20 proteins that bind to STAT proteins to inhibit the immunological responses mediated by cytokine signalling. In addition, TSG16 is located in a region of restricted LOH seen in breast and prostate cancer and mutations in cancer cell lines from these tissues have been identified. 25 TSG16 is also expressed in many tissues including those of WO 01/32861 PCT/AUOO/01329 19 the immune system. These findings determine that TSG16 is associated with not only cancer but also with immune diseases including autoimmune/inflammatory disorders. With the identification of the TSG16 nucleotide and protein 5 sequence, probes and antibodies raised to the gene can be used in a variety of hybridisation and immunological assays to screen for and detect the presence of either a normal or mutated gene or gene product. The invention enables therapeutic methods for the 10 treatment of all diseases associated with TSG16, including cancer and immune/autoimmune/inflammatory disorders, screening, and also enables methods for the diagnosis of all diseases associated with TSG16. In the treatment of diseases associated with 15 decreased TSG16 expression or activity, it is desirable to increase the expression or activity of TSG16. In the treatment of disorders associated with increased TSG16 expression or activity, it is desirable to decrease the expression or activity of TSG16. 20 Therefore, in another aspect the invention provides a method for the treatment of a disorder associated with decreased expression or activity of TSG16, comprising administering a polypeptide as described above, or an agonist thereof, to a subject in need of such treatment. 25 Typically, TSG16 is administered to a subject to WO 01/32861 PCT/AUOO/01329 20 treat or prevent a disorder associated with decreased expression or activity of TSG16. In another aspect the invention provides the use of a polypeptide as described above, or an agonist thereof, in 5 the manufacture of a medicament for the treatment of a disorder associated with decreased expression or activity of TSG16. Examples of such disorders include, but are not limited to, cancers such as adenocarcinoma, leukemia, 10 lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, and stomach. Other cancers may include those of the adrenal 15 gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland. Immune/autoimmune/inflammatory disorders include acquired immunodeficiency syndrome (AIDS), Addison's disease, adult 20 respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis. autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact 25 dermatitis, Crohn's disease, cystic fibrosis, atopic WO 01/32861 PCT/AUOO/01329 21 dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, 5 gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid 10 arthritis, scleroderma, Siogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of wound healing (eg scarring), cancer, hemodialysis, and extracorporeal 15 circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma. In a further aspect of the invention there is provided a pharmaceutical composition comprising a polypeptide as described above, typically a substantially 20 purified TSG16, and a pharmaceutically acceptable carrier may be administered. The pharmaceutical composition may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of TSG16 including, but 25 not limited to, those provided above. Pharmaceutical WO 01/32861 PCT/AUOO/01329 22 compositions in accordance with the present invention are prepared by mixing TSG16 or active fragments or variants thereof having the desired degree of purity, with acceptable carriers, excipients, or stabilizers which are 5 well known. Acceptable carriers, excipients or stabilizers are nontoxic at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including absorbic acid; low molecular weight (less than about 10 residues) 10 polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates 15 including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitrol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG). 20 According to still another aspect of the invention there is provided a method of treating a disorder associated with increased expression or activity of TSGl6, comprising administering an antagonist of TSG16 to a subject in need of such treatment. 25 In still another aspect of the invention there is WO 01/32861 PCT/AUOO/01329 23 provided the use of an antagonist of TSG16 in the manufacture of a medicament for the treatment of a disorder associated with increased expression or activity of TSG16. 5 Such disorders may include, but are not limited to, those discussed above. In one aspect, an antibody, which specifically binds TSG16, may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or 10 tissues that express TSG16. In a further aspect of the invention there is provided an antibody to a polypeptide as described above, particularly an antibody to TSG16. The antibody may be a monoclonal antibody or polyclonal antibody as would be 15 understood by the person skilled in the art. The gene, or fragments thereof, may be delivered to affected cells in a form in which it can be taken up and can code for sufficient protein to provide effective function. Alternatively, in some mutants, it may be 20 possible to prevent malignancy by introducing another copy of the homologous gene bearing a second mutation in that gene, or to alter the mutation, or to use another gene to block any negative effect. According to still another aspect of the present 25 invention there is provided a method of treating a WO 01/32861 PCT/AUOO/01329 24 disorder associated with decreased expression or activity of TSG16, comprising administering an isolated DNA molecule as described above to a subject in need of such treatment. 5 In a further aspect there is provided the use of an isolated DNA molecule as described above in the manufacture of a medicament for the treatment of a disorder associated with decreased expression or activity of TSG16. 10 Typically, a vector capable of expressing TSG16 or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of TSG16 including, but not limited to, those described above. Transducing 15 retroviral vectors are often used for somatic cell gene therapy because of their high efficiency of infection and stable integration and expression. The targeted cells must be able to divide and the expression level of normal protein should be high. The full length TSG16 gene, or 20 portions thereof, can be cloned into a retroviral vector and driven from its endogenous promoter or from the retroviral long terminal repeat or from a promoter specific for the target cell type of interest. Other viral vectors can be used and include, as is known in the art, 25 adeno-associated virus, vaccinia virus, bovine papilloma WO 01/32861 PCT/AUOO/01329 25 virus, or a herpes virus such as Epstein Barr virus. Gene transfer using non-viral methods of infection in vitro can also be used. These methods include direct injection of DNA, uptake of naked DNA in the presence of calcium 5 phosphate, electroporation, protoplast fusion or liposome delivery. Gene transfer can also be achieved by delivery as a part of a human artificial chromosome or receptor mediated gene transfer. This involves linking the DNA to a targeting molecule that will bind to specific cell 10 surface receptors to induce endocytosis and transfer of the DNA into mammalian cells. One such technique uses poly-L-lysine to link asialoglycoprotein to DNA. An adenovirus is also added to the complex to disrupt the lysosomes and thus allow the DNA to avoid degradation and 15 move to the nucleus. Infusion of these particles intravenously has resulted in gene transfer into hepatocytes. In a further aspect of the invention there is provided a method of treating a disorder associated with 20 increased activity or expression of TSG16, comprising administering an isolated DNA molecule which is the complement of any one of the DNA molecules described above and which encodes a mRNA that hybridizes with the mRNA encoded by TSG16. 25 In a still further aspect of the invention there is WO 01/32861 PCT/AUOO/01329 26 provided the use of an isolated DNA molecule which is the complement of a DNA molecule of the invention and which encodes a mRNA that hybridizes with the mRNA encoded by TSG16, in the manufacture of a medicament for the 5 treatment of a disorder associated with increased expression or activity of TSG16. Typically, a vector expressing the complement of the polynucleotide encoding TSG16 may be administered to a subject to treat or prevent a disorder associated with 10 increased expression or activity of TSG16 including, but not limited to, those described above. Antisense strategies may use a variety of approaches including the use of antisense oligonucleotides, injection of antisense RNA and transfection of antisense RNA expression vectors. 15 Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into 20 that same patient. Delivery by transfection, by -liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (For example, see Goldman et al., 1997). In further embodiments, any of the proteins, 25 antagonists, antibodies, agonists, complementary WO 01/32861 PCT/AUOO/01329 27 sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents may be made by those skilled in the art, according to conventional 5 pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, therapeutic efficacy with lower dosages of each agent may be possible, thus reducing the potential 10 for adverse side effects. Using methods well known in the art, an antagonist of TSG16 may be produced. In particular, purified TSG16 may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those that specifically 15 bind TSG16. Antibodies to TSG16 may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies. 20 For the production of antibodies, various hosts including rabbits, rats, goats, mice, humans, and others may be immunized by injection with TSG16 or with any fragment or oligopeptide thereof, which has immunogenic properties. Various adjuvants may be used to increase 25 immunological response and include, but are not limited WO 01/32861 PCT/AUOO/01329 28 to, Freund's, mineral gels such as aluminum hydroxide, and surface-active substances such as lysolecithin. Adjuvants used in humans include BCG (bacilli Calmette-Guerin) and Corynebacterium parvum. 5 It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to TSG16 have an amino acid sequence consisting of at least about 5 amino acids, and, more preferably, of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or 10 fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of TSG16 amino acids may be fused with those of another protein, such as KLH, and 15 antibodies to the chimeric molecule may be produced. Monoclonal antibodies to TSG16 may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma 20 technique, the human B-cell hybridoma technique,- and the EBV-hybridoma technique. (For example, see Kohler et al., 1975; Kozbor et al., 1985; Cote et al., 1983; Cole et al., 1984). Antibodies may also be produced by inducing in vivo 25 production in the lymphocyte population or by screening WO 01/32861 PCT/AUOO/01329 29 immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (For example, see Orlandi et al., 1989; Winter et al., 1991). Antibody fragments which contain specific binding 5 sites for TSG16 may also be generated. For example, such fragments include, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be 10 constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (For example, see Huse et al., 1989). Various immunoassays may be used for screening to identify antibodies having the desired specificity. 15 Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between TSG16 and its 20 specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering TSG16 epitopes is preferred, but a competitive binding assay may also be employed. According to still another aspect of the invention 25 peptides of the invention, particularly purified TSG16 WO 01/32861 PCT/AUOO/01329 30 polypeptide, and cells expressing these are useful for screening of candidate pharmaceutical agents in a variety of techniques. Such techniques include, but are not limited to, high-throughput screening for compounds having 5 suitable binding affinity to the TSG16 polypeptides. Large numbers of small peptide test compounds can be synthesised on a solid substrate and can be assayed through TSG16 polypeptide binding and washing. Bound TSG16 polypeptide is then detected by methods well known in the art. An 10 additional method for drug screening involves the use of host eukaryotic cell lines or cells which carry mutations in the TSG16 gene. The host cell lines or cells are also defective at the TSG16 polypeptide level. The host cell lines or cells are grown in the presence of various drug 15 compounds and the rate of growth of the host cells is measured to determine if the compound is capable of regulating the growth of TSG16 defective cells. Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, 20 for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans. Polynucleotide sequences encoding TSG16 may be used for the diagnosis of disorders associated with TSG16 and the use of the DNA molecules of the invention in disorders 25 associated with TSG16, or a predisposition to such WO 01/32861 PCT/AUOO/01329 31 disorders, is therefore contemplated. Examples of such disorders include, but are not limited to, cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of 5 the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, and stomach. Other cancers may include those of the adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, 10 parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland. Immune/autoimmune/inflammatory disorders include acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, 15 anemia, asthma, atherosclerosis. autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, 20 dermatomyositis, diabetes mellitus, emphysema, -episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, 25 irritable bowel syndrome, multiple sclerosis, myasthenia WO 01/32861 PCT/AUOO/01329 32 gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, 5 systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of wound healing (eg scarring), cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, 10 protozoal, and helminthic infections, and trauma. Such qualitative or quantitative methods are well known in the art. In another embodiment of the invention, the polynucleotides that may be used for diagnostic purposes 15 include oligonucleotide sequences, genomic DNA and complementary RNA and DNA molecules. The polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which abnormal expression of TSG16 or mutations in TSG16 may be correlated with disease. Genomic 20 DNA used for the diagnosis may be obtained from body cells, such as those present in the blood, tissue biopsy, surgical specimen, or autopsy material. The DNA may be isolated and used directly for detection of a specific sequence or may be amplified by the polymerase chain 25 reaction (PCR) prior to analysis. Similarly, RNA or cDNA WO 01/32861 PCT/AUOO/01329 33 may also be used, with or without PCR amplification. To detect a specific nucleic acid sequence, direct nucleotide sequencing, hybridization using specific oligonucleotides, restriction enzyme digest and mapping, PCR mapping, RNase 5 protection, and various other methods may be employed. Oligonucleotides specific to particular sequences can be chemically synthesized and labeled radioactively or nonradioactively and hybridized to individual samples immobilized on membranes or other solid-supports or in 10 solution. The presence, absence or excess expression of TSG16 may then be visualized using methods such as autoradiography, fluorometry, or colorimetry. In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences 15 described herein may be used as targets in a microarray. The microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to 20 understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents. Microarrays may be prepared, used, and analyzed using methods known in the art. (For example, see Schena et al., 1996; Heller et al., 1997). 