MXPA99004677A - Survivin, a protein that inhibits cellular apoptosis, and its modulation - Google Patents

Abstract

The present invention provides the amino acid of a protein that inhibits cellular apoptosis, herein termed the Survivin protein and nucleic acid molecules that encode Survivin. Based on this disclosure, the present invention provides isolated Survivin protein, isolated Survivin encoding nucleic acid molecules, methods of isolating other members of the Survivin family of proteins, methods for identifying agent that blocks Survivin mediated inhibition of cellular apoptosis, methods of using agent that block Survivin mediated inhibition or Survivin expression to modulate biological and pathological processes, and methods of assaying Survivin activity.

Description

SURVIVINA. A PROTEIN THAT INHIBITS CELLULAR APOPTOSIS. AND ITS MODULATION DECLARATION OF RELATED REQUESTS This application is based on the provisional US application Serial No. 60/031, 435, filed on November 20, 1996, the description of which is incorporated herein by reference.

FIELD OF THE INVENTION The present invention relates to the field of modulating cellular apoptosis, particularly to agents useful for inhibiting apoptosis, as well as to diagnostic and prognostic analyzes involving conditions mediated by the expression of apoptosis inhibitors. The invention relates specifically to the identification of a novel human gene, tentatively called Survivin. Survivin encodes a protein, survivin, that inhibits cellular apoptosis, particularly in cancer cells and in embryonic cells.

BACKGROUND OF THE INVENTION Regulation of cell proliferation by programmed cell death (apoptosis) maintains tissue homeostasis during development and differentiation (Raff, MD, Nature (1992) 356: 397-400; Vaux, D.L and co-authors, Ce // J1994) 76: 777-779). This process involves a cascade of many steps, conserved in an evolutionary manner (Oltvai, Z and coauthors, Cell (1994) 79: 189-192) and is controlled by proteins that promote or counteract apoptotic cell death. Apoptosis also involves cell surface receptors (Smith, A. and coauthors, Cell (1994), 76, 959-962) and associated signal transducers (Tartaglia, LA, and co-authors Immunol. Today, (1992) 13: 151 -153), protease gene families (Martin, SJ and co-authors Cell (1995) 82: 349-352), second intracellular messengers (Kroemer, G and co-authors, FASEB J. (1995) 9: 1277-1287), genes tumor suppressors (Haffner, R. Y co-authors, Curr Op Gen Dev, (1995) 5: 84-90), and negative regulatory proteins that counteract apoptotic cell death (Hockenbery, D. Y co-authors, Nature (1990) 348: 334-336). Aberrantly increased apoptosis, or abnormally prolonged survival of cells (Oltvai, ZN, and co-authors, Cell (1994) 79: 189-192) can both contribute to the pathogenesis of human diseases, including autoimmune disorders, neurodegenerative processes and cancer (Steller, H, Science (1995) 267-1445-1449; Thompson, CB, Science (1995) 267: 1456: 1462). Specifically, for example, apoptosis inhibitors, most notably from the bcl-2 family (Reed, JJ Cell Biol (1994) 124: 1-6, and Yang, E., and co-authors, Blood (1995) 88: 386- 401), maintain lymphoid homeostasis and morphogenesis in adult tissues (Hockenbery, D., and co-authors, Proc. Nati, Acad. Sci., USA (1991) 88: 6961-6965) and fetal (LeBrun, D., and co-authors ( 1993), 142: 743.753). The deregulated expression of Jbc / -2 has also been implicated in cancer, aberrantly prolonging the survival of the cells and facilitating the appearance of transformative mutations. In addition to bcl-2, several members of a new family of genes, of apoptosis inhibitors, related to the baculovirus IAP gene have been identified (Birnbaum, MJ, and co-authors, J. Virology (1994) 58: 2521-2528; Clem, RJ, and co-authors, Mol. Cell Biol. (1994) 14: 5212-5222) in Drosophila and mammalian cells (Duckett, O S. Y coauthors EMBO J. (1996), 15: 2685-2694; , BA and coauthors, Cell (1995) 83: 1253-1262; Liston, P. and co-authors, Nature (1996) 379: 349-353; Rothe, M. And co-authors, Cell 83: 1243-1252; Roy, N and coauthors, Cell (1995) 80: 167-178.These molecules are evolutionarily highly conserved, they share a similar architecture, organized in two or three repeats of IAP in baculovirus (BIR) of Cys-His end, of approximately 70 amino acids and by a carboxy-terminal zinc binding domain, designated RING index (Duckett, CS, and co-authors, EMBO J. (1996) 15: 2685-2694; Hay, BA and co-authors, Cell (1995) 83: 1253-1262 Lath, P. and co-authors Nature (1996) 379: 349-353; Rothe, M. and co-authors Cell (1995) 83: 1243-1252; Roy N, and co-authors, Cell (1995) 80: 167-178). Recombinant expression of IAP proteins blocks apoptosis induced by various stimuli in vitro (Duckett, CS Y co-authors, EMBO J (1996) 15: 2685-2694; Listón, P, and co-authors, Nature (1996) 379: 349-353 ) and promotes the abnormally prolonged survival of cells in the regulated Drosophila eye model in development, in vivo (Hay, B. A, and coauthors, Cell (1995) 83: 1253-1262). Finally, omissions were reported in a neuronal IAP inhibitor of apoptosis, NAIP, in 75% of patients with spinal muscular atrophy; thus suggesting a potential role of this gene family in human diseases (Roy, N, and coauthors Cell (1995) 80: 167-178). The therapeutic and diagnostic uses of the nucleic acids encoding various inhibitors of apoptosis, which refer to a member of the IAP family, have been described in the patent literature. See, for example, international patent applications No. WO 97/06255, WO 97/26331 and WO 97/32601. In particular, the uses of said genes and generic products are contemplated for the novel protein and its encoding nucleic acid, discussed below. Recently, a novel gene encoding an apoptotic inhibitor IAP of unique structure, designated Survivin, has been identified. Survivin is a cytoplasmic protein of about 16.5 kD, which contains a single BIR and a spiral region, with carboxyl terminus, with a strong charge, instead of a RING index, which inhibits apoptosis induced by growth factor withdrawal ( IL-3) when transferred into B cell precursors (Ambrosini, G and co-authors, Nature Med. (1997) 3: 917-921). In variation with bcl-2 or other IAP proteins, survivin is not detectable in adult tissues, but becomes prominently expressed in all very common human cancers of the lung, colon, breast, pancreas and prostate, and in about 50% of high-grade non-Hodgkin lymphomas, in vivo. It is intriguing that the filament coding for the survivin gene is highly homologous to the effector cell protease receptor-1 (EPR-1) sequence (Altieri, D.C.

FASEB J (1995) 9: 860-865), but oriented in the opposite direction; suggesting the existence of two separate genes, duplicated in a head-to-head configuration. The present invention is based on the identification of a novel human gene that is almost identical to EPR-1, but which is oriented in the opposite direction. The antisense gene product EPR-1, designated survivin, is a distantly related member of the IAP family of apoptosis inhibitors (Duckett, CS and co-authors, EMBO J (1996) 15: 2685-2694; Hay, B. A , and co-authors Cell (1995) 83: 1253-1262; Listón, P. and co-authors, Nature 1996) 379: 349-353; Rothe, M and co-authors Cell (1995) 83: 1243-1252; Roy N and coauthors, Cell (1995) 80: 167-178) and is prominently expressed in actively proliferating transformed cells, and in common human cancers, in vivo, but not in adjacent normal cells. Functionally, the inhibition of the expression of survivin upregulating its EPR-1 transcript of opposite, natural sense, resulted in massive apoptosis and decrease in cell development.

BRIEF DESCRIPTION OF THE INVENTION The present invention is based, in part, on the isolation and identification of a protein that is expressed in most cancer cells and inhibits cellular apoptosis, hereinafter referred to as survivin or. Survivin protein. Based on this observation, the invention provides purified survivin protein. The present invention additionally provides nucleic acid molecules that encode the survivin protein. Said nucleic acid molecules may be in isolated form or they may be operably linked to expression control elements or vector sequences. The present invention further provides methods for identifying other members of the survivin protein family. Specifically, the survivin nucleic acid sequence can be used as a probe, or to generate PCR sensitizers, in methods for identifying nucleic acid molecules that encode other members of the survivin family of proteins. The present invention additionally provides antibodies that bind to survivin. Said antibodies can be polyclonal or monoclonal. Anti-survivin antibodies can be used in a variety of diagnostic formats and for a variety of therapeutic methods. The present invention further provides for the isolation of the survivin binding partners. Survivin binding partners are isolated using the survivin protein as a capture probe. Alternatively survivin can be used as a bait in the two-hybrid yeast system to select an expression bank and identify genes that encode proteins that bind to the survivin protein. The binding partners isolated by these methods are useful in the preparation of antibodies and also serve as targets or targets for drug development. The present invention also provides methods for identifying agents that can block or modulate the association of survivin with a binding partner. Specifically, an agent can be tested for its ability to block, reduce or otherwise modulate the association of survivin with a binding partner, by contacting survivin or a fragment thereof, and a binding partner, with a binding agent. proof; and determining whether the test agent blocks or reduces the binding of the survivin protein to the binding partner. The present invention further provides methods for reducing or blocking the association of survivin with one or more of its binding partners. Specifically, the association of survivin with a binding partner can be blocked or reduced by contacting survivin or the binding partner with an agent that blocks the binding of survivin to the binding partner. The method can use an agent that binds survivin or the binding partner. The present invention further provides methods for regulating the expression of survivin within a cell. The expression of survivin within a cell can be regulated so that it produces or inhibits the production of survivin. Blocking associations of survivin / binding partner or the expression of survivin can be used to modulate biological and pathological processes that require survivin. For example, methods that reduce the production of survivin induce the apoptosis of tumor cells. Stimulation of survivin production can be used as a means to prolong the possibility of cell or tissue culture. Biological or pathological processes that require survivin or survivin / binding partner interactions can be modulated using gene therapy methods. Additional genetic manipulation within the organism can be used to alter the expression of a survivin gene or the production of a survivin protein in an animal model. For example, the survivin gene can be altered to correct a genetic deficiency; Peptide modulators of survivin activity can be produced within a target or target cell, using genetic transformation methods to introduce a modulator encoding nucleic acid molecules, into a target or target cell, etc. The use of said nucleic acids for therapies and interventions of the opposite direction and triple helix, is expressly contemplated. The present invention further provides methods for reducing the severity of pathological processes that require survivin. Since the expression of survivin or the association of survivin with a binding partner is necessary for biological processes mediated by survivin, agents that block the expression of survivin, the activity of survivin or the association of survivin with a binding partner can be used. , in therapeutic methods.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the identification of a complementary EPR-1 gene. A, B. Chromosomal location. A human P1 genomic clone, labeled with digoxigenin, selected by hybridization with the EPR-1 cDNA, was incubated with metaphase chromosomes isolated from PBMC stimulated by phytohemagglutinin in 50% formamide, 10% dextran sulfate and 2X SSC. The hybridizing gene EPR-1 was mapped in monochromatic labeling with the long arm of a group E chromosome (A, spotted with green) and in bichromatic spotting with probe D17Z1, specific for the centromere of chromosome 17 (B, stained in red) , to the long arm of chromosome 17 (B, stained green), to band 17q26. C. Map of the opposite EPR-1 gene. A contig covering 14796 base pairs, two P1 hybridizing clones of EPR-1, subcloned in pBSKS, was derived and sequenced completely in both strands.The orientation of the map is 5'- 3 'with respect to the position of the intron-exon boundaries (see below) Exons are solid boxes, a putative CpG island upstream of exon 1 is an open cell, the translation start codon (ATG) is indicated. are: B, SamHI, H, HincnW; P, Psñ; S, Smal, X, Xbal, D. Intron-exon boundaries of the opposite-sided EPR-1 gene.The positions of intron-exon boundaries in base pairs, Figure 2 shows the complexity and evolutionary conservation of the sequences related to EPR-1 A. Southern blot of human genomic DNA Samples were digested with the indicated restriction enzymes, transferred to GeneScreen nylon membranes, and hybridized them with the cDNA of EPR-1 in 5X SSC, 0.5% SDS, 5X Denhardt and 0.1% sodium pyrophosphate, at 65 ° C. The radioactive bands indicated by an arrow (fragments 7.6 kb BamHI, 7.5 kb Xbal and Hindll of 15, 7.5, 6.4 and 3.7 kb) are not derived from the gene EPR-1 opposite direction in Figure 1C. B. Southern blot of gel electrophoresis, pulsed field. Human genomic DNA, of high molecular weight, was digested with the indicated restriction enzymes, separated by pulsed field gel electrophoresis, for 20 hours at 200 V with a pulse time of 75 seconds, transferred to nylon membrane and hybridized with the EPR-1 cDNA, as described in A. C- Southern blot of multiple species. Genomic DNA, digested with EcoRI, of the indicated species was hybridized with a 3 'fragment of 548 base pairs of the EPR-1 cDNA, as described in A. For all panels, left-hand markers are shown on the left. molecular weight in kb. Figure 3 shows the discordant tissue distribution of EPR-1 transcripts of equal sense / opposite direction. Northern hybridization was performed in an adult or fetal multiple-tissue mRNA, stained with specific probes for a single strand, in 5X SSPE, 10X Denhardt's solution, 2% SDS, 100 mg / ml DNA Denatured salmon sperm at 60 ° C, for 14 hours. After washing in 2X SSC at 60 ° C and in 0.2X SSC at 22 ° C, the radioactive bands were visualized by autoradiography. A. Single-filament probe, specific for EPR-1. B.- Probe of a single filament, specific for EPR-1 of opposite direction.- C- Control actin probe. The molecular weight markers, in kb, are shown on the left. Figure 4 shows the sequence analysis of survivin and the expression in cell lines. A. Predicted translation of the opposite-sided EPR-1 gene product (survivin) (SEQ ID NO: 34). B.- Alignment of the sequence of the BIR in survivin (SEQ ID NO: 8 and 21) and in other IAP proteins, by means of the Clustal method. The IAP proteins are identified by accession number L49433 (SEQ ID NO: 9 and 22), IAP associated with the signaling complex TNFR2-TRAF; L49441 (SEQ ID NO: 10 and 23), apoptosis inhibitor 2 (Drosophila); P41436 (SEQ ID NO: 11 and 24), IAP gene of granulosis virus of Cydia pomonella; P41437 (SEQ ID NO: 12 and 25) and IAP gene from nuclear polyhedrosis virus of Orgya pseudotsugata; U19251 (SEQ ID NO: 13 and 26), NAIP, neuronal inhibitor of apoptosis; U32373 (SEQ ID NO: 14 and 27), ILP protein similar to IAP, from Drosophila melanogaster; U32974 (SEQ ID NO: 15 and 28), ILP protein similar to human IAP; U36842 (SEQ ID NO: 16 and 29), inhibitor of apoptosis in mouse; U45878 (SEQ ID NO: 17 and 30), human apoptosis inhibitor 1; U45879 (SEQ ID NO: 18 and 31); inhibitor of human apoptosis 2; U45880 (SEQ ID NO: 19 and 32), inhibitor of apoptosis linked in X; U45881 (SEQ ID NO: 20 and 33); apoptosis inhibitor in Drosophila. The conserved residues are pigeonholed, the identities between Survivina and NAIP (U19251) (SEQ ID NO: 13 and 26) are pigeonholed and shaded. C- Immunoblotted with anti-survivin JC700 antibody. Aliquots of normalized protein were separated from SDS extracts from HEL cell lines (erythroleukemia), Daudi and JY (lymphoma B), THP-1 (monocytic), Jurkat and MOT13 (T leukemia) or untransformed human lung Lu18 fibroblasts, HUVEC or PBMC, by electrophoresis, in a gel with 5-20% gradient SDS, they were transferred to Immobilon and immunostained with non-immune control rabbit IgG (RblgG) or anti-survivin JC700 antibody (survivin). The protein bands were visualized by goat anti-rabbit IgG, conjugated with alkaline phosphatase and with tetrazolium salts. The molecular weight markers in kDa are shown on the left. Figure 5 shows the regulation of survivin expression by cell development / differentiation. HL-60 cells were terminally differentiated to a mature monocytic phenotype, through a 72-hour culture with 0.1 mM vitamin D3, plus 17.8 mg / ml indomethacin. Expression of survivin was detected before or after differentiation of vitamin D3 by immunostaining with JC700 antibody, or by Northern hybridization with a single-filament probe, specific for survivin. Non-immune rabbit IgG, control, RblgG. The molecular weight markers of the protein, in kDa and the position of the ribosomal bands, are shown to the left of each spot. Figure 6 shows the excess expression of survivin in human cancer, in vivo. A.- Immunohistochemical staining of human lung adenocarcinoma with anti-survivin antibody purified by affinity JC700 (20 μg / ml). B.- Inhibition of spotting with lung adenocarcinoma JC700, by preabsorption with peptide 3-19 d survivin, immunizer. C- Immunohistochemical expression of survivin in squamous cell carcinoma, but not in adjacent normal glandular epithelium of the lung (C, arrow). D.- In situ hybridization of survivin mRNA in squamous cell carcinoma of the lung, with a specific riboprobe for survivin. E.- Expression of survivin in pancreatic adenocarcinoma by immunohistochemistry with JC700. F.- Normal pancreas, negative for the expression of survivin in immunohistochemistry. G.- In situ hybridization of survivin mRNA, expression in colon adenocarcinoma, except H, non-neoplastic, non-adjacent colon glandular epithelium (H, arrow). The extensions are x200, except G, which is x400. Figure 7 shows the effect of survivin on apoptosis / proliferation. A.- Regulation of the expression of survivin with EPR-1. HeLa cells were transfected with the control vector pML1 or the cDNA of EPR-1 (which is opposite in direction to survivin), by electroporation and selected in hygromycin (0.4 mg / ml). Was aliquots of HeLa d cells vector control (Vector) or transfectants of opposite sense of survivin (opposite sense) with 200 mM of ZnS? , it was solubilized with detergent and immunostained with the JC700 anti-survivin antibody. The molecular weight markers are shown on the left. B.- Effect of survivin on apoptosis. Transfectants (1, 2) of opposite sense of survivin were induced, or vector control HeLa cells (3, 4) with Zn2 + ions in 0% FBS for 24 hours and stained by the AptoTag method with dUTP labeling catalyzed with TdT, that of the ends 3'-OH of DNA and with immunoperoxidase (1, 3) or by hematoxilin-eosin (HE) (2,4). 1.- Prominent fragmentation of nuclear DNA, detected by spotting AptoTag in transfectants opposite to survivin, with exhausted serum; 2.- The HE staining of transfectants of opposite direction reveals the presence of numerous apoptotic bodies (arrows). 3. - The AptoTag staining of vector control HeLa cells detects a few scarce apoptotic cells (arrow) .- 4.- HE staining of vector control HeLa cells. The arrow indicates a single apoptotic body. Expansion x400.

