Abstract:
Tumor Suppressor Activated Pathway (TSAP) genes and nucleotide sequences therefore as well as vectors and cells containing such nucleotide sequences and various uses therefore are described. The mechanism by which TSAP 3 activates apoptosis also is described. Pharmaceutical compositions and methods for preventing tumorigenesis also is described.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional application of U.S. Ser. No. 09/134,873, filed Aug. 17, 1998 now abandoned which in turn, is a continuation-in-part application of U.S. Ser. No. 09/091,647, filed Jun. 22, 1998, abandoned Jul. 29, 1999, which in turn is a 371 of PCT/FR96/02061, filed Dec. 20, 1996. This application also claims foreign priority to French Patent Application No. 96 04853, filed Apr. 18, 1996 and French Patent Application No. 95 15146, filed Dec. 20, 1995. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to the demonstration of genes which are involved in the molecular pathways of tumor suppression and to the use of the genes which have thus been demonstrated for treating certain genetic malfunctions, in particular cancers. 
     BACKGROUND OF THE INVENTION 
     The present invention was made possible by isolating cDNA which corresponded to the messenger RNAs which are expressed or repressed during the process of apoptosis which is induced by the p53 suppressor gene. 
     A global analysis of the molecular events which take place during the cell cycle at the time of development and cell apoptosis is required in order to better understand the importance of the p53 gene in the process of tumor suppression or, on the contrary, of canceration. 
     The transformation of a normal cell into a tumor cell is a process which takes place in several stages and which requires a sequence of molecular events. At the physiological level, these events find expression in the tumor cell becoming independent of external signals and in an internal deregulation which leads to uncontrolled growth. 
     Two groups of genes are responsible for this so-called “malignant” transformation; on the one hand oncogenes and, on the other hand, suppressor genes or anti-oncogenes. Because of their deregulation in cancer (resulting most frequently from a mutation or a translocation), oncogenes induce a positive signal which promotes neoplastic growth. By contrast, the suppressor genes are unable, either because they have been deleted, because they are not being expressed due to mutation of the promoter, for example, or because of mutations which modify the structure and function of the protein, to supply, in the cancer, the signal which would normally retard this abnormal growth. As a consequence, malfunction of the suppressor genes contributes to neoplastic transformation. 
     SUMMARY OF THE INVENTION 
     The object of the present invention is to isolate genes which normally play a part in tumor suppression and any possible malfunctions of which can then be monitored and treated. 
     In particular, isolation of these genes makes it possible to carry out a gene replacement therapy or else to synthesize protein or non-protein pharmacological agents which, directly or indirectly, induce activation and expression of these genes by acting on the promoters, or else to synthesize pharmacological agents which mimic the physiological effect of these suppressor genes. 
     The final objective is either to inhibit tumor growth or, even better, induce the apoptotic process in these tumor cells, that is to cause the tumor cells to “commit suicide”. 
     Thus, in one embodiment the invention relates to an isolated DNA molecule comprising a sequence selected from the group consisting of: 
     (a) a nucleotide sequence of one of SEQ ID Nos. 4 to 11, 
     (b) a nucleotide sequence that hybridizes with one of the sequences of 
     (a), and 
     (c) a nucleotide sequence that is at least 80% homologous with a 
     sequence of either (a) or (b). Expression of this DNA molecule may activate cell apoptosis and/or tumor suppression. 
     In another embodiment, the invention relates to an isolated DNA molecule comprising a sequence selected from the group consisting of: 
     (a) a nucleotide sequence of one of SEQ ID Nos. 1 or 3; 
     (b) a nucleotide sequence that hybridizes with one of the sequences of 
     (a), and 
     (c) a sequence that is at least 80% homologous with a sequence of either 
     (a) or (b). Tumor suppression induces expression of this DNA molecule. 
     In another embodiment, the invention relates to an isolated DNA molecule comprising a sequence selected from the group consisting of: 
     (a) SEQ ID No. 2, 
     (b) a nucleotide sequence that hybridizes with SEQ ID No. 2, and 
     (c) a nucleotide sequence that is at least 80% homologous with either (a) or (b); wherein cell apoptosis induces expression of this DNA molecule. 
     In a preferred embodiment, the DNA molecule of this invention is SEQ ID No. 11 or fragment thereof. 
     In yet another embodiment, the invention relates to a biologically functional vector comprising one of the above described DNA molecules. Another embodiment relates to a host cell stably transformed with this vector. In yet another embodiment, the invention relates to a protein obtained by culturing the host cell under appropriate nutrient conditions so as to allow the cell to express the protein. 
     In another embodiment, the invention relates to a pharmaceutical composition comprising the above described vector and a pharmaceutically acceptable carrier. Another embodiment relates to a method of preventing tumorigenesis, the method comprising contacting cells with a tumorigenesis inhibiting amount of the pharmaceutical composition. In a preferred embodiment, the pharmaceutical composition comprises the DNA SEQ ID NO. 11 or TSAP 3. 
     In other embodiments, the invention relates to a DNA probe or a PCR amplification primer comprising a nucleotide sequence selected from the group consisting of SEQ ID No. 1-11, or a fragment thereof. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention relates to demonstrating genes which are involved in this apoptosis, particularly TSAP 3, which has been discovered to directly activate apoptosis. Thus, each cell contains within itself a program of physiological death. This is also a physiological process which is involved in development for the purpose of maintaining homeostasis of the body and of preventing abnormal cell proliferations from becoming established even if, for all that, they are not malignant in nature. 
     One of the most important suppressor genes involved in apoptosis is the p53 gene. In its normal function, this gene controls cell growth and the apoptotic process; in particular, it is this gene which blocks cell growth and which is responsible for inducing the apoptotic process in order to avoid the development of a cancer. Thus, it has been demonstrated that mice which are nullizygous for p53 are much more sensitive to the formation of tumors. The fact has also been demonstrated that, in cancers, the p53 gene is very often altered and leads to the production of proteins which are unable to serve as a vehicle for the apoptotic message. 
     It is this distinctive feature which has been employed within the context of the present invention. 
     Thus, the present invention is based on the observation that it is not possible, or that it at least appears very difficult, to institute a direct replacement therapy when the p53 gene is malfunctioning. Thus, when p53 is mutated as it is in cancer, it nullifies the physiological effect of the normal p53. 
     It was therefore necessary, at least initially, to abandon a replacement therapy which acted directly at the level of p53. 
     The present invention is therefore linked to studying the genes which are situated downstream of p53 in order to bypass the abovementioned difficulty. 
     In order to isolate the genes which are activated or inhibited by normal p53 (wild-type p53), a global screening was carried out of gene expression in a cell in which apoptosis had been induced and in the same malignant cell, more specifically in a cell which was expressing a p53 whose function was normal and in a cell which was expressing a p53 which was mutated and whose function was oncogenic. Comparison of the expressed genes (messenger RNAs expressed in the two types of cell) made it possible to identify genes which were expressed differentially, that is which were expressed in one of the cells but not the other (the genes can be activated or inhibited). 
     It was readily deduced that these genes are involved in the process of canceration, in the one case by their absence, and in the other case by their presence. 
     The method used for carrying out this differential study is the method described in 1992 by Liang and Pardee (Differential display of eucaryotic mRNA by means of a polymerase chain reaction)  Science  257: 967-971 (1992), which is herewith incorporated by reference. 
     Until now, genes involved in suppression have been isolated either by positional cloning or by using double hybrids. The first method has made it possible, by making a statistical computation, to calculate the greatest probability of where a suppressor gene which was a candidate for a rather specific type of cancer, in particular those of familial origin, might be located within the chromosome. The double hybrid system enables the proteins which interact with a given gene to be isolated one by one. 
     The approach to the problem which was adopted in accordance with the present invention made it possible to isolate sequences which were directly linked to a function. As a result, in contrast to the random sequencing of the ESTs, the sequences are sequences whose function is known and which arc involved in the apoptosis process which is induced by the p53 suppressor gene. 
     More precisely, this method was used on a cell model described by Moshe Oren; this model involves mouse myeloid tumor cells which have been transfected with a stable mutant of the p53 gene. Expression of this gene is temperature-sensitive, i.e. when the cells are cultured at 37° C., the protein which is produced is a mutated protein, that is to say it cannot act as a tumor suppressor and the corresponding cell line therefore develops in the form of malignant cells; by contrast, at a temperature of 32° C., the p53 protein which is expressed is able, like the natural protein, to act as a suppressor, and prevents the corresponding cell line from becoming malignant. 
     This systematic study made it possible to identify the genes which are involved in the suppression cascade which is induced by p53. More particularly, the inventors discovered that one gene, TSAP3, is responsible for apoptosis and/or tumor suppression. 
     For this reason, the present invention relates to these novel sequences and the genes which comprise them, as well as to the use of these sequences, both in diagnosis and therapy, and also for creating models for testing antineoplastic products. 
     The present invention relates, first of all, to a nucleotide sequence which corresponds to a gene which comprises: 
     (a) a sequence according to one of the SEQ ID Nos 1 to 10, or an equivalent gene which comprises: 
     (b) a sequence which hybridizes with one of the sequences according to (a), 
     (c) a sequence which exhibits at least 80% homology with (a) or (b), or 
     (d) a sequence which encodes a protein which is encoded by a gene according to (a), (b) or (c), or which encodes an equivalent protein, 
     and their application, in particular in the suppression of cancer and in the therapeutic follow-up. 
     In addition, the present invention relates to a human gene which is involved in the suppression cascade induced by p53, and to the use of the sequences of this gene, both in diagnosis and in therapy, and also for creating models for testing antineoplastic products and their application as antiviral agents. 
     The present invention therefore also relates to a nucleotide sequence which corresponds to a gene which comprises: 
     (a) a sequence according to SEQ ID Nos 11, corresponding to the human TSAP 3 gene or HUMSIAH (Human Homologue of the Drosophila seven in absentia gene), or an equivalent gene which comprises: 
     (b) a sequence which hybridizes with one of the sequences according to (a), 
     (c) a sequence which exhibits at least 80% homology with (a) or (b), or 
     (d) a sequence which encodes a protein which is encoded by a gene according to (a), (b) or (c), or which encodes an equivalent protein, 
     and their application, in particular in the suppression of cancer and in the therapeutic follow-up. 
     With regard to sequences 1 to 11, the present invention covers both the nucleotide sequence which corresponds to the entire gene and fragments of this gene, in particular when they encode an equivalent protein, as will be described below. 
     The nucleotide sequences can equally well be DNA sequences or RNA sequences or sequences in which some of the nucleotides are unnatural nucleotides, either in order to improve their pharmacological properties or to enable them to be identified. 
     The sequences mentioned in (b) (for SEQ ID Nos 1 to 11) are essentially sequences which are totally or partially complementary (in particular in the previously mentioned cases). 
     The (a) and (b) sequences (for SEQ ID Nos 1 to 10) provide access not only to the murine gene from which they are derived but also, by homology, to the corresponding human genes. 
     Thus, the invention also relates to the nucleotide sequences of the genes which exhibit strong homology with the previously mentioned genes, preferably a homology which is greater than 80% over the essential parts of the said genes, or, in general, at least 50% of the sequence; preferably, the homology over these parts is greater than 90%. “Homology” means the degree to which the sequences contain the same nucleotides when two nucleotide sequences are aligned and compared, using methods well known in the art of the invention. 
     Finally, when the said genes encode a protein, the present invention also relates to the sequences which encode the same protein, taking into account the degeneracy of the genetic code, and also equivalent proteins, that is to say which produce the same effects, in particular proteins which have been deleted and/or which have undergone point mutations. 
     The sequences according to the present invention are, more specifically, the sequences which are induced or inhibited at the time of cell apoptosis, in particular those which are induced by p53, or which are responsible for apoptosis, as in the case of TSAS 3. 
     The said genes are grouped together in the TSAP or “Tumor Suppressor Activated Pathway” and designated TSAP 1 to TSAP 8 and human TSAP 3, corresponding to SEQ ID Nos 1 to 8 and 11 (HUMSIAH) respectively, and the TSIP or “Tumor Suppressor inhibited Pathway” and designated TSIP 1 and TSIP 2, corresponding to SEQ ID Nos 9 and 10. 
     The characteristics of the sequences which correspond to SEQ ID Nos 1 to 10 are compiled in the appended table. 
     The nucleotide sequences which correspond to the TSAP genes (including human TSAP 3 or HUMSIAH) are sequences which are expressed during the apoptosis process, whereas the process of oncogenesis takes place when they are not expressed. It is therefore of interest: 
     to detect any anomaly in the corresponding gene which might lead to greater susceptibility to oncogenesis, and 
     to be able to plan a replacement therapy. 
     It must also be recalled that these genes are able to intervene in other processes besides oncogenic processes; thus, p53 is, as it were, the guardian of the integrity of the genome; under these conditions, the TSAP or TSIP genes are doubtless also involved in this control function; the previously mentioned detection and therapy can therefore cover all the possible alterations of the genome. By contrast, the TSIP genes are expressed during oncogenesis and not during apoptosis; it is therefore also of interest in this case to detect any possible anomalies in the TSIP genes and to plan an inhibition/blocking therapy. 
     The replacement therapy can be effected by means of gene therapy, that is by introducing the TSAP gene together with the elements which enable it to be expressed in vivo. The principles of gene therapy are known. Specific viral or nonviral vectors can be used, for example adenovirus, retrovirus, herpesvirus or poxvirus vectors. Most of the time, these vectors are used in defective forms which serve as TSAP-expressing vehicles, with or without integration. The vectors can also be synthetic vectors, that is to say which mimic viral sequences, or else consist of naked DNA or RNA in accordance with the technique developed by the VICAL company, in particular. 
     In most cases, it is necessary to provide targeting elements which ensure expression which is specific for tissues or organs; thus, it is not possible to consider activating a phenomenon of uncontrolled apoptosis. 
     The present invention therefore relates to all the previously described vectors. 
     The present invention also relates to the cells which are transformed by an expression vector such as previously described as well as to the protein which can be obtained by culturing transformed cells. 
     The expression systems for producing proteins can be either eucaryotic systems, such as the preceding vectors, or procaryotic systems in bacterial cells. 
     One of the important features of the present invention is that it has demonstrated the involvement of several genes in apoptosis; thus, the use of gene therapy to over-express one of the genes may, for some of the genes, only lead to apoptosis of the cells in which other deregulated genes are already being expressed, that is malignant cells. 
     The present invention also relates to a compound, as a medicament, which ensures cellular expression of at least one of the previously mentioned nucleotide sequences, in particular of the TSAP 1 to TSAP 8 and human TSAP 3 genes, when it is induced during cell apoptosis, or, on the contrary, which ensures inhibition of the cellular expression of at least one cell sequence such as previously described, in particular TSIP 1 and TSIP 2, when it is inhibited during cell apoptosis. 
     It is, for example, possible to envisage approaches other than gene therapy, in particular the use of nucleotide sequences in a sense or antisense strategy, that is to say sequences which are able to block TSIP expression or which, on the contrary, acting upstream, promote TSAP expression. 
     It is also possible to envisage a direct replacement strategy which involves supplying proteins which correspond to TSAP or inhibitory antibodies which correspond to TSIP. 
     Finally, it is possible to envisage using non-protein molecules whose activity is to activate TSAP or to mimic the action of its expression product or else to inhibit TSIP or else to block the action of its expression product. 
     These products can be easily tested on modified cells, which are described in the examples, by introducing the products to be tested into the cell culture and detecting the appearance of the apoptotic phenomenon. In the strategies using DNA, RNA or protein, the products are, of course, developed in accordance with the sequences which are described. 
     The present invention relates, in particular, to the use of the abovementioned medicaments as antineoplastic agents. 
     However, the product of the human TSAP 3 gene (HUMSIAH) may also be used as an antiviral agent, as will be apparent from reading Example 2. The present invention therefore also relates to the use of the abovementioned medicaments as antiviral agents. 
     The present invention also relates to all or part of the sequences according to the invention for use, in the role of a diagnostic agent for determining predisposition to cancer, as a nucleotide probe or as an amplification primer, and, also in the role of diagnostic agent for determining predisposition to cancer, to an antigen which corresponds to all or part of the proteins encoded by the sequence according to the invention or to the corresponding antibodies, in particular monoclonal antibodies, where appropriate following culture. 
     The diagnostic methods are known; they can, for example, be techniques for microsequencing variable parts following isolation and possible amplification, or detection methods of the RFLP type, or straightforward amplification in particular. The differential techniques can, in particular, make it possible to demonstrate the divergence between normal and abnormal TSAP or TSIP. 
     The invention also relates to models which make use of the abovementioned sequences. The PCR method, or other amplification methods, may be employed, in particular, to isolate the human TSAP 3 gene (HUMSIAH) by utilizing the structure of the gene. It is also possible to synthesize this gene bit by bit, if required. 
     Finally, the invention relates to an improvement to the method of Liang and Pardee,  Science  257: 967-971 (1992), which involves carrying out a stepwise decrease (“touch down”), as described in Don et al. Nucl. Acids Res. 19: 4008 (1991), in the PCR amplification. Liang et al and Don Et al are herewith incorporated by reference. 
    