25 In a particular aspect, the nucleotide sequences WO 01/32861 PCT/AUOO/01329 34 encoding TSG16 may be useful in assays that detect the presence of associated disorders, particularly those mentioned previously. The nucleotide sequences encoding TSG16 may be labelled by standard methods and added to a 5 fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is 10 significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding TSG16 in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular 15 therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient. In order to provide a basis for the diagnosis of a disorder associated with abnormal expression of TSG16, a 20 normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding TSG16, under conditions suitable for hybridization or 25 amplification. Standard hybridization may be quantified by WO 01/32861 PCT/AUOO/01329 35 comparing the values obtained from normal subjects with values f rom an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values 5 obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder. Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may 10 be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several 15 days to months. In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding TSG16 or closely related molecules may be used to identify nucleic acid 20 sequences which encode TSG16. The specificity_ of the probe, whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the 25 probe identifies only naturally occurring sequences WO 01/32861 PCT/AUOO/01329 36 encoding TSG16, allelic variants, or related sequences. Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the TSG16 encoding sequences. 5 The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:1 or from genomic sequences including promoters, enhancers, and introns of the TSG16 gene (SEQ ID Numbers: 3-12, 124). 10 Means for producing specific hybridization probes for DNAs encoding TSG16 include the cloning of polynucleotide sequences encoding TSG16 or TSG16 derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, and are commercially available. Hybridization 15 probes may be labeled by radionuclides such as 3P or 5, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, or other methods known in the art. According to a further aspect of the invention there 20 is provided the use of a polypeptide as described above in the diagnosis of a disorder associated with TSG16, or a predisposition to such disorders. When a diagnostic assay is to be based upon the TSG16 protein, a variety of approaches are possible. For 25 example, diagnosis can be achieved by monitoring WO 01/32861 PCT/AUOO/01329 37 differences in the electrophoretic mobility of normal and mutant proteins. Such an approach will be particularly useful in identifying mutants in which charge substitutions are present, or in which insertions, 5 deletions or substitutions have resulted in a significant change in the electrophoretic migration of the resultant protein. Alternatively, diagnosis may be based upon differences in the proteolytic cleavage patterns of normal and mutant proteins, differences in molar ratios of the 10 various amino acid residues, or by functional assays demonstrating altered function of the gene products. In another aspect, antibodies that specifically bind TSG16 may be used for the diagnosis of disorders characterized by abnormal expression of TSG16, or in 15 assays to monitor patients being treated with TSG16 or agonists, antagonists, or inhibitors of TSG16. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for TSG16 include methods that utilize the antibody 20 and a label to detect TSG16 in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labelled by covalent or non-covalent attachment of a reporter molecule. 25 A variety of protocols for measuring TSG16, including WO 01/32861 PCT/AUOO/01329 38 ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of TSG16 expression. Normal or standard values for TSG16 expression are established by combining body fluids or cell extracts 5 taken from normal mammalian subjects, preferably human, with antibody to TSG16 under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of TSG16 10 expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease. The present invention also provides for the 15 production of genetically modified, knock-out or knock-in non-human animal models transformed with the DNA molecules of the invention. These animals are useful for the study of the TSG16 gene function, to study the mechanisms of disease as related to the TSG16 gene, for the screening of 20 candidate pharmaceutical compounds, for the creation of explanted mammalian cell cultures which express the protein or mutant protein and for the evaluation of potential therapeutic interventions. The TSG16 gene may have been inactivated by knock-out 25 deletion, and knock-out genetically modified non-human WO 01/32861 PCT/AUOO/01329 39 animals are therefore provided. Animal species which are suitable for use in the animal models of the present invention include, but are not limited to, rats, mice, hamsters, guinea pigs, 5 rabbits, dogs, cats, goats, sheep, pigs, and non-human primates such as monkeys and chimpanzees. For initial studies, genetically modified mice and rats are highly desirable due to their relative ease of maintenance and shorter life spans. For certain studies, transgenic yeast 10 or invertebrates may be suitable and preferred because they allow for rapid screening and provide for much easier handling. For longer term studies, non-human primates may be desired due to their similarity with humans. To create an animal model for mutated TSG16 several 15 methods can be employed. These include generation of a specific mutation in a homologous animal gene, insertion of a wild type human gene and/or a humanized animal gene by homologous recombination, insertion of a mutant (single or multiple) human gene as genomic or minigene cDNA 20 constructs using wild type or mutant or artificial promoter elements or insertion of artificially modified fragments of the endogenous gene by homologous recombination. The modifications include insertion of mutant stop codons, the deletion of DNA sequences, or the 25 inclusion of recombination elements (lox p sites) WO 01/32861 PCT/AUOO/01329 40 recognized by enzymes such as Cre recombinase. To create a transgenic mouse, which is preferred, a mutant version of TSG16 can be inserted into a mouse germ line using standard techniques of oocyte microinjection or 5 transfection or microinjection into embryonic stem cells. Alternatively, if it is desired to inactivate or replace the endogenous TSG16 gene, homologous recombination using embryonic stem cells may be applied. For oocyte injection, one or more copies of the 10 mutant or wild type TSG16 gene can be inserted into the pronucleus of a just-fertilized mouse oocyte. This oocyte is then reimplanted into a pseudo-pregnant foster mother. The liveborn mice can then be screened for integrants using analysis of tail DNA for the presence of human TSG16 15 gene sequences. The transgene can be either a complete genomic sequence injected as a YAC, BAC, PAC or other chromosome DNA fragment, a cDNA with either the natural promoter or a heterologous promoter, or a minigene containing all of the coding region and other elements 20 found to be necessary for optimum expression. According to still another aspect of the invention there is provided the use of genetically modified non human animals for the screening of candidate pharmaceutical compounds. 25 In a still further aspect of the invention there is WO 01/32861 PCT/AUOO/01329 41 provided a nucleic acid encoding a mutant TSG16 polypeptide which cannot form a complex with a wild-type protein with which wild-type TSG16 does form a complex. Typically the protein is a member of the protein inhibitor 5 of activated signal transducer and activator of transcription (PIAS) family of proteins, more particularly PIAS1. According to a still further aspect of the invention there is provided a mutant TSG16 polypeptide which cannot 10 form a complex with a wild-type protein with which wild type TSG16 does form a complex. Typically the protein is a member of the PIAS family, particularly PIAS1. While not wishing to be bound by theory, it is believed that the mutation in TSG16 occurs in one or more 15 ankyrin repeat domains. According to a still further aspect of the present invention there is provided a complex of wild-type TSG16 and a PIAS protein. Typically the PIAS protein is PIASI. 20 In a still further aspect of the present invention there is provided the use of a complex as described above in screening for candidate pharmaceutical compounds. It will be clearly understood that, although a number of prior art publications are referred to herein, this 25 reference does not constitute an admission that any of WO 01/32861 PCT/AUOO/01329 42 these documents forms part of the common general knowledge in the art, in Australia or in any other country. Throughout this specification and the claims, the words "comprise", "comprises" and "comprising" are used in a 5 non-exclusive sense, except where the context requires otherwise. Brief Description of the Drawings Figure 1. Schematic representation of tumours with 10 interstitial and terminal allelic loss on chromosome arm 16q in the two series of tumour samples. Polymorphic markers are listed according to their order on 16q from centromere to telomere and the markers used for each series are indicated by X. Tumour identification numbers 15 are shown at the top of each column. At the right of the figure, the three smallest regions of loss of heterozygosity are indicated. Figure 2. Portion of the physical map at 16q24.3 containing the TSG16 gene. The complete genomic structure 20 is represented by black boxes numbered 1 to 13. Exons that were trapped from TSG16 and physically mapped to the clone contig are shown as filled circles. The restriction map is indicated by the thick line with individual fragment sizes shown in kilobases. E: EcoRI; Ea: EagI. The location of WO 01/32861 PCT/AUOO/01329 43 cosmid, BAC and PAC clones are shown below the restriction map. Figure 3. Confirmation of TSG16 gene sequence and structure. The upper figure shows the gene structure of 5 TSG16 with vertical lines indicating the position of exon boundaries. Exons are numbered 1 to 13 and the shaded regions indicate the 3' untranslated region (UTR) and the 5' UTR. The location of the trapped exons is shown above the figure. Numbered arrows indicate the locations of the 10 primers used for the RT-PCR analysis and those products sequenced are represented by thin horizontal lines. All primer sequences are shown in Table 1 and correspond to SEQ ID Numbers:13-43. Regions of TSG16 used to probe Northern blots are indicated by thick horizontal lines. 15 The autoradiograph of each Northern is presented below the corresponding probe. The position of size standards in kilobases is shown on the left of each Northern. The blots contained the following tissues: 1: heart; 2: brain; 3: placenta; 4: lung; 5: liver; 6: skeletal muscle; 7: 20 kidney; 8: pancreas. Figure 4A-4G. Genomic sequence of TSG16. Intron sequences are indicated in lowercase letters whereas exonic sequences are in uppercase. Intervals between larger introns are shortened by stretches of v's.
WO 01/32861 PCT/AUOO/01329 44 Figure 5. The complex 5' region of TSG16 is shown indicating the TSG16 isoforms and overlapping genes. Two overlapping genes originate in intron 4 and their nucleotide and amino acid sequences are represented by the 5 SEQ ID Numbers: 125-128. Three TSG16 isoforms originate in intron 7 with two of these containing an additional exon (exon 5E). Of these two, one isoform also contains further sequence (exon 5E+) due to the use of an alternative 3' splice acceptor site. The nucleotide and amino acid 10 sequences of these isoforms are represented by SEQ ID Numbers: 129-134.
WO 01/32861 PCT/AUOO/01329 45 EXAMPLES EXAMPLE 1: Collection of breast cancer patient material Two series of breast cancer patients were analysed for this study. Histopathological classification of each 5 tumour specimen was carried out by our collaborators according to World Health Organisation criteria (WHO, 1981). Patients were graded histopathologically according to the modified Bloom and Richardson method (Elston and Ellis, 1990) and patient material was obtained upon 10 approval of local Medical Ethics Committees. Tumour tissue DNA and peripheral blood DNA from the same individual was isolated as previously described (Devilee et al., 1991) using standard laboratory protocols. Series 1 consisted of 189 patients operated on 15 between 1986 and 1993 in three Dutch hospitals, a Dutch University and two peripheral centres. Tumour tissue was snap frozen within a few hours of resection. For DNA isolation, a tissue block was selected only if it contained at least 50% of tumour cells following 20 examination of haematoxilin and eosin stained tissue sections by a pathologist. Tissue.blocks that contained fewer than 50% of tumour cells were omitted from further analysis. Series 2 consisted of 123 patients operated on 25 between 1987 and 1997 at the Flinders Medical Centre in WO 01/32861 PCT/AUOO/01329 46 Adelaide, Australia. Of these, 87 were collected as fresh specimens within a few hours of surgical resection, confirmed as malignant tissue by pathological analysis, snap frozen in liquid nitrogen, and stored at -70 0 C. The 5 remaining 36 tumour tissue samples were obtained from archival paraffin embedded tumour blocks. Prior to DNA isolation, tumour cells were microdissected from tissue sections mounted on glass slides so as to yield at least 80% tumour cells. In some instances, no peripheral blood 10 was available such that pathologically identified paraffin embedded non-malignant lymph node tissue was used instead. EXAMPLE 2: LOH analysis of chromosome 16q markers in breast cancer samples. 15 A total of 45 genetic markers were used for the LOH analysis of breast tumour and matched normal DNA samples. Figure 1 indicates for which tumour series they were used and their cytogenetic location. Details regarding all markers can be obtained from the Genome Database (GDB) at 20 http://www.gdb.org. The physical order of markers with respect to each other was determined from a combination of information in GDB, by mapping on a chromosome 16 somatic cell hybrid map (Callen et al., 1995) and by genomic sequence information. 25 WO 01/32861 PCT/AUOO/01329 47 Four alternative methods were used for the LOH analysis: 1) For RFLP and VNTR markers, Southern blotting was used to test for allelic imbalance. These markers were 5 used on only a subset of samples. Methods used were as previously described (Devilee et al., 1991). 2) Microsatellite markers were amplified from tumour and normal DNA using the polymerase chain reaction (PCR) incorporating standard methodologies (Weber and May, 10 1989; Sambrook et al., 1989). A typical reaction consisted of 12 pl and contained 100 ng of template, 5 pmol of both primers, 0.2 mM of each dNTP, 1 pCurie [0X- 32 P dCTP, 1.5 mM MgCl 2 , 1.2 pl Supertaq buffer and 0.06 units of Supertaq (HT biotechnologies). A Phosphor Imager type 445 SI 15 (Molecular Dynamics, Sunnyvale, CA) was used to quantify ambiguous results. In these cases, the Allelic Imbalance Factor (AIF) was determined as the quotient of the peak height ratios from the normal and tumour DNA pair. The threshold for allelic imbalance was defined as a 40% 20 reduction of one allele, agreeing with an AIF of 1.7 or ;0.59. This threshold is in accordance with the selection of tumour tissue blocks containing at least 50% tumour cells with a 10% error-range. The threshold for retention has been previously determined to range from 0.76 to 1.3 WO 01/32861 PCT/AUOO/01329 48 (Devilee et al., 1994). This leaves a range of AIFs (0.58 - 0.75 and 1.31 - 1.69) for which no definite decision has been made. This "grey area" is indicated by grey boxes in Figure 1 and tumours with only "grey area" values were 5 discarded completely from the analysis. 