C- Effect of survivin on the growth of cells. Twenty thousand vector control HeLa cells (Vector) or transfectants of opposite sense of survivin (opposite direction) were plated in plates of 24 concavities, they were induced with ZnSO-t, harvested at the indicated time points and the proliferation of the cells was determined microscopically by means of a direct count of the cells. The data are the mean ± SEM of duplicates of a representative experiment of seven independent determinations. Figure 8 shows the expression of survivin in HL-60 cells. HL-60 cells were examined by Western and Northern staining for the expression of survivin. * Figure 9 presents a structural analysis of survivin. The survivin protein was analyzed using the analysis methods of Chou-Faman, Gamier-Robson, Kyle-Doolittle, Eisenberg, Karplus-Schultz, Majeson-Wolf and Emini. Figure 10 shows the nucleotide sequence of survivin.

Figure 11 shows the expression of survivin and the generation and characterization of anti-survivin mAb 8E2, by ELISA and immunoblotting. Figure 12 shows the site-directed mutagenesis of survivin and the identification of the key functional residues involved in the inhibition of apoptosis. Figure 13 shows the cytoprotective effect of the addition of survivin on endothelial cell apoptosis. Figure 14 shows that the presence of survivin is a negative predictive-prognostic factor in neurobiastomas. Figure 15 shows that the presence of survivin is a negative predictive-prognostic factor in high-grade non-Hodgkin lymphoma. Figure 16 shows the down regulation of survivin induced by inflammatory and cytostatic cytokines. Figure 17 shows the effect of survivin or XI AP constructs on apoptosis induced in NIH3T3 cells by hydrogen peroxide.

DETAILED DESCRIPTION OF THE PREFERRED MODALITY I.- GENERAL DESCRIPTION The present invention is based, in part, on the identification of a novel protein that is expressed in tumor cells and that inhibits cellular apoptosis, hereinafter referred to as the survivin or survivin protein. It is also found that survivin is expressed in embryonic tissues. The survivin protein can be used as an agent, or serves as a target for agents, which can be used to inhibit or stimulate the inhibition of cell apoptosis, mediated by survivin, for example, to block abnormal cell growth or extensive cell development. in crops. As used herein, the modulation of apoptosis means increasing or decreasing the number of cells that would otherwise suffer apoptosis in a given population of cells. This can be done by increasing or decreasing the amount of survivin present in a cell or by increasing or decreasing the activity of survivin. Preferably the given population of cells in which apoptosis is to be modulated, is found in a tumor or other tissue or group of cells in which a beneficial effect of modulation results. Also, preferably, the increase or decrease in the number of cells that would otherwise suffer apoptosis in a given population of cells is at least about 10%, 20%, 40% or more, preferably at least about of 50% of the cells of that population.

The present invention is further based on the development of methods for isolating proteins that bind to survivin. Probes, based on the survivin protein or survivin fragments, which are discussed below, are used as capture probes to isolate the proteins that bind to survivin. The dominant negative proteins, the DNAs that encode those proteins, the antibodies for those binding proteins, the peptide fragments of those proteins or the mimics (mimics) of those proteins can be introduced into the cells to affect the function of survivin. Additionally, these proteins give novel targets for selecting small, synthetic molecules, and libraries of combinatorial or naturally occurring compounds, to discover novel therapeutics to regulate the function of survivin.

II.- IDENTIFICATION. GENERAL CHARACTERIZATION AND DISTRIBUTION OF SURVIVINA IN THE TISSUE The present invention is based on the identification, on chromosome 17q25, of a novel member of the family of apoptosis inhibitors, designated survivin, which may confer a selective advantage for the development of cancer cells. Relevant aspects of the survivin gene include its regulated expression in terms of development and in terms of differentiation, its DNA sequence almost identical and complementary, with the EPR-1 receptor of factor Xa, and its abundant expression in vivo in common human malignancies. , but not in the adjacent non-neoplastic population. As described below, determining the expression of survivin by the induction by metallothionein of the mRNA of EPR-1, resulted in apoptosis and inhibition of the proliferation of HeLa cell transfectants. In addition to their contribution to hemostasis, cellular protease receptors in blood have recently emerged as pleiotropic signaling molecules, which play a crucial role in embryological development (Connolly, A. J and co-authors, Nature (1996) 381-516 -519), and vasculogenesis (Carmeliet, P and co-authors, Nature (1996) 383: 73-75). In this context, the survivin gene was isolated by hybridization with the cDNA for EPR-1, a receptor for factor Xa that contributes to the procoagulant activity (Altieri, D. C, FASEB J (1995) 9: 860-865) and activation of T cells (Duchosal, MA and co-authors, Nature, (1996) 380: 352-356). Although the survivin coding sequence was found to be almost identical to the EPR-1 cDNA, its orientation was unambiguously assigned to the opposite-sense EPR-1 strand for the position of the consensus splice sites in the limits of intron-exon (Padgett, R. A and co-authors, Ann. Rev. Biochem, (1986) 55: 1119-1150). On the other hand, the authenticity of the "same-like" filament of EPR-1 was demonstrated in previous studies, when mammalian cells, transfected with EPR-1 cDNA, or with chimeric constructions of EPR-1 (Ambrosini, G and co-authors , J. Biol. Chem., (1996) 271-1243-1248 and Altieri, DC FASEB J (1995) 9: 860-865), were recognized by anti-EPR-1 mAbs and the Xa binding factor in a reaction specific and saturable.

These discoveries could be reconciled by the existence of multiple transcripts of EPR-1, highly homologous, and oriented in opposite directions. The heterogeneity of the EPR-1 mRNA and the complex pattern of Southern hybridization support this hypothesis. Previously, the double-filament EPR-1 probes detected three strongly hybridizing bands of 1.9, 3.4 and around 1.5 kb in the EPR-1 cell mRNA.

(Altieri, D. C, FASEB J (1995) 9: 860-865). Here the specific probes of a single filament confirmed the presence of multiple messages related to EPR-1 mature and polyadenylated, and revealed that the bands of 1.9 and 3.4 kb corresponded with two transcripts of EPR-1 of opposite direction, highly regulated, while the band of 1.5 kg, more precisely defined as 1. 2 kb, coincided with an authentic encoder message from EPR-1. Although the 1.9 kb opposite sense transcript originated clearly from the survivin gene described here, a gene encoding the "normal sense" 1.2-Kb EPR-1 message has not yet been identified. However, (i) the presence of several genomic EPR-1 hybridizing bands, unrelated to the survivin gene, (ii) the different restriction pattern of the EPR-1 sequences in various species, and (iii) the numerous label database annotations in the expressed sequence, coincident (P = 0.018 -7x10"11) with the positive filament (Access No. W46267) or negative (Accession No. W34764, W83810, T29149), suggest, as a whole, the existence of less a second gene EPR-1, highly related, oriented in the opposite direction to that described here, and which encodes the Xa receptor of the previously characterized factor (Altieri, D. C, FASEB J (1995) 9: 860-865). similar situation could arise from the event (s) of gene duplication that in they carry EPR-1 sequences. Interestingly, the unique hybridization signal, detected on chromosome 17q25, and the individual hybridizing bands identified in a Southern spotting of high molecular weight genomic DNA, suggest that the sequences related to EPR-1, potentially oriented in opposite directions , they can be adjacent, very close, within a physical range of 75-130 kb. The presence of multiple EPR-1 transcripts oriented in opposite directions implies a reciprocal regulatory mechanism, in the opposite sense that occurs naturally. This is consistent with the predominantly discordant and mutually exclusive distribution of normal-sense and opposite-sense EPR-1 messages, in developing or adult tissues, in vivo, and during terminal differentiation of HL-60 cells. Although regulation of the opposite direction is common in prokaryotes (Green, P. J and co-authors, Annu, Rev. Biochem. (1986) 55: 560-597), a growing number of eukaryotic gene products have recently been characterized for occurrence of opposite-sense, functional transcripts that participate in the generic regulation, including the basic fibroblast development factor (Kimmelman, D and co-authors, Cell (1989) 59: 687-696; Murphy, PR and co-authors, Molecular Endocrinology ( 1995) 8: 852-859), collagen Al (l) (Farrell, C. M and co-authors, J. Biol. Chem (1995) 270: 3400-3408 and Lukens, 1995), n-myc (Krystal, G. W and co-authors, Mol. Cell Biol. (1990) 10: 4180-4191), c-myc (Celano, P. and co-authors, J. Biol. Chem., (1992) 267-15092-15096), p53 (Khochbin, S and co-authors, EMBO J (1989) 8: 4107-4114), c-erbAa (Lazar, M.A. and co-authors, Mol.Cell Biol. (1989), 9: 1128-1136) and the CD3 site (dzet / eta / theta) (Lerner, A and coauthors, J. Immunol, (1993) 151: 3152-3162). As described below, the existence of a genetic equilibrium EPR-1 / survivin, regulated by opposite functional sense, was demonstrated in HeLa cell transfectants, when metallothionein-induced transcription, of the "normal-sense" filament of EPR-1 it suppressed survivin expression and profoundly influenced apoptosis / cell proliferation (see below). This regulatory mechanism was not due to a potential protein association between EPR-1 and survivin, since the EPR-1 construct used for these experiments lacked a translation initiation codon. Other experiments have evaluated the ability of an opposite sense of survivin to inhibit the development of the cell. This was done by transiently cotransfecting the opposite sense of survivin with a plasmid reported as iacZ, and making the determination of cell viability after transfection for 48 hours in cells expressing β-galactosidase. The results indicated that the viability of the transfectants of sense laid of survivin was <20% of the control cells transfected with the empty vector. An opposite sense of control, of ICAM-1 (intracellular adhesion molecule 1) cotransfected in a similar manner in HeLa cells was ineffective.

Survivin was found to be a small protein, 142 amino acids (about 16.5 kDa) with no homology to the amino acid sequence with EPR-1, and was designated survivin by the presence of a homologous domain with BIR (Bimbaum, M. J and coauthors, J. Virology, (1994) 68: 2521-2528; CIem, RJ and co-authors, Mol Cell Biol. (1994) 14: 5212-5222), found in IAP inhibitors of apoptosis (Duckett, CS Y co-authors, EMBO J (1996) 15: 2685-2694; Hay, B. A. and co-authors, Cell (1995) 83: 1253-1262; Listón, P. and co-authors, Nature (1996), 379: 349-353; Rothe, M and co-authors Cell (1995) 83: 1243-1252; Roy, N and coauthors, Cell (1995) 80: 167-178). Based on general sequence conservation, the absence of a carboxy-terminal RING index and the presence of a single partially conserved BIR domain, survivin is the most distantly related member of the IAP family, sharing the highest degree of similarity with NAIP. (Roy, N and co-authors, Cell (1995) 80: 167-178). So, unlike £ > c / -2 or other IAP proteins, survivin is undetectable in adult tissues, but becomes prominently expressed in all very common human cancers of the lung, colon, breast, pancreas and prostate, and in about 50% of lymphomas high grade, which are not Hodgkin's, in vivo. Additionally, unlike other IAP proteins (Deveraux, Q and co-authors Nature (1997) 388: 300-304), survivin does not bind to caspases in a cell-free system (Roy, N. and co-authors, Blood (1997) 595-2645). Consistent with the anti-apoptosis properties of IAP proteins in vitro (Duckett, C. S et al., EMBO J (1996) 14: 2685-26904; Listón, P. and co-authors Nature (1996) 379: 349-353), and inhibition in vivo (Hay, BA and co-authors, Cell (1995) 83: 1253-1262) of the expression of survivin by the EPR-1 transcript (which is naturally opposite in sense to survivin) resulted in increased apoptosis, as determined by the fragmentation of intemucleosomal DN in situ in HeLa cell transfectants. The capacity of an IAP protein without a RING index to counteract apoptosis is not without precedent, as demonstrated by the suppression of apoptosis mediated by NAIP (Ribbon, P and coauthors, Nature (1996) 379: 349-353) and by the increase in in vivo function of a Drosophila IAP protein after the omission of the RING index (Hay, B. A et al., Cell (1995). ) 83: 1253-1262). While it is believed that anti-apoptosis genes play an indirect role in cell development, favoring the accumulation of oncogene mutation (s) in aberrantly prolonged life cells (Reed, J. C, J. Cell Biol. (1994) 124: 1-6), down-regulation of survivin resulted in profound inhibition of HeLa cell proliferation. While this may be derived from the rapid disappearance of HeLa cells, which express the highest levels of opposite-sense transcripts, by apoptosis, a similar decrease in tumor cell proliferation has been reported in vivo, after inhibition of the opposite direction of bcl-2 (Reed, JC and co-authors, Proc. Nati, Acad. Sci. USA (1990) 87: 3660-3664). The possibility that IAP proteins play a more general role in cell proliferation, not restricted exclusively to the inhibition of apoptosis, has been proposed previously. Rothe and coauthors have recently shown that amino-terminal BIR in two IAP proteins (clAP) physically interacts with the signal transducers associated with the 75 kDa TNF receptor (Rothe, M and others Cell (1995) 83: 1243-1252) , a molecule primarily involved in the proliferation and survival of cells instead of apoptotic signaling (Tartaglia, LA and co-authors, Immunol. Today (1992) 13: 151-153). Although it is not known if survivin is physically linked to signaling molecules (Rothe, M and co-authors, Cell (1995) 83: 1243-1252), the structural divergence of their BIR, compared to other IAP proteins (Duckett, CS and coauthors, EMBO J (1996) 15: 2685-2694; Hay, BA and coauthors, Cell (1995 ) 83-1253-1262; Listón, P. and coauthors Nature (1996) 379: 349-353; Rothe, M and coauthors, Cell (1995) 83: 1243-1252; Roy, N Y co-authors, Cell (1995) 80: 167-178), can confer specificity for one or more supramolecular interactions, potentially relevant to their particular mechanism of inhibition of apoptosis / cell development. Recently the dysregulation of programmed cell death (apoptosis) has emerged as a primary mechanism that contributes to the pathogenesis of various human diseases, including cancer (Steller, H., Science (1995) 267: 1446-1449; Thompson, CB , Science (1995) 267: 1456-1462). Although the impact of the anti-apoptosis gene (s) on neoplasia is highlighted by the role of bcl-2 in follicular lymphoma (Korsmeyer, S. J, Blood (1992) 80: 879-886), a distribution IAP protein potential in cancer had not been previously investigated. In this context, one of the most outstanding characteristics of survivin was its abundant expression in transformed cell lines, actively proliferating, and in all the most common human malignancies of the lung, colon, pancreas and breast, in vivo, but not in the not neoplastic, adjacent to the cell population. This distribution in multiple human cancers may indicate a fundamental role of this molecule in the mechanisms of apoptosis / cell proliferation in neoplasms. By analogy with the paradigm of £ > c / -2, excess expression of survivin in cancer can lead to aberrantly prolonged cell viability (Vis, DJ Y coauthors, Cell (1993) 75: 229-240), increased resistance to apoptosis induced by chemotherapy (Miyashita , T and coauthors Blood (1993) 81: 151-157) and, as suggested by the in vitro studies reported above, a direct advantage for the proliferation of transformed cells. On the other hand, due to its presence in normal PBMC and in benign breast adenomas, the expression of survivin in vivo (unpublished observations) per se can not be interpreted as a marker of malignant transformation; but it may reflect a more general response to certain stimuli, specific to the development or type of cells. This is consistent with the presence of survivin during normal embryonic development (observations by the inventors, unpublished) and normal fetal development, and its rapid disappearance in arrested cell types (ie, HL-60 treated with vitamin D3), and terminally differentiated tissues, in vivo. Unlike other IAP proteins, which are found constitutively in adult mature tissues (Duckett and coauthors, EMBO J (1996) 15: 2685-2694; Listón, P and co-authors, Nature (1996) 379: 349-353; Rothe, M and co-authors (Cell (1995) 83: 1243-1252), this expression pattern is reminiscent of the distribution of bcl-2 in fetal tissues (LeBrun, D. P. and co-authors, Am. J. Pathol. (1993) 142-743-753) and its more restricted presence in differentiated cells, which correlates with the susceptibility to apoptosis (Hockenbery, D.M. and coauthors, Proc. Nati. Acad.