    
     DESCRIPTION OF THE FIGURES 
     FIG.  1 —Quantification of the differential expression of the mRNAs using a 1200 β imager. Hybridization to mRNAs derived from LTR6 cells at 37° C. and LTR6 cells after 4 hours at 32° C. The numbers on the ordinates from 0 to 500 correspond to counts detected per 0.15 mm and are proportional to the hybridization signal. 
     C1: mRNA also expressed using a clone without differential expression; 
     C2: positive control using Cyclin G and showing induction of the mRNAs corresponding to 32° C.; 
     MER-LTR: showing induction of this sequence at 32° C.; 
     TSAP 1 to TSAP 8: differential expression of the 8 activated mRNAs in the first 4 hours following the induction of apoptosis; 
     TSIP 1 and TSIP 2: differential expression of the 2 mRNAs which are inhibited in the first 4 hours following the induction of apoptosis. 
     FIGS.  2 A- 2 C: Northern blot analysis. 
     A: hybridization with the TSAP 3 probe; 
     B: hybridization with the mouse siah 1b probe; 
     tracks 1 and 2: polyA+ mRNA of M1 myeloid leukemic cells (clone S6) cultured at 37° C. and 32° C., respectively; 
     tracks 3 and 4: polyA+ mRNA of LTR6 cells cultured at 37° C. and 32° C., respectively; 
     the arrow indicates the differential expression of the TSAP 3 1.9 kb transcript—mouse siah 1b; 
     lower panels: GAPDH; 
     C: tissue distribution using TSAP 3 as a probe; 
     1: heart, 2: brain, 3: spleen, 4: lung, 5: liver, 6: skeletal muscle, 7: kidney, 8: testicle; 
     the arrows indicate the 1.9 and 2.4 kb transcripts; lower panel: β-actin. 
     FIG.  3 —Analysis of in-situ hybridization using the TSAP 3 probe; 
     A: M1 cells incubated at 32° C. for 4 hours and hybridized with an antisense TSAP 3 probe; 
     B: LTR6 cells incubated at 32° C. for 4 hours and hybridized with a sense TSAP 3 probe; 
     C: LTR6 cells incubated at 37° C. and hybridized with an antisense TSAP 3 probe; 
     D to F: LTR6 cells cultured at 32° C. for 1, 2 and 4 hours, respectively, and hybridized to an antisense TSAP 3 probe; 
     the bar in panel A: 10 μm; 
     the arrows indicate accumulation of the TSAP 3 mRNAs in the cytoplasm. 
     FIG.  4 —Comparison of the TSAP 1 cDNA sequence (SEQ ID NO: 1) and the nucleotide sequence corresponding to rat beta 4 phospholipase C (SEQ ID NO: 13). 
     FIG.  5 —Comparison of the TSAP 2 cDNA sequence (SEQ ID NO: 2) and the nucleotide sequence corresponding to the zinc finger protein (ZFM 1) (SEQ ID NO: 14) which is located in the multiple endocrine neoplasia (MEN 1) locus. 
     FIG.  6 —Comparison of the TSAP 3 cDNA sequence and the nucleotide sequence corresponding to the murine homolgoue, MMSIAH 1B gene. 
     FIG.  7 —Comparison of the product of the sina genes of different species, human (SEQ ID NO: 17)(HUMSIAH), murine (SEQ ID NO: 18 &amp; 19 respectively ) (MMSIAH 1B) 1A and Drosophila (SEQ ID NO: 20) (DROSINA). 
     FIG.  8 —Comparison of the TSIP 2 cDNA sequence (SEQ ID NO: 10) and the cDNA sequence of the murine S182 transcript (SEQ ID NO: 16) of the AD3 gene, which is involved in Alzheimer&#39;s disease. 
     FIGS.  9 A- 9 C—Shows the biological effects of TSAP 3 expression in U937 cells. FIG. 9A shows the results of a FACS analysis of the DNA content in U937 cells transfected with the vector alone (RSV-C) and those transfected with TSAP 3 (RSV-7S. Respectively, 3% and 12% of the cell population is in the sub G1 phase. FIG. 9B shows the results of a FACS analysis of the TUNEL assay with 3% of the U937 cells transfected with the vector along (RSV-C), as compared to 15% of the U937 cells transfected with TSAP 3 (RSV-7S) being positive. 
     FIG. 9C shows the results of a tumorigenicity assay in SCID/SCID mice. After injection with either U937 cells transfected with the control vector alone (—O— RSV C), mice form large tumors in 20 out of the 20 injection sites and appear early. The U937 cells stably transfected with TSAP 3 (-φ- RSV-75) form smaller tumors. * indicates the statistical significance: p≦0.001. 
     FIGS.  10 A- 10 C—This figure shows the characterization of the TSAP 3 protein. FIG. 10A shows expression of TSAP 3 during wt-p53 induction of apoptosis. Specifically this is the results of a Western blot analysis with anti-TSAP3 antibodies generated against the first 16 amino acids of TSAP. Lane 1- LTR-6 cells at 37° C. Lane 2- LTR6 cells after 4 hours of incubation at 32° Lane 3- LTR-6 cells after 7 hours of incubation at 32° C. Lane 4- LTR-6 cells after 9 hours of incubation at 32° C. Lane 5- LTR-6 cells after 16 hours of incubation at 32° C. Lane 6- LTR-6 cells after 24 hours of incubation at 32° Arrow indicates the 30 kDa TSAP 3 protein. FIG. 10B shows the subcellular localization of TSAP 3, via Western blot with anti-TSAP 3 antibodies. Lane 1- nuclear fraction of LTR-6 cells after 4 hours of incubation at 32° C. Lane 2-membrane fraction of LTR-6 cells after 4 hours of incubation at 32° C. Lane 3-cytoplasmic fraction of LTR-6 cells after 4 hours of incubation at 32° C. Arrow indicates the 30 kDa TSAP 3 protein. FIG. 10C shows expression of TSAP 3 in U937 cells with a suppressed malignant phenotype. Lane 1- U937 cells transfected with the control vector alone. Lane 2- US cells derived from U937 cells but displaying a suppressed malignant phenotype. Lane 3- U937 cells stably transfected with TSAP 3 (clone RSV-7S). Lane 4- U937 cells stably transfected with TSAP 3 (clone RSV-8S). Lane 5- U937 cells stably transfected with TSAP 3 (clone RSV-10S). 
    