3) The third method for determining allelic imbalance was similar to the second method above, however radioactively labelled dCTP was omitted. Instead, PCR of polymorphic microsatellite markers was done with one of 10 the PCR primers labelled fluorescently with FAM, TET or HEX. Analysis of PCR products generated was on an ABI 377 automatic sequencer (PE Biosystems) using 6% polyacrylamide gels containing 8M urea. Peak height values and peak sizes were analysed with the GeneScan programme 15 (PE Biosystems). The same thresholds for allelic imbalance, retention and grey areas were used as for the radioactive analysis. 4) An alternative fluorescent based system was also used. In this instance PCR primers were labelled with 20 fluorescein or hexachlorofluorescein. PCR reaction volumes were 20 pl and included 100 ng of template, 100 ng of each primer, 0.2 mM of each dNTP, 1-2 mM MgCl 2 , 1X AmpliTaq Gold buffer and 0.8 units AmpliTaq Gold enzyme (Perkin Elmer). Cycling conditions were 10 cycles of 94 0 C for 30 seconds, 25 60*C for 30 seconds, 72 0 C for 1 minute, followed by 25 WO 01/32861 PCT/AUOO/01329 49 cycles of 94'C 30 seconds, 55 0 C for 30 seconds, 72 0 C for 1 minute, with a final extension of 72 0 C for 10 minutes. PCR amplimers were analysed on an ABI 373 automated sequencer (PE Biosystems) using the GeneScan programme (PE 5 Biosystems). The threshold range of AIF for allele retention was defined as 0.61 - 1.69, allelic loss as 0.5 or 2.0, or the "grey area" as 051 - 0.6 or 1.7 - 1.99. The first three methods were applied to the first tumour series while the last method was adopted for the 10 second series of tumour samples. For statistical analysis, a comparison of allelic imbalance data for validation of the different detection methods and of the different tumour series was done using the Chi-square test. The identification of the smallest region of overlap 15 (SRO) involved in LOH is instrumental for narrowing down the location of a putative tumour suppressor gene targeted by LOH. Figure 1 shows the LOH results for tumour samples, which displayed small regions of loss (ie interstitial and telomeric LOH) and does not include samples that showed 20 complex LOH (alternating loss and retention of markers). When comparing the two sample 'sets at least three consistent regions emerge with two being at the telomere in band 16q24.3 and one at 16q22.1. The region at 16q22.1 is defined by the markers D16S398 and D16S301 and is based WO 01/32861 PCT/AUOO/01329 50 on the interstitial LOH events seen in three tumours from series 1 (239/335/478) and one tumour from series 2 (237). At the telomere (16q24.2 - 16q24.3), the first region is defined by the markers D16S498 and D16S3407 and is based 5 on four tumours from series 2 (443/75/631/408) while the second region (16q24.3) extends from D16S3407 to the telomere and is based on one tumour from series 1 (559) and three from series 2 (97/240/466). LOH limited to the telomere but involving both of the regions identified at 10 this site could be found in an additional 17 tumour samples. Other studies have shown that the long arm of chromosome 16 is also a target for LOH in prostate, lung, hepatocellular, ovarian, rhabdomyosarcoma and Wilms' 15 tumours. Detailed analysis of prostate carcinomas has revealed an overlap in the smallest regions of LOH seen in this cancer to that seen with breast cancer which suggests that 16q harbours a multi-tumour suppressor gene. 20 EXAMPLE 3: Construction of a physical map of 16q24.3 To identify novel candidate tumour suppressor genes mapping to the smallest regions of overlap at 16q24.3, a clone based physical map contig covering this region was needed. At the start of this phase of the project the most 25 commonly used and readily accessible cloned genomic DNA WO 01/32861 PCT/AUOO/01329 51 fragments were contained in lambda, cosmid or YAC vectors. During the construction of whole-chromosome 16 physical maps, clones from a number of YAC libraries were incorporated into the map (Doggett et al., 1995). These 5 included clones from a flow-sorted chromosome 16-specific YAC library (McCormick et al., 1993), from the CEPH Mark I and MegaYAC libraries and from a half-telomere YAC library (Riethman et al., 1989). Detailed STS and Southern analysis of YAC clones mapping at 16q24.3 established that 10 very few were localised between the CY2/CY3 somatic cell hybrid breakpoint and the long arm telomere. However, those that were located in this region gave inconsistent mapping results and were suspected to be rearranged or deleted. Coupled with the fact that YAC clones make poor 15 sequencing substrates, and the difficulty in isolating the cloned human DNA, a physical map based on cosmid clones was the initial preferred option. A flow-sorted chromosome 16 specific cosmid library had previously been constructed (Longmire et al., 1993), 20 with individual cosmid clones gridded in high-density arrays onto nylon membranes. These filters collectively contained -15,000 clones representing an approximately 5.5 fold coverage of chromosome 16. Individual cosmids mapping to the critical regions at 16q24.3 were identified by the 25 hybridisation of these membranes with markers identified WO 01/32861 PCT/AUOO/01329 52 by this and previous studies to map to the region. The strategy to align overlapping cosmid clones was based on their STS content and restriction endonuclease digestion pattern. Those clones extending furthest within each 5 initial contig were then used to walk along the chromosome by the hybridisation of the ends of these cosmids back to the high-density cosmid grids. This process continued until all initial contigs were linked and therefore the region defining the location of the breast cancer tumour 10 suppressor genes would be contained within the map. Individual cosmid clones representing a minimum tiling path in the contig were then used for the identification of transcribed sequences by exon trapping, and for genomic sequencing. 15 Chromosome 16 was sorted from the mouse/human somatic cell hybrid CY18, which contains this chromosome as the only human DNA, and Sau3A partially digested CY18 DNA was ligated into the BamHI cloning site of the cosmid sCOS-1 vector. All grids were hybridised and washed using methods 20 described in Longmire et al. (1993). Briefly,- the 10 filters were pre-hybridised in 2 large bottles for at least 2 hours in 20 ml of a solution containing 6X SSC; 10 mM EDTA (pH8.0); 1OX Denhardt's; 1% SDS and 100 ptg/ml denatured fragmented salmon sperm DNA at 65 0 C. Overnight WO 01/32861 PCT/AUOO/01329 53 hybridisations with [Oc-"P]dCTP labelled probes were performed in 20 ml of fresh hybridisation solution at 65 0 C. Filters were washed sequentially in solutions of 2X SSC; 0.1% SDS (rinse at room temperature), 2X SSC; 0.1% SDS 5 (room temperature for 15 minutes), 0.1X SSC; 0.1% SDS (room temperature for 15 minutes), and 0.1X SSC; 0.1% SDS (twice for 30 minutes at 500C if needed). Membranes were exposed at -70'C for between 1 to 7 days. Initial markers used for cosmid grid screening were 10 those known to be located below the somatic cell hybrid breakpoints CY2/CY3 and the long arm telomere (Callen et al., 1995). These included three genes, CMAR, DPEP1, and MC1R; the microsatellite marker D16S303; an end fragment from the cosmid 317E5, which contains the BBC1 gene; and 15 four cDNA clones, yc8leO9, yh09aO4, D16S532E, and ScDNA C113. The IMAGE consortium cDNA clone, yc8leO9, was obtained through screening an arrayed normalised infant brain oligo-dT primed cDNA library (Soares et al., 1994), with the insert from cDNA clone ScDNA-A55.. Both the ScDNA 20 A55 and ScDNA-C113 clones were originally isolated from a hexamer primed heteronuclear cDNA library constructed from the mouse/human somatic cell hybrid CY18 (Whitmore et al., 1994). The IMAGE cDNA clone yh09aO4 was identified from direct cDNA selection of the cosmid 37B2 which was WO 01/32861 PCT/AUOO/01329 54 previously shown to map between the CY18A(D2) breakpoint and the 16q telomere. The EST, D16S532E, was also mapped to the same region. Subsequent to these initial screenings, restriction fragments representing the ends of 5 cosmids were used to identify additional overlapping clones. Contig assembly was based on methods previously described (Whitmore et al., 1998). Later during the physical map construction, genomic libraries cloned into 10 BAC or PAC vectors (Genome Systems or Rosewell Park Cancer Institute) became available. These libraries were screened to aid in chromosome walking or when gaps that could not be bridged by using the cosmid filters were encountered. All BAC and PAC filters were hybridised and washed 15 according to manufacturers recommendations. Initially, membranes were individually pre-hybridised in large glass bottles for at least 2 hours in 20 ml of 6X SSC; 0.5% SDS; 5X Denhardt's; 100 ptg/ml denatured salmon sperm DNA at 65 0 C. Overnight hybridisations with - 32 p]dCTP labelled 20 probes were performed at 65'C in 20 ml of a solution containing 6X SSC; 0.5% SDS; 100 ptg/ml denatured salmon sperm DNA. Filters were washed sequentially in solutions of 2X SSC; 0.5% SDS (room temperature 5 minutes), 2X SSC; 0.1% SDS (room temperature 15 minutes) and 0.1X SSC; 0.5% WO 01/32861 PCT/AUOO/01329 55 SDS (37 0 C 1 hour if needed). PAC or BAC clones identified were aligned to the cosmid contig based on their restriction enzyme pattern. As the microsatellite D16S303 was known to be the 5 most telomeric marker in the 16q24.3 region (Callen et al., 1995), fluorescence in situ hybridisation (FISH) to normal metaphase chromosomes using whole cosmids mapping in the vicinity of this marker, was used to define the telomeric limit for the contig. Whole cosmid DNA was nick 10 translated with biotin-14-dATP and hybridised in situ at a final concentration of 20 ng/tl to metaphases from 2 normal males. The FISH method had been modified from that previously described (Callen et al., 1990). Chromosomes were stained before analysis with both propidium iodide 15 (as counter-stain) and DAPI (for chromosome identification). Images of metaphase preparations were captured by a cooled CCD camera using the CytoVision Ultra image collection and enhancement system (Applied Imaging Int. Ltd.). The cosmid 369E1 showed clear fluorescent 20 signals at the telomere of the long arm of chromosome 16. However, this probe also gave clear signal at the telomeres of chromosomal arms 3q, 7p, 9q, 11p, and 17p. Conversely, the cosmid 439G8, which mapped proximal to D16S303, gave fluorescent signals only at 16qter with no WO 01/32861 PCT/AUOO/01329 56 consistent signal detected at other telomeres. These results enabled us to establish the microsatellite marker D16S303 as the boundary of the transition from euchromatin to the subtelomeric repeats, providing a telomeric limit 5 to the contig (Whitmore et al., 1998). A minimum tiling path consisting of 35 cosmids, 1 BAC and 2 PAC clones, which extends approximately 1.1 Mb from the telomere of the long arm of chromosome 16 was produced. 10 EXAMPLE 4: Identification of candidate tumour suppressor genes by exon trapping At the start of this phase of the project international efforts in the physical mapping of 15 individual cDNA clones (ESTs) were not yet underway. This necessitated the identification of transcribed sequences by other means. At this time, the two most commonly used procedures for the identification of transcribed sequences within relatively large genomic intervals were direct cDNA 20 selection and exon amplification. The latter procedure was chosen because it is not limited by the tissue or developmental expression of genes. Instead, the expression of cloned genomic DNAs is driven by a promoter in tissue culture cells, which allows for coding sequences to be 25 identified without prior knowledge of their expression WO 01/32861 PCT/AUOO/01329 57 profile. This therefore circumvents the need to analyse multiple cDNA libraries as with cDNA selection. Exon trapping using the pSPL3B vector relies on the presence of a functional splice acceptor and donor site flanking an 5 exon. As a result, the terminal 5' and 3' exons of a gene are not selected, and similarly, transcripts that contain less than three exons will not be identified. However, as a general procedure for identifying coding sequences within large genomic regions, this technique has been 10 shown to be very effective. Clones forming a minimum tiling . path in the established physical map were used for exon trapping using either the pSPL3B vector, for trapping from BAC clones or a modified version, pSPL3BCAM for trapping from cosmid or 15 PAC clones (Burn et al., 1995). The pSPL3B-CAM vector has had the ampicillin resistance gene replaced with a gene conferring chloramphenicol resistance. This assists in the initial step of sub-cloning cosmid or PAC DNA restriction enzyme fragments into the trapping vector by allowing 20 selection against contaminating re-ligated ampicillin resistant cosmid or PAC vector clones. It has also been noted that increasing the complexity of the DNA used for each trapping experiment dramatically decreases the exon recovery (Yaspo et al., 1995). For this reason, the 25 maximum number of clones used in any one trapping WO 01/32861 PCT/AUOO/01329 58 experiment was three, with the majority of traps involving a single clone. The exon trapping procedures adopted have been described previously (Whitmore et al., 1998). Template DNA 5 was double digested at 37 0 C overnight in a volume of 50 pl with BamHI and BglII, or with PstI alone, in the presence of BSA at a concentration of 100 pg/ml. The next day, 150 pl of water was added to each digest and the samples were phenol/chloroform extracted using standard procedures 10 (Sambrook et al., 1989). Dried DNA pellets were resuspended in 10 pl of sterile water. Vector DNA was digested at 37 0 C overnight in a volume of 50 [L1 with either BamHI or PstI. The following day, 50 pl of water was added to the digests and they were cleaned with QlAquick columns 15 according to manufacturers instructions (Qiagen). The cleaned vector digests were then treated with 2.5 units of calf intestinal alkaline phosphatase (Boehringer Mannheim) in a volume of 100 pl using the buffer supplied. The dephosphorylation reactions were carried out at 37'C for 1 20 hour. Following this, the samples were again cleaned by QIAquick columns before ligation to template fragments. Cleaned template and vector digests were ligated overnight at 15 0 C. Typical reactions used 50 ng of vector together with 100 ng of template. A control reaction was WO 01/32861 PCT/AUOO/01329 59 always included consisting of a vector alone ligation with no template present. Ligations were then transformed into competent XL-1 Blue cells (Stratagene) prepared by the method of Chung et al., (1989). For each reaction, 100 pl 5 of competent XL-1 Blue cells were thawed on ice and 5 pl of the ligation reaction was added. Following gentle mixing, the cells were placed at 4'C for 30 minutes. During this step, 880 gl of L-Broth was placed in a 10 ml tube and glucose was added to a concentration of 20 mM. The cells 10 and DNA mix were then added, and the tube was incubated at 37 0 C for 1 hour. The cells were then spun down and the pellet resuspended in 100 pl of L-Broth. The entire 100 pl was then plated onto an L-Agar plate containing the appropriate antibiotic and incubated at 37'C overnight. 15 Subcloning of the cosmids into the trapping vector was deemed successful if the number of colonies on the experimental plate exceeded the number of colonies on the vector-only ligation control plate by 10 fold. For each successful ligation plate, 3 ml of L-Broth plus antibiotic 20 was aliquoted onto the plate, and the cells were scraped off into the solution with a sterile glass spreader. The solution was then transferred to a sterile 10 ml tube and DNA was isolated using a Qiagen Tip-20 column according to manufacturers conditions (Qiagen).