Sci. USA (1991) 88: 6961-6965). In summary, these findings identify survivin as a novel link between IAP proteins and cancer, in vivo. A key implication of the data presented below is the possibility of balancing the effect of this potent anti-apoptosis gene, by manipulating a regulatory mechanism of cells, normal, entered on the expression of EPR-1 (Altieri, D. C, FASEB J (1995) 9: 860-865). Directing the target or target to survivin may "eliminate a selective advantage for the development of transformed cells, and may be therapeutically beneficial to increase the susceptibility of cancer cells to chemotherapeutically induced apoptosis." Following the same line, the identification of polymorphic markers and the construction of extensive aplotypes in and around the EPR-1 / survivin site, new possibilities in the population genetics of susceptibility to chemotherapy can be provided.

III.- SPECIFIC MODALITIES A.- SURVIVINE PROTEIN The present invention provides isolated survivin protein, as well as allelic variants of the survivin protein, and conservative amino acid substitutions of the survivin protein. As used herein, the survivin protein (or survivin) refers to a protein having the amino acid sequence of human survivin, which is illustrated in Figure 4. The term "survivin protein" also includes the allelic variants that occur naturally, of survivin, the naturally occurring proteins, which have a slightly different amino acid sequence than the one specifically mentioned above. The allelic variants, although possessing a slightly different amino acid sequence than those mentioned above, still have the capacity required to inhibit cellular apoptosis. As used herein, the survivin family of proteins refers to survivin proteins that have been isolated from organisms other than humans. The methods used to identify and isolate other members of the survivin family of proteins are described below. Survivin is a member of the IAP family (protein acronym for its designation in English: Inhibitory Apoptosis Proteins). However, survivin is the first member of a unique subfamily of IAP proteins that differ from other IAP proteins in significant ways. Despite the homology and sequence conservation in the BIR module, between survivin and other members of this family of genes, there are important structural differences that are unique to members of the survivin family of proteins.

Firstly, unlike other IAP proteins, survivin has only one BIR module (most other molecules have 2 or 3).

Additionally, survivin does not contain a carboxy-terminal RING index, but instead has a predicted coiled helix. Only the inhibitory neuronal apoptosis protein (NAIP, acronym for its designation in English: Neuronal Apoptosis Inhibitory Protein) in the IAP family, lacks a RING index, but does not contain a coiled, carboxy-terminal helix. Finally, there is no similarity in the DNA sequence between survivin and other IAP proteins (the RCP sensitizers, designed on survivin, are unable to detect other IAP proteins and vice versa). The survivin proteins of the present invention are preferably in isolated form. As used herein, a protein is said to be isolated when physical, mechanical or chemical methods are employed to separate the survivin protein from cellular constituents that are normally associated with the survivin protein. One skilled in the art can easily employ common and current purification methods to obtain an isolated survivin protein. The survivin proteins of the present invention additionally include conservative variants of the survivin proteins described herein. As used herein, a "conservative" variant refers to alterations in amino acid sequence that do not adversely affect the ability of the survivin protein to bind to a survivin binding partner, and / or to inhibit cellular apoptosis. A substitution, insertion or omission is said to adversely affect the survivin protein when the altered sequence prevents the survivin protein from being associated with a survivin binding partner and / or prevents the survivin protein from inhibiting cellular apoptosis. For example, the total charge, structure or hydrophobic / hydrophilic properties of survivin can be altered without adversely affecting the activity of survivin. Accordingly, the amino acid sequence of survivin can be altered, for example, to render the peptide more hydrophobic or hydrophilic, without adversely affecting the activity of survivin. Allelic variants, conservative substitution variants and members of the survivin family of proteins, will have the ability to inhibit cellular apoptosis. Said proteins will ordinarily have an amino acid sequence having at least about 75% identity in the amino acid sequence with the human survivin sequence, more preferably, at least about 80 $, still more preferable, at least about 90% and, what is most preferred, at least about 95%. The identity or homology with respect to said sequences is defined here as the percentage of amino acid residues in the candidate sequence, which are identical with the known peptides, after aligning the sequences and introducing the separations, if necessary, to obtain the maximum percentage homology, and that include any conservative substitutions as if they were homologous. The N-terminal, C-terminal or internal extensions, omissions or insertions in the peptide sequence should not be considered as affecting the homology. Thus, the survivin proteins of the present invention include molecules that have the amino acid sequences described in Figure 1; its fragments having a consecutive sequence of at least about 3, 5, 10 or 15 amino acid residues of the survivin protein; variants of the amino acid sequence of said sequence, in which an amino acid residue has been inserted at the N-termini or C-terminal position with, or within, the described sequence of survivin; the amino acid sequence variants of the described sequence of survivin, or its fragments as defined above, which have been replaced by another residue. The contemplated variants further include those which contain predetermined mutations, for example, by homologous recombination, site-directed mutagenesis or PCR, and corresponding survivin proteins from other animal species, including, but not limited to, rabbit, rat, murine, porcine, bovine, ovine, equine and non-human primates, and alleles and other naturally occurring variants of the survivin family of proteins; and derivatives in which the survivin protein has been covalently modified by substitution, chemical, enzymatic or other appropriate means, with a portion other than the naturally occurring amino acid (eg, a detectable portion, such as an enzyme or a radioisotope). The recombinant survivin protein can also be used to solve the molecular structure of survivin by 2D NMR, circular dichroism and X-ray crystallography, thus integrating the site-directed mutagenesis approach and the rational design of the small molecule-specific inhibitors. . As described below, members of the survivin family of proteins can be used: 1) as a target or target to block the inhibition of cellular apoptosis, mediated by survivin; 2) to identify and isolate binding partners to which survivin binds; 3) in methods for identifying agents that block the association of survivin with a binding partner; 4) as a target or target to analyze the inhibition of cellular apoptosis, mediated by survivin; 5) as an agent to block cellular apoptosis, administered alone or as part of a combination therapy; 6) as a binding partner in an analysis to quantify the circulating levels of anti-survivin antibodies; 7) as an antigen to trigger the production of anti-survivin antibodies which, in turn, can be used in an analysis to quantify circulating levels of survivin and / or can be used for immunohistochemical purposes; and 8) as an anti-cancer therapeutic vaccine, or as a component of a polyvalent vaccine.

B.- ANTI-SURVIVINA ANTIBODIES The present invention further provides antibodies that selectively bind to a survivin protein. Particularly contemplated anti-survivin antibodies include monoclonal and polyclonal antibodies, as well as fragments that contain the antigen binding domain and / or one or more complement determining regions. Antibodies are generally prepared by immunizing a suitable mammalian host, using a survivin protein, a fragment, an isolated or immunoconjugated form (Harlow, Antibodies, Cold Sp Harbor).

Press, NY (1989)). Figure 9 provides a Jameson-Wolf diagram of the antigenic index of various regions of survivin. Said regions, in combination with other structural analyzes provided in Figure 9, give suitable fragments for use in the generation of antibodies specific for survivin. Methods for prepa immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier proteins, are well known in the art. In some circumstances, direct conjugation can be used using, for example, carbodiimide reagents; in other cases, the binding reagents, such as those supplied by Pierce Chemical Co., Rockford, IL, E. U. A., can be effective. In general, the administration of the survivin immunogen is effected by injection, for an appropriate period of time, and with the use of a suitable adjuvant, as is generally understood in the art. Du the immunization program, antibody titers can be taken to determine the adequacy of antibody formation. While the polyclonal antisera produced in this way may be satisfactory for some applications, monoclonal antibody preparations are preferred for pharmaceutical compositions. The immortalized cell lines that secrete a desired monoclonal antibody can be prepared using the common and current method of Kohler and Milstein, or modifications that effect the immortalization of lymphocytes or spleen cells, as is generally known. The immortalized cell lines that secrete the desired antibodies are selected by immunoassay, in which the antigen is the survivin peptide. When the appropriate immortalized cell culture, which secretes the desired antibody, is identified, the cells can be cultured either in vitro, or by production of the ascites fluid. The desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of monoclonal or polyclonal antisera that contain the immunologically significant portion can be used as antagonists, as well as intact antibodies. The use of immunologically reactive fragments, such as Fab, Fab 'fragments, of F (ab') 2 fragments is often preferred, especially in a therapeutic context, since these fragments are generally less immunogenic than whole immunoglobulin . Antibodies or fragments can also be produced using current technology, by recombinant means. It is also possible to produce the regions that bind specifically to the desired regions of the receptor, in the context of chimeras or antibodies grafted by RDC, of multiple origin of species.

The antibodies thus produced are useful not only as modulators of the survivin association with the survivin binding partner, but are also useful in immunoassays to detect the expression / activity of survivin and for the purification of survivin and the associated binding partners. .

C- NUCLEIC ACID MOLECULES THAT CODE SURVIVINA The present invention further provides nucleic acid molecules encoding survivin, and related survivin proteins, described herein, preferably in isolated form. For convenience, all survivin-encoding nucleic acid molecules will be referred to in the present survivin-encoding nucleic acid molecule.; the survivin gene or Survivina. As used herein, "nucleic acid" is defined as the RNA or DNA that encodes a peptide as defined above, or that is complementary to a nucleic acid sequence encoding said peptides, or hybridizes to said nucleic acid and remains stably bound to it, under stringent conditions, or encodes a polypeptide that shares at least the 75% sequence identity, preferably at least 80% and, more preferably, at least 85%, with the peptide sequences. Specifically contemplated are genomic DNA, cDNA, mRNA and molecules of opposite direction, as well as nucleic acids based on an alternative skeleton or including alternative bases, either derived from natural sources or synthesized. Said hybridizing or complementary nucleic acid, however, is further defined as being novel and non-obvious with respect to any prior art nucleic acid, including that which codes, hybridizes under appropriate stringent conditions, or is complementary to, a nucleic acid which encodes a survivin protein, according to the present invention. As used herein, "stringent conditions" are the conditions in which hybridization produces a clear and detectable sequence. Strict conditions are those that (1) employ low ionic concentration and high temperature for washing, for example, 0.015 M NaCl, 0.0015 M sodium citrate, 0.1% SDS at 50 ° C or (2) used during hybridization a denaturing agent, such as formamide, for example, 50% formamide (in volume / volume), with 0.1% bovine serum albumin; 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5, with 750 mM NaCl, 75 mM sodium citrate at 42 ° C. Another example is the use of 50% formamide, 5X SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, Denhardt 5X solution, Salmon sperm DNA treated sonically (50 μg / ml), 0.1% SDS and 10% dextran sulfate at 42 ° C, washed at 42 ° C in 0.2X SSC and 0.1% SDS. A person skilled in the art can easily determine and vary the strict conditions appropriately, to obtain a clear and detectable hybridization signal.

As used herein, a nucleic acid molecule is said to be "isolated" when the nucleic acid molecule is substantially separated from the contaminating nucleic acid, encoding other polypeptides, from the nucleic acid source. The present invention additionally provides fragments of the nucleic acid molecule encoding survivin. As used herein, a fragment of a nucleic acid molecule encoding survivin refers to a small portion of the total protein coding sequence. The size of the fragment will be determined by the use it is intended for. For example, if the fragments are selected to encode an active portion of the survivin protein, such as the C-terminal beta turns of the IAP motif, the fragment will need to be large enough to encode one or more functional regions of the survivin protein. If the fragment is to be used as a nucleic acid probe or as a PCR sensitizer, then the length of the fragment is selected so that a relatively small number of false positives is obtained during the probe / sensitization. Figure 1 identifies fragments of the survivin gene that are particularly useful as selective hybridization probes or as PCR sensitizers. The fragments of nucleic acid molecules encoding survivin, of the present invention (ie, synthetic oligonucleotides) that are used as probes or as specific sensitizers for the polymerase chain reaction (PCR) or to synthesize the gene sequences they encode Survivin proteins can be easily synthesized by chemical techniques; for example, the phosphotriester method of Matteucci and coauthors, J. Am. Chem. Soc. (1981) 103: 3185-3191, or using automated synthesis methods. Additionally, larger segments of DNA can easily be prepared by well-known methods, such as the synthesis of a group of oligonucleotides that define several modular segments of the survivin gene, followed by ligation of the oligonucleotides to constitute the complete survivin gene., modified. The nucleic acid molecules encoding survivin, of the present invention, can be further modified so as to contain a detectable label for diagnosis and for use as a probe. As described above, such probes can be used to identify other members of the survivin family of proteins and, as described below, such probes can be used to detect the expression of survivin and the potential for tumor development. A variety of such labels are known in the art and can be easily employed with the survivin coding molecules described herein. Suitable labels include, but are not limited to: biotin, radiolabeled nucleotides and the like. Whoever is skilled in the art can use any of the labels known in the art, to obtain a labeled nucleic acid molecule of survivin. Since the survivin gene is in an opposite sense orientation or inverse orientation of the EPR-1 gene, single-ended probes are particularly preferred for use in diagnostic purposes.

Specifically, single-stranded diagnostic probes can be used to selectively hybridize to mRNA that encodes survivin. Single probes can finally be generated using known methods in which a single filament or double filament probe is isolated, or in which a single filament RNA probe is generated. Modifications can be made in the primary structure itself by omission, addition or alteration of the amino acids incorporated in the protein sequence during translation, without destroying the activity of the protein. Such substitutions or other alterations result in proteins having an amino acid sequence encoded by DNA that falls within the scope contemplated by the present invention.

D.- ISOLATION OF OTHER MOLECULES OF NUCLEIC ACID THAT CODE SURVIVINA As described above, the identification of the nucleic acid molecule encoding human survivin allows one skilled in the art to isolate nucleic acid molecules encoding other members of the survivin family of proteins, in addition to the human sequence described herein. Essentially one skilled in the art can easily use the amino acid sequence of survivin to generate antibody probes to select expression banks prepared from cells. Typically, polyclonal antiserum from mammals, such as rabbits immunized with the purified survivin protein (which is described below) or monoclonal antibodies can be used to probe mammalian cDNA or a genomic expression bank, such as a lambda gtll, to obtain the appropriate coding sequence of survivin or other members of the survivin family of proteins. The cloned cDNA sequence can be expressed as a fusion protein, it can be expressed directly using its own control sequence, or it can be expressed by constructs using appropriate control sequences for the particular host used for the expression of the enzyme. Figure 1 identifies important antigenic and / or operating domains, putative, found in the survivin protein sequence. Said regions are preferred sources of antigenic portions of survivin protein for the production of probe, diagnostic and therapeutic antibodies. Alternatively, a portion of the survivin coding sequence described herein can be synthesized, and used as a probe to recover DNA encoding a survivin family member of proteins, from any mammalian organisms, that protein contains. Oligomers containing about 18 to 20 nucleotides (which encode stretches of about 6 to 7 amino acids) are prepared and used under stringent conditions or sufficiently stringent conditions to eliminate an undue level of false positives. Additionally, pairs of oligonucleotide sensitizers can be prepared for use in the polymerase chain reaction (PCR) to selectively clone a nucleic acid molecule encoding survivin. A cycle of denaturation / fixation / extension of PCR for use with said PCR sensitizers is well known in the art and can be easily adapted for use in the isolation of other nucleic acid molecules encoding survivin. Figure 1 identifies regions of the human survivin gene that are particularly well suited for use as a probe or as sensitizers.