    
     EXAMPLES 
     The following general methods were used in the examples which follow and which are not intended to limit the invention in anyway. 
     MATERIALS AND METHODS 
     Cell Cultures 
     M1 myeloid leukemia cells (clone S6) and M1 cells which are stably transfected with a temperature-sensitive mutant, val 135 p53 (LTR6) (3), Yonish-Rouach et al.,  Nature  352: 345-347 (1991). 
     These cells are cultured on RPMI 1640 medium containing 10% FCS at 5% CO 2  and 37° C. In order to change the temperature, the cultures are placed in a second incubator at 32° C. In all the assays carried out in this study, the cells are tested for the presence of apoptosis after 12 and after 24 hours. 
     Study of the Differential cDNAs 
     The following modifications of the original protocol by Liang et at. (1) were made in order to carry out the tests under standard experimental conditions and obtain total reproducibility of the results. 
     Use is always made of polyA+ mRNAs which have been purified twice on an oligodT column making use of Fast Track (Invitrogen, San Diego Calif.). After reverse transcription (M-MLV Reverse Transcriptase, Gibco BRL) on 0.05 μg of polyA+ using 20 μm of each of the dNTPs (Boehringer-Mannheim), no additional dNTP is added to the final PCR mixture. A “hot start” at 94° C. is carried out for 5 minutes before the PCR (GeneAmp PCR system 9600, Perkin Elmer Cetus). The samples are cooled down rapidly in ice water. A “touch down” (Don et at., supra)(2) of 10 cycles or 50° C. to 40° C. is carried out (94° C. 30 seconds −50° C. 1 minute −72° C. 30 seconds), followed by 35 cycles (94° C. 30 seconds −40° C. 1 minute −72° C. 30 seconds) and a final extension of 5 minutes at 72° C. The PCR products are separated on non-denaturing 6% polyacrylamide gels (Bauer et al., Nucl. Acids Res. 21: 4272-4280 (1993)(4). The gels are exposed without drying. Each differential presentation is performed by comparing M1S6 and LTR6 at 37° C., and after incubating the two cell lines at 32° C. for 4 hours. 
     The differential presentation procedure is repeated in 3 different experiments in order to confirm complete reproducibility. 
     The bands which are expressed differentially are excised from the gel, eluted and reamplified (1). The PCR products are subcloned using the TA-cloning system (Invitrogen, San Diego, Calif.) in accordance with the instructions supplied. 
     For each ligation reaction, 10 recombinant clones are sequenced using the automated ABI system. 
     Extraction of the RNAs, and Northern Blot Probes and Analyses 
     The total RNA is extracted using Trizol (Life Technologies). The polyA+ RNAs are prepared using an OligotexdT kit (Qiagen, Calif.). 30 μg of total RNA or 2 μg of polyA+ RNA are separated on a 1% agarose/1×MOPS/2% formaldehyde gel, transferred onto a nylon membrane (Hybond N+, Appligene, France) as has been previously described (Sambrook et at.,  Molecular Clotting: a laboratory manual  (1989) (5). The Northern blots are hybridized with P 32 -labeled probes on the TSAP and TSIP inserts and washed as previously described (Sambrook et at, supra). In order to check that the function of the wild-type p53 has been induced, the Northern blots are hybridized with a cyclin G probe (Okamoto, et al.,  EMBO J ., 13: 4816-4822 (1994). As a control for the quantity of mRNA which has been loaded on, the blots are hybridized with a GAPDH probe. Different Northern blots (Clontech, Calif.) are used under identical conditions and hybridized, as a control, with a β-actin probe. The RT-PCR products in the case LTR6 are amplified using the following siah 1b primers (SEQ ID NOS 21 &amp; 22, respectively): 5′CAGTAAACCACTGAAAAACC3′ and 5′CAAACCAAACCAAAACCAC3′. The subcloned PCR product is used as a control siah 1b probe. The Northern blots are exposed at −80° C. for 10 days. 
     Slot Blots 
     The reproducibility of the results obtained by the Northern blot analyses. The blots are prepared (bio-Rad, Hercules, Calif.) by placing the PCR products (200 ng of Zeta-Probe Blotting Membranes, Bio-Rad, in accordance with the manufacturer&#39;s instructions) of TSAP clones and hybridized with a P 32 -labeled cDNA probe (Superscript II Gibco-BRL, Life Technologies) corresponding to the RNA of LTR6 cells which have been incubated at 37° C. and then at 32° C. for 4 hours. The PCR product of the clone containing cyclin G is also deposited on the membranes and used as a positive control. The slot blots are exposed at −80° C. overnight. 
     Quantitative Image Analysis 
     This is performed on the two Northern blots (for TSIP 1 and TSIP 2), and on the slot blots for all the control cDNAs and TSAP 1 to 8, using a 1200 β imager (Biospace instruments, Paris, France). For the quantitative analysis represented in the graphs in FIG. 1, a constant number was subtracted from each peak. This constant is calculated by measuring the mean value of the background noise in the slots which do not contain any cDNA. The β imager results were obtained by counting the slot blots overnight and by confirming them by means of autoradiography using variable exposure times. These autoradiograms show the same relative qualitative variations between the activities at 32° C. and 37° C. as do the measurements obtained using the β imager. 
     In-situ Hybridization (7, 8) 
     The cells are washed 3 times in a saline phosphate buffer (PBS), “cytospinned” and fixed with 4% paraformaldehyde in PBS for 10 minutes, and then stored in 70% ethanol. RNA transcripts of TSAP 3 which are labeled with digoxigenin-11-uridine-5′-triphosphate (DIG) and biotin-11-UTP are used in the analyses in accordance with the previously described procedure (Boehringer-Mannheim). In order to detect strains labeled with hybridized digoxigenin, the slices are incubated in SAD-10 (10 nm of gold-labeled sheep anti-DIG antibody diluted 1/1000, Biocell UK). The analysis is performed using confocal laser microscopy. See  Atigerer  et al.,  Methods in cell biology: functional organization of the nucleus , 35: 37-71 (1991) and Linares-Cruz et al.,  J. Microsc . 173: 27-38 (1994). 
     Example 1 
     Differential study of the cDNAs using the Liang and Pardee method provides a very powerful and efficient tool for detecting variations in gene expression. Nevertheless, it was necessary to modify the original protocol, as has previously been pointed out, in order to eliminate some problems of reproducibility which were observed when applying the method as originally described. 
     Complete reproducibility was found when a “hot start” followed by a “touch down” were introduced into the PCR method. 
     Nevertheless, after having been isolated and reamplified, the differentially expressed bands are often contaminated with bands arising from the RNAs which migrate into the regions adjacent to the cDNA; errors result if these probes are used directly on Northern blots. The second PCR products were therefore subcloned and Northern blot analyses, used for lack of a single probe recombinant, were carried out. The systematic sequencing of at least 10 recombinant subclones in the case of each selected band showed that this was very effective for selecting the clones of interest. 
     In die current state of knowledge, the p53 gene is the tumor suppressor which is mutated in the largest number of cancers of very diverse origin, and use of the temperature-sensitive mutant val-135 p53 has already previously been shown to provide a very considerable amount of information regarding the function of the wild-type p53 in inducing either cessation of cell growth in the G-1 phase or initiation of the program of cell death. 
     Until now, the molecular pathways upstream and downstream of p53 which lead to tumor suppression have been very unclear. 
     A certain number of genes downstream of p53 have previously been identified; these are, in particular, gadd 45, mdm 2, mck, mouse endogenous retrovirus LTR, p21-waf and cyclin G. 
     The present invention has demonstrated the existence of 11 genes which are expressed differentially in cells which are expressing p53 in its active suppressor form or else in tumor cells which are expressing the inactive p53 gene. 
     FIG. 1 shows a quantification of the hybridization signals which correspond to the differential expression of 8 of these genes which are activated at 32° C., that is to say in which the wild-type p53 function is activated and therefore leads to apoptosis of the cells; in that which follows, these activated genes will be designated TSAP (for tumor suppressor activated pathway); by contrast, it is observed that, in two experiments, 2 genes which are expressed at 37° C. are partially inhibited at 32° C., implying that they are inhibited during programmed cell death; these genes were designated TSIP (for tumor suppressor inhibited pathway). 
     Analysis of the homologies of the different activated sequences of TSAP 1 to TSAP 3 showed that these genes were already known. By contrast, the other cDNAs, i.e. the TSAP 4 to TSAP 8 cDNAs, do not show any significant homology with known genes. 
     The cDNA corresponding to TSIP 1, whose expression is inhibited during apoptosis, does not exhibit any homology with known genes. 
     The cDNA corresponding to TSIP 2, whose expression is also inhibited during apoptosis, shows a high degree of homology with the S182 transcript of the AD3 gene, which is involved in the metabolic pathways of Alzheimer&#39;s disease (Sherrington et al.,  Nature  375: 754-760 (1995)) (FIG.  8 ). 
     Consequently, it is possible to act on the metabolic pathways of Alzheimer&#39;s disease by acting on the p53-dependant metabolic pathways. 
     The present invention therefore also relates to a compound, as a medicament, which ensures the cellular expression of TSIP 2 and which is intended for treating Alzheimer&#39;s disease, and to all or part of the TSIP 2 sequence for use, in the role of a diagnostic agent for determining predisposition to Alzheimer&#39;s disease, as a nucleotide probe or as an amplification primer, and also to an antigen which corresponds to all or part of the proteins encoded by TSIP 2, or to the antibodies, in particular the corresponding monoclonal antibodies, where appropriate after culture. 
     The hypothesis which can be put forward with regard to the genes whose expression is inhibited by wild-type p53 is that they may encode oncogenic sequences which are regulated downstream of the process of tumor suppression or else that it is a matter of structural or cytoskeletal proteins, the regulation of which downstream of expression occurs concomitantly with cell death by apoptosis. 
     TSAP 1 is homologous with rat beta 4 phospholipase C. The TSAP 1 sequence exhibits 100% identity with PLC between nucleotides 3967 and 3985; 82% identity between nucleotides 3986 and 4116 and 85% identity between nucleotides 4070 and 4220 (FIG.  4 ). PLC is known to be involved in the tyrosine kinase receptor signalling pathway and to catalyze the hydrolysis of phosphatidylinositol-4,5-biphosphate to diacylglycerol and inositol-1,4,5-triphosphate. However, the present studies suggest that PLC is a downstream target in p53-mediated apoptosis. 