WO 01/32861 PCT/AUOO/01329 60 Subcloned DNA was then transfected into COS-7 cells (ATCC CRL-1651) grown to a confluence of 40 to 60% using the LipofectACE reagent (Life Technologies). Methods used were those given by the manufacturer. Transfected cells 5 were initially grown overnight followed by a 48 hour incubation at 37 0 C in a 5% CO 2 incubator. Following this, culture media was removed and the cells were washed 3 times in cold phosphate buffered saline (PBS) and were then removed from the culture flask using a sterile 10 scraper. RNA was subsequently isolated from the cells using the Trizol reagent (Life Technologies) and methods supplied by the manufacturer. The precipitated RNA was washed and dried and resuspended in 50 pl of RNAse free water. 15 Isolated RNA was reverse transcribed with the Superscript enzyme (Life Technologies) according to standard techniques (Sambrook et al., 1989) combined with those supplied by the manufacturer. A total of 3 pg of RNA was used per reaction, while the reverse transcription was 20 initiated from a trapping vector specific - primer. Following this reaction, 2 units of RNAseH (Promega) was added and the reaction incubated for a further 10 minutes at 55 0 C. Products were then amplified by standard PCR reactions (Weber and May, 1989; Sambrook et al., 1989) 25 using trapping vector specific primers that spanned the WO 01/32861 PCT/AUOO/01329 61 cloning site of the vector. Typical cycling reactions were 6 cycles of 94'C for 1 minute, 60'C for 1 minute, 72 0 C for 5 minutes followed by a 10 minute incubation at 72'C. To prevent the amplification of false-positive (vector-only) 5 products in subsequent PCR reactions, 25 units of BstXI were added to the PCR reaction and incubated overnight at 55'C. The following day, a further 5 units of BstXI was added to each tube and incubated for a further 2 hours at 55 0 C. In the secondary amplification stage, nested vector 10 specific primers were used in PCR reactions consisting of 30 cycles of 94'C for 1 minute, 60 0 C for 1 minute, 72 0 C for 3 minutes, followed by a 10 minute incubation at 72 0 C. Primers used for this amplification step contained dUMP residues at their 5' ends such that PCR products generated 15 could be sub-cloned into the pAMP1O vector using Uracil DNA glycosylase. The methods for this procedure were described in the CloneAMP pAMP10 kit (Life Technologies). Products that had been ligated into the pAMP10 vector were transformed into competent XL-1 Blue cells using 20 standard techniques (Chung et al., 1989; Sambrook et al., 1989). A total of 24-135 random clones from each transformation were analysed by colony PCR using pAMP1O specific primers to determine the size of the products trapped. Clones containing trapped products, based on WO 01/32861 PCT/AUOO/01329 62 comparison with a "vector-only" control product, were grouped according to size, and representative clones from each group were selected for further analysis. DNA from potential trapped exons was sequenced using Dye Primer 5 cycle sequencing with AmpliTaq DNA polymerase, FS (Perkin Elmer) according to manufacturers instructions. As the amplified trapped products were cloned non-directionally into pAMP10, the DNA sequence obtained was aligned in the sense orientation, and the vector sequences were removed. 10 This left sequence corresponding to the trapped product alone. Exons that had been trapped were mapped back to their clone of origin within the physical map using Southern blotting techniques (Sambrook et al., 1989). The BLASTN 15 program (Altschul et al., 1997) was used to search for nucleotide sequence homology between the trapped products and sequences in the GenBank non-redundant and EST databases (http: / /www.ncbi.nlm.nih. gov/index.html) . In the majority of cases, the critical P value indicating 20 significant homology was taken to be 10~5 (1.Ge-5). To reveal any open reading frames within the sequences of the trapped products, the Applied Biosystems SeqEd (version 1.0.3) software was used. Homologous IMAGE Consortium cDNA clones were purchased from Genome Systems and were 25 sequenced. In many instances trapped exons that lay in WO 01/32861 PCT/AUOO/01329 63 close proximity within the physical map contig were found to belong to the same cDNA clone. These longer stretches of sequence were then compared to known genes by nucleotide and amino acid sequence comparisons using the 5 above procedures. Any sequences that are expressed in the breast are considered to be candidate tumour suppressor genes. Those genes whose function could implicate it in the tumourigenic process, as predicted from homology searches with known proteins, were treated as the most 10 likely candidates. Evidence that a particular candidate is the responsible gene comes from the identification of defective alleles of the gene in affected individuals. EXAMPLE 5: Identification of the TSG16 sequence 15 A detailed map of transcripts was developed for the 1.1 Mb physical map contig established at 16q24.3 (Whitmore et al., 1998). The integrated physical and transcription map for a part of this region is shown in Figure 2. The trapped exons 561-4, 561-13, 561-72 and 561 20 114 mapped in close proximity suggesting that they may all belong to the same gene. From homology searches with trapped exon sequences, 561-72 identified the human heart cDNA clone R41095 while 561-114 identified a human T-cell cDNA clone AA312702. Both of these cDNA clones overlap 25 with the mouse IMAGE cDNA clone 585695, which suggests WO 01/32861 PCT/AUOO/01329 64 that the trapped exons probably belong to the same transcript. Further homology searches of the EST database with each of these clones identified additional -overlapping human cDNAs such that a contig of clones could 5 be established. Sequencing of the entire insert for a number of these clones provided a large contig of sequence, which confirmed that 561-72 and 561-114 did belong to the same gene. Surprisingly this contig of sequences could be extended to include the trapped exon 10 ET24.35. This exon maps a distance of up to 100 Kb away from 561-114, which suggests the presence of a large intron in the gene containing these trapped products. BLAST analysis of 561-4 and 561-13 sequences failed to identify any human cDNA clones but both exons 15 identified the same overlapping mouse IMAGE cDNA clones 1123879, 1197009 and 935574. Additional BLAST homology searches with the mouse mammary gland cDNA clone 935574 identified an overlapping IMAGE human cDNA clone 1753804. This clone was purchased from Genome Systems and the 20 insert was sequenced which established that this clone contained the trapped exon 561-4 but did not extend to include the 561-13 sequence. 25 WO 01/32861 PCT/AUOO/01329 65 Northern Analysis To determine the size of the gene to which the trapped exons clustered at this point of the physical map belonged to, Northern blots obtained from Clontech were 5 probed in sequential hybridisations with the insert from a cDNA clone which contained both 561-72, 561-114 and ET24.35, and a clone which contained 561-4. Hybridisations were conducted in 10 ml of ExpressHyb solution (Clontech) overnight at 65 0 C. Filters were washed according to 10 manufacturers conditions. Figure 3 shows the result of these hybridisations. Both probes detected a band of approximately 9.5 Kb in size with the most prominent expression in fetal heart, brain and pancreas. Weaker expression was detected in all other tissues including 15 placenta, lung, liver, skeletal muscle and kidney. This provided evidence for the first time that the trapped exons ET24.35, 561-4, 561-13, 561-72 and 561-114 may belong to the same gene. To identify additional sequence for this gene, 5' and 3' 20 RACE were initiated along with the sequencing of genomic DNA from the PAC74015 clone, which would assist in genomic characterisation of the gene. 5' RACE To extend the sequence 5' to that obtained so far, 25 the 5' RACE procedure was used. Methods adopted for these WO 01/32861 PCT/AUOO/01329 66 experiments were those recommended by the supplier of the 5' RACE kit (Life Technologies) . One hundred ng of PolyA* mRNA from normal mammary gland (Clontech) was reverse transcribed with a gene specific primer located towards 5 the 5' end of the sequence obtained so far, followed by nested PCR reactions to amplify gene specific extension fragments. Products were cloned into the pGEM-T vector (Promega) based on manufacturers protocols and were subsequently sequenced using vector specific primers. 10 Results of these experiments showed that the sequence already obtained from cDNA clone sequencing and dbEST database information could not be extended further indicating that the 5' end of the gene had been identified and that there must be additional 3' sequence to obtain. 15 Genomic Sequence Analysis PAC74015 was sequenced to assist in the genomic structure characterisation of the transcript. This sequence would also aid in the prediction of 3' exons not 20 yet identified. DNA was prepared from this PAC- and was sheared by nebulisation (10psi for 45 seconds). Sheared DNA was then blunt ended using standard methodologies (Sambrook et al., 1989) and run on an agarose gel in order to isolate DNA in the 2-4 Kb size range. These fragments 25 were cleaned from the agarose using QIAquick columns WO 01/32861 PCT/AUOO/01329 67 (Qiagen), ligated into puc18 and used to transform competent XL-1 Blue E. coli cells. DNA was isolated from transformed clones and was sequenced using vector specific primers on an AB1377 sequencer. Analysis of genomic 5 sequence was performed using PHRED, PHRAP and GAP4 software on a SUN workstation. The genomic sequence was masked for repeats and analysed using the BLASTN algorithm or was analysed for predicted gene structure using the GENSCAN program. 10 The GENSCAN program was successful in predicting a number of additional 3' exons to those already identified by exon trapping and cDNA sequencing, with one of the internal exons predicted to be 6.5 Kb in size. Confirmation that the predicted exons were indeed part of 15 the gene was determined by RT-PCR experiments with primers specific for the predicted coding sequence. Figure 3 indicates the primers used for the analysis and Table 1 shows the primer sequences used and their location in the gene. Primer sequences for RT-PCR analysis are also set 20 forth in SEQ ID Numbers: 13-43. In each of the 17 RT-PCR amplifications, products were cloned and sequenced in order to verify that the coding sequence was correct. One of the RT-PCR products specific for the large 6.5 Kb internal exon was also used as a probe on Northern blots 25 to verify it represented a transcribed part of the gene.