E.- rDNA MOLECULES CONTAINING A NUCLEIC ACID MOLECULE CODIFIES SURVIVINA The present invention additionally provides recombinant DNA molecules (rDNA) that contain a survivin coding sequence. As used herein, a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation, in vitro. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook and coauthors, Molecular Cloning (1989). In the preferred rDNA molecules, a survivin coding DNA sequence is operably linked to expression control sequences and / or vector sequences. The selection of the vector and / or expression control sequences to which one of the survivin coding sequences of the present invention is operably linked directly depends, as is well known in the art, on the desired functional properties, for example , the expression of protein and the host cell that is to be transformed. A vector contemplated by the present invention is capable of at least directing the reproduction or insertion into the host chromosome and, preferably, also the expression, of the survivin gene included in the rDNA molecule. Expression control elements that are used to regulate the expression of an operably linked protein coding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretory signals and other elements regulators. Preferably the inducible promoter is easily controlled, for example, by being sensitive to a nutrient in the medium of the host cell. In one embodiment, the vector containing a survivin-encoding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence that has the ability to direct autonomous reproduction and maintenance of the recombinant DNA molecule in an environment outside the chromosome, in a prokaryotic host cell, such as a bacterial host cell, transformed with it. Said replicons are well known in the art. Additionally, vectors that include a prokaryotic replicon may also include a gene whose expression confers a detectable label, such as resistance to a drug. Typical genes for bacterial resistance to a drug are those that confer resistance to ampicillin or tetracycline.

Vectors that include a prokaryotic replicon may additionally include a prokaryotic or viral promoter, capable of directing the expression (transcription and translation) of the gene sequences encoding survivin, in a bacterial host cell, such as E. coli. A promoter is an expression controlling element, formed by a DNA sequence that allows RNA polymerase binding and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for the insertion of a DNA segment of the present invention. Typical of such vector plasmids are: pUC8, pUC9, pBR322 and pBR329, obtainable from Biorad Laboratories (Richmond, CA, USA), and pKK223, obtainable from Pharmacia, Piscataway, NJ, USA. Cell-compatible expression vectors can also be used eukaryotic, preferably those compatible with vertebrate cells, to form rDNA molecules containing a survivin coding sequence. Eukaryotic cell expression vectors are well known in the art and are available from various sources. Typically, such vectors containing convenient restriction sites for insertion of the desired DNA segment are provided. Typical of such vectors are PSVL and pKSV-10 (Pharmacia), pBPV-1 / pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC # 31255), the pCDMd vector described herein, and all eukaryotic expression vectors.

Eukaryotic cell expression vectors, used to construct the rDNA molecules of the present invention, may additionally include a selectable marker that is effective in a eukaryotic cell, preferably a selection marker for drug resistance. A preferred drug resistance marker is the gene whose expression results in resistance to neomycin, ie, a (neo) gene of neomycin phosphotransferase. Southern and co-authors, J. Mol Anal. Genet (1982) 1: 327-341. Alternatively, the selectable marker can be present in a separate plasmid and the two vectors are introduced by cotransfection of the host cell, and selected by culturing on the appropriate drug for the selectable marker.

F.- HOST CELLS CONTAINING A NUCLEIC ACID MOLECULE CODIFIES SURVIVINE. SUPPLIED EXOGENOUSLY The present invention further provides host cells transformed with a nucleic acid molecule encoding a survivin protein of the present invention. The host cell can be prokaryotic or eukaryotic. Eukaryotic cells, useful for the expression of a survivin protein, are not limited, as long as the cell line is compatible with cell culture methods and is compatible with the propagation of the expression rector and with the expression of the gene product. Survivin Preferred eukaryotic host cells include, but are not limited to: yeast, insect and mammalian cells, preferably vertebrate cells, such as mouse, rat, monkey or a human fibroblast cell line; cells being most preferred that do not naturally express a survivin protein. Preferred eukaryotic host cells include the BaF3 cell line, dependent on murine IL-3 and similar eukaryotic tissue culture cell lines. Any prokaryotic host can be used to express the rDNA molecule encoding survivin. The preferred prokaryotic host is E. i or coli. The transformation of the appropriate cellular hosts with a rDNA molecule of the present invention is achieved by well-known methods, which typically depend on the type of vector used and the host system employed. With respect to the transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed.; see, for example, Cohen and co-authors, Proc. Nati Acad. Sci. USA (1972) 60: 2110, and Maniatis and co-authors, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). With respect to the transformation of vertebrate cells with rDNA containing vectors, electroporation or methods of treatment with cationic or salt lipid are typically employed; see, for example, Graham and coauthors, Virol. (1973) 52: 456; Wigler and co-authors, Proc. Nati Acad. Sci USA, (1979) 76: 1373-76.

Successfully transformed cells, i.e., cells containing an rDNA molecule of the present invention, can be identified by well known techniques. For example, cells that result from the introduction of a rDNA of the present invention can be cloned to produce individual colonies. The cells from these colonies can be harvested, lysed and their DNA content examined for the presence of rDNA, using a method such as that described by Southern, J. Mol. Biol. (1975) 98: 503, or Berent and co-authors, Biotech., (1985) 3: 208, or the proteins produced from the cell can be analyzed by an immunological method.

G.- SURVIVINA PRODUCTION USING A RNAD MOLECULE CODIFIES A SURVIVINA PROTEIN The present invention further provides methods for producing a survivin protein using one of the nucleic acid molecules encoding survivin, described herein. In general terms, the production of a recombinant form of a survivin protein typically involves the following steps. First, a nucleic acid molecule encoding a survivin protein is obtained, such as the nucleic acid molecule illustrated in Figure 1. If the survivin coding sequence is interrupted by introns, it is directly suitable for expression in any host. If not, then a spliced form of the nucleic acid molecule encoding survivin can be generated, and the nucleic acid molecule containing the intron can be used, in a compatible eukaryotic expression system. The nucleic acid molecule encoding survivin in operable linkage is then preferably placed with suitable control sequences, as described above, to form an expression unit containing the survivin coding sequences. The expression unit is used to transform an appropriate host and the transformed host is cultured under conditions that allow production of the survivin protein. Optionally, the survivin protein is isolated from the medium or from the cells; the recovery and purification of the protein may not be necessary in some cases, when certain impurities can be tolerated. Each of the preceding steps can be carried out in a variety of ways. For example, coding sequences can be obtained from genomic fragments and can be used directly in appropriate hosts. The construction of expression vectors that are operable in a variety of hosts is achieved using appropriate replicons and control sequences, as discussed above. Control sequences, expression vectors and transformation methods depend on the type of host cell to express the gene, and were discussed in detail further back. Suitable restriction sites, if not normally available, can be added to the ends of the coding sequence, in order to provide a removable gene for insertion into those vectors. One skilled in the art can easily adapt any host / expression system known in the art for use with the survivin coding sequences to produce a survivin protein.

H.- INHIBITION OF THE DEATH OF THE CELL USING SURVIVINA As provided hereinbefore, survivin has been shown to inhibit cellular apoptosis. Consequently, survivin can be used in methods to prolong the life of cells. In general, apoptosis of the cells can be inhibited by contacting a cell with survivin. There are numerous situations in which it is convenient to inhibit cellular apoptosis. For example, cell death can be inhibited in tissues and organs that are being prepared for transport and transplantation, using the survivin protein. Alternatively, cell lines can be established for long-term culture, using nucleic acid molecules encoding survivin, expressed in the cell line. Accordingly, the survivin protein or the expression of the survivin gene can be used as a means to inhibit cellular apoptosis. In cell culture systems, the survivin protein can be introduced into a cell, for example, via liposome, by penetrin-1 delivery or by inclusion in cell development media, to inhibit apoptosis.

Alternatively, the survivin gene can be introduced and expressed in cells to increase the longevity of the cells in the culture. They provide means and methods for increasing the ability of cultured cells to produce desired compounds, as well as providing methods for establishing long-term culture of primary cell and tissue explants. 5 In tissue transplantation, tissues and organs are typically stored and transported before transplantation. Cell death, by mechanisms similar to apoptosis, can lead to loss of viability of tissues or organs. In this disclosure, an infusion with the survivin protein can be used as a method to inhibit the death of the cells in the said tissues and organs. There are pathological conditions characterized by premature and undesirable cellular apoptosis, for example, in disorders accelerated by age. It is already known that inactivating mutations in an IAP protein can cause human diseases. The example is for NAIP (see above).

Studies of patients with SMA (spinal muscular atrophy, a neurodegenerative disease believed to be caused by aberrantly increased apoptosis) have shown that the NAIP gene is inactivated and is omitted in 75% of these patients (Roy and co-authors, 1995, Cell 80: 167). By extension, inactivating mutations in survivin can result in diseases degenerative diseases characterized by aberrantly increased cell death. Haplotypic markers can be used within and around the survivin site on chromosome 17q25, in population genetics studies, to determine if the site had already been implicated in diseases with increased apoptosis. In those cases, the survivin gene or the survivin protein can be used to treat the conditions. Consequently, the survivin protein or a nucleic acid molecule encoding survivin is administered to an individual as a means to treat abnormal apoptosis.

I.- METHODS TO IDENTIFY SURVIVINA UNION PARTNERS Another embodiment of the present invention provides methods for use in isolating and identifying survivin binding partners. Specifically, the survivin protein can be used as a capture probe to identify survivin binding partners. As used herein, a survivin binding partner is a biomolecule (such as a protein, a DNA or other cofactor) that binds to survivin and mediates survivin inhibition of cellular apoptosis. In detail, a survivin protein is mixed with an extract or fraction of a cell expressing survivin under conditions that allow the association of a binding partner with survivin. After mixing, the peptides that have been associated with survivin are separated from the mixture. The binding partner that binds to survivin can then be removed and further analyzed.

To identify and isolate a binding partner, the entire survivin protein can be used. Alternatively, a fragment of a survivin protein can be used. As used herein, a "cell extract" refers to a preparation or fraction that is made from a cell that is lysed or broken. The preferred source of cell extracts will be cells that naturally express survivin. Examples of such cells include, but are not limited to, tumor cells and embryonic tissues. A variety of methods can be used to obtain an extract from a cell. The cells can be broken using physical or chemical breaking methods. Examples of physical breakdown methods include, but are not limited to, sonic treatments and mechanical shear stress. Examples of chemical lysis methods include, but are not limited to, detergent lysis and enzymatic lysis. Additionally, the cell extract may be prepared from cells that have recently been isolated from a subject and from cells or cell lines that have been cultured. One skilled in the art can easily adapt methods for preparing cell extracts in order to obtain extracts for use in the methods herein. Once an extract is prepared from a cell, the extract is mixed with the survivin protein, under conditions in which the association of survivin with the binding partner can occur. A variety of conditions can be used, conditions being highly preferred those conditions of great resemblance to those found in the cytoplasm of a cell expressing survivin. The aspects, such as osmolarity, pH, temperature and concentration of cell extract used, can be varied to maximize the association of survivin with the binding partner. After mixing under appropriate conditions, the survivin is separated from the mixture. A variety of techniques can be used to separate the mixture. For example, antibodies specific to survivin can be used to immunoprecipitate survivin and the associated binding partner.

Alternatively, common separation techniques and currents can be used, such as chromatography and density / sediment centrifugation. After removing the non-associated cellular constituents found in the extract, the binding partner of the survivin protein can be dissociated, using conventional methods. For example, dissociation can be achieved by altering the salt concentration or the pH of the mixture. To help separate the survivin / binding partner pairs from the mixed extract, the survivin protein can be immobilized on a solid support. For example, survivin can be fixed to a nitrocellulose matrix or to acrylic granules. The binding of survivin to a solid support further aids in separating the peptide / binding partner pair from other constituents found in the extract. Alternatively, the nucleic acid molecule encoding survivin can be used in a yeast two-hybrid system. The yeast two-hybrid system has been used to identify other protein / partner pairs and can be easily adapted to employ the survivin-encoding molecules described herein.

J.- USE OF SURVIVINA UNION PARTNERS Once isolated, the participants in the survivin binding, obtained using the methods described above, can be used for a variety of purposes. The binding partners can be used to generate antibodies that bind to the survivin binding partner, using techniques known in the art. Antibodies that bind to the survivin binding partner can be used to analyze the activity of survivin, as a therapeutic agent to modulate a biological or pathological process mediated by survivin, or to purify the binding partner. These uses are described in detail later.

K, - METHODS TO IDENTIFY AGENTS THAT BLOCK THE INTERACTIONS SURVIVINA PARTICIPATE UNION Another embodiment of the present invention provides methods for identifying agents that reduce or block the association of survivin with a survivin binding partner. Specifically, survivin is mixed with a survivin binding partner, in the presence and absence of an agent to be tested. After mixing under conditions that favor the association of survivin with the survivin binding partner, the two mixtures are analyzed and compared to determine whether the agent reduced or blocked the association of survivin with the survivin binding partner. Agents that block or reduce the association of survivin with the survivin binding partner will be identified as the amount of association present in the sample containing the tested agent decreases. As used herein, an agent is said to reduce or block the survivin / survivin binding partner association, when the presence of the agent decreases the extent to which the survivin binding partner becomes associated with survivin, or prevents such binding. . One class of agents will reduce or block the association by joining the survivin binding partner, while another class of agents will reduce or block the association, joining survivin. The survivin binding partner used in the above analysis can be an isolated and fully characterized protein, or it can be a partially characterized protein that binds survivin or a survivin binding partner that has been identified as being present in an extract cell phone. It will be apparent to one of ordinary skill in the art that, as long as the survivin binding partner has been characterized by an identifiable property, eg, molecular weight, the present analysis can be used. The agents that are analyzed in the above method can be randomly selected or rationally selected or designed.

As used here, it is said that an agent is randomly selected when the agent is chosen at random, without considering the specific sequences involved in the association of survivin with the survivin binding partner. An example of the randomly selected agents is the use of a chemical bank or of a combinatorial bank of peptides, or of a developing broth of an organism. As used herein, it is said that an agent is selected or rationally designed when the agent is chosen on a non-random basis, taking into account the sequence of the destination site and / or its conformation, in relation to the action of the agent . As described above, there are two action sites for agents that block the survivin / survivin binding partner interaction: the contact site of the survivin binding partner, and the survivin contact site in the survivin partner. survivin binding. The agents can be rationally selected or rationally designed using the peptide sequences that constitute the contact sites of the survivin pair / survivin binding partner. For example, a rationally selected peptide agent may be a peptide whose amino acid sequence is identical to the survivin contact site in the survivin binding partner. Said agent will reduce or block the association of survivin with the binding partner, joining the survivin binding agent. The agents of the present invention can be, for example, peptides, small molecules, vitamin derivatives, as well as carbohydrates.

One skilled in the art can easily recognize that there is no limit to the structural nature of the agents of the present invention. One class of agents of the present invention are peptide agents whose amino acid sequences are selected based on the amino acid sequence of the survivin protein. The peptide agents of the invention can be prepared using common and current, solid-phase (or phase-in-solution) peptide synthesis methods, as is known in the art. In addition, the DNA encoding those peptides can be synthesized using commercially available instrumentation for oligonucleotide synthesis, and recombinantly produced using common and current recombinant production systems. Production that uses peptide synthesis in solid phase is necessary if it is not going to include amino acids not encoded by gene. Another class of agents of the present invention are immunoreactive antibodies with critical positions of survivin or the survivin binding partner. As described above, antibodies are obtained by immunization of suitable mammalian subjects with the peptides which contain, as antigenic regions, those portions of the survivin or binding partner, intended to be the target of the antibodies. The critical regions include the contact sites involved in the association of survivin with the survivin binding partner. As discussed previously here, the important minimal sequence of residues involved in survivin activity defines a functional linear domain that can be effectively used as a bait for selection by two hybrids and identification of potential molecules associated with survivin. The use of said survivin fragments will significantly increase the specificity of the selection, as opposed to the use of the full length molecule, or the entire BIR domain and, therefore, is preferred. Similarly, this linear sequence can also be used as an affinity matrix, also to isolate the survivin binding proteins, using a biochemical affinity purification strategy.