     TSAP 2 exhibits sequences which are conserved (92% identity between nucleotides 259 and 299; 100% identity between nucleotides 418 and 458 and 92% identity between nucleotides 645 and 685) between it and the zinc finger protein (ZFM 1), which is located in the multiple endocrine neoplasia (MEN 1) locus (FIG.  5 ). MEN 1 is an autosomal dominant disorder which is associated with the development of tumors which affect the anterior lobe of the pituitary and parathyroid glands and the cells of the pancreatic ilots. It is particularly interesting to have demonstrated that both ZFM and an isoenzyme of PLC are co-located in the same chromosomal region, i.e. 11q13, which contains the gene for susceptibility to MEN 1. In mice, the homologous regions are located on chromosome 19B. The fact that TSAP 1 and TSAP 2 are found to be activated in response to p53 may suggest that these genes belong to a more global tumor suppression pathway and that p53 is able to cooperate with MEN 1. 
     TSAP 3 is identical to Siah 1b. This gene is the vertebrate homologue of the Drosophila seven in absentia (sina) gene. The described clone exhibits 94% identity with the murine homologue (nucleotides 1496 to 1634) (FIG.  6 ). Differential expression of a 1.9 kb messenger from this gene has been detected by means of Northern blot analysis using a TSAP 3 probe (FIG.  2 A). This is confirmed by using a second probe which corresponds to the same region of the described siah 1b sequence (FIG.  2 B). FIG. 2C shows the tissue distribution of this gene, using a TSAP 3 probe which detects, at one and the same time, mRNAs of 1.9 and 2.4 kb in size, corresponding to the previously mentioned results when a siah probe is used. The in-situ hybridization shows that the TSAP 3 mRNA is rapidly induced 1 hour after inducing apoptosis (FIG.  3 D). Its expression increases after 2 and 4 hours (FIGS.  3 E and  3 F). No signal is detected in the cells which have entered mitosis. 
     Carthew and Rubin have shown that seven in absentia is required for eye development in drosophila. On the other hand, mutants of this gene exhibit a much more general role in development in drosophila. The murine homologue is subdivided into two groups, i.e. siah 1 and siah 2, and these proteins exhibit a degree of conservation in relation to drosophila seven in absentia which is altogether unusual. 
     Results have shown that TSAP 3/siah 1b is activated in the cell death program in M1 cells which are induced by the p53 tumor suppressor gene. Since this gene encodes a nuclear zinc finger protein, it could be a regulatory transcription factor which is downstream of the p53 signal. The results also show a direct link between the genes which are concerned with development in drosophila and a major tumor suppression pathway. 
     Example 2 
     The above-described murine cDNA fragment (TSAP 3), which was obtained by differential analysis of mRNA, was used to make a probe for isolating a 1.1 kb fragment from a human cDNA library, which fragment was then expanded to encompass the entire coding region by means of RACE-PCR. 
     FIG. 7 shows the cDNA and the amino acid sequence of the human sina gene (TSAP 3). 
     This sequence encodes a 282 amino acid protein which has a C3HC4 zinc finger motif. This protein also exhibits analogies with proteins which are able to attach to RNA. The amino acid sequence is very highly conserved between the drosophila, mouse and human genes (FIG.  7 ). The tissue distribution indicates that human sina is expressed ubiquitously and encodes an mRNA of 2.3 kb; in the placenta, there is an additional transcript of 2.5 kb in size. 
     It was possible to isolate 8 YACs (350-1000 kb) and 2 BACs (100 and 125 kb) by analyzing the YACs of the CEPH and BAC libraries by means of PCR using specific human sina primers. 
     Using the YAC and BAC clones, fluorescence by in-situ hybridization (FISH) shows that the seven in absentia is located on chromosome 16q12-13, that is in a region which contains the genes which are candidate tumor suppressor genes in various cancers, in particular: breast cancer (Bieche, et al.,  Genes Chromosomes and Cancer  14: 227-251 (1995)), Wilm&#39;s tumor (Wang-Wuu, et at.  Cancer Res . 50: 2786-2793 (1990), Maw, et al.  Cancer Res . 52: 3094-3098 (1992), Austruy et al.,  Genes, Chromosomes and Cancer  14: 285-294 (1995)), Laurence, Moon, Bard and Biedl&#39;s syndrome (Kuytek-Black et al.  Nat. Genet . 5(4): 392-396 (1993)) and Beckwith and Wiederman&#39;s syndrome (Newsham et al.  Genes, Chromosomes and Cancer  12(1): 1-7 (1995)). 
     As was pointed out in French patent application No. 95 15 146, it was found that murine M1 cells which were stably transfected with mutant temperature-sensitive p53 exhibited activation of seven in absentia following induction of apoptosis at 32° C. Given the fact that the murine TSAP 3 was isolated in an apoptosis model induced with the p53 gene, it was logical to extend the analysis of the TSAP 3 (HUMSIAH) gene in a model of human physiological apoptosis. 
     This model is described in the intestine, where the cells migrate from the bottom of the crypt toward the apical region of the villosities, where they die by apoptosis before being released into the lumen. These apoptotic cells are specifically labeled by means of the TUNEL technique. Moreover, in physiological apoptosis in man, these same cells are positive for the TSAP 3 (HUMSIAH) gene by in-situ hybridization. 
     Finally, in order to investigate the involvement of the human TSAP 3 gene in tumor suppression, use was made of a model which is based on all the genes rather than on a single gene. This model is based on the biological properties of the H-1 parvovirus. 
     Very exhaustive research in this area has demonstrated over the last 20 years that parvovirus preferentially kills tumor cells while sparing their normal counterpart. 
     So as to construct a model, the following hypothesis was put forward: if it were possible to select cells which were resistant to the cytopathic effect of H-1 parvovirus from a tumor which is sensitive to this effect, this resistance might be due to a change in the malignant phenotype of these resistant cells. It was possible to demonstrate this in the case of KS cells which were selected from human K562 erythroleukemic cells. While the parental K562 cells are sensitive to the cytopathic effect of the H-1 parvovirus, the KS cells are resistant. These resistant cells re-express the wild-type p53 and have a phenotype which is suppressed both in vitro and in vivo. 
     In order to confirm these observations on other cells, daughter US3 and US4 cells were selected from a monoclone of a human U937 monocytic leukemia. These clones are resistant to the cytopathic effect of H-1 parvoviruses and exhibit in vivo reversion of the malignant phenotype. Analysis of the surface markers on 20 cells indicates that there is no shift between U937 and the US clones in the stage of differentiation, in turn indicating that suppression of the malignant phenotype is not due to terminal differentiation. 
     Neither the K562 nor the U937 cells express p53. In contrast to the KS cells, which re-express p53, the US3 and US4 cells do not re-express p53. Nevertheless, it was possible to show that the US3 and US4 cells exhibited activation of WAF-1 as compared with the malignant parental U937 cells. Such activation of WAF-1, in an alternative, p53-independent pathway, has recently been described, and the current results show that the US3 and US4 clones appear to use this alternative WAF-1 pathway. 
     The sina gene is activated by the wild-type p53 which can be induced in M1 cells as well as in the KS cells which re-express the wild-type p53. 
     While the parental U937 cells only express sina mRNA to a very low degree, expression of this mRNA is activated in the daughter US3 and US4 clones, whose malignant phenotype has reverted and which are re-expressing p21 waf-1 . 
     Interestingly, sina is activated in cells which become apoptotic, as has been demonstrated by means of double labeling using a sina probe for in-situ hybridization combined with a TUNEL assay. 
     This demonstrates that the human sina gene, which is very conserved in phylogeny, is involved in apoptosis and tumor suppression. 
     Still more importantly, sina is located at the intersection of the p53 and WAF-1 pathways. 
     In addition, it was possible, using the U937, US3 and US4 model, to demonstrate that the suppressor molecules are functionally linked by using a global biological model which compares parental malignant cells and directly derived daughter cells at molecular levels. These experiments indicate that it is not necessary to transfer specific human tumor suppressor genes so as to confer on them the suppressor phenotype and that, on the contrary, tumor reversion is under the control of a regulatory system which is always present in the genetic material of the tumor cells even if it is necessary to reactivate it. 
     The results depicted in FIG. 9 establish the direct effect of TSAS 3 on apoptosis. FIG. 9A shows the results of a FACS analysis of the DNA content in U937 cells transfected with the vector alone (RSV-C) and those transfected with TSAP 3 (RSV-7S). Respectively, 3% and 12% of the cell population is in the sub G1 phase. FIG. 9B shows the results of a FACS analysis of the TUNEL assay with 3% of the U937 cells transfected with the vector alone (RSV-C), as compared to 15% of the U937 cells transfected with TSAP 3 (RSV-7S) being positive. FIG. 9C shows the results of a tumorigenicity assay in SCID/SCID mice. After injection with either U937 cells transfected with the control vector alone (—O— RSV C), mice form large tumors in 20 out of the 20 injection sites and appear early. The U937 cells stably transfected with TSAP 3 (-•- RSV-75) form smaller tumors. (* indicates the statistical significance: p≦0.001) This tumorigeneicity test shows the TSAP 3 gene&#39;s suppression of tumorigenicity in vivo 
     FIG. 10 shows the characterization of the TSAP 3 protein. FIG. 10A shows expression of TSAP 3 during wt-p53 induction of apoptosis. Specifically this is the results of a Western blot analysis with anti-TSAP3 antibodies generated against the first 16 amino acids of TSAP 3. Lane 1- LTR-6 cells at 37° C. Lane 2- LTR-6 cells after 4 hours of incubation at 32° Lane 3- LTR-6 cells after 7 hours of incubation at 32° C. Lane 4- LTR-6 cells after 9 hours of incubation at 32° C. Lane 5- LTR-6 cells after 16 hours of incubation at 32° C. Lane 6- LTR-6 cells after 24 hours of incubation at 32° Arrow indicates the 30 kDa TSAP 3 protein. FIG. 10B shows the subcellular localization of TSAP 3, via Western blot with anti-TSAP 3 antibodies. Lane 1-nuclear fraction of LTR-6 cells after 4 hours of incubation at 32° C. Lane 2-membrane fraction of LTR-6 cells after 4 hours of incubation at 32° C. Lane 3-cytoplasmic fraction of LTR-6 cells after 4 hours of incubation at 32° C. Arrow indicates the 30 kDa TSAP 3 protein. FIG. 10C shows expression of TSAP 3 in U937 cells with a suppressed malignant phenotype. Lane 1- U937 cells transfected with the control vector alone. Lane 2- US cells derived from U937 cells but displaying a suppressed malignant phenotype. Lane 3- U937 cells stably transfected with TSAP 3 (clone RSV-7S). Lane 4- U937 cells stably transfected with tSAP 3 (clone RSV-8S). Lane 5- U937 cells stably transfected with TSAP 3 (clone RSV-10S). In sum, it was possible to identify in the MI/LTR6 model, a 30 kDA TSAP 3 protein hat induces apopotosis. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 
               