WO 01/32861 PCT/AUOO/01329 68 Figure 3 indicates that this probe identified the same 9.5 Kb RNA fragment as detected previously indicating this large exon does belong to the coding sequence of the gene. BLAST analysis of the sequence of the GENSCAN 5 predicted 3' terminal exon against the dbEST database failed to identify any human cDNA clones. However, mouse clones homologous to this terminal exon and further 3' genomic sequence of PAC74015 were identified. To confirm that this sequence was also part of the gene in humans, 3' 10 RACE was initiated. 3' RACE A procedure adapted from Gecz et al. (1997) was used with primers specific for the predicted 3' terminal exon 15 of the gene. First strand cDNA was synthesised from polyA* mRNA from normal mammary gland using an anchored oligo-dT primer, 5' -CCATCCTAATACGACTCACTATAGGGCTCGAGCGGC (T) 18 VN-3', in which V was any of the four A, C, G, and T nucleotides and N was C, G, or A. Second strand cDNA was synthesised 20 in a linear PCR using a biotinylated gene specific primer. Gene specific cDNAs were then captured on streptavidin coated magnetic beads (Dynal) using manufacturers methods, and the 3' end was subsequently amplified using nested primers, followed by an additional nested PCR. Resulting 25 products were then cloned into the pGEM-T vector and WO 01/32861 PCT/AUOO/01329 69 sequenced. Results from these experiments confirmed that the predicted 3' terminal exon could be extended further 3' in line with the sequence identified in overlapping mouse cDNA clones. 5 In combination, these experiments have established that the gene, termed TSG16, is 9,063 base pairs in length (SEQ ID NO:1) and is composed of 13 exons that span approximately 150 Kb of genomic DNA. Table 2 shows the genomic structure of the gene indicating the size of 10 introns and exons. Analysis of exons 1 to 13 indicate an open reading frame of 7,989 nucleotides with a start codon in exon 3 at base 221 and a stop codon in exon 13 at base 8,210. This defines a protein of 2,663 amino acids (SEQ ID NO:2). Partial genomic DNA sequences indicating 15 exon/intron junctions for TSG16 are set forth in Figure 4A-G and SEQ ID Numbers: 3-12. EXAMPLE 6: Characteristics of TSG16 Nucleotide Sequence 20 A large number of cDNA clones are present in dbEST which belong to the TSG16 gene. An observation of the tissues these cDNA clones were derived from indicates that the gene is also expressed in Germinal B-cells, melanocytes, infant brain, fetal heart, Jurkat T-Cells, 25 gall bladder, ovary, muscle, head-neck, colon, uterus, WO 01/32861 PCT/AUOO/01329 70 testis and stomach. These tissues are in addition to those shown to express TSG16 from Northern analysis and RACE procedures. The TSG16 nucleotide sequence also detects a number of mouse cDNA clones. The homology is as high as 5 90%, which suggests that this gene is highly conserved. - TSG16 also appears to exist as a truncated pseudogene on the X chromosome. Homology searches with the non redundant database identified 99% homology with the last 2,026 bases of TSG16 (part of exon 9 through to exon 13) 10 to sequence from PAC203P18, which maps to chromosome Xq27 . 1-Xq27 .3. Amino Acid Sequence The amino acid sequence of TSG16 was used for in 15 silico analysis to identify homologous proteins in order to establish the function of the gene product. Initially the BLASTP program was used to search for homologous sequences in the GenBank non-redundant protein database (http://www.ncbi.nlm.nih.gov/index.html). The search 20 identified a number of proteins that exhibited homology to TSG16 in a region containing an ankyrin repeat motif (ANK). Analyses of the TSG16 protein using the PfScan program (http: / /www. isrec .isb 25 sib.ch/software/PFSCANform.html) confirmed the presence WO 01/32861 PCT/AUOO/01329 71 of an ankyrin repeat domain with a PfScan score of 37.109. In comparison, Ankyrin 1 contains 23 ANK repeats and exhibits a PfScan score of 249.73, BARD1 with 3 ANK repeats exhibits a score of 36.2 and IK3-< with 4 ANK 5 r epeats a score of 41.9. Ankyrin repeats have been identified in over 400 proteins ranging from transcription factors to toxins. The main function of ANK domains is to provide a site for protein-protein interactions. The ANK repeat unit contains 33 amino acids with a conserved 10 consensus of XGXTPLHXAAXXGHXXXV/AXXLLXXGAXXN/DXXXX (where X can be any amino acid). The number of repeats within a protein can vary widely from 3 in the rat Vip to 23 in the human Ankyrin protein. TSG16 contains 3 ANK repeat units. X-ray structural analysis of the human p53 binding protein 15 (53BP2), IKB-X, and the yeast protein Swi6 ankyrin domains indicate that the ANK domain is an L-shaped structure, which consists of P-hairpins and a-helices. The a-helices create a pit which is surrounded by P-helical protrusions thus providing a docking site for interacting proteins. 20 The highest BLAST homology scores for TSG16 were obtained with the ANK domains present in the proteins BARD1 and IKB-R. In addition to the conserved amino acids, which define the ANK repeat, homology extended to those residues, which are often non-conserved within the ANK WO 01/32861 PCT/AUOO/01329 72 motif. This suggests that TSG16, BARD1 and/or IKB-R may have common protein interaction partners. PfScan analysis also identified ten putative bipartite nuclear localisation signals (BNLS) within 5 TSG16. BNLS are characterised as stretches of sequence that consist of two leading basic amino acids - either arginine or lysine, separated from another three basic residues by ten amino acids. These sequences are recognised by nuclear importing machinery and allow the 10 protein to be localised within the nucleus. The presence of a large number of BNLS within proteins indicates that there does not necessarily need to be one dominating BNLS. It has been suggested that nuclear localisation can be due to the cumulative effect of all BNLS present rather than 15 the effect of one signal. Using the Pestfind program six potential PEST sequences were identified in the TSG16 protein. PEST sequences are regions found in proteins that have short half-lives. These include p53, ornithine decarboxylase, c 20 myc, HMG-CoA reductase and the aX and P caseins. PEST sequences are characterised by a high content of the amino acids proline (P), glutamic acid (E), serine (S), and threonine (T). PEST sequences have been found to 'label' proteins for proteolytic degradation most commonly via WO 01/32861 PCT/AUOO/01329 73 ubiquitination and subsequent degradation by the 26S proteosome pathway (Rechsteiner and Rogers, 1996). Other degradation methods have been postulated with calpain directed proteolysis of c-Fos, and the cAMP dependent 5 protein kinase. Five of these PEST sequences in TSG16 are located at the carboxy terminus, a common feature of proteins containing PEST sequences. The Pestfind scores for the TSG16 PEST motifs range from +5.97 to +20.42. In 10 comparison, two proteins known to contain PEST sequences, IKB-a and Bcl-3 exhibit Pestfind scores of +5.13 and +16.66 respectively. Taken collectively the in silicon analyses indicates that TSG16 is a nuclear protein that contains an ANK 15 domain which may mediate interactions with proteins that also interact with the proteins BARD1 and/or IKB-R. BARDI has been shown to interact with the tumour suppressor protein BRCA1 via its RING finger motif. The ankyrin domain of BARD1 is responsible for the interaction 20 with CstF-50, a member of the Cleavage stimulation factor complex. This complex, along with RNA polymerase II, has been shown to be involved in polyadenylation of mRNA precursors whereby the CstF complex specifies the site of processing (Takagaki et al., 1989). BARD1 and BRCA1 also WO 01/32861 PCT/AUOO/01329 74 interact with RNA polymerase II and the BARD1/CstF-50 interaction has been shown to inhibit polyadenylation in vitro (Kleiman and Manley, 1999). It has been proposed that BARD1 as part of the polyadenylation apparatus, 5 senses sites of DNA damage and repair, and the inhibitory interaction with Cst ensures that nascent RNAs are not erroneously polyadenylated at such sites until the DNA has been repaired. IKB-R is involved in modulating the function of the 10 transcription factor NF-KB. NF-KB transcription factors include a collection of proteins conserved from humans to Drosophila (reviewed in Gilmore, 1999). These transcription factors are notably absent in yeast and C. elegans, probably as a result of the primary function of 15 these factors, which is to control a variety of physiological aspects of immune responses, inflammation, and growth and development. The NF-KB proteins are related through a highly conserved DNA-binding/dimerisation domain called the Rel homology (RH) domain. NF-KB transcription 20 factors bind to 10 base pair DNA sites (KB sites) as dimers. The activity of NF-KB is tightly regulated by interaction with inhibitory IKB proteins. There are several IKB proteins, each of which contains six to seven ANK repeats. However, each IKB protein has different affinities WO 01/32861 PCT/AUOO/01329 75 for individual NF-KB complexes and each are expressed in a tissue specific manner. The binding of IKB-x to NF-KB blocks the ability of NF-KB to enter the nucleus and bind to DNA. From structural studies it is clear that IKBU 5 binding masks the nuclear localization sequence of NF-KB and also interferes with sequences important for DNA binding (Chen and Ghosh, 1999). Therefore, in most cells, NF-KB is present as a latent and inactive IKB-bound complex in the cytoplasm. When a cell receives an extracellular 10 signal, NF-KB rapidly enters the nucleus and activates gene expression. Virtually all signals that lead to activation of NF-KB converge on a complex that contains a serine specific IKB kinase (IKK). Activation of IKK leads to the phosphorylation of two specific serine residues near the N 15 terminus of IKB-a, which targets IKB-a for ubiquitination and degradation by the proteasome. The unmasked NF-KB can then enter the nucleus to activate target gene expression. Evidence linking deregulated NF-KB activity to oncogenesis in mammalian systems has been - observed 20 recently (reviewed in Gilmore et al., 1999). In addition, alterations affecting the expression or function of the IKB family members Bcl-3, IKB-a and IKB-E have also been observed in several cancers. Together, these studies have WO 01/32861 PCT/AUOO/01329 76 identified tumour cells that display constitutively high levels of nuclear NF-KB activity due to hyperactivation of the NF-KB signaling pathway or to inactivating mutations in the regulatory IKB subunits (reviewed in Rayet and Gelinas, 5 1999). The IKB-R protein was originally cloned by differential expression from a human lung epithelial cell line and has been shown to inhibit the DNA binding ability of an NF-KB complex present in nuclear extracts prepared 10 from interleukin-1 activated HeLa cells (Ray et al., 1995). It is therefore possible that this member of the IKB family may play an important role in the regulation of NF KB function in epithelial cells. While not wishing to be bound by theory, the high 15 homology of TSG16 ANK repeats to the ANK repeats of BARD1 and IKB-R suggests that TSG16 may be a key protein in either or both of the pathways to which these important proteins belong, particularly in epithelial cells. Based on past studies it is possible that TSG16 may in actual 20 fact form a link connecting the BARD1/BRCA1 and NF-KB/IKB pathways.