L.- USES FOR AGENTS THAT BLOCK THE SURVIVINA ASSOCIATION WITH A PART OF SURVIVINA As established in the background section, survivin inhibits cellular apoptosis. Agents that reduce or block the interactions of survivin with the survivin binding partner can be used to modulate the biological and pathological processes associated with the function and activity of survivin. In detail, a biological or pathological process mediated by survivin can be modulated by administering to a subject an agent that blocks the interaction of survivin with a survivin binding partner. As used herein, a subject can be any mammal, as long as the mammal needs the modulation of a pathological or biological process, mediated by survivin. The term "mammal" is used to mean any individual that belongs to the class of mammals. The invention is particularly useful in the treatment of human subjects. As used herein, a biological or pathological process, mediated by survivin or survivin that binds to a survivin binding partner, refers to a wide variety of cellular events mediated by survivin. Pathological processes refer to a category of biological processes that produce a harmful effect. For example, a pathological process mediated by survivin is the inhibition of cellular apoptosis in tumor cells. This pathological process can be modulated using agents that reduce or block the survival / survivin binding partner association, or block the expression of survivin. As used herein, an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process. For example, an agent is said to modulate the proliferation of tumor cells when the agent decreases the rate or extent of cell division.

M.- ADMINISTRATION OF SURVIVINA OR OF AGENTS THAT AFFECT THE ACTIVITY OF SURVIVINA The agents of the present invention, whether they are agents that block the survivin / binding partner association, or the survivin protein, can be administered parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally or buccally. Alternatively or concurrently, the administration can be carried out by oral route. The dose administered will depend on the age, health and weight of the recipient; of the concurrent treatment class, if any; of the frequency of the treatment and the nature of the desired effect. For example, to treat tumor cells as a means to block the inhibition of apoptosis by survivin, an agent that blocks the expression of survivin or the interaction of survivin with a binding partner, is administered systemically or locally to an individual being treated. . As described below, there are many methods that can be easily adapted to administer said agents. The present invention further provides compositions containing survivin or one or more agents that block the survivin / binding partner association. While individual needs vary, it is within the knowledge of those skilled in the art to determine the optimal scales of effective amounts of each component. Typical doses comprise 0.1 to 100 μg / kg of body weight. Preferred doses comprise 0.1 to 10 μg / kg of body weight. Very preferred doses comprise 0.1 to 1 μg / kg of body weight. In addition to the pharmacologically active agent, the compositions of the present invention may contain pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds, to preparations that can be used pharmaceutically to deliver them to the site of action. Formulations suitable for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water soluble salts. In addition, suspensions of the active compounds, such as oily injection suspensions, may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil; or the synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous suspensions for injection may contain substances which increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol and / or dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent to deliver it to the cell. The pharmaceutical formulation for systemic administration according to the invention can be formulated for enteral, parenteral or topical administration. In fact, all three types of formulations can be used simultaneously to obtain systemic administration of the active ingredient. Formulations suitable for oral administration include hard or soft gelatin capsules; pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations, and their controlled release forms. In practicing the methods of this invention, the compounds of this invention can be used alone or in combination, or in combination with other therapeutic or diagnostic agents. In certain preferred embodiments, the compounds of this invention may be coadministered together with other compounds typically prescribed for these conditions, in accordance with generally accepted medical practice, such as chemotherapeutic agents.

N.- COMBINATION THERAPY Survivin, as well as the agents of the present invention that modulate the activity of survivin, can be provided alone or in combination with other agents that modulate a particular biological or pathological process. For example, an agent of the present invention can be administered that reduces apoptosis inhibited by survivin, in combination with other anti-cancer agents, to * control the growth of cancer cells. Alternatively, survivin may be administered with other protective agents, as a means to reduce cellular apoptosis. As used herein, it is said that two agents are to be administered in combination when the two agents are administered simultaneously or are administered independently, such that the agents act at the same time. The inhibition of the activity / expression of survivin can be used in combination with conventional chemotherapies. The time to use the chemotherapeutic agent in combination with the inhibition of activity / expression of survivin depends on the chemotherapeutic agent used and the type of tumor cell treated. Examples of chemotherapeutic agents that can be used in combination with agents that affect the activity / expression of survivin inc, but are not limited to: alkylating agents, such as cyclophosphamide (CTX); cytoxan), chlorambucil (CHL, leukeran), cisplatin (CisP, platinol), busulfan (myleran), melphalan, carmustine (BCNU), streptozotocin, triethylenemelamine (TEM), mitomycin C and similar alkylating agents; antimetabolites, such as methotrexate (MTX), etoposide (VP16), vepesid) 6-mercaptopurine (6MP), 6-thioguanine (6TG), cytarabine (Ara-C), 5-fluorouracil (5FU), dacarazine (DTIC) and similar antimetabolites; antibiotics, such as actimonicin D, doxorubicin (DXR, adriamycin) daunorubicin (daunomycin), bleomycin, mithramycin and similar antibiotics; alkaloids, such as vinca alkaloids, such as vincristine (VCR), vinblastine and the like; and other antitumor agents, such as taxol and taxol derivatives; glucocorticoid cytostatic agents, such as dexamethasone (DEX, decadron) and corticosteroids, such as prednisone, nucleoside enzyme inhibitors, such as hydroxyurea, amino acid-depleting enzymes, such as asparaginase, and various, similar anti-tumor agents. The use of cytotoxic agents described above in chemotherapeutic regimens is generally well characterized in the arts of cancer therapy, and their use here remains under the same considerations to monitor tolerance and effectiveness and to control routes of administration and dosages. , with some adjustments. For example, current doses of cytotoxic agents may vary, depending on the response of the cultured cells of the patient, determined using the methods of histoculture herein. In general, the dose will be reduced compared to the amount used in the absence of agents to affect the activity / expression of survivin. Typical doses of an effective cytotoxic agent may be in the scans recommended by the manufacturer, and when indicated by in vitro responses or responses in animal models, it may be reduced to approximately one order of magnitude of concentration or amount. Thus, the actual dose will depend on the judgment of the doctor, the condition of the patient and the effectiveness of the therapeutic method based on the in vitro response capacity of the cultured primary malignant cells, or on the sample of histocultivated tissue, or the observed responses in appropriate animal models.

O.- METHODS TO IDENTIFY THE EXPRESSION OF SURFICINE AND THE INHIBITION OF MEDIATED APOPTOSIS BY SURVIVINA The present invention further provides methods for identifying cells involved in the inhibition of survivin-mediated apoptosis, as well as techniques that can be applied to diagnose biological and pathological processes associated with the activity of survivin, the progress of said conditions, the susceptibility of said conditions to the treatment and the effectiveness of the treatment for said conditions. Specifically, the inhibition of survivin-mediated apoptosis can be identified by determining whether the survivin protein is expressed in a cell. It is considered that cells expressing survivin inhibit natural cellular apoptosis. A variety of immunological and nucleic acid techniques can be used to determine whether the survivin protein, or a mRNA that encodes survivin, is produced in a particular cell. In one example, a cell extract is prepared. The extract is then analyzed to determine if survivin is expressed in the cell. The degree of expression provides a measure of the degree of inhibition of apoptosis. An increase in expression is a measure of an increased inhibition of apoptosis. The measure of survivin expression can be used as a marker for a variety of purposes. In tumors, the presence of survivin expression correlates with the proliferating potential of the tumor. In the examples it is shown that lymphomas exhibit varying degrees of expression of survivin; lymphomas that show little to no expression of survivin are low-grade lymphomas, while lymphomas that show high levels of survivin expression are aggressive, high-grade lymphomas, which typically can not be effectively treated. As a consequence, the level of expression of survivin in a lymphoma or in another tumor can be used as a predictive measure of aggression and the possibility of tumor treatment.; the higher the level of expression of survivin, the greater the aggressiveness of the tumor and the greater the difficulty for the treatment. For example, to determine the proliferating potential of the tumor and the ease / prognosis of the treatment, an extraction of tumor cells is performed and the extract is analyzed, for example, by gel electrophoresis to determine if a survivin protein is present. The presence and level of survivin correlate with the proliferating potential of the cancer and with the ease of treatment. Alternatively, as described above, single-filament probes can be used to identify the survivin-encoding mRNA in the cell extracts. In addition to being a marker of the aggressiveness of the tumor and the potential of its treatment, the expression of survivin can be used as a measure of the effectiveness of anti-tumor therapy. In the examples it is shown that HL-60, a promilocitic cell line had high levels of survivin expression. The treatment of the HL-60 cells with retinoic acid and with an anti-cancer agent which acts to differentiate the tumor cells, resulted in the reduction and elimination of survivin expression. The reduction in expression was correlated with the degree of differentiation: the greater the differentiation, the lower the level of expression of survivin. Consequently, the expression of survivin can be used to measure the effectiveness of anti-tumor treatment: if the expression of survivin decreases during treatment, the treatment protocol is effective and can be continued, whereas if the expression of survivin remains unchanged, it is necessary to a different therapeutic regimen or protocol.

P.- OTHER METHODS TO CONTROL THE EXPRESSION OF SURVIVINA The present invention additionally provides additional methods that can be used to control the expression of survivin in a cell. As discussed further back and below, the survivin promoter has a CPG island upstream of its promoter. It is known that CPG islands are known targets for DNA methylation. The DNA methylation sites on the CPG island serve as a means to regulate the expression of survivin: the methylation of the CPG islands results in the suppression of transcription of the gene found downstream of the promoter. Consequently, agents that cross-link DNA, such as DNA-methylase and agents that stimulate the production of endogenous methylases, can be used to control the expression of survivin. Specifically, the expression of survivin in a cell can be reduced or eliminated, causing the cell to increase the level of DNA methylation, particularly in the CPG island found upstream of the survivin gene. In another method, the expression of survivin can be reduced by increasing the expression level of EPR-1. As shown in the examples, the expression of survivin and the expression of EPR-1 are generally mutually exclusive; the expression of EPR-1 results in a decrease or elimination in the expression of survivin, and vice versa. Consequently, the expression of survivin can be reduced by causing a cell to increase the expression of EPR-1.

Q.- ANIMAL MODELS The almost complete structure of the mouse survivin gene has been isolated. The gene is highly conserved with the human counterpart, including sizes of introns, exons and intron-exon boundaries. The coding regions of the mouse survivin gene are 88%, with respect to the sequenced extension, identical to the human protein, thus demonstrating strong evolutionary conservation. The differential and regulated distribution has also been determined in relation to the development of survivin during human development and during mouse development. The availability of the complete structure of the mouse survivin gene and the protein will allow the preparation of target vectors for gene annihilation experiments and a more rational approach for the generation of transgenic mice that express survivin under the control of specific promoters for the tissue. The survivin gene and the survivin protein can serve as targets for gene therapy in a variety of contexts. For example, in one application, non-human animals deficient in survivin can be generated, using common and current annihilation procedures, to inactivate a survivin gene or, if such animals are not viable, molecules of opposite sense of survivin can be used, for regulate the activity / expression of survivin. Alternatively, an animal may be altered so as to contain a survivin or survivin expression unit of the opposite direction, which directs the expression of survivin or the opposite direction molecule, in a tissue-specific manner. In those uses, a non-human mammal is generated, for example, a mouse or a rat, in which the expression of the survivin gene is altered by inactivation or by activation. This can be achieved using a variety of procedures known in the art, such as target or target recombination. Once the survivin-deficient animal is generated, the animal expressing survivin can be used in a tissue-specific manner, or an animal expressing the opposite-sense molecule, 1) to identify biological and pathological procedures mediated by survivin; 2) identify proteins and other genes that interact with survivin; 3) identify agents that can be delivered exogenously to overcome survivin deficiency; and 4) serve as an appropriate screen to identify mutations within survivin that increase or decrease activity. For example, it is possible to generate transgenic mice expressing the human minigene for survivin, in a tissue-specific manner, and test the effect of excess protein expression where survivin is not normally contained. This strategy has been used successfully for another family of apoptosis inhibitors, ie, bcl-2 (Veis and co-authors, Cell (1993) 75: 229). Such an approach can easily be applied to the survivin protein and can be used to obtain a potential beneficial effect of survivin in a specific tissue area to protect the cells against apoptosis (transplantation).

R.- THERAPY WITH THE SURVIVIAN GENE In another embodiment, genetic therapy can be used as a means to modulate biological or pathological processes mediated by survivin. For example, in tumor therapy, it may be convenient to introduce into the subject being treated, a gene expression unit encoding a modulator of survivin expression, such as a nucleic acid molecule encoding an opposite sense. Said modulator can be constitutively produced or can be inducible within a specific cell or target cell or target cell. This allows a continuous or inducible delivery of a survivin expression modulator within the subject. The blocking of the expression of survivin allows the control of the development of tumor cells. Similarly, cells can be genetically engineered to express survivin, for example, in pancreatic, allograft, β cells for transplantation. The level of survivin gene expression can be correlated with the level of resistance to apoptosis. Thus, the survivin genes also have use in therapy with anti-apoptosis genes. In particular, a functional survivin gene can be used to support neuronal cells undergoing apoptosis in the course of a neurodegenerative disease, lymphocytes (i.e., T cells and B cells) or cells that have been damaged by ischemia. Retroviral vectors, adenoviral vectors, adeno-associated viral vectors or other viral vectors with appropriate tropism for cells that are likely to be involved in apoptosis (eg, epithelial cells) can be used as a delivery system for gene transfer, for a therapeutic construction of survivin gene. The many useful factors for this purpose are generally known (Miller, Human Gene Therapy 15-14, 1990, Friedman, Science 244: 1275-1281, 1989, Eglitis and Anderson, Bio Techniques 6: 608-614, 988, Tolstoshev and Andreson , Current opinion in biotechnology, 1: 55-61, 1990; Sharp, The Lancet, 337: 1277-1278, 1991; Cometia and co-authors, Nucleic Acid Research and Molecular Biology 36: 311-322, 1987; Anderson, Science 226: 401-409, 1984, Moen, Blood Cells 17: 407-416, 1991, Miller and co-authors, Biotechniques, 7: 980-990, 1989, Le Gal La Salle and co-authors, Science, 259: 988-990, 1993; Johnson, Chest 107: 77S-83S, 1995). The retroviral rectors are particularly well developed and have been used in clinical settings (Rosenberg and co-authors, N. Engl. J. Med., 323-370, 1990, Anderson and co-authors, US Patent No. 5399,346). Non-viral approaches can also be employed for the introduction of therapeutic DNA into cells that have been otherwise predicted to suffer from apoptosis. For example, survivin can be introduced into a neuron or a T cell by lipofection (Feigner and co-authors, Proc. Nati, Acad. Sci. USA, 84: 7413-1987; Ono and co-authors, Neurosci. Lett., 117: 259 , 190; Brigham and co-authors, Meth. Enz., 101: 512, 1983); conjugation asialorrosonucoide-pollisina (Wu and coauthors, J. Biol. Chem., 263: 14621, 1988; Wu and co-authors, J. Biol. Chem., 265: 16985, 1989); or, less preferable, by microinjection under surgical conditions (Wolff et al., Science, 247: 1465, 1990).

For any of the above-described application methods, the therapeutic construction of survivin nucleic acid is preferably applied to the site of the predicted apoptosis event (eg, by injection). However, it can also be applied to tissue in the vicinity of the predicted apoptosis event or to a blood vessel that feeds the predicted cells suffering from apoptosis. In the described constructs, survivin cDNA expression can be directed from any suitable promoter (e.g., human cytomegalovirus (CMV) promoters, simian virus 40 (SV40) or metalloionein promoters) and can be regulated by any appropriate mammalian regulatory element. For example, if desired, the enhancers known to preferentially direct gene expression in neural cells, T cells or B cells, can be used to direct the expression of survivin. The enhancers used could include, but are not limited to, those that are characterized as specific for tissue or for cell, in their expression. Alternatively, if a survivin genomic clone is used as a therapeutic construct (eg, after isolation by hybridization with the survivin cDNA described above), regulation can be mediated by cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, which includes any of the promoter or regulatory elements described above.

S.- USE OF THE PROMOTER OF SURVIVINA TO DIRECT THE EXPRESSION OF GENES The present invention further provides the survivin gene promoter in a form that can be used in the generation of expression vectors. Specifically, the survivin promoter, identified as being 5 'from the initial ATG codon of survivin, can be used to direct the expression of a DNA sequence encoding the protein, operably linked. Since the survivin promoter does not have a box TATA, who is skilled in the art will use a 5 'fragment, such as nucleotides 2560-2920 (which include exon 1). The survivin promoter is expressed in fetal tissues and, therefore, can be used as an expression of the target protein in specific cell types during specific stages of development. As discussed below, transfection of 3T3 cell with * ^ the oncogene c-myc, results in up-regulation of the mRNA of survivin, detected by Northern blotches. Consequently, the DNA encoding anti-tumor polypeptides under the control of the survivin promoter could be used to transfect tumor cells, in which it would be expressed. Anyone who is an expert in art can easily use the Survivin promoter in expression vectors, using methods known in the art.