             
             
               
                   
               
               
                 CHARACTERISTICS OF THE CLONES 
               
             
          
           
               
                   
                 Differentially 
                 3′ and 5′ 
                 Size of the 
                   
               
               
                   
                 expressed clone 
                 primers* 
                 mRNA in kb 
                 Homology 
               
               
                   
                   
               
               
                   
                 TSAP 1 
                 T11GC-16 
                 2.0 and 4.5 
                 PLC # 
               
             
          
           
               
                   
                 TSAP 2 
                 T11GC-5 
                 5.9 
                 MEN1 § 
               
               
                   
                 TSAP 3 (IDS No.3) 
                 T11CG-4 
                 1.9 
                 siah 1b ¶ 
               
               
                   
                 TSAP 4 
                 T11GC-6 
                 5.0 
                 No 
               
               
                   
                 TSAP 5 
                 T11CG-5 
                 1.2 
                 No 
               
               
                   
                 TSAP 6 
                 T11AG-1 
                 2.8 
                 NO 
               
               
                   
                 TSAP 7 
                 T11GC-16 
                 &gt;8.0 
                 No 
               
               
                   
                 TSAP 8 
                 T11GC-6 
                 &gt;10.0 
                 No 
               
               
                   
                 TSIP 1 
                 T11CG-8 
                 3.0 
                 No 
               
               
                   
                 TSIP 2 
                 T11AA-5 
                 3.1 
                 AD3  
               
               
                   
                   
               
               
                   
                 *the figures and the sequences of the 5′ primers correspond to those reported by Bauer et al. ,supra (4)  
               
               
                   
                 # rat beta 4 phospholipase C mRNA (RATPHOSCB)  
               
               
                   
                 § human mRNAs (HUMMEN1C: HUMZFM1C: HUMZFM1A: HUMMEN1A)  
               
               
                   
                 ¶ siah-1B mRNA (MMSIAH1B)  
               
               
                   
                  AD3, murine S182 mRNA transcript (human S182 mRNA homologue) (Sherrington et al., supra).  
               
             
          
         
       
     
     Applicants hereby incorporate by reference all journal articles, patents and patent applications referred to above. 
     (1) Liang P. &amp; Pardee A. B. (1992) Science 257, 967-971. 
     (2) Don R. H., Cox P. T., Wainwright B. J., Baker K. &amp; Mattick J. S. (1991) Nucl. Acids Res., 19, 4008. 
     (3) Yonish-Rouach E., Resnitzky D., Lotem J., Sachs L., Kimchi A. &amp; Oren M. (1991) Nature 352, 345-347. 
     (4) Bauer D., Muller H., Reich J., Riedel H., Ahrenkiel V., Warthoe P. &amp; Strauss M. (1993) Nucl. Acids Res. 21, 4272-4280. 
     (5) Sambrook J., Fritsch E. F. &amp; Maniatis T. (1989) Molecular Cloning: a laboratory manual. 
     (6) Okamoto K. &amp; Beach D. (1994) EMBO J., 13, 4816-4822. 
     (7) Angerer L. &amp; Angerer R. C. (1991) Methods in cell biology: functional organization of the nucleus, 35, 37-71. 
     (8) Linares-Cruz G., Rigaut J. P., Vassy J., De Oliveira T. C., De Cremoux P., Olofsson B. &amp; Calvo F. (1994) J. Microsc., 173, 27-38. 
     (9) Bieche I. and Lidereau R., Genes Chromosomes and Cancer 14, 227-251 (1995). 
     (10) Wang-Wuu S., Soukup S., Bove K., Gotwals B. and Lampkin B., Cancer Research 50, 2786-2793 (1990). 
     (11) Maw M. A. et al., Cancer Research 52, 3094-3098 (1992). 
     (12) Austruy E. et al., Genes, Chromosomes and Cancer, 14, 285-294 (1995). 
     (13) Kuytek-Black A. E. et al., Nat. Genet 5(4)n 392-396 (1993). 
     (14) Newsham I. et al., Genes Chromosomes and Cancer 12(1), 1-7, (1995). 
     (15) Sherrington et al., Nature, vol. 375, p. 754-760 (1995) 
     
       
         
           
             22 
           
           
             1 
             230 
             DNA 
             TSAP 1 
           
            1
tgatcacgta cacacacaca cacagagaga gagagagaga gagagagggg gagagagaga     60
gagagagaga tcccctattc ctgacaggca gagttgaatc atgatatatg gcttaaacat    120
gtttgctatg agacagcatc acaagccagt gggcttggtg ataacaactc tgctttgtgg    180
tgcattagga catttttgag ctgctgctgc tgcaaaaaaa ataagagccg               230
 
           
             2 
             143 
             DNA 
             TSAP 2 
           
            2
gcttggaacc aatctacaac agcgagggga agcggcttaa cactcgagag ttccgtaccc     60
gcaaaaaaaa aaatctcttg tgttttccta agcttttccc tgtgctaggg aaagatcagt    120
aagtccgtgg ttatagattg gtt                                            143
 
           
             3 
             146 
             DNA 
             TSAP 3 
           
            3
tttttttttt tgcggggtgg gggtgtgcct gcacacatgc gtgcacgtgt gtgcttggtt     60
ttcctttaac aagccatcta cgtgtcatag cccactgttt tccccttgtg agtcaacaca    120
tagtgctgct gtggtttggg tttggt                                         146
 
           
             4 
             202 
             DNA 
             TSAP 4 
           
            4
aactccgtcg tgggtgtggg gacctaattc cttatatttt tacaacaagc actgtacaaa     60
ctgtgccttt ccctaatgca gttatactat ttccattaag atgggtaacc ttagttaagg    120
ctttatattc actgccatgg gtaggaatgc tcacggtgaa tgggccaact tgtcatggaa    180
gaagccctca ttttcagttg gc                                             202
 
           
             5 
             1309 
             DNA 
             TSAP 5 
           
            5
taacaaggat attcaggttc gggattggtt tcctaagcga tgatctcaac ctccacgtgg     60
aactgatttc ccaagggaca gaaatggtct ttgatctttc tgaaccactt gtcttcaaac    120
tctttggagg acgcaaccac catggcagtc agggctccgg ggcccacaca cttcacctcc    180
gaatgaagct cctcttttat cttttctggg acaatgtctt cccccatagc ctcctccatc    240
aacagcaaag taccttccct aaagttgaag tccttcactt tccctgcaat ttcctgctga    300
gtcctcaagt tcttctccaa cgcgaatgat gtttgctgag actgggcgag ctgaagcagg    360
agcctggcgc ggagcaaaaa ggcgcatgct ttcctccgag cctccatctg tgcctcttcc    420
ctccgccttg ccagggaagg catattctcc tgagcactac cactcgcttc cacggagagc    480
agtgcattct caggcaaggt cgtgggcaaa gacaaaagag agcctgttcc cgagtgtaca    540
gaggagggac cgacggcctt gtcacttgag gcagaactct tctgtccctg cggtgacacc    600
ctgctggcag gccgggccct ggactcaggt atgcctctgc cagcttacac cagctccacg    660
ggttgagcgg gtgcaaagca atcagcttgt gcaggcagaa gatcgtgtgc tcccggctct    720
gcaggctgga aaagacggcc aggtggaggt ggagcaccac ggtcagatgg tctgtgttgg    780
tggctttgct ttccaagtct gccgccatct ccagcgcctc ctcatgcctc ccaagtgagc    840
cagacaccga gcctggcctt cttggacatc ccttttcatg gcaaaattag tagatggtaa    900
tgttcggaga tatggagtat tcctgcaggg ctttctcgta ttcctgtcgt ctgtaggcca    960
ggtcccctct gaatttcttg agagtgagaa cttcaatatc gtcactacat tctgtctctt   1020
cataaaacca tgcggctcgc agagcttggc gcggtagggg gagggcggct cgggccggcg   1080
ctccggcctc tgctcgaaca ccgagtcctc aaattcgccg cccagcaccc agcatccggt   1140
ctccatcgcg cggaagtgca actggacctc gaaacgaggc gacacctaga gcgacgccca   1200
tcacccagcc tccaaagcgc gcgacagcag ccgcgccaag gctgccgagg caaggtagag   1260
acctgcccgg gcggccgctc gagccctata gtgagtcgta ttaggatgg               1309
 
           
             6 
             1203 
             DNA 
             TSAP 6 
             
               unsure 
               (1)..(1203) 
               applicants are unsure of various bases
                         designated as “n” 
             