WO 01/32861 PCT/AUOO/01329 77 Alternative Splicing and Overlapping Transcripts Figure 5 illustrates that the 5' end of TSG16 appears to be very complex. Two clusters of cDNA clones have 3' 5 origins in intron 4 of TSG16. The first cluster consists of 20 human cDNA clones which have the same orientation as TSG16. These clones do not appear to have been primed from polyadenosine sequences present in this intron. Sequence analysis of a representative clone from this group (IMAGE 10 clone 291690) showed the presence of an open reading frame but no methionine start codon indicating more 5' sequence needs to be identified. Northern analysis of multiple tissue blots (Clontech) identified a single RNA species of approximately 3 Kb expressed in adult heart, brain, 15 placenta, lung, liver, skeletal muscle, kidney and pancreas. Previously, when TSG16 coding sequence located 5' to intron 4 was used for Northern analysis, the 9.5 Kb TSG16 specific band was identified, together with an additional band of approximately 3 Kb (Figure 3). The 20 common 3 Kb RNA identified from both hybridisations suggests these clones also contain TSG16 specific sequence in their 5' region and therefore may represent an alternatively spliced variant of TSG16. The partial nucleotide and amino acid sequences of this potential WO 01/32861 PCT/AUOO/01329 78 alternative TSG16 isoform are listed as SEQ ID NO: 125 and SEQ ID NO: 126 respectively. The second cluster of cDNAs originating from intron 4 comprises an overlapping set of 6 clones which correspond 5 to the UniGene cluster Hs.26975. These clones again do not appear to originate from polyadenosine sequences present in this intron. The orientation of the clones is opposite to that of TSG16 so they most likely do not represent an alternatively spliced variant of TSG16. The partial 10 nucleotide and amino acid sequences of this overlapping gene are listed as SEQ ID NO: 127 and SEQ ID NO: 128 respectively. A group of 59 cDNA clones corresponding to the UniGene cluster Hs.10238 originate from intron 7 of the 15 TSG16 gene and are transcribed in the same orientation. RT-PCR experiments showed that these transcripts contained exons 1 to 5 of the TSG16 gene spliced with an additional exon present in intron 7. This additional exon contained a stop codon and polyadenylation signal. Additional versions 20 of this transcript were identified which - utilized alternative exons 5E and 5E+ (Figure 5) present in intron 4 of TSG16. These alternative spliced forms of TSG16 are predicted to code for proteins varying in length from 162 to 250 amino acids. The nucleotide and amino acid 25 sequences of these alternatively spliced variants of TSG16 WO 01/32861 PCT/AUOO/01329 79 are listed as SEQ ID Numbers:129-131 and SEQ ID Numbers: 132-134 respectively. EXAMPLE 7: Analysis of tumours and cell lines for TSG16 5 mutations The TSG16 gene was screened by SSCP analysis in DNA isolated from both series of tumour samples. Due to the size of the TSG16 gene, mutation analysis from samples in series 2 was limited to tumours that displayed loss of the 10 whole long arm of chromosome 16 due to larger amounts of DNA being available. In total 55 primary breast tumours with 16q LOH were examined for mutations. A number of cell lines were also screened for mutations. These included 22 breast cancer cell lines, 2 15 prostate cancer cell lines and three normal breast epithelial cell lines. All cell lines were purchased from ATCC, grown according to manufacturers conditions, and DNA isolated from cultured cells using standard protocols (Wyman and White, 1980; Sambrook et al., 1989). 20 TSG16 exons were amplified by PCR using - flanking intronic primers, which were labele< at their 5' ends with HEX. Exon 9 was an exception due to its large size. Instead, this exon was split into 30 overlapping PCR amplimers which were individually analysed for mutations. 25 Table 3 lists the sequence of each primer used and the WO 01/32861 PCT/AUOO/01329 80 expected PCR amplimer size for each amplification. Typical PCR reactions were performed in a volume of 10 pl using 30 ng of template DNA with final MgCl 2 concentrations of 1.5 mM. Exceptions were exon 9-15 and 9-16 which used 1 mM 5 MgCl 2 and exons 9-18 and 9-28 which used 0.5 mM MgCl 2 Reactions were performed in 96 well plates and cycling conditions were an initial denaturation step at 94 0 C for 3 minutes followed by 35 cycles of 94 0 C for 30 seconds, 60'C for 11/2 minutes and 72 0 C for 1112 minutes. A final extension 10 step of 72*C for 10 minutes followed. Twenty pl of loading dye comprising 50% (v/v) formamide, 12.5 mM EDTA and 0.02% (w/v) bromophenol blue were added to completed reactions which were subsequently run on 4% polyacrylamide gels and analysed on the GelScan 2000 system (Corbettt Research, 15 Australia) according to manufacturers specifications. Table 4 shows a list of the polymorphisms detected by the SSCP analysis. Amino acid changes were detected in exon 9 for one breast cancer cell line and one prostate cancer cell line. These changes were not detected in 56 20 normal Caucasian individuals. A total of seven rare variants were identified in which no more than 2 tumour samples had the bandshift. However, where the bandshift occurred in a primary tumour sample, the matching normal DNA also showed the same 25 change. In six of the seven variants, the SSCP change was WO 01/32861 PCT/AUOO/01329 81 not detected in the 56 normal samples either. Sequencing of five of these variants showed 3 gave rise to amino acid changes while the remaining two were non-sense changes. All five variants occurred in exon 9. Due to the presence 5 of the changes in both the tumour and matching normal sample, it suggests either a very rare polymorphism or the possibility that the individuals are carrying germ-line mutations in the TSG16 gene. Frequent nucleotide polymorphisms were also seen in 10 exon 9. Seven separate amplimers from this exon showed bandshifts in tumour and matching normal DNA in 5 or more samples. To date, three of these have been sequenced and shown to give rise to amino acid changes. In two cases, the SSCP bandshifts were identified in a number of normal 15 population samples indicating common polymorphisms not related to disease. Examination of the genomic sequence surrounding TSG16 shows that the 5' end including exon 1 is extremely G-C rich suggesting the presence of a CpG island. While not 20 wishing to be bound by theory, this raises the possibility that epigenetic mechanisms to inactivate TSG16 function may exist. Abnormal methylation at this site may result in a down-regulation of TSG16 transcription of the remaining copy of the gene. Recent studies have shown that this 25 mechanism has been responsible for the inactivation of WO 01/32861 PCT/AUOO/01329 82 other tumour suppressor genes such as RB1 (Ohtani-Fujita et al., 1997), VHL (Prowse et al., 1997), MLH1 (Herman et al., 1998) and BRCA1 (Esteller et al., 2000). Methods to detect the level of expression of TSG16 in cancer samples 5 compared with normal controls will need to be done. This will involve isolation of RNA from cancer cell lines and tumour/matching normal specimens along with appropriate cell line controls and normal mammary gland tissue. RNA is reverse transcribed using standard techniques (Sambrook et 10 al., 1989) and cDNA templates generated are normalised with respect to each other following PCR amplification with a house-keeping gene such as Esterase D or HPRT. Primers specific for the TSG16 gene are then used in PCR reactions to determine the presence of TSG16 RNA from the 15 original source. With the incorporation of SYBR green into the proceeding PCR reaction (standard kits include those from Perkin Elmer), the level of template amplification can be measured in real-time. The number of cycles taken to reach the linear amplification range is a direct 20 reflection of the amount of starting RNA present in each sample. Other methods to detect TSG16 expression levels may be used. These include the generation of polyclonal or monoclonal antibodies, which are able to detect relative amounts of both normal and mutant forms of TSG16 using WO 01/32861 PCT/AUOO/01329 83 various immunoassays such as ELISA assays (See Example 9 and 10). To determine if TSG16 is involved in immune/autoimmune/inflanmatory disorders, similar SSCP 5 studies can be undertaken in affected individuals as well as examining the expression levels of TSG16 in these patients. EXAMPLE 8: Analysis of the TSG16 gene 10 The following methods are used to determine the structure and function of TSG16. Biological studies Mammalian expression vectors containing TSG16 cDNA can be transfected into breast, prostate or other 15 carcinoma cell lines that have lesions in the gene. Phenotypic reversion in cultures (eg cell morphology, growth of transformants in soft-agar, growth rate) and in animals (eg tumourigenicity in nude mice) is examined. These studies can utilise wild-type or mutant forms of 20 TSG16. Deletion and missense mutants of TSG16 can be constructed by in vitro mutagenesis.. Molecular biological studies The ability of TSG16 protein to bind known and 25 unknown protein has been examined. Due to the presence of WO 01/32861 PCT/AUOO/01329 84 an ANK domain region in TSG16 it is most likely that this gene participates in protein/protein interactions and procedures such as the yeast two-hybrid system are used to discover and identify any functional partners. The 5 principle behind the yeast two-hybrid procedure is that many eukaryotic transcriptional activators, including those in yeast, consist of two discrete modular domains. The first is a DNA-binding domain that binds to a specific promoter sequence and the second is an activation domain 10 that directs the RNA polymerase II complex to transcribe the gene downstream of the DNA binding site. Both domains are required for transcriptional activation as neither domain can activate transcription on its own. In the yeast two-hybrid procedure, the gene of interest or parts 15 thereof (BAIT), is cloned in such a way that it is expressed as a fusion to a peptide that has a DNA binding domain. A second gene, or number of genes, such as those from a cDNA library (TARGET), is cloned so that it is expressed as a fusion to an activation domain. Interaction 20 of the protein of interest with its binding partner brings the DNA-binding peptide together, with the activation domain and initiates transcription of the reporter genes. The first reporter gene will select for yeast cells that contain interacting proteins (this reporter is usually a 25 nutritional gene required for growth on selective media) .
WO 01/32861 PCT/AUOO/01329 85 The second reporter is used for confirmation and while being expressed in response to interacting proteins it is usually not required for growth. For TSG16 yeast two-hybrid analysis the displayGREEN 5 BASIC" Two-Hybrid System kit (Display Systems Biotech) was used. All standard yeast manipulation procedures were conducted as recommended by the kit supplier. In this approach, the ANK domain of TSG16 was cloned in-frame into the supplied BAIT vector containing the LexA protein as a 10 DNA binding domain. The TARGET vector contained inserts from a human breast cDNA library (Invitrogen). Following yeast colony selection procedures, a number of positive clones were identified and plasmid DNA from each of the interacting TARGET clones was obtained. 15 From sequence analysis of these clones, members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family of proteins were identified to be interacting with the ANK domain of TSG16. The PIAS family of proteins has been shown to specifically 20 inhibit STAT protein signaling (Liu et al., 1998). STAT proteins are a family of latent cytoplasmic transcription factors that become activated, by tyrosine phosphorylation, following the binding of cytokines to their cell surface receptors. After receptor activation, 25 phosphorylated STATs dimerise, translocate to the nucleus WO 01/32861 PCT/AUOO/01329 86 and bind specific DNA elements in the promoters of responsive genes to activate transcription. STAT1 for example, plays an important role in mediating interferon gamma (IFN-y), interleukin-6 (IL-6) type cytokine and 5 epidermal growth factor (EGF)-dependant biological responses. IFN-y is a cytokine that plays a fundamental role in several aspects of the immune response (Boehm et al., 1997). Other properties include stimulation of 10 bactericidal activity of phagocytes, stimulation of antigen presentation through class I and II major histocompatability complex molecules, as well as affects on cell proliferation and apoptosis. At the cellular level IFN-y is able to mediate activation of an antiviral state 15 and cause cell growth arrest at the G, phase of the cell cycle. The IFN-y response has recently been postulated to be part of an endogenous tumour surveillance system (Kaplan et al., 1998). To add support to this claim, additional 20 experiments have shown that STAT1 interacts with the tumour suppressor BRCA1. This leads to differential activation of transcription of a subset of IFN-y target genes leading to growth inhibition by this cytokine, with one of these genes being the cyclin-dependent kinase WO 01/32861 PCT/AUOO/01329 87 inhibitor, p21WAF1 (Ouchi et al., 2000). It has been further shown that p21WAF1 activation is impaired in breast cancer cells lacking a functional BRCA1 protein. Thus it is possible that the disturbance of the p2lWAFl 5 induction provides an early growth advantage to nascent tumour cells, which allows them to bypass the initial antitumour actions of IFN-7. EGF and IL-6 type cytokines also mediate their actions through the STAT pathway. While EGF is a mitogen 10 for many cells, growth of some cultured cell lines, containing high numbers of EGF receptors, are inhibited by EGF. This growth inhibition has been shown for A431 cells to be mediated by the activation of STATI (via specific receptor kinase activity) and NF-KB (via IKB degradation), 15 which drive p21WAF1 gene expression (Ohtsubo et al., 2000). The IL-6 type cytokines signal through the common receptor subunit gp130 and are involved in the regulation of many processes including gene expression, cell proliferation and differentiation. IL-6 has also been 20 shown to stimulate inflammatory responses during wound healing. In fetal skin, this process is characterized by minimal inflammation and scarless repair and it has been suggested that diminished inflammation (due to diminished production of inflammatory cytokines such as IL-6) may WO 01/32861 PCT/AUOO/01329 88 provide a permissive environment for scarless wound healing (Liechty et al., 2000). Two members of the PIAS family, PIAS1 and PIAS3, have been shown to be negative regulators of STAT signaling. 5 PIAS1 binds to the STATI dimer, which has been proposed to mask the DNA-binding activity of STAT1 (Liao et al., 2000). Recent studies have suggested that the PIAS family of proteins may function to regulate other transcriptional responses (Moilanen et al., 1999). Therefore the 10 recruitment of PIAS1 to different transcription factors only after ligand stimulation may allow the targeting of PIAS1 to a specific transcriptional response induced by the corresponding signal. The binding of PIASI to the ANK domain of TSG16 may represent one of these novel 15 transcriptional responses, and like STAT1, may be linked to immunological responses, including those associated with tumour suppression. Given the interaction of more than one member of the PIAS family with the TSG16 ANK domain, TSG16 may play a role in all PIAS associated 20 functions. The nature of the TSG16 interacting genes and proteins can also be studied such that these partners can also be targets for drug discovery.
WO 01/32861 PCT/AUOO/01329 89 Structural studies TSG16 recombinant proteins can be produced in bacterial, yeast, insect and/or mammalian cells and used in crystallographical and NMR studies. Together with 5 molecular modeling of the protein, structure-driven drug design can be facilitated. EXAMPLE 9: Generation of polyclonal antibodies against TSG16 The knowledge of the nucleotide and amino acid 10 sequence of TSG16 allows for the production of antibodies, which selectively bind to TSG16 protein or fragments thereof. Following the identification of mutations in the gene, antibodies can also be made to selectively bind and distinguish mutant from normal protein. Antibodies 15 specific for mutagenised epitopes are especially useful in cell culture assays to screen for malignant cells at different stages of malignant development. These antibodies may also be used to screen malignant cells, which have been treated with pharmaceutical agents to 20 evaluate the therapeutic potential of the agent. To prepare polyclonal antibodies, two short peptides were designed to the 5' end of TSG16 (Peptide 1: CPKAPQQEELPLSS; Peptide 2: HQQGGEAAAAVRR) in regions of least homology to the mouse orthologue. Both peptides were 25 conjugated to biotin using Sulfo-NHS-LC Biotin and the WO 01/32861 PCT/AUOO/01329 90 PIERCE" kit (PIERCE) using manufacturers protocols. The biotin to peptide concentration ratios for the conjugations were 0.57 mM biotin:1 mM peptide 1 and 0.65mM biotin:1 mM peptide 2. Both biotinylated peptides were 5 subsequently complexed with avidin in solution at ratios of 1 mg of avidin per 26 nM biotin conjugated peptide. For each peptide complex, 2 rabbits were immunized with 4 doses of antigen (200 pg per dose) in intervals of three weeks between doses. The initial dose was mixed with 10 Freund's Complete adjuvant while subsequent doses were combined with Freund's Immuno-adjuvant. After completion of the immunization, rabbits were test bled and reactivity of sera was assayed by dot blot with serial dilutions of the two peptides. All four rabbits showed significant 15 reactivity compared with pre-immune sera. The rabbits were then sacrificed and the blood collected and immune sera was separated for further experiments. EXAMPLE 10: Generation of monoclonal antibodies specific 20 for TSG16 Monoclonal antibodies can be prepared for TSG16 in the following manner. Immunogen comprising intact TSG16 protein or TSG16 peptides (wild type or mutant) is injected in Freund's adjuvant into mice with each mouse 25 receiving four injections of 10 to 100 ug of immunogen.