T.- PREVENTIVE ANTI-APOPTOTIC THERAPY In a patient who was diagnosed as being heterozygous for a survivin mutation or who was susceptible to survivin mutations (even if those mutations still do not produce an alteration or loss of the biological activity of survivin), or a patient who was diagnosed with a degenerative disease (for example, motor neuron degenerative diseases, such as SMA or ALS diseases) , or to those who are ^ fc diagnosed HIV-positive, any of the therapies can be administered - 10 described, before the phenotype of the disease occurs. For example, therapies can be provided in a patient who is HIV positive, but who still does not show a decrease in T cell count or other obvious signs of the disease.

AIDS. In particular, compounds can be administered which demonstrate that they increase the expression of survivin or the biological activity of survivin, f ^ by any normal dose and any usual route of administration.

Alternatively, gene therapy can be performed using a survivin expression construct, to reverse or prevent the cell defect, prior to the development of the degenerative disease. The methods of the present invention can be used to reduce or diagnose the disorders described here in any mammal, for example, humans, domestic pets or livestock. When a non-human mammal is treated or diagnosed, the survivin polypeptide, the nucleic acid or the antibody, specific for that species, is preferably used.

U.- EXAMPLES OF ANALYSIS OF ADDITIONAL APOPTOSIS In addition to the preceding discussion, specific examples of apoptosis analysis are also provided in the following references. Analyzes for lymphocyte apoptosis are described by: Li and coauthors, Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein, Science, 268: 429-431, 1995; Gibellini and coauthors, Tat-expressing Jurkat cells show an increased resistance to different apoptotic stimulin, including acute human immunodeficiency virus-type 1 (HIV-1) infection, Br. J. Haematol., 8: 24-33, 1995; Martin and co-authors, HIV-1 infection of human CD4 * T cells in vitro. Differential induction of apoptosis in these cells, J. Immunol., 152: 330-42, 1994; Terai and co-authors Apoptosis as a mechanism of cell death in cultured T lymphoblasts acutely infected with HIV-1, J. Clin. Invest, 87: 1710-5, 1991; Dhein and co-authors, Autocrne T-cell suicide mediated by APO-1 / (Fas / CD95), Nature, 373: 438-441, 1995; Katsikis and coauthors, Fas antigen stimulation induced apoptosis of T lymphocytes in human immunodeficiency virus infected individuáis, J. Exp. Med., 1815: 2029-2036, 1995; Westendorp and co-authors, Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120, Nature 375: 497, 1995; DeRossi and co-authors, Virology, 198: 234-44, 1994. Analyzes for apoptosis in fibroblasts are described by: Vossbeck and coauthors, Direct transforming activity of TGF-beta on rat fibroblasts, Int. J. Cancer, 61; 92-97, 1995; Goruppi and coauthors, Dissection of c-myc domains in volved in S phase induction of HIH3T3 fibroblast, Oncogene, 9: 1537-44, 1994; Fernández and co-authors, Differential sensitivity of normal and Ha-ras transformed C3H mouse embryo fibroblasts tumor necrosis factor; induction of gcl-2, c-myc and manganese superoxide dismutase, in resistant cells, Oncogene, 9: 2009-17, 1994; Harrington and coauthors, Myc-induced apoptosis in fibroblasts is inhibited by specific cytokines, EMBO J., 13: 3286-3295, 1994; Itoh and coauthors, A novel protein domain required for apoptosis. Mutational analysis of human Fas antigen, J. Biol. Chem., 268: 10932-7, 1993. Analyzes for apoptosis in neuronal cells are described by: Melino and co-authors, Tissue transglutaminase and apoptosis: sense and antisense transfection studies with human neuroblastoma cells, Mol. Cell Biol., 14: 6584-6596, 1994; Resembleum and co-authors, Evidence for hypoxia-induced, programmed cell death of cultured neurons, Ann. Neurol. 36: 864-870, 1994; Sato and coauthors, Neuronal differentiation of PC12 cells as a result of cell death death by bcl-2, J. Neurobiol., 25: 1227-1234, 1994; Ferrari and co-authors, N-acetylcysteine D- and L-stereoisomers prevent apoptotic death of neuronal cells J. Neurosci., 1516: 2857-2866, 1995; Tallery and co-authors, Tumor necrosis factor alpha-induced apoptosis in human neuronal cells: protection by the antioxidant N-acetylcysteine and the genes bcl-2 and crma, Mol. Cell. SW/. 585: 2359-2366, 1995; Talley and co-authors, Tumor Necrosis factor alpha-induced apoptosis in human neuronal cells: protection by the antioxidant N-acetylcysteine and the genes bcl-2 and crma, Mol. Cell. Biol., 15: 2359-2366, 1995; Waikinshaw and co-authors, Induction of apoptosis in cataecholaminergic PC12 cells by L- DOPA. Implications for the treatment of Parkinson's Disease, J. Clin, Invest, 95: 2458-2464, 1995. 5 Analyzes for apoptosis in insect cells are described by: CIem and coauthors: Prevention of apoptosis by a baculovirus gene during infection on insect Cells, Science, 254: 1388-90, 1991; Crook and co-authors, An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif, J. Virol., ^ Fc, 67: 2168-74; Rabizadeh and coauthors, Expression of the baculovirus p35 gene 0 inhibits mammalian neural cell death, J. Neurochem., 61: 2318-21, 1993; Birmbaum and coauthors, An apoptosis inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys / His sequence motrf 's, J. Virol., 68: 2521-8, 1994; CIem and co-authors, Mol. Cell Biol, 14: 5212-5222, 1994.

V.- THE USE OF SURVIVINA IN TISSUE AND ORGAN TRANSPLANTATION The present invention includes methods for inhibiting or preventing rejection of tissue or organ transplantation in a subject, comprising the local administration of a survivin polypeptide. The polypeptide or survivin fragment of a peptidomimetic thereof, inhibitor of apoptosis, of a transgene encoding a survivin polypeptide or of a transgene encoding a survivin polypeptide fragment to the tissue, organ or site next to the transplant. The local delivery of polypeptides, peptidomimetics to tissue, organ or a site close to transplantation, is achieved by any commonly available means, including, but not limited to, local perfusion, injection, microsponges, microcapsules, liposomes or delivery vehicles released for a time. . Local delivery of a transgene encoding a survivin polypeptide or a transgene encoding a survivin polypeptide fragment to the tissue, organ, or site near the transplant can be achieved with any available vector, by lipofection, or by direct injection of plasmid DNA. See Quin and co-authors (1995), Transplantation 59 (6): 809-816; Le Coultre and coauthors (1997) Eur. J. Pediatr. Surg., 7 (4): 221-226; Wang and co-authors, (19092) Transplantation (53 (3): 703-705; Wang and co-authors (1996) Transplantation 61 (12): 1726-1729; Schmid and co-authors (1997) Eur. J. Cardiothorac, Surg. (6): 1023-28, and Boasquevisque, C. and coauthors (1997) Ann Thorac Surg., 63 (6): 1556-1561 The vectors encoding the transgene include replicable replication and defective vectors, such as retroviral vectors, adenovirus vectors or other vectors with appropriate tropism for cells, which are probably involved in apoptosis, or cells near the site of apoptosis In transgenic constructs, expression can be directed from any suitable promoter, including tissue-specific promoters that direct gene expression in specific cell types, such as the human insulin promoter Local delivery of transgene to the tissue, organ, or site near the transplant is obtained by commonly available means, including , but without limitation, direct local perfusion, injection, microsponges, microcapsules, liposomes or delivery vehicles over time. Without further description, it is believed that one of ordinary skill in the art can, using the foregoing description and the following illustrative examples, make and use the compounds of the present invention and practice the claimed methods. Accordingly, the following working examples specifically indicate the preferred embodiments of the present invention, but should not be construed as limiting the remainder of the description in any way. Other generic configurations will be apparent to those skilled in the art. All newspaper articles and other published documents, such as patents and patent applications, are hereby incorporated by reference, in their entirety.

EXAMPLES EXAMPLE 1 EXPERIMENTAL PROCEDURES AND CLONING CELLS AND CELL CULTURE The following cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA): HEL lymphoma B, Daudi and JY B lymphoma, THP-1 monocytic, Jurkat T leukemia, HeLa epithelial carcinoma, HL-60 promyelocytes and Lu18 fibroblasts of human lung, untransformed. The T leukemia cell line MOLT13 was previously characterized (Altieri, DC FASEB J (1955) 9: 860-865. Cells were maintained in culture in RPMI 1640 complete medium or DMEM (HeLa, Lu18) (BioWhitaker, Walkersville, MD, USA), supplemented with 10% heat inactivated fetal bovine serum, 10% (FBS, Whitaker), 2 mM L-glutamine and 10 mM HEPES, endothelial cells were isolated from the human umbilical vein (HUVEC) by treatment with collagenase and kept in culture in DMEM medium, supplemented with 20% FBS, 2 mM L-glutamine and '10 endothelial cell growth factor (Biomedical Technologies, Stoughton, MA, E. U. A.). Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood, collected from normal informed volunteers, by differential centrifugation in Ficoll-Hypaque (Pharmacia, Piscataway, NJ, USA) at 400 g and 22 ° C, and washed in phosphate regulated salt (PBS), pH 7.4. In some experiments HL-60 cells were terminally differentiated to a mature monocyclic phenotype by a 72 hour culture in the presence of 0.1 μM of 1, 25-dihydroxy-vitamin D3 and 17.8 μg / ml of indomethacin (Sigma Chemical Co., St Louis, MO, USA). The induction again of markers dependent on differentiation, on HL-60 cells treated with vitamin D3, including integrin CD11b / CD18 (Hickstein DD Y co-authors, J. Immunol. (1987), 138: 513-519) was determined by flow cytometry with mAb LM2 / 1 anti-CD11b.

GENOMIC CLONING AND cDNA. CHROMOSOMAL LOCATION AND SPOTS OF SOUTHERN A genomic bank of human P1 was selected (Genome Systems, St. Louis, MO, E. U. A.) by hybridization with a 1.6 kb fragment containing the complete human EPR-1 cDNA (Altieri, D. C, FASEB J (1995) 9: 860-865). Three translapant clones were isolated, purified and confirmed by Southern hybridization with the EPR-1 cDNA. j ^ The hybridizing fragments generated were cloned by digestion by restriction with BamHl, Hindlll and Xbal (Boehringer Mannheim, Indianapolis, IN, USA, in pBluescript (pBSKS ", Stratagene, San Diego, CA, USA) for further analysis, an overlapping contig covering 14796 base pairs was arranged , from two hydrolyzing P1 clones of EPR-1, characterized by extraction analysis, and completely sequenced in both strands by f ^ automated sequencing based on Taq FS polymerase, using the automatic sequencer Applied BioSystem Prism 377 (Foster City, CA, USA) In some experiments, 10 mg of total RNA extracted from HeLa cells by the guanidinium isothiocyanate method was sensitized with oligonucleotide C3 / 27"sense" positive, EPR-1 (base pairs 80 -102) and was reverse transcribed in the presence of 200 U of Superscript II (Life Science, Grand Island, NY, E.U.A.), for 50 minutes at 42 ° C. The resulting cDNA was amplified by means of PCR in the presence of 0.5 mg of T5 / 27 sensitizers derived from EPR-1 (base pairs 161-184) and G11 / 16 (1124-1098, numbering of the coding sequence of EPR-1 ), 200 mM dNTP (New England Biolabs, Beverly, MA, USA) and 2 U of Vent DNA-polymerase (New England Biolabs) in a total volume of 50 ml.

After 35 cycles of amplification, with fixation at 58 ° C for one minute, denaturation at 94 ° C for 1 minute and extension at 72 ° C for 1 minute, the product was analyzed by agarose gel electrophoresis, subcloned in pCRM (Invitrogen Corp., San Diego, CA, USA) and completely sequenced in both strands. The analysis of the "contig" assembly of the DNA and the ~ g ^ protein sequence were performed using the packages of '10 Lasergene programming (DNASTAR, Madison, Wl, E. U. A.), and Mac Vector (Eastman Kodak, Rochester, NY, E. U. A.). The chromosomal location of the EPR-1 hybridization gene was carried out by fluorescence in situ hybridization. The purified DNA from a P1 cloning hydride of EPR-1 was labeled with digoxigenin dUTP (Amersham Corp., Arlington Heights, IL, E. U.

A.), by nick translation. The labeled probe was combined with human DNA subjected to shear stress and hybridized to normal metaphase chromosomes, from PBMC stimulated with phytohemagglutinin in a solution containing 50% formamide, 10% dextran sulfate and 2X SSC. For dyeing 20 in two colors, the D17Z1 probe conjugated with biotin, specific for the centromere of chromosome 17, was cohybridized, with the clone P1 marked with digoxigenin. Specific spotting was detected by incubating the hybridized slides with fluorescein anti-digoxigenin antibodies and Texas avidin red. The slides were counter-stained with propidium iodide for marking with one color, or with DAPI for marking with two colors. A total of 80 metaphase cells were analyzed, exhibiting 59 specific labeling cells. For Southern hybridization, human genomic DNA was extracted from HeLa cells, according to published protocols, digested with EcoRI, BamHl, Xbal or Hindlll, separated on a 0.8% agarose gel and transferred to membranes of GeneScreen nylon (New England Nuclear, Boston, MA, USA). After UV entanglement (Stratalinker, Stratagene, San Diego, CA), the membrane was prehybridized with 100 mg / ml sperm DNA from s? denatured salmon (Promega Corp., Madison, Wl, USA) in 5X SSC, 0.5% SDS, 5X Denhardt's solution and 0.1% sodium pyrophosphate, at 65 ° C in a roller hybridization oven (Hoefer Scientific , San Francisco, CA). Hybridization was carried out with gel-purified ^ P-dCTP (Amersham) (GeneClean Bio 101, Vista, CA, USA), 1.6 kb EPR-1 cDNA, labeled and f randomly sensitized (Boehringer Mannheim, Indianapolis, IN , USA), for 16 hours at 65 ° C. After two washes in 2X SCC, 1% SDS for 30 minutes at 65 ° C and 0.2X SSC at 22 ° C, the radioactive bands were visualized by autoradiography, using an X-ray film X-Omat AR of 20 Kodak, and intensifying the filters (DuPont de Nemours, Wilmington, DE, USA). In other experiments, lymphoblastoid cells cultured in LMP agarose (Bio Rad, Richmond, CA) were imbibed at a concentration of 2x10 6 / block of 220 μl and DNA was extracted by common procedures. After block digestion with Mlul or Notl, the samples were separated by pulsed-field gel electrophoresis, on a 1% agarose gel, for 20 hours, at 200 V, with a pulse time of 75 seconds, using a Bio-Rad CHEF DRII device (Hercules, CA). After transferring to nylon membranes and interlacing with UV, hybridization was carried out with the EPR-1 cDNA and the washings were performed as described above. In another series of experiments, a blot containing aliquots of genomic DNA, isolated from various species (Clontech, San Francisco, CA) was hybridized with a 3 '548 base pair fragment of EPR-1 cDNA, as described earlier.

STAINED NORTHERN Single-filament probes, specific for normal-sense or opposite-sense EPR-1 sequences, were generated by asymmetric PCR amplification of a fragment of 301 base pairs of EPR-1 cDNA. The template, which comprised the first 226 base pairs 5 'of the coding sequence of EPR-1, plus 75 base pairs of the retained regulatory intron (Altieri, DC FASEB J (1995) 9: 860-865) was generated by digestion by restriction of the EPR-1 cDNA with EcoRI (cloning site) and Sacll, it was gel purified and mixed in a total volume of 10 ml with 15 pmoles of dNTP (New England Biolabs), 7.5 pmol of dCTP and 25 mCi of ^ P-dCTP (Amersham), in the presence of 20 mM Tris HCl, 50 mM KCI, pH 8.4, 1.5 mM MgCl2 and 2.5 U Taq DNA polymerase (Life Science). The generation of a probe of opposite direction, specific for EPR-1, was carried out by the addition of 0.2 mg / ml of an oligonucleotide inverse "Sacll", 5TGCTGGCCGCTCCTCCCTC3 ', while extending the positive strand of EPR-1 and generating a specific probe for survivin, using 0.2 mg / ml of the positive sense oligonucleotide F11 5-ATGACCTCCAGAGGTTTC3'. Twenty-five cycles of amplification were performed with denaturation at 94 ° C for one minute, fixation at 52 ° C • 10 for one minute and extension at 72 ° C for one minute. The normal sense or opposite direction probes of EPR-1 were centrifuged through a Sephadex G-50 centrifugal column (Worthington Biochemical Corp., Freehold, NJ) at 14,000g for 5 minutes, to separate the free radioactivity from the incorporated, was heated at 100 ° C for two minutes and added immediately to the hybridization reaction. Identical filament-specific probes were used for hybridization of multiple tissue spots, adult or fetal human mRNA (Clontech) in 5X SSPE, 10X Denhardt's solution 2% SDS, 100 mg / ml sperm DNA of denatured salmon at 60 ° C for 14 hours, and washed at 60 ° C, as described above. Aliquots of total RNA, extracted from undifferentiated or terminally differentiated HL-60 cells with vitamin D3, were processed for Northern hybridization with survivin-specific single-filament probe, as described above.