           
            6
gtgagtacat atcacatgta tggggtgtca ttctgagtat gtcagtttac acctgcatcc     60
caggaattag gatctcagcc acccacgcat atatcatcac ctcgctgtgc agcatccaga    120
aaagagaccc gaacccagct cagggccccc acaagccatc tccacttcca gggcctcaca    180
cgtggcttgt tttctccccc tgtgtgtggt cgccggacag catgaacttg acagccccat    240
ctttctccca gcccctgcgg atcttggtga gtctgcggtt tgaggcaggg caggaggaag    300
aggcccttgg ccaggatgat tcacacaggg gcagggagca gcgtgagtgt ggaatgtggg    360
gcgggcaggt agaacttgkt agtggttttt cctncaaaag gcacgggtcc agccgtaggt    420
gagtgtgtgc attgtgctga gtatcagggc cacgaagccc agtgtggact gcacgaagct    480
gaactccttc cagttgaggg aattagcaat ggacgggagc gaggtgacag ccagcagcga    540
caacatgccc agggccagca cacccaggga caggtatatc tccatcctcc agacttcttc    600
ctcagcccag aggcggctct tgttggccag gacctgcttc acagccagat tgaccaggtc    660
gtaggcggtg ggagcggcgc agcggcaggc agaagctgta gagagcgtgc agcatcgcga    720
agaagaagct gagcagcccg atctgcttgc gatgctgcag ccagtggtcc agccagtctg    780
ggaagcgctg gtacttggtc cccctccgca gctgaagcgc agctgccagc acaccgggca    840
ggtacactag ggacagcagc acataagcca cacagggtag tgtggtgttg accacagaca    900
agggcatctt gtaaaacttg ttctcatctt tccgaatgtn tggctgtana acgtcccgga    960
tgaaattgta ggtgtanaan cacacaaaga ccccagtgcc caggaaggtg ggccccttcc   1020
agaatggaag gaagcncagg ggtttngctt ctacctccct cnctgaaggc canggatcca   1080
tntccagggg ttnaaaccat ngggcgtgca tctctgaaaa tggtcncttg gnttctggtk   1140
gatcamtgca aataacncct gcctgttccn tcccttgggg ccaccctntn ggggccatgc   1200
caa                                                                 1203
 
           
             7 
             140 
             DNA 
             TSAP 7 
           
            7
gcccatccag tcattcttta tttcagtgtg tgaaagcctc ctacgcattt tcccccaaat     60
taatttttaa tccattttca aaccagcctt tactgtggcc ttttctgcta tttttgatat    120
atgttagcac gtgtgcatag                                                140
 
           
             8 
             257 
             DNA 
             TSAP 8 
             
               unsure 
               (1)..(257) 
               applicants are  unsure of  various bases
                         designated as “n” 
             
           
            8
cacgtnaaag taccacatcc ncccccattg gtagatattg anagagtata tanataggnc     60
gaagcacaat ctcttccctt cctntgtaca cctcanaccc agtgacttcc naccnaagcn    120
cntgantgtn tttgtngata tgagtgtctg ngtgtgtgna tntgcgtctc acatgtatgg    180
gacgaccnac cccaccccca gcggccttca ngcacaatng aggacgccta tngtggatac    240
gngcatcggt aaanagc                                                   257
 
           
             9 
             111 
             DNA 
             TSIP 1 
           
            9
ggagggggtc tagctttctc tttagttatc actctgaggt gctcaggtca cagagaaggc     60
acttaattgg gaaggtcatc tgattccggc catcttctct ccctttacca a             111
 
           
             10 
             2681 
             DNA 
             TSIP 2 
           
            10
caccggtgag acctctaggg cggggcctag gacgacctgc tccgtgggcc gcgagtattc     60
gtcggaaaca aaacagcggc agctgaggcg gaaacctagg ctgcgagccg gccgcccggg    120
cgcggagaga gaaggaacca acacaagaca gcagcccttc gaggtcttta ggcagcttgg    180
aggagaacac atgagagaaa gaatcccaag aggttttgtt ttctttgaga aggtatttct    240
gtccagctgc tccaatgaca gagatacctg cacctttgtc ctacttccag aatgcccaga    300
tgtctgagga cagccactcc agcagcgcca tccggagcca gaatgacagc caagaacggc    360
agcagcagca tgacaggcag agacttgaca accctgagcc aatatctaat gggcggcccc    420
agagtaactc aagacaggtg gtggaacaag atgaggagga agacgaagag ctgacattga    480
aatatggagc caagcatgtc atcatgctct ttgtccccgt gaccctctgc atggtcgtcg    540
tcgtggccac catcaaatca gtcagcttct atacccggaa ggacggtcag ctaatctaca    600
ccccattcac agaagacact gagactgtag gccaaagagc cctgcactcg atcctgaatg    660
cggccatcat gatcagtgtc attgtcatta tgaccatcct cctggtggtc ctgtataaat    720
acaggtgcta caaggtcatc cacgcctggc ttattatttc atctctgttg ttgctgttct    780
ttttttcgtt catttactta ggggaagtat ttaagaccta caatgtcgcc gtggactacg    840
ttacagtagc actcctaatc tggaattttg gtgtggtcgg gatgattgcc atccactgga    900
aaggccccct tcgactgcag caggcgtatc tcattatgat cagtgccctc atggccctgg    960
tatttatcaa gtacctcccc gaatggaccg catggctcat cttggctgtg atttcagtat   1020
atgatttggt ggctgtttta tgtcccaaag gcccacttcg tatgctggtt gaaacagctc   1080
aggaaagaaa tgagactctc tttccagctc ttatctattc ctcaacaatg gtgtggttgg   1140
tgaatatggc tgaaggagac ccagaagccc aaaggagggt acccaagaac cccaagtata   1200
acacacaaag agcggagaga gagacacagg acagtggttc tgggaacgat gatggtggct   1260
tcagtgagga gtgggaggcc caaagagaca gtcacctggg gcctcatcgc tccactcccg   1320
agtcaagagc tgctgtccag gaactttctg ggagcattct aacgagtgaa gacccggagg   1380
aaagaggagt aaaacttgga ctgggagatt tcattttcta cagtgttctg gttggtaagg   1440
cctcagcaac cgccagtgga gactggaaca caaccatagc ctgctttgta gccatactga   1500
tcggcctgtg ccttacatta ctcctgctcg ccattttcaa gaaagcgttg ccagccctcc   1560
ccatctccat caccttcggg ctcgtgttct acttcgccac ggattacctt gtgcagccct   1620
tcatggacca acttgcattc catcagtttt atatctagcc tttctgcagt tagaacatgg   1680
atgtttcttc tttgattatc aaaaacacaa aaacagagag caagcccgag gaggagactg   1740
gtgactttcc tgtgtcctca gctaacaaag gcaggactcc agctggactt ctgcagcttc   1800
cttccgagtc tccctagcca cccgcactac tggactgtgg aaggaagcgt ctacagagga   1860
acggtttcca acatccatcg ctgcagcaga cggtgtccct cagtgacttg agagacaagg   1920
acaaggaaat gtgctgggcc aaggagctgc cgtgctctgc tagctttgac cgtgggcatg   1980
gagatttacc cgcactgtga actctctaag gtaaacaaag tgaggtgaac caaacagagc   2040
tgccatyctt ccacaccatg ttggaaataa aaccgtccta gctggaaccc ttactgtccc   2100
aggaggttcc gtgtgggggt ggcactgggc cgggcctccc tctcaggctc ctttgctgcc   2160
cacttgtaag tttaaataag gacaccgccc tacacaaacc tcacccctgt cacatccagt   2220
gactctgacc actttagttc tcaaactctc tcactattat ctgtggttgc cgtttcttcc   2280
caaggccagc ctggacgaat ttggggttgc tctatcctga gagttgtaac ctcaacttcc   2340
aaagtttata ttttcttgaa atgatggatc tattgctcaa cagtccctgt catccttaag   2400
tgacttctgg gtttcccaca aattcctcac ttttagacac actctaagct tacttctggc   2460
ctggatgctt cctctccctg tctctccctt gccccacagc ggttccctga cagcagacaa   2520
ggcagctctg ggaggtagct agtatccaat aacccagggg tttcctcatg tgatgcaaat   2580
actacgtgtc caaccaatca gtgctgtcaa cgggctgcca tagctccttc gatggcaaat   2640
aggatgtgtg cccaaagaat taaagcgatc agtggctggt g                       2681
 
           
             11 
             1884 
             DNA 
             TSAP 3 
             
               CDS 
               (1)..(846) 
             