WO 01/32861 PCT/AUOO/01329 91 After the fourth injection blood samples taken from the mice are examined for the presence of antibody to the immunogen. Immune mice are sacrificed, their spleens removed and single cell suspensions are prepared (Harlow 5 and Lane, 1988). The spleen cells serve as a source of lymphocytes, which are then fused with a permanently growing myeloma partner cell (Kohler and Milstein, 1975). Cells are plated at a density 'of 2X10 5 cells/well in 96 well plates and individual wells are examined for growth. 10 These wells are then tested for the presence of TSG16 specific antibodies by ELISA or RIA using wild type or mutant TSG16 target protein. Cells in positive wells are expanded and subcloned to establish and confirm monoclonality. Clones with the desired specificity are 15 expanded and grown as ascites in mice followed by purification using affinity chromatography using Protein A Sepharose, ion-exchange chromatography or variations and combinations of these techniques. 20 Industrial Applicability The tumour suppressor gene, TSG16, is implicated not only in breast cancer, but in the tumourigenic process in general. In addition, this gene is implicated in other disease states due to the presence of specific functional 25 domains within its encoded protein. The novel DNA WO 01/32861 PCT/AUOO/01329 92 molecules of the present invention are therefore useful in methods for the early detection of disease susceptible individuals as well as in diagnostic, prognostic and therapeutic procedures associated with these disease 5 states.
WO 01/32861 PCT/AUOO/01329 93 References References cited herein are listed on the following pages, and are incorporated herein by this reference. Altschul, SF. et al. (1997). Nucleic Acids Res. 25: 3389 5 3402. Boehm, U. et al. (1997). Annu. Rev. .Immunol. 15: 749-795. Brenner, AJ. and Aldaz CM. (1995). Cancer Res. 55: 2892 2895. Burn, TC. et al. (1995). Gene 161: 183-187. 10 Callen, DF. et al. (1990). Ann. Genet. 33: 219-221. Callen, DF. et al. (1995). Genomics 29: 503-511. Cavenee, WK. et al. (1983). Nature 305: 779-784. Chen, T. et al. (1996). Cancer Res. 56: 5605-5609. Chen, FE. and Ghosh, G. (1999). Oncogene 18: 6845-6852. 15 Chung, CT. et al. (1989). Proc. Natl. Acad. Sci. USA 86: 2172-2175. Cleton-Jansen, A-M. et al (1995). Br. J. Cancer 72: 1241 1244. Cole, SP. et al. (1984). Mol. Cell Biol. 62: 109-120. 20 Cote, RJ. et al. (1983). Proc. Natl. Acad. Sci.- USA 80: 2026-2030. Devilee, P. et al. (1991). Oncogene 6: 1705-1711. Devilee, P. and Cornelisse, CJ. (1994). Biochimica et Biophysica Acta 1198: 113-130. 25 Devilee, P. et al. (1994). Genes Chrom. Cancer 11: 71-78.
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Claims (118)
1. An isolated DNA molecule comprising the nucleotide sequence set forth in SEQ ID NO:1, 5
2. An isolated DNA molecule as claimed in claim 1, comprising the nucleotide sequence set forth in SEQ ID NO:1, or a fragment thereof, which encodes a polypeptide active in suppressing cellular proliferation. 10
3. An isolated DNA molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:1 and which encodes a polypeptide active in suppressing cellular proliferation. 15
4. An isolated DNA molecule as claimed in claim 3 that is at least 85% identical.
5. An isolated DNA molecule as claimed in claim 4 that is at least 95% identical. 20
6. An isolated DNA molecule as claimed in claim 3 wherein sequence identity is determined using the BLASTN algorithm and the BLOSSUM62 default matrix. 25 WO 01/32861 PCT/AUOO/01329 99
7. An isolated DNA molecule that encodes a polypeptide active in suppressing cellular proliferation, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth 5 in SEQ ID NO:1.
8. An isolated DNA molecule as claimed in claim 7 wherein the stringent conditions comprise hybridization at 42 0 C in 750 mM NaCl, 75 mm trisodium citrate, 2% SDS, 50% 10 formamide, 1x Denhart's, 10% (w/v) dextran sulphate and 100 pg/ml denatured salmon sperm DNA.
9. An isolated DNA molecule which encodes a polypeptide having the amino acid sequence set forth in 15 SEQ ID NO:2.
10. An isolated DNA molecule which encodes a polypeptide active in suppressing cellular proliferation, the polypeptide having an amino acid sequence with at 20 least 70% identity to that set forth in SEQ ID NO:.2.
11. An isolated DNA molecule as claimed in claim 10 wherein the amino acid sequence has at least 85% sequence identity. 25 WO 01/32861 PCT/AUOO/01329 100
12. An isolated DNA molecule as claimed in claim 11 wherein the amino acid sequence has at least 95% sequence identity. 5
13. An isolated .DNA molecule as claimed in claim 10 wherein sequence identity is determined using the BLASTP algorithm and the BLOSSUM62 default matrix.
14. An isolated gene comprising the nucleotide 10 sequence set forth in SEQ ID NO:1 and TSG16 control elements.
15. An isolated gene as claimed in claim 14 wherein the TSG16 control elements are those which mediate 15 expression in breast tissue.
16. An expression vector which comprises a DNA molecule as defined in any one of claims 1 to 13 operably linked to suitable control elements. 20
17. Host cells transformed with the expression vector of claim 16.
18. An isolated polypeptide, comprising the amino 25 acid sequence set forth in SEQ ID NO:2. WO 01/32861 PCT/AUOO/01329 101
19. An isolated polypeptide as claimed in claim 19 comprising the amino acid sequence set forth in SEQ ID NO:2, or a fragment thereof, active in suppressing cellular proliferation. 5
20. An isolated polypeptide active in suppressing cellular proliferation and having at least 70% identity with the amino acid sequence set forth in SEQ ID NO:2. 10
21. An isolated polypeptide as claimed in claim 20 with at least 85% sequence identity.
22. An isolated polypeptide as claimed in claim 21 with at least 95% sequence identity. 15
23. An isolated polypeptide as claimed in claim 20 wherein sequence identity is determined using the BLASTP algorithm and the BLOSSUM62 default matrix. 20
24. A method of preparing a polypeptide as defined in any one of claims 18 to 23, comprising the steps of (1) culturing the host cells of claim 17 under conditions effective for production of the polypeptide; 25 and WO 01/32861 PCT/AUOO/01329 102 (2) harvesting the polypeptide.
25. An antibody which is immunologically reactive with a polypeptide as defined in any one of claims 18 to 5 23.
26. An antibody as claimed in claim 25 which is a monoclonal antibody. 10
27. An antibody which is immunologically reactive with TSG16.
28. An antibody as claimed in claim 27 which is a monoclonal antibody. 15
29. An isolated DNA molecule comprising the nucleotide sequence set forth in SEQ ID NO:1, or a fragment thereof, which encodes a polypeptide active in suppressing cellular function. 20
30. An isolated DNA molecule as claimed in claim 29 wherein cellular functions mediated through the STAT pathway are suppressed. 25 WO 01/32861 PCT/AUOO/01329 103
31. An isolated DNA molecule that is at least 70% identical to a DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:1 and which encodes a polypeptide active in suppressing cellularfunction. 5
32. An isolated DNA molecule as claimed in claim 31 wherein cellular functions mediated through the STAT pathway are suppressed. 10
33. An isolated DNA molecule as claimed in claims 31 or 32 that is at least 85% identical.
34. An isolated DNA molecule as claimed in claim 33 15 that is at least 95% identical.
35. An isolated DNA molecule as claimed in claim 31 or 32 wherein sequence identity is determined using the BLASTN algorithm and the BLOSSUM62 default matrix. 20
36. An isolated DNA molecule that encodes a polypeptide active in suppressing cellularfunction, and which hybridizes under stringent conditions with a DNA molecule consisting of the nucleotide sequence set forth 25 in SEQ ID NO:1. WO 01/32861 PCT/AUOO/01329 104
37. An isolated DNA molecule as claimed in claim 36 wherein cellular functions mediated through the STAT pathway are suppressed. 5
38. An isolated DNA molecule as claimed in claims 36 or 37 wherein the stringent conditions comprise hybridization at 42 0 C in 750 mM NaCl, 75 mM trisodium citrate, 2% SDS, 50% formamide, 1X Denhart's, 10% (w/v) 10 dextran sulphate and 100 pg/ml denatured salmon sperm DNA.
39. An isolated DNA molecule which encodes a polypeptide active in suppressing cellularfunction, the 15 polypeptide having an amino acid sequence with at least 70% identity to that set forth in SEQ ID NO:2.
40. An isolated DNA molecule as claimed in claim 39 wherein cellular functions mediated through the STAT 20 pathway are suppressed.
41. An isolated DNA molecule as claimed in claims 39 or 40 wherein the amino acid sequence has at least 85%. 25 sequence identity. WO 01/32861 PCT/AUOO/01329 105
42. An isolated DNA molecule as claimed in claim 41 wherein the amino acid sequence has at least 95% sequence identity. 5
43. An isolated DNA molecule as claimed in claims 39 or 40 wherein sequence identity is determined using the BLASTP algorithm and the BLOSSUM62 default matrix. 10
44. An expression vector which comprises a DNA molecule as defined in any one of claims 29 to 43 operably linked to suitable control elements.
45. Host cells transformed with the expression vector 15 of claim 44.
46. An isolated polypeptide, comprising the amino acid sequence set forth in SEQ ID NO:2, or a fragment thereof, active in suppressing cellular function. 20
47. An isolated polypeptide as claimed in claim 46 wherein cellular functions mediated through the STAT pathway are suppressed. 25
48. An isolated polypeptide active in suppressing WO 01/32861 PCT/AUOO/01329 106 cellular function and having at least 70% identity with the amino acid sequence set forth in SEQ ID NO:2.
49. An isolated polypeptide as claimed in claim 48 5 wherein cellular functions mediated through the STAT pathway are suppressed.
50. An isolated polypeptide as claimed in claims 48 or 49 with at least 85% sequence identity. 10
51. An isolated polypeptide as claimed in claim 50 with at least 95% sequence identity.
52. An isolated polypeptide as claimed in claims 48 15 or 49 wherein sequence identity is determined using the BLASTP algorithm and the BLOSSUM62 default matrix.
53. A method of preparing a polypeptide as defined in any one of claims 46 to 52, comprising the steps of 20 (1) culturing the host cells of claim 45 under conditions effective for production of the polypeptide; and (3) harvesting the polypeptide. 25 WO 01/32861 PCT/AUOO/01329 107
54. An antibody which is immunologically reactive with a polypeptide as defined in any one of claims 46 to 52.
55. An antibody as claimed in claim 54 which is a 5 monoclonal antibody.
56. An antibody which is immunologically reactive with TSG16. 10
57. An antibody as claimed in claim 56 which is a monoclonal antibody.
58. A pharmaceutical composition comprising a polypeptide according to any one of claims 18 to 23 or 15 claims 46 to 52, and a pharmaceutically acceptable carrier.
59. A method of treatment of a disorder associated with decreased expression or activity of TSG16, comprising 20 administering a polypeptide as defined in any one of claims 18 to 23 or claims 46 to 52, or an agonist thereof, to a subject in need of such treatment.