EXAMPLE 2 PRODUCTION OF ANTI-SURVIVINE ANTIBODIES An antibody specific for the survivin sequence, designated JC700, was produced and characterized in the following manner. A 17mer peptide was synthesized, corresponding to the survivin sequence A3PTLPPAWQPFLKDHRI19, and characterized by mass spectrometry. 100 mg of the survivin peptide was coupled in a ratio of 1: 1, to keyhole limpet hemocyanin, and injected subcutaneously into a rabbit, in complete Freund's adjuvant. After an interval of 4 weeks, the animals were boosted with a subcutaneous injection of 100 mg of peptide in complete Freund's adjuvant and sequentially reinforced and bled in alternate weeks. Purification of the anti-survivin antibody was performed by affinity chromatography on a peptide-Sepharose matrix (5 mg / ml peptide) with elution of the specific IgG fraction in 1 mM glycine, pH 2.5. The specificity of the affinity purified anti-survivin antibody, designated JC700, was determined by ELISA against the immobilized survivin peptide or a control EPR-1 peptide by absorbance at OD405.

EXAMPLE 3 PRODUCTION OF A MONOCLONAL ANTIBODY AGAINST A SURVIVAL FUSION PROTEIN The survivin cDNA was expressed as a fusion protein with GST in E. coli strain BL21, and was purified to homogeneity with GST elimination. Purified protein was used to inject mice and generate monoclonal antibodies using the common hybridoma technology. Three independent mAbs were isolated, cloned twice by limiting dilution and further characterized. One of the new anti-survivin mAbs, designated 8E2, recognized immobilized and purified recombinant survivin, by ELISA and bound survivin to immunoblots, as shown in Figure 11.

EXAMPLE 4 IMMUNOMANCHED AND HYBRIDIZATION IN SITU For immunoblotting, aliquots of extracts solubilized with SDS, of several transformed cell lines, untransformed HUVEC, PBMC or Lu18, or undifferentiated or differentiated HL cells with vitamin D3, were normalized in terms of protein content, by absorbance to OD ^ or, it was separated by electrophoresis in a gel with gradient of 5-20% SDS-polyacrylamide, under non-reducing conditions, and electrowinged on Immobilon membranes (Millipore Corp., New Bedford, MA, USA), at 1.1 A for 30 minutes. minutes, at 22 ° C. The membrane was blocked in TBS, pH 7.4, plus 5% milk, and incubated with 20 mg / ml non-immune rabbit IgG, control, or anti-survivin JC700 antibody, for one hour, at 22 ° C, collected by washes in TBS, pH 7.4 and addition of a 1: 7500 dilution of goat anti-rabbit IgG, conjugated with alkaline phosphatase (Promega) for 30 minutes at 22 ° C. The binding of the primary antibody was revealed by the addition of 75 mg / ml of nitrotetrazolium blue in 70% dimethylformamide (Sigma Chemical Co., St. Louis, MO, USA), plus 50 mg / ml of 5-bromine phosphate -4-chloro-3-indolyl (Sigma) in 100% dimethylformamide.

TISSUE SAMPLES. IMMUNOHISTOCHÍMICA AND HYBRIDIZATION IN SITU Colonic adenocarcinoma tissue samples (6 cases), squamous cell carcinoma of the lung (6 cases), lung adenocarcinoma (9 cases), adenocarcinoma of the pancreas (2 cases), invasive adenocarcinoma of the breast (7 cases), from the archives of the Yale-New Have Hospital, and were used in the present study. Samples were also obtained from 44 tissues of high grade lymphoma and 7 from low grade lymphoma tissue. The tissue samples were fixed in formalin, embedded in paraffin, cut into sections of 5 μm, deparaffinized in xylene and rehydrated in grade alcohol, followed by inactivation of endogenous peroxidase activation by treatment with 2% of H2O2 in methanol.

For immunostaining the slides were boiled for 5 minutes in an ordinary pressure cooker, blocked in 10% normal goat serum and incubated with affinity purified anti-survivin JC700 antibody (20 μg / ml) for 14 minutes. hours at 4 ° C. After washes in PBS, pH 7.4, the slides were further incubated with goat anti-rabbit IgG, conjugated with biotin (Vector Laboratories, Burlingame, CA, E. U. A.), minutes at 22 ° C, and washed in PBS, pH 7.4. After the addition of peroxidase conjugated with streptavidin-biotin (Boehringer Mannheim) during "^ fe 30 minutes at 22 ° C, the slides were washed and the binding of mAbs was revealed • 10 primaries by the addition of 3'-3'-diaminobenzidine (DAB) and counterstained with hematoxylin. Negative controls were carried out replacing the primary antibody with normal goat serum, under the same experimental conditions. In some experiments, aliquots of JC700 antibody were pre-adsorbed with 25 • f mg / ml of the survivin peptide 3-19, before staining the tissue. For in situ hybridization, 1 μg of the survivin cDNA containing the complete coding sequence plus 271 base pairs of the 3 'untranslated region in pcDNA3 (Invitrogen) was completely digested with EcoRI and transcribed in the opposite sense orientation using RNA -polymerase of T7, in the presence of 11-uridine-5'-triphosphate of digoxigenin (Boehringer Mannheim). Tissue slides were coated with 1% gelatin, 0.1% chromium-alumina, baked at 120 ° C for 2 hours and stored, free of dust, at 22 ° C. Sections were deparaffinized and rehydrated by grade alcohol, digested with proteinase K (1 μg / ml in 100 mM Tris HCl, pH 8.7, 50 mM EDTA) for 30 minutes at 37 ° C and acetylated at 0.25% of acetic anhydride and 100 mM of triethanoiamine, pH 8.0 for 10 minutes at 22 ° C. The detection of survivin mRNA in human tissues was carried out, by in situ hybridization of the riboprobe of the opposite sense of survivin, in a regulator containing 4X SSC, 1X Denhardt's solution, 50% deionized formamide, 250 μg. / ml of yeast tRNA, 500 μg / ml salmon sperm DNA and 5% dextran, for 16 hours, at 50 ° C. After washing in 2X SSC for 90 minutes at 48 ° C, immobilized digoxigenin was detected using an anti-digoxigenin mAb (Boehringer Mannheim) at a dilution of 1: 3000, and revealed as alkaline phosphatase, staining with cytochemical stain NGT / BCIP EXAMPLE 5 EXPRESSION OF SURVIVINA IN HUMAN CANCER Survivin is expressed prominently in human cancer. Due to its abundant distribution in types of transformed cells, a potential expression of survivin in neoplasia was investigated, in vivo. Immunohistochemical analysis of formalin-fixed tissue sections, embedded in paraffin, with anti-survivin antibody JC700, purified by affinity, showed abundant expression of survivin in all the examined cases of human lung cancer, including adenocarcinoma (Figure 6a) and Squamous cell carcinoma (Figure 6C). Consistent with the topography of other IAP proteins (Duckett, O S. Y coauthors, EMBO J (1996) 15: 2685-2694), the expression of the protein was localized exclusively in the cytoplasm of the tumor cells, whereas the glandular epithelium Normal, adjacent, lung, did not express survivin (Figure 6C, arrow). No staining was observed when the anti-survivin antibody was replaced with control goat serum (not shown) or after pre-adsorption with the surviving peptide 3-19 immunizer (FIG. 6B), thereby confirming the specificity of the recognition observed. The prominent accumulation of survivin mRNA in squamous cell carcinoma of the lung was independently demonstrated by in situ hybridization with a single-stranded riboprobe (FIG. 6D). Extensive survivin was also detected in all the examined cases of pancreatic adenocarcinoma (figure 6E) and breast (not shown) by immunohistochemistry and colon (figure 6G) by in situ hybridization. However, consistent with its absence in the non-transformed cell types HUVEC and Lu18 (Figure 4C), in the mature tissues (Figure 3) and in terminally differentiated HL-60 cells (Figure 5) no reactivity of the JC700 antibody was observed anti-survivin, with normal, exocrine pancreatic epithelial cells, by immunohistochemistry (FIG. 6F), and no survivin mRNA was found in adjacent non-neoplastic colon glandular epithelium by in situ hybridization (FIG. 6H).

EXPRESSION OF SURVIVINE IN LYMPHOMA TISSUE Tissue samples were obtained from 44 patients with aggressive, high-grade lymphoma and seven samples were obtained from seven patients with non-aggressive, low-grade lymphoma. The sample was treated as described above and examined for the expression of survivin. None of the low-grade lymphoma samples exhibited survivin expression, while 27 samples (61%) of patients with high-grade lymphoma expressed survivin.

EXAMPLE 6 EXPRESSION OF SURVIVINA IN OTHER CANCERS In addition to the malignant forms of cancer discussed above, the expression of survivin was investigated in other types of cancer, in the laboratory of the inventors, or in collaboration with other academic researchers. Survivin was found to be prominently expressed in the most aggressive and metastatic forms of malignant thymoma (around 100 cases tested), in squamous cell carcinoma of the head and neck (around 140 cases) and in all forms of prostate cancer (15 cases). ), including the transitional lesion of benign prostatic hyperplasia. The most aggressive forms of neuroblastoma are also positive for survivin, as discussed below.

EXAMPLE 7 EXPRESSION OF SURVIVINA SPECIFIC TO THE TISSUE Survivin was recently found in all cancers most common humans, but not in adult tissues, terminally differentiated. The expression of survivin in embryonic and fetal development was investigated. Immunohistochemistry and in situ hybridization studies demonstrated strong survivin expression in several fetal tissues regulated by apoptosis, including the stratified epithelial stem cell layer, pancreas "10 endocrine and thymic medulla, with a pattern that does not mesh with that of another inhibitor of apoptosis, that is, bcl-2. A sequence-specific antibody to survivin immunostained a single survivin band of about 16.5 kD in human fetal lung, liver, heart, kidney and gastrointestinal tract.

In mouse embryos, a prominent and almost ubiquitous distribution of survivin to embryonic date (E) 11.5 was found, while at E15-21, expression of survivin was restricted to the distal brochiolar epithelium of the lung and cells derived from the crest. neural, including ganglion neurons in the dorsal root, pituitary, and chorioid plexus. These data suggest that the expression of survivin in fetal and embryonic development may contribute to homeostasis and to the differentiation, independently of oc / -2.

EXAMPLE 8 PREPARATION OF SURVIVINA TRANSFECTANTS TRANSFECTORS OF OPPOSITE SENSE. OF SURVIVINA. INDUCIBLES. AND EXPERIMENTS OF APOPTOSIS / PROLIFERATION A 708 base pair Smal-EcoRI fragment, comprising nucleotides 379-1087 of the EPR-1 cDNA, was directionally cloned in the normal sense orientation into the mammalian cell expression vector pML1 (generously provided by the Dr. R. Pytela, Cardiovascular Research Institute, University of California, San Francisco, CA, USA). The vector of the episomal mammalian cell expression pCEP4 vector is derived, replacing the cytomegalovirus promoter cell with the mMT1 promoter, directing the Zn2 + -dependent expression of the recombinant proteins in mammalian cells (Lukashev, ME and co-authors, J. Biol. Chem. (1994) 269: 18311-18314). Ten million HeLa cells were incubated with 10 mg of pML1 DNA, which contained the opposite-sense survivin construct, plus 50 mg of salmon sperm DNA, for 15 minutes, on ice, followed by a single electrical pulse supplied by a Gene Pulser device (Bio-Rad), at 350 V at 960 μF. Forty-eight hours after transfection, the cells were diluted fifteen times, plaque was formed on 100 mm diameter tissue culture dishes and selected for 4 weeks in complete growth medium containing 0.4 mg / ml hygromycin. Apoptosis was evaluated in the control cultures of the HeLa cell transfectants from the opposite direction of Survivin, by in situ detection of the degradation of the intemucleosomal DNA after induction of the transcription of Zn2 + -dependent EPR-1, under exhaustion conditions. of serum. Briefly, control or opposite-sense survivin transfectants were treated with 200 mM ZnSO4 in 0% FBS for 24 hours at 37 ° C.

The cells were harvested, centrifuged at 800g for 10 minutes at 4 ° C and fixed * ^ pellet in formaina ai 10% overnight, dehydrated, imbibed paraffin blocks and sections of 3-5 mm were placed on highly adhesive slides. The samples were treated with 20 mg / ml proteinase K for 15 minutes at 22 ° C, washed in distilled water, inactivated with endogenous peroxidase in 2% H2O2, in PBS, and subsequently mixed with digoxigenin-labeled dUTP, in the presence of terminal deoxynucleotidyl transferase (TdT), -f followed by anti-digoxigenin antibody conjugated with peroxidase. Nuclear staining in apoptotic cells was detected by DAB, according to the manufacturer's instructions (AptoTag, Onco, Gaithersburg, MD, E. U. A.). Control experiments were carried out omitting the step of incubation of the enzyme. The morphological aspects of 20 apoptotic cells (apoptotic bodies) were detected under the various conditions tested, by staining with hematoxylin / eosin, on the same slides.

For proliferation experiments, vector control HeLa cells or opposite-sense transfectants of survivin, in tissue culture plates of 24 concavities, at 20 × 10 4 / concavity, (Costar), was induced with 200 mM of ZnSO 4 for 16 hours. at 37 ° C, it was harvested at 24 hour intervals, and cell proliferation was determined under the various conditions tested, by means of a microscope, directly counting the cells. The up-regulation of survivin expression under these experimental conditions was determined by immunostaining with JC700 antibody.

EXAMPLE 9 IDENTIFICATION OF THE COMPLEMENTARY GENE EPR-1 Three overlapping clones were isolated by hybridization selection of a human P1 plasmid genomic library with the EPR-1 cDNA, and confirmed by Southern blotting. This gene was placed with respect to the long arm of chromosome 17, to the band 17q25, by in situ hybridization by fluorescence (figure 1A, B). A "contig" of P1 fragments covering 14796 base pairs in pBSKS "was cloned and completely sequenced in both strands (figure 1 C) .There were three putative splice sites, which perfectly coincide with the consensual sequences for the limits. eukaryotic intron-exons (Padgett, RA and coauthors, Ann.Rev. Biochem (1986) 55: 1119-1150), in positions 2922, 3284 and 5276 (donor) and 3173, 5157 and 11954 (recipient), defining that Thus, a gene organization in four exons and three introns of 252, 1874 and 6678 base pairs, respectively (Figure 1 D), The sequence analysis of the putative coding regions showed almost complete identity with the EPR-1 cDNA (Figure 1 D). Altieri, D.C., FASEB J (1995) 9: 860-865), except for five nucleotide changes and six nucleotide insertions, however, the three splice sites were found in the opposite direction filament, complementary to the EPR-1 that encodes the sequence. Consistent with is In the unexpected orientation, the complementary EPR-1 gene revealed a GC-rich 5 'region, comprising nucleotides 2560-29290 and including the exon (see below) that satisfies the base composition criteria of a CpG island ( Gardiner-Garde, M and coauthors, J. Mol. Biol. (1987) 196: 261-282 and Frommer, 1987). Sequencing of the 2.5 kb upstream of the CpG island revealed a less TATA promoter with numerous Sp1 sites (not shown).

PATTERN OF COMPLEX HYBRIDIZATION AND CONSERVATION EVOLUTIONARY OF SEQUENCES EPR-1 The probing of human genomic DNA with the EPR-1 cDNA revealed several hybridizing fragments (Figure 2a). Of these, it was not possible to recapitulate an Xbal fragment of around 7.5 kg, a BamHl fragment of 7.6 kb and four Hindlll fragments of around 15, 7.5, 6.4 and 3.7 kb, respectively (figure 2a, arrows), by the map of restriction of the opposite-side EPR-1 gene (Figure 1 C). In contrast, other bands of comparable intensity, which included an Xbal fragment of 5.15 kb and a BamH1 fragment of 7.1 kb, originated genuinely from the EPR-1 gene of opposite direction, and consisted of two or three exons, respectively (Figure 2a) . In contrast to this complex hybridization pattern, the Southern blot of human genomic DNA, of high molecular weight, digested with Mlul or Notl, and separated by pulsed-field gel electrophoresis, revealed individual hybridizing bands of EPR-1, about 75 kb and 130 kb, respectively (Figure 2B). Finally, Southern blots of multiple species genomic DNA revealed significant evolutionary conservation of EPR-1 related sequences (Figure 2C), with numerous strongly hybridizing bands in mammalian species and weaker signals in rabbit or chicken genomic DNA , under very strict hybridization conditions (figure 2C).