           
            11
atg agc cgt cag act gct aca gca tta cct acc ggt acc tcg aag tgt       48
Met Ser Arg Gln Thr Ala Thr Ala Leu Pro Thr Gly Thr Ser Lys Cys
  1               5                  10                  15
cca cca tcc cag agg gtg cct gcc ctg act ggc aca act gca tcc aac       96
Pro Pro Ser Gln Arg Val Pro Ala Leu Thr Gly Thr Thr Ala Ser Asn
             20                  25                  30
aat gac ttg gcg agt ctt ttt gag tgt cca gtc tgc ttt gac tat gtg      144
Asn Asp Leu Ala Ser Leu Phe Glu Cys Pro Val Cys Phe Asp Tyr Val
         35                  40                  45
tta ccg ccc att ctt caa tgt cag agt ggc cat ctt gtt tgt agc aac      192
Leu Pro Pro Ile Leu Gln Cys Gln Ser Gly His Leu Val Cys Ser Asn
     50                  55                  60
tgt cgc cca aag ctc aca tgt tgt cca act tgc cgg ggc cct ttg gga      240
Cys Arg Pro Lys Leu Thr Cys Cys Pro Thr Cys Arg Gly Pro Leu Gly
 65                  70                  75                  80
tcc att cgc aac ttg gct atg gag aaa gtg gct aat tca gta ctt ttc      288
Ser Ile Arg Asn Leu Ala Met Glu Lys Val Ala Asn Ser Val Leu Phe
                 85                  90                  95
ccc tgt aaa tat gcg tct tct gga tgt gaa ata act ctg cca cac aca      336
Pro Cys Lys Tyr Ala Ser Ser Gly Cys Glu Ile Thr Leu Pro His Thr
            100                 105                 110
gaa aaa gca gac cat gaa gag ctc tgt gag ttt agg cct tat tcc tgt      384
Glu Lys Ala Asp His Glu Glu Leu Cys Glu Phe Arg Pro Tyr Ser Cys
        115                 120                 125
ccg tgc cct ggt gct tcc tgt aaa tgg caa ggc tct ctg gat gct gta      432
Pro Cys Pro Gly Ala Ser Cys Lys Trp Gln Gly Ser Leu Asp Ala Val
    130                 135                 140
atg ccc cat ctg atg cat cag cat aag tcc att aca acc cta cag gga      480
Met Pro His Leu Met His Gln His Lys Ser Ile Thr Thr Leu Gln Gly
145                 150                 155                 160
gag gat ata gtt ttt ctt gct aca gac att aat ctt cct ggt gct gtt      528
Glu Asp Ile Val Phe Leu Ala Thr Asp Ile Asn Leu Pro Gly Ala Val
                165                 170                 175
gac tgg gtg atg atg cag tcc tgt ttt ggc ttt cac ttc atg tta gtc      576
Asp Trp Val Met Met Gln Ser Cys Phe Gly Phe His Phe Met Leu Val
            180                 185                 190
tta gag aaa cag gaa aaa tac gat ggt cac cag cag ttc ttc gca atc      624
Leu Glu Lys Gln Glu Lys Tyr Asp Gly His Gln Gln Phe Phe Ala Ile
        195                 200                 205
gta cag ctg ata gga aca cgc aag caa gct gaa aat ttt gct tac cga      672
Val Gln Leu Ile Gly Thr Arg Lys Gln Ala Glu Asn Phe Ala Tyr Arg
    210                 215                 220
ctt gag cta aat ggt cat agg cga cga ttg act tgg gaa gcg act cct      720
Leu Glu Leu Asn Gly His Arg Arg Arg Leu Thr Trp Glu Ala Thr Pro
225                 230                 235                 240
cga tct att cat gaa gga att gca aca gcc att atg aat agc gac tgt      768
Arg Ser Ile His Glu Gly Ile Ala Thr Ala Ile Met Asn Ser Asp Cys
                245                 250                 255
cta gtc ttt gac cca gca ttg cac agc ttt ttg cag aca aat ggc aat      816
Leu Val Phe Asp Pro Ala Leu His Ser Phe Leu Gln Thr Asn Gly Asn
            260                 265                 270
tta ggc atc aat gta act att tcc atg tgt tgaaatggca atcaaacatt        866
Leu Gly Ile Asn Val Thr Ile Ser Met Cys
        275                 280
ttctggccag tgtttaaaac ttcagtttca cagaaaataa ggcacccatc tgtctgccaa    926
cctaaaactc tttcggtagg tagaagctcg acatgaaggc caataaaaag aaagactgct    986
aaatacagga aacagttcca tgtagtaaca ctaatatatt taaaaataag tcaacagtaa   1046
accactgaaa aaatatatgt atatacaccc aagatgggca tcttttgtat taagaaagga   1106
agcattgtaa aataattctg agttttgtgt ttgttgtaga ttgattgtat tgttgaaaaa   1166
gtttgttttt gcgtgggagt gtgtgcctgc gtgggtgtgt gcgtgtttgg gtttttttcc   1226
tttaactgac aagccatctt gagtggtcat gggccactgc ttttcccttt gtgagtcaat   1286
acatagtgct gctgtaagcc gtttttgtgt gtatttgcta atttttatta attttagttt   1346
ttcattaaat aaatttgact tttctgtaat tcaggttttt cctttttttg taccatttta   1406
aagttagtat cttttgatat ggcatatttg tttatggtaa aaaatttata acgggttcaa   1466
tattttcttt tcccccatta atcaagtcca ttggaaatat tttaaaacca gcctattttg   1526
gtgaacccat gagttcccag aaagtaaagg tgacacccgg aaaaataatc caaaagccta   1586
tttaaagcca cctataaggt gccccccttt cctgtcttcc tacagatgag tcacaccttt   1646
gagccttaac ctttgaaagg ttagagaata aattgatttt tataaatact gcaaatccag   1706
gcttttgttt cctttttcca gatatccttg gacaaatcac atattttaaa atttgttctt   1766
gtatttattg gttttgcaga agaaggcatc gtcatgcaca gtatttgtaa ttaaaagcaa   1826
attcatttgt ttaaaaaggc agtttgcaaa aaatgttttt ggtcttttat aattctca     1884
 
           
             12 
             282 
             PRT 
             TSAP 3 
           
            12
Met Ser Arg Gln Thr Ala Thr Ala Leu Pro Thr Gly Thr Ser Lys Cys
  1               5                  10                  15
Pro Pro Ser Gln Arg Val Pro Ala Leu Thr Gly Thr Thr Ala Ser Asn
             20                  25                  30
Asn Asp Leu Ala Ser Leu Phe Glu Cys Pro Val Cys Phe Asp Tyr Val
         35                  40                  45
Leu Pro Pro Ile Leu Gln Cys Gln Ser Gly His Leu Val Cys Ser Asn
     50                  55                  60
Cys Arg Pro Lys Leu Thr Cys Cys Pro Thr Cys Arg Gly Pro Leu Gly
 65                  70                  75                  80
Ser Ile Arg Asn Leu Ala Met Glu Lys Val Ala Asn Ser Val Leu Phe
                 85                  90                  95
Pro Cys Lys Tyr Ala Ser Ser Gly Cys Glu Ile Thr Leu Pro His Thr
            100                 105                 110
Glu Lys Ala Asp His Glu Glu Leu Cys Glu Phe Arg Pro Tyr Ser Cys
        115                 120                 125
Pro Cys Pro Gly Ala Ser Cys Lys Trp Gln Gly Ser Leu Asp Ala Val
    130                 135                 140
Met Pro His Leu Met His Gln His Lys Ser Ile Thr Thr Leu Gln Gly
145                 150                 155                 160
Glu Asp Ile Val Phe Leu Ala Thr Asp Ile Asn Leu Pro Gly Ala Val
                165                 170                 175
Asp Trp Val Met Met Gln Ser Cys Phe Gly Phe His Phe Met Leu Val
            180                 185                 190
Leu Glu Lys Gln Glu Lys Tyr Asp Gly His Gln Gln Phe Phe Ala Ile
        195                 200                 205
Val Gln Leu Ile Gly Thr Arg Lys Gln Ala Glu Asn Phe Ala Tyr Arg
    210                 215                 220
Leu Glu Leu Asn Gly His Arg Arg Arg Leu Thr Trp Glu Ala Thr Pro
225                 230                 235                 240
Arg Ser Ile His Glu Gly Ile Ala Thr Ala Ile Met Asn Ser Asp Cys
                245                 250                 255
Leu Val Phe Asp Pro Ala Leu His Ser Phe Leu Gln Thr Asn Gly Asn
            260                 265                 270
Leu Gly Ile Asn Val Thr Ile Ser Met Cys
        275                 280
 
           
             13 
             287 
             DNA 
             RAT 
           
            13
cttcttctac ttaacaattt gactattgaa tttctttggc caaccaaaag tagctatgta     60
cacacacaca cacacacaca cacacacaca cacacacaca cacagaaatc ccctattcct    120
gacaggcaga gttgaaccat aatccacaac ttaaacatgt tggctagggg acagcatcac    180
aagccagtgg gcttggtgat aacaactctg ctttgtggtg cattaggaca tgttcgagct    240
cctgctggaa aaggaaaatt agtgcattag tactttaatg gcaagcc                  287
 
           
             14 
             170 
             DNA 
             HUMAN 
           
            14
cccctgagcc catctacaat agcgagggga agcggcttaa cacccgagag ttccgcaccc     60
gcaaaaagct ggaagaggag cggcacaacc tcatcacaga gatggttgca ctcaatccgg    120
atttcaagcc acctgcagat tacaaacctc cagcaacacg tgtgagtgat               170
 
           
             15 
             240 
             DNA 
             MOUSE 
           
            15
ttgtaaaata tttctgaact ttgtatttgt tgtagattga ttgtattgtt gacaattttt     60
cggggtgggg gtgtgcctgc acacatgcgt gcacgtgtgt gcttggtttt cctttaacaa    120
gccatctacg tgtcatagcc cactcttttc cccttgtgag tcaacacata gtgctgctgt    180
ggttttggtt tggtttgctt ttggtttttg atgtgtgtgt atttgataat ttttattcta    240
 
           
             16 
             1636 
             DNA 
             MOUSE 
             
               unsure 
               (1)..(1636) 
               applicants are unsure of various bases
                         designated as “n” 
             
           
            16
accanacanc ggcagctgag gcggaaacct aggctgcgag ccggccgccc gggcgcggag     60
agagaaggaa ccaacacaag acagcagccc ttcgaggtct ttaggcagct tggaggagaa    120
cacatgagag aaagaatccc aagaggtttt gttttctttg agaaggtatt tctgtccagt    180
tgctccaatg acagagatac ctgcaccttt gtcctacttc cagaatgccc agatgtctga    240
ggacagccac tccagcagcg ccatccggag ccagaatgac agccaagaac ggcagcagca    300
gcatgacagg cagagacttg acaaccctga gccaatatct aatgggcggc cccagagtaa    360
ctcaagacag gtggtggaac aagatgagga ggaagacgaa gagctgacat tgaaatatgg    420
agccaagcat gtcatcatgc tctttgtccc ccgtgaccct ctgcatggtc gtcgtcgtgg    480
ccaccatcaa atcagtcagc ttctataccc ggaaggacgg tcagctaatc tacaccccat    540
tcacagaaga cactgagact gtaggccaaa gagccctgca ctcgatcctg aatgcggcca    600
tcatgatcag tgtcattgtc attatgacca tcctcctggt ggtcctgtat aaatacaggt    660
gctacaaggt catccacgcc tggcttatta tttcatctct gttgttgctg ttcttttttt    720
cgttcattta cttaggggaa gtatttaaga cctacaatgt cgccgtggac tacgttacag    780
tagcactcct aatctggaat tttggtgtgg tcgggatgat tgccatccac tggaaaggcc    840
cccttcgact gcagcaggcg tatctcatta tgatcagtgc cctcatggcc ctggtattta    900
tcaagtacct ccccgaatgg accgcatggc tcatcttggc tctgatttca gtatatgatt    960
tggtggctgt tttatgtccc aaaggcccac ttcgtatgct ggttgaaaca gctcaggaaa   1020
gaaatgagac tctctttcca gctcttatct attcctcaac aatggtgtgg ttggtgaata   1080
tggctgaagg agacccagaa gcccaaagga gggtacccaa gaaccccaag tataacacac   1140
aaagagcgga gagagagaca caggacagtg gttctgggaa cgatgatggt ggcttcagtg   1200
aggagtggga ggcccaaaga gacagtcacc tggggcctca tcgctccact ccccgagtca   1260
agagctgctg tccaggaact ttctgggagc attctaacga gtgaagaccc ggaggaaaga   1320
ggagtaaaac ttggactggg agatttcatt ttctacagtg ttctggttgg taaggcctca   1380
gcaaccgcca gtggagactg gaacacaacc atagcctgct ttgtagccat actgatcggc   1440
ctgtgcctta cattactcct gctcgccatt ttcaagaaag cgttgccagc cctccccatc   1500
tccatcacct tcgggctcgt gttctacttc gccacggatt accttgtgca gcccttcatg   1560
gaccaacttg cattccatca gttttgagat ttacccgcac tgtgaactct ctaaggtaaa   1620
caaagtgagg tgaacc                                                   1636
 