60. A method according to claim 59 wherein the 25 disorder is selected from the group consisting of cancers WO 01/32861 PCT/AUOO/01329 108 such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, 5 uterus, testis, stomach, adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland; immune/autoimmune/inflammatory disorders including 10 acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis 15 ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic 20 gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, 25 polymyositis, psoriasis, Reiter's syndrome, rheumatoid WO 01/32861 PCT/AUOO/01329 109 arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis and Werner syndrome; and complications of wound 5 healing (eg scarring), cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma. 10
61. The use of a polypeptide as defined in any one of claims 18 to 23 or claims 46 to 52, or an agonist thereof, in the manufacture of a medicament for the treatment of a disorder associated with decreased expression or activity of TSG16. 15
62. The use as claimed in claim 61 wherein the disorder is selected from the group consisting of cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the breast, prostate, liver, ovary, head and neck, heart, 20 brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, stomach, adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland; 25 immune/autoimmune/inflammatory disorders including WO 01/32861 PCT/AUOO/01329 110 acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune 5 thyroiditis, autoimmune polyenodocrinopathy-candidiasis ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, 10 erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial 15 inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, 20 uveitis and Werner syndrome; and complications of wound healing (eg scarring), cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma. 25 WO 01/32861 PCT/AUOO/01329 111
63. A method of treating a disorder associated with increased expression or activity of TSG16, comprising administering an antagonist of TSG16 to a subject in need of such treatment. 5
64. A method according to claim 63 wherein the antagonist of TSG16 is an antibody to TSG16.
65. The use of an antagonist of TSG16 in the 10 manufacture of a medicament for the treatment of a disorder associated with increased expression or activity of TSG16.
66. The use as claimed in claim 65 wherein the 15 antagonist of TSG16 is an antibody to TSG16.
67. A method of treating a disorder associated with decreased expression or activity of TSG16, comprising administering an isolated DNA molecule as defined in any 20 one of claims 1 to 13 or claims 29 to 43 to a subject in need of such treatment.
68. A method as claimed in claim 67 wherein an expression vector comprising the isolated DNA molecule 25 operably linked to suitable control elements is WO 01/32861 PCT/AUOO/01329 112 administered.
69. A method as claimed in claim 67 or claim 68 5 wherein the disorder is selected from the group consisting of cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, 10 colon, uterus, testis, stomach, adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin', spleen, thymus and thyroid gland; immune/autoinmune/inflammatory disorders including 15 acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis 20 ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease,. cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic 25 gastritis, glomerulonephritis, Goodpasture's syndrome, WO 01/32861 PCT/AUOO/01329 113 gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, 5 polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis and Werner syndrome; and complications of wound 10 healing (eg scarring), cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma. 15
70. The use of an isolated DNA molecule as defined in any one of claims 1 to 13 or claims 29 to 43 in the manufacture of a medicament for the treatment of a disorder associated with decreased expression or activity of TSG16. 20
71. The use as claimed in claim 70 wherein the DNA molecule is a part of an expression vector which also includes suitable control elements. 25
72. The use as claimed in claim 70 or claim 71 WO 01/32861 PCT/AUOO/01329 114 wherein the disorder is selected from the group consisting of cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the breast, prostate, liver, ovary, head and neck, 5 heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, stomach, adrenal gland, bladder, bone, bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid, penis, salivary glands, skin, spleen, thymus and thyroid gland; 10 immune/autoimmune/inflammatory disorders including acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune 15 thyroiditis, autoimmune polyenodocrinopathy-candidiasis ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, 20 erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial 25 inflammation, osteoarthritis, osteoporosis, pancreatitis, WO 01/32861 PCT/AUOO/01329 115 polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, 5 uveitis and Werner syndrome; and complications of wound healing (eg scarring), cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma. 10
73. A method of treating a disorder associated with increased activity or expression of TSG16, comprising administering an isolated DNA molecule which is the complement of a DNA molecule as defined in any one of 15 claims 1 to 13 or claims 29 to 43 and which encodes a mRNA that hybridizes with the mRNA encoded by TSG16.
74. A method as claimed in claim 73 wherein an expression vector comprising the isolated DNA molecule and 20 suitable control elements is administered.
75. The use of an isolated DNA molecule which is the complement of a DNA molecule as defined in any one of claims 1 to 13 or claims 29 to 43 and which encodes a mRNA 25 that hybridizes with the mRNA encoded by TSG16, in the WO 01/32861 PCT/AUOO/01329 116 manufacture of a medicament for the treatment of a disorder associated with increased activity or expression of TSG16.
76. The use as claimed in claim 75 wherein the DNA 5 molecule is a part of an expression vector which also includes suitable control elements.
77. The use of an isolated DNA molecule as claimed in any one of claims 1 to 13 or claims 29 to 43 for the 10 diagnosis of disorders associated with TSG16, or a predisposition to such disorders.
78. The use as claimed in claim 77 wherein the disorder is selected from the group consisting of cancers 15 such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the breast, prostate, liver, ovary, head and neck, heart, brain, pancreas, lung, skeletal muscle, kidney, colon, uterus, testis, stomach, adrenal gland, bladder, bone, 20 bone marrow, cervix, gall bladder, ganglia, gastrointestinal tract, parathyroid,. penis, salivary glands, skin, spleen, thymus and thyroid gland; immune/autoimmune/inflammatory disorders including acquired immunodeficiency syndrome (AIDS), Addison's 25 disease, adult respiratory distress syndrome, allergies, WO 01/32861 PCT/AUOO/01329 117 ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis ectodermal dystrophy (APECED), bronchitis, cholecystitis, 5 contact dermatitis, Crohn's disease, cystic fibrosis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, 10 gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid 15 arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis and Werner syndrome; and complications of wound healing (eg scarring), cancer, hemodialysis, and 20 extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma.
79. The use of a polypeptide as defined in any one of 25 claims 18 to 23 or claims 46 to 52 in the diagnosis of a WO 01/32861 PCT/AUOO/01329 118 disorder associated with TSG16, or a predisposition to such disorders.
80. The use of an antibody as defined in any one of 5 claims 25 to 28 or claims 54 to 57 in the diagnosis of a disorder associated with abnormal expression of TSG16, or a predisposition to such disorders.
81. A method for the diagnosis of a disorder 10 associated with abnormal expression of TSG16, or a predisposition to such disorders, comprising the steps of: (1) establishing a profile for normal expression of TSG16 in unaffected subjects; (2) measuring the level of expression of TSG16 15 in a person suspected of abnormal expression of TSG16; and (3) comparing the measured level of expression with the profile for normal expression.
82. A method as claimed in claim 81 wherein a 20 hybridisation assay using a probe derived from TSG16, or a fragment thereof, is employed to measure levels of expression.
83. A method as claimed in claim 82 wherein the probe 25 has at least 50% sequence identity to a nucleotide WO 01/32861 PCT/AUOO/01329 119 sequence encoding TSG16, or a fragment thereof.
84. A method as claimed in claim 83 wherein the probe 5 is selected from the group consisting of nucleotide sequences as set forth in any one of SEQ ID NO:13-43.
85. A method for the diagnosis of a disorder associated with abnormal expression of TSG16, or a 10 predisposition to such disorders due to mutations in TSG16, comprising the steps of: (1) establishing a physical property of wild type TSG16; (2) obtaining TSG16 from a person suspected of 15 abnormal expression of TSG16; and (3) measuring the property for the TSG16 expressed by the person and comparing it to the established property for wild-type TSG16 in order to establish whether the person expresses a mutant TSG16. 20
86. A method as claimed in claim 85 wherein the property is the electrophoretic mobility.
87. A method as claimed in claim 86 wherein the 25 property is the proteolytic cleavage pattern. WO 01/32861 PCT/AUOO/01329 120
88. A method as claimed in claim 85 wherein the property is the activity of TSG16 as measured by a functional assay. 5
89. A method as claimed in claim 85 wherein the property is binding of an antibody as defined in any one of claims 25 to 28 or claims 54 or 57.
90. A genetically modified non-human animal 10 transformed with an isolated DNA molecule as defined in any one of claims 1 to 13 or claims 29 to 43.
91. A genetically modified non-human animal as claimed in claim 90 in which the animal is selected from 15 the group consisting of rats, mice, hamsters, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs and non-human primates such as monkeys and chimpanzees.
92. A genetically modified non-human animal as 20 claimed in claim 91 wherein the animal is a mouse.
93. A genetically modified non-human animal in which TSG16 gene function has been knocked out by homologous recombination. 25 WO 01/32861 PCT/AUOO/01329 121
94. A genetically modified non-human animal as claimed in claim 93 in which the animal is selected from the group consisting of rats, mice, hamsters, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs and non-human 5 primates such as monkeys and chimpanzees.
95. A genetically modified non-human animal as claimed in claim 94 wherein the animal is a mouse. 10
96. The use of a genetically modified non-human animal as claimed in any one of claims 90 to 95 in screening for candidate pharmaceutical compounds.
97. The use of a host cell as defined in claim 17 or 15 claim 45 in screening for candidate pharmaceutical compounds.
98. The use of an isolated polypeptide as defined in any one of claims 18 to 23 or claims 46 to 52 in screening for candidate pharmaceutical compounds. 20
99. A nucleic acid encoding a mutant TSG16 polypeptide which cannot form a complex with a wild-type protein with which wild-type TSG16 does form a complex. 25
100. A nucleic acid as claimed in claim 99 wherein the WO 01/32861 PCT/AUOO/01329 122 wild-type protein is a protein selected from the group consisting of members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family of proteins. 5
101. A nucleic acid as claimed in claim 100 wherein the wild-type protein is PIAS1.
102. A nucleic acid as claimed in any one of claims 99 to 101 wherein the mutant TSG16 polypeptide contains a 10 mutation in an ankyrin repeat domain.
103. A mutant TSG16 polypeptide which cannot form a complex with a wild-type protein with which wild-type TSG16 does form a complex. 15
104. A mutant TSG16 as claimed in claim 103 wherein the wild-type protein is selected from the group consisting of members of the protein inhibitor of activated signal transducer and activator of transcription 20 (PIAS) family of proteins.
105. A mutant TSG16 as claimed in claim 104 wherein the wild-type protein is PIAS1. 25
106. A mutant TSG16 polypeptide as claimed in any one WO 01/32861 PCT/AUOO/01329 123 of claims 103 to 105 containing a mutation in an ankyrin repeat domain. 5
107. The use of mutant TSG16 polypeptide as defined in any one of claims 103 to 106 in the diagnosis of a disorder associated with expression of TSG16.
108. The use of mutant TSG16 polypeptide as defined in 10 any one of claims 103 to 106 for the screening of candidate pharmaceutical compounds.
109. A complex of wild-type TSG16 and a protein selected from the group consisting of members of the 15 protein inhibitor of activated signal transducer and activator of transcription (PIAS) family of proteins.
110. A complex as claimed in claim 109 wherein the protein is PIAS1. 20
111. The use of a complex as claimed in either one of claims 109 or 110 for the screening of candidate pharmaceutical compounds. 25
112. An isolated DNA molecule consisting of the DNA WO 01/32861 PCT/AUOO/01329 124 sequence set forth in any one of SEQ ID NOS:3-12 and 124.
113. An isolated DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:125. 5
114. An isolated polypeptide consisting of the amino acid sequence set -forth in SEQ ID NO:126.
115. An isolated DNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO:127. 10
116. An isolated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:128.
117. An isolated DNA molecule consisting of the 15 nucleotide sequence set forth in any one of SEQ ID NOS:129 to 131.
118. An isolated polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOS:132 to 20 134.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AUPQ3771 | 1999-10-29 | ||
AUPQ3771A AUPQ377199A0 (en) | 1999-10-29 | 1999-10-29 | Tumour suppressor genes from chromosome |
PCT/AU2000/001329 WO2001032861A1 (en) | 1999-10-29 | 2000-10-30 | Tumour suppressor genes from chromosome 16 |
Publications (1)
Publication Number | Publication Date |
---|---|
AU1118301A true AU1118301A (en) | 2001-05-14 |
Family
ID=3817922
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AUPQ3771A Abandoned AUPQ377199A0 (en) | 1999-10-29 | 1999-10-29 | Tumour suppressor genes from chromosome |
AU11183/01A Withdrawn AU1118301A (en) | 1999-10-29 | 2000-10-30 | Tumour suppressor genes from chromosome 16 |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AUPQ3771A Abandoned AUPQ377199A0 (en) | 1999-10-29 | 1999-10-29 | Tumour suppressor genes from chromosome |
Country Status (2)
Country | Link |
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AU (2) | AUPQ377199A0 (en) |
WO (1) | WO2001032861A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
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AUPR204900A0 (en) * | 2000-12-14 | 2001-01-11 | Bionomics Limited | A novel gene |
CN115501340B (en) * | 2021-06-07 | 2024-04-12 | 中山大学附属第三医院(中山大学肝脏病医院) | Application of CircPIAS1 as target in preparation of liver cancer diagnostic reagent or therapeutic drug |
-
1999
- 1999-10-29 AU AUPQ3771A patent/AUPQ377199A0/en not_active Abandoned
-
2000
- 2000-10-30 AU AU11183/01A patent/AU1118301A/en not_active Withdrawn
- 2000-10-30 WO PCT/AU2000/001329 patent/WO2001032861A1/en not_active Application Discontinuation
Also Published As
Publication number | Publication date |
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AUPQ377199A0 (en) | 1999-11-25 |
WO2001032861A1 (en) | 2001-05-10 |
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