           
             17 
             282 
             PRT 
             HUMAN 
           
            17
Met Ser Arg Gln Thr Ala Thr Ala Leu Pro Thr Gly Thr Ser Lys Cys
  1               5                  10                  15
Pro Pro Ser Gln Arg Val Pro Ala Leu Thr Gly Thr Thr Ala Ser Asn
             20                  25                  30
Asn Asp Leu Ala Ser Leu Phe Glu Cys Pro Val Cys Phe Asp Tyr Val
         35                  40                  45
Leu Pro Pro Ile Leu Gln Cys Gln Ser Gly His Leu Val Cys Ser Met
     50                  55                  60
Cys Arg Pro Lys Leu Thr Cys Cys Pro Thr Cys Arg Gly Pro Leu Gly
 65                  70                  75                  80
Ser Ile Arg Asn Leu Ala Met Glu Lys Val Ala Asn Ser Val Leu Phe
                 85                  90                  95
Pro Cys Lys Tyr Ala Ser Ser Gly Cys Glu Ile Thr Leu Pro His Thr
            100                 105                 110
Glu Lys Ala Asp His Glu Glu Leu Cys Glu Phe Arg Pro Tyr Ser Cys
        115                 120                 125
Pro Cys Pro Gly Ala Ser Cys Lys Trp Gln Gly Ser Leu Asp Ala Val
    130                 135                 140
Met Pro His Leu Met His Gln His Lys Ser Ile Thr Thr Leu Gln Gly
145                 150                 155                 160
Glu Asp Ile Val Phe Leu Ala Thr Asp Ile Asn Leu Pro Gly Ala Val
                165                 170                 175
Asp Trp Val Met Met Gln Ser Cys Phe Gly Phe His Phe Met Leu Val
            180                 185                 190
Leu Glu Lys Gln Glu Lys Tyr Asp Gly His Gln Gln Phe Phe Ala Ile
        195                 200                 205
Val Gln Leu Ile Gly Thr Arg Lys Gln Ala Glu Asn Phe Ala Tyr Arg
    210                 215                 220
Leu Glu Leu Asn Gly His Arg Arg Arg Leu Thr Trp Glu Ala Thr Pro
225                 230                 235                 240
Arg Ser Ile His Glu Gly Ile Ala Thr Ala Ile Met Asn Ser Asp Cys
                245                 250                 255
Leu Val Phe Asp Thr Ser Ile Ala Gln Leu Phe Ala Glu Asn Gly Asn
            260                 265                 270
Leu Gly Ile Met Val Thr Ile Ser Met Cys
        275                 280
 
           
             18 
             282 
             PRT 
             MOUSE 
           
            18
Met Ser Arg Gln Thr Ala Thr Ala Leu Pro Thr Gly Thr Ser Lys Cys
  1               5                  10                  15
Pro Pro Ser Gln Arg Val Pro Ala Leu Thr Gly Thr Thr Ala Ser Asn
             20                  25                  30
Asn Asp Leu Ala Ser Leu Phe Glu Cys Pro Val Cys Phe Asp Tyr Val
         35                  40                  45
Leu Pro Pro Ile Leu Gln Cys Gln Ser Gly His Leu Val Cys Ser Met
     50                  55                  60
Cys Arg Pro Lys Leu Thr Cys Cys Pro Thr Cys Arg Gly Pro Leu Gly
 65                  70                  75                  80
Ser Ile Arg Asn Leu Ala Met Glu Lys Val Ala Asn Ser Val Leu Phe
                 85                  90                  95
Pro Cys Lys Tyr Ala Ser Ser Gly Cys Glu Ile Thr Leu Pro His Thr
            100                 105                 110
Glu Lys Ala Glu His Glu Glu Leu Cys Glu Phe Arg Pro Tyr Ser Cys
        115                 120                 125
Pro Cys Pro Gly Ala Ser Cys Lys Trp Gln Gly Ser Leu Asp Ala Val
    130                 135                 140
Met Pro His Leu Met His Gln His Lys Ser Ile Thr Thr Leu Gln Gly
145                 150                 155                 160
Glu Asp Ile Val Phe Leu Ala Thr Asp Ile Asn Leu Pro Gly Ala Val
                165                 170                 175
Asp Trp Val Met Met Gln Ser Cys Phe Gly Phe His Phe Met Leu Val
            180                 185                 190
Leu Glu Lys Gln Glu Lys Tyr Asp Gly His Gln Gln Phe Phe Ala Ile
        195                 200                 205
Val Gln Leu Ile Gly Thr Arg Lys Gln Ala Glu Asn Phe Ala Tyr Arg
    210                 215                 220
Leu Glu Leu Asn Gly His Arg Arg Arg Leu Thr Trp Glu Ala Thr Pro
225                 230                 235                 240
Arg Ser Ile His Glu Gly Ile Ala Thr Ala Ile Met Asn Ser Asp Cys
                245                 250                 255
Leu Val Phe Asp Thr Ser Ile Ala Gln Leu Phe Ala Glu Asn Gly Asn
            260                 265                 270
Leu Gly Ile Met Val Thr Ile Ser Met Cys
        275                 280
 
           
             19 
             282 
             PRT 
             MOUSE 
           
            19
Met Ser Arg Gln Ala Ala Thr Ala Leu Ser Thr Gly Thr Ser Lys Cys
  1               5                  10                  15
Pro Pro Ser Gln Arg Val Pro Ala Leu Thr Asp Thr Thr Ala Ser Asn
             20                  25                  30
Asn Asp Leu Ala Ser Leu Phe Glu Cys Pro Val Cys Phe Asp Tyr Val
         35                  40                  45
Leu Pro Pro Ile Leu Gln Cys Gln Ser Gly His Leu Val Cys Ser Asn
     50                  55                  60
Cys Arg Pro Lys Leu Thr Cys Cys Pro Thr Cys Arg Gly Pro Leu Gly
 65                  70                  75                  80
Ser Ile Arg Asn Leu Ala Met Glu Lys Val Ala Asn Ser Val Leu Phe
                 85                  90                  95
Pro Cys Lys Tyr Ser Ala Ser Gly Cys Glu Ile Thr Leu Pro His Thr
            100                 105                 110
Lys Lys Ala Glu His Glu Glu Leu Cys Glu Phe Arg Pro Tyr Ser Cys
        115                 120                 125
Pro Cys Pro Gly Ala Ser Cys Lys Trp Gln Gly Ser Leu Asp Ala Val
    130                 135                 140
Met Pro His Leu Met His Gln His Lys Ser Ile Thr Thr Leu Gln Gly
145                 150                 155                 160
Glu Asp Ile Val Phe Leu Ala Thr Asp Ile Asn Leu Pro Gly Ala Val
                165                 170                 175
Asp Trp Val Met Met Gln Ser Cys Phe Gly Phe His Phe Met Leu Val
            180                 185                 190
Leu Glu Lys Gln Glu Lys Tyr Asp Gly His Gln Gln Phe Phe Ala Ile
        195                 200                 205
Val Gln Leu Ile Gly Thr Arg Lys Gln Ala Glu Met Phe Ala Tyr Arg
    210                 215                 220
Leu Glu Leu Asn Gly His Arg Arg Arg Leu Thr Trp Glu Ala Thr Pro
225                 230                 235                 240
Arg Ser Ile His Glu Gly Ile Ala Thr Ala Ile Met Asn Ser Asp Cys
                245                 250                 255
Leu Val Phe Asp Thr Ser Ile Ala Gln Leu Phe Ala Glu Asn Gly Asn
            260                 265                 270
Leu Gly Ile Met Val Thr Ile Ser Met Cys
        275                 280
 
           
             20 
             314 
             PRT 
             DROSINA 
           
            20
Met Ser Asn Lys Ile Met Pro Lys Arg Arg Glu Pro Thr Ala Ala Ala
  1               5                  10                  15
Ala Gly Ala Gly Ala Thr Gly Val Ala Thr Asn Thr Ser Thr Ser Thr
             20                  25                  30
Gly Ser Ser Ser Ala Gly Asn Thr Ser Ser Ala Met Thr Ser Ser Ser
         35                  40                  45
Ser Ser Ser Ser Leu Ser Ser Ala Gly Gly Gly Gly Ala Gly Met Ser
     50                  55                  60
Ala Asp Leu Thr Ser Leu Phe Glu Cys Pro Val Cys Phe Asp Tyr Val
 65                  70                  75                  80
Leu Pro Pro Ile Leu Gln Cys Ser Ser Gly His Leu Val Cys Val Ser
                 85                  90                  95
Cys Arg Ser Lys Leu Thr Cys Cys Pro Thr Cys Arg Gly Pro Leu Ala
            100                 105                 110
Met Ile Arg Asn Leu Ala Met Glu Lys Val Ala Ser Asn Val Lys Phe
        115                 120                 125
Pro Cys Lys His Ser Gly Tyr Gly Cys Thr Ala Ser Leu Val Tyr Thr
    130                 135                 140
Glu Lys Thr Glu His Glu Glu Thr Cys Glu Cys Arg Pro Tyr Leu Cys
145                 150                 155                 160
Pro Cys Pro Gly Ala Ser Cys Lys Trp Gln Gly Pro Leu Asp Leu Val
                165                 170                 175
Met Gln His Leu Met Met Ser His Lys Ser Ile Thr Thr Leu Gln Gly
            180                 185                 190
Glu Asp Ile Val Phe Leu Ala Thr Asp Ile Asn Leu Pro Gly Ala Val
        195                 200                 205
Asp Trp Val Met Met Gln Ser Cys Phe Gly His His Phe Met Leu Val
    210                 215                 220
Leu Glu Lys Gln Glu Lys Tyr Asp Gly His Gln Gln Phe Phe Ala Ile
225                 230                 235                 240
Val Gln Leu Ile Gly Ser Arg Lys Glu Ala Glu Asn Phe Val Tyr Arg
                245                 250                 255
Leu Glu Leu Asn Gly Asn Arg Arg Arg Leu Thr Trp Glu Ala Met Pro
            260                 265                 270
Arg Ser Ile His Glu Gly Val Ala Ser Ala Ile His Met Ser Asp Cys
        275                 280                 285
Leu Val Phe Asp Thr Ser Ile Ala Gln Leu Phe Ala Asp Met Gly Met
    290                 295                 300
Leu Gly Ile Met Val Thr Ile Ser Leu Val
305                 310
 
           
             21 
             20 
             DNA 
             primer 
           
            21
cagtaaacca ctgaaaaacc                                                 20
 
           
             22 
             19 
             DNA 
             primer 
           
            22
caaaccaaac caaaaccac                                                  19