DISTRIBUTION OF EPR-1 TRANSCRIPTS OF NORMAL SENSE / OPPOSITE SENSE IN DISCORDING TISSUE We investigated the potential expression of different transcripts of EPR-1 of normal sense or opposite direction, in Northern blots, with specific probes of a single filament. Consistent with the size of the spliced EPR-1 message (Altieri, DC FASEB J (1995) 9: 860-865), a specific filament probe of EPR-1 detected a prominent band of about 1.2 kb in mRNA extracted from all adult tissues and terminally differentiated examined (figure 3A). In contrast, no specific band hybridized with the single-stranded probe, specific to the opposite direction, of EPR-1, in adult tissues, under the same experimental conditions (Figure 3B). A similar band was detected, of about 1.2 kb, using the EPR-1 specific probe, of a single filament, in the fetal kidney and, to a lesser extent, in the fetal liver, lung and brain (Figure 3A). Unlike the absence of hybridization in adult tissues, the specific probe for the opposite direction of EPR-1 recognized a band of about 1.9 kb, prominent, and a larger species, 3.2 kb, which corresponded to the size of an incompletely processed transcript , in fetal liver, at the same time that weaker hybridization bands were also seen in fetal kidney, lung and brain (Figure 3B). A control hybridization with an actin probe confirmed comparable loading of mRNA in adult and fetal samples (Figure 3C).

CHARACTERIZATION OF THE GENE EPR-1 PRODUCT OF OPPOSITE SENSE Inspection of the 5'-CpG island in the opposite-sense EPR-1 gene revealed an ATG start codon at position 2811, surrounded by a sequence (CGGCATGG) that conformed well to consensus for the eukaryotic start of the translation (Kozak, M., Nucleic Acids Res. (1984) 12: 857-872).

Analysis of the opposite direction EPR-1 sequence, in the 5 '- 3' direction, dictated by the position of the intron-exon boundaries, revealed an open reading frame of 426 base pairs, spanning the four exons and ending with a TGA codon at position 12042 in exon 4. A canonical polyadenylation signal (AATAAA) was found at position 13166. PCR products, amplified from reverse transcribed HeLa cell RNA, sensitized with oligonucleotides "Normal sense" of EPR-1, coincided perfectly with the genomic sequence and confirmed the open reading frame and predicted intron-exon boundaries (not shown). Two Iambdagt11 cDNA clones, isolated by hybridization of a HEL bank with the EPR-1 cDNA, also coincided with the consensus genomic sequence and revealed a homopolymer tail A in the opposite-sense EPR-1 filament, at position 13186 , 14 base pairs downstream of the polyadenylation signal, generating a 3 'untranslated region of 1144 base pairs. In those clones, the untranslated 5 'region, upstream of the initiating AG, was 49 base pairs, starting at position 2762 in the genomic sequence and containing a stop codon within the frame. The translation of the open reading frame of EPR-1 in the opposite direction, predicted a new protein of 142 amino acids, with an estimated molecular weight of 16,389 and a pl acid of 5.74, lacking the amino-end signal peptide, or a stretch hydrophobic carboxy end for insertion into the membrane (Figure 4A).

A coiled helix was predicted for the last 40 carboxy-terminal residues (Lupas, A. and co-authors, Science (1991) 252: 1162-1164). Investigations with the BLAST database revealed a significant degree of similarity between residues 18-88 of the opposite-side EPR-1 gene product and the BIR module in the IAP family of apoptosis inhibitors (Birmbaum, M. J et al. , J. Virology (1994) 68: 2521-2528; CIem, RJ and coauthors, Mol Cell Biol. (1994) 14: 5212-5222). By this analogy, the gene product EPR-1 of opposite sense survivin was designated. In contrast to other IAP proteins, survivin contained only one BIR, encoded by the first three exons of the gene, and lacked a carboxy-end RING index, with no additional / alternative exons potentially coding for this domain (Figure 1C). An alignment by the Clustal method, between the BIR of survivin and that of other known IAP proteins, is shown in Figure 4B. Despite the overall agreement of consensus substitutions and several conservative substitutions, phylogenetic analysis suggested that survivin is a distantly related member of the IAP family, more closely related to NAIP, which also lacks a RING index (Figure 4B, shaded boxes) (Roy, N. Y co-authors, Cell (1995) 80: 167-178). A polyclonal rabbit antiserum, designated JC700, was established against residues A3PTLPPAWQPFLKDHRI19 (SEQ ID NO: 3) of survivin, purified by affinity chromatography, on a peptide-Sepharose column, and used in Western blots. Consistent with the predicted molecular weight of survivin, the JC700 antibody immunostained a single band of about 16.5 kDa of detergent solubilized extracts from all the transformed cell lines examined, including B lymphoma, Daudi and JY, Leukemia T Jurkat and MOLT13, THP-1 monocytic and HEL of erythroleukemia (Figure 4C). Survivin was also found in isolated peripheral blood mononuclear cells (PBMC). In contrast, survivin expression was not detected in untransformed Lu-18 human lung fibroblasts, or in human umbilical vein endothelial cells (HUVEC) (Figure 4C). Specific bands were not immunoblotted by non-immune, control rabbit IgG under the same experimental conditions (Figure 4C).

IDENTIFICATION OF AGENTS THAT MODULATE THE TRANSCRIPTION OF THE GENE EPR-1 The agents that increase the transcription of the EPR.1 gene can be identified by conventional techniques. Preferably a candidate agent is brought into contact with a cell expressing the EPR-1 gene product and the level of expression of that product or the level of transcription is determined, and the agents that increase or decrease the transcripts of the EPR-1 gene. Alternatively, transcriptional regulatory elements of EPR-1 upstream of a reporter gene can be placed, such as CAT or β-galactosidase.

EXAMPLE 10 REGULATION OF THE EXPRESSION OF SURVIVINA THROUGH DEVELOPMENT / DIFFERENTIATION OF CELLS Consistent with the expression of survivin in transformed cell lines (see Figure 4C), actively proliferating promyelocytic HL-60 cells constitutively expressed high levels of survivin, as demonstrated by single-band immunostaining of about 16.5 kDa with the JC700 antibody, and by Northern blotting of «10 a transcript of about 1.9 kb, with a specific probe of a single filament (figure 5). In contrast, specific bands were not recognized by non-immune, control IgG of rabbit, under the same experimental conditions (Figure 5). The terminal differentiation of HL-60 cells, induced by vitamin (f D3, to a mature monocytic phenotype, resulted in the arrest of the development of these cells and the induction again of specific markers for differentiation, which included the expression, increased by about 200-fold, of CD11 b / CD18 leukocyte integrin. , detected by flow cytometry (not shown) and in agreement with the previous 20 observations (Hickstein, DD Y co-authors, J. Immunol. (1987) 138: 513-519) Under these experimental conditions, the anti-survivin JC700 antibody. it could not immunolabell specific bands of HL-60 extracts treated with vitamin D3, and no survivin transcript was detected by Northern hybridization with a single-filament specific probe (Figure 5) In contrast, a polyclonal anti-EPR antibody -1 immunoassayed an individual band of around 62 kDa, which corresponded to EPR-1, in extracts of HL-60, differentiated with vitamin D3, under the same experimental conditions is (not shown). In addition, the down-regulation of survivin in HL-60 cells differentiated with vitamin D3 was accompanied by an increased surface expression of 5 to 10 times of EPR-1 in these cells, as detected by flow cytometry with monoclonal antibodies. B6 or 12H12 anti-EPR-1 (figure 8). It is also shown in Figure 16 that survivin is down regulated by the combination of gamma cytokine interferon and tumor necrosis factor alpha, but not by any cytokine alone. Similarly, transfection of 3T3 cells with the c-myc oncogene resulted in the up-regulation of survivin mRNA, as detected by Northern blots.

EXAMPLE 11 PROMOTION OF APOPTOSIS WITH SURVIVINA DIRECT WHITE LA SURVIVINA PROMOTES APOPTOSIS AND INHIBES 5 THE PROLIFERATION OF CELLS Transfection of survivin cDNA in mouse or hamster cell lines (NIH 3T3, CHO) was not adequate for the presence of homologs "Endogenous genes are immunochemically indistinguishable in these cells (not shown)." Similarly, initial attempts to hit the target with the survivin gene in stable opposite sense transfectants were not successful because of the slow development of the cells and rapid loss of viability (not shown) Therefore, survivin * HeLa cells were transfected with the 3 'end of the EPR-1 cDNA (opposite sense of survivin) under • ^ P the control of an inducible promoter with metallothionein (Lukashev, ME and co-authors, J. Biol. Chem. (1994) 269: 18311-18314) selected in hygromycin, and analyzed for apoptosis and cell proliferation after activation of transcription, dependent on Zn2 +. Consistent with the expression of survivin in cell lines transformed (FIG. 4C), the JC700 antibody immunostained a single molecular species of about 16.5 kDa in extracts of control HeLa cells, transfected with the vector alone (FIG. 7a). In contrast, no specific band was recognized by the JC700 antibody in metallothionein-induced HeLa cells, transfected with the EPR-1 cDNA (opposite sense of survivin) (Figure 7A). Under these experimental conditions, in situ analysis of the fragmentation of the intemucleosomal DNA by Apto Tag staining revealed only a few apoptotic cells in HeLa vector control cells, induced with Zn2 +, with depleted serum (Figure 7B). In contrast, as discussed above, the inhibition of survivin expression in opposite-sided HeLa cell transfectants, induced with Zn2 +, was associated with prominent nuclear staining in the vast majority '^ of the cells examined (Figure 7B). No nuclear staining was detected in »10 absence of TdT label application of dUTP labeled with digoxigenin (not shown). Typical morphological aspects of apoptosis, including numerous apoptotic bodies, were also demonstrated in HeLa cell transfectants of opposite direction, by staining with hematoxylin / eosin, whereas only occasional apoptotic bodies were observed in cultures of ß) HeLa of control, vectors, under the same experimental conditions (figure 7B). A potential effect of survivin on cell development was also investigated. In these experiments the inhibition of survivin expression, dependent on EPR-1, controlled by metallothionein, caused a profound reduction in the proliferation of HeLa cells, dependent on the serum (Figure 7C). Three days after induction with Zn2 +, the cell count in the HeLa control vector cultures increased by 288%, as opposed to only a 20% increase in the transfectants opposite from survivin, under the same experimental conditions (figure 7C).

EXAMPLE 12 RELATION ESRUCTURA-FUNCION DE LA SURVIVINA The minimal structural requirements involved in the inhibition of survivin-mediated apoptosis have been identified through a strategy '^ Mutagenesis of Ala substitutions for residues very evolutionary conserved in the individual BIR survivin module (IAP repeat of baculovirus). These residues included the amino-terminal half of BIR of survivin, Arg18, Phe22, Trp25, Pro26, Pro35, Ala39, Ala41, Gly42, and Cys46. In the carboxy-terminal half of survivin BIR, the Ala mutants were first identified in the putative zinc binding motif. Other * ^ Additional residues localized by mutagenesis included Asp53, Leu64, Trp67, Pro69, Asp71, Asp72 and Pro73. Survivin mutants are characterized in stable and transiently transfected cells, BaF3 cells dependent on IL-3 and NIH3T3, respectively. In addition to these point mutants, a chimeric survivin molecule containing the carboxy-terminal RING index has also been generated and selected for inhibition of apoptosis (the RING index is a domain found in most other IAP proteins, but not in survivin). Secondly, a truncated form of survivin has also been generated, in which the last 40 carboxyl end residues, containing the predicted coiled helix structure, have been omitted. As shown in Figure 12, Ala mutagenesis of key residues conserved in survivin Trp ^ -Pro ^ -Cys84, produced a recombinant molecule lacking the ability to protect BaF3 cells against apoptosis induced by IL-3 withdrawal. .

EXAMPLE 13 CYVOPROTECTING EFFECTS OF SURVIVINA The classic examples of cell damage in stable cell populations, mediated by apoptosis, include allograft rejection by lymphocyte infiltration; Alzheimer's disease and reperfusion damage after myocardial infarction. In addition to expressing yourself in cancer, functioning in that way as a developmental advantage factor for cancer cells, the sought expression of survivin is useful to protect populations of stable cells against apoptosis and other cellular damage. This application of survivin was tested by adding increasing concentrations of purified recombinant survivin to monolayers of damaged human endothelial cells with hydrogen peroxide (H2O2), a classic apoptosis-inducing stimulus. The results are summarized in Figure 13. Increasing the concentrations of added survivin resulted in a significantly increased viability of the treated cells, as opposed to the control cultures treated with myoglobin, a control protein. Similarly, survivin protected NIH3T3 cells against apoptosis induced by hydrogen peroxide, after transient cotransfection with a lacZ reporter gene, as shown in Figure 17.

EXAMPLE 14 SURVIVINA AS A PREDICTION-FORECAST FACTOR 'g The presence of survivin can be used as a factor Negative predictive-prognosis in neuroblastoma and in non-Hodgkin's lymphoma, and in other cancers. NEUROBLASTOMA: A large series of cases of neuroblastoma (72) was selected for the expression of survivin in a multicenter study. As shown in Figure 14, the expression of survivin increased dramatically when the patients contained at least one negative prognostic factor for aggressive disease and progressing rapidly. Secondly, the expression of survivin was strongly correlated with a more aggressive disease and with an unfavorable histology. It was important that the expression of survivin was not a more sensitive prognostic index than simple histology. It was found that cases positive for survivin, with early diagnosis of favorable histology, contained at least one negative prognostic factor for the progression and spread of the disease.

THE LINFQMA OF HODGKIN. A similar multicenter study on high-grade, non-Hodgkin's lymphoma survivin expression analysis (n = 48) had recently been completed. The results are similar to those observed for neuroblastoma. As shown in Figure 15, the expression of survivin was strongly correlated with a more widespread disease, predominantly in stage IV. Clinically, patients expressing survivin had fewer episodes of complete remission and more episodes of incomplete remission, lack of emission or relapse, compared with patients negative for survivin. POTENTIAL IMPLICATIONS. The demonstrated role of survivin as a predictive and negative prognostic factor in these two embryologically different types of cancer, iterates the potential use of these molecules as a diagnostic tool to monitor the progress and response to therapy. It can also be used for the purpose of determining and identifying populations of patients potentially susceptible to multidrug resistance (groups without remissions or with incomplete remissions). In addition, survivin-derived sensitizers easily designed from the complete survivin gene sequence can be used as a screening tool to identify potential cancer cases in which the survivin gene has been omitted, or mutated. These cases will be very important to identify because the desired inactivation of the survivin gene would confer a favorable prognostic factor for cancer patients, eliminating a gene of potential resistance to the drug. Inactivation of mutations in the survivin gene can indicate the same key residues identified in the initial discrimination of the present, of Ala-based mutagenesis, or result in an abortive or truncated protein, for premature termination of the translation.

EXAMPLE 15 CANCER VACCINE AGAINST SURVIVINA Survivin-directed vaccines, such as those found in various types of cancer, can be developed, as with other intracellular protein targets, related to other diseases. These techniques are commonly available and representative approaches are described by the references cited below. Vaccines may also include the systemic administration of survivin peptide fragments and the use of vectors to deliver minigenes encoding survivin peptides to tumor cell targets is contemplated. As mentioned previously, survivin is not expressed in normal cells, even in proliferating stem cells, in the bone marrow. This guarantees that the immune response mounted against survivin will be highly selective and specific and will not involve normal cells.

DEVELOPMENT AND ADMINISTRATION OF VACCINES BASED ON POLYPEPTIDES Methods of using the peptide components in a polyvalent vaccine-vaccine product for cancer are described by Nardi, N., and coauthors, Mol. Med., (1995) 1 (5): 563-567. Additional references that discuss the different cancer vaccines and immunotherapies for cancer that are currently used, include: N. P. Restifo and M. Sznol Cancer Vaccines, in Cancer: Principies & amp; amp;; Practice of Oncology, DeVita, 30-23-3043 (Lippincott-Raven, Philadelphia, 1997); J. Galea-Lauri and co-authors, Cancer Gene Ther. (1996) 3 (3): 202-214; D. C. Linehan and co-authors, Ann. Surg. Oncol., (1996) 3 (2): 219-228; and J. Vieweg and co-authors, Cancer I nvest. (1995) 13 (2): 193-201. Consistent with the previous approach, Survivin polypeptides or full length Survivin are chemically synthesized by known techniques or recombinantly expressing the appropriate prokaryotic or eukaryotic cells cDNA. The survivin proteins thus produced are then purified, as necessary, to remove contaminating proteins, such as serum or bacterial proteins. Survivin can be further purified using columns containing antibodies that bind to survivin, such as the monoclonal antibody 8E2 antibody JC700 or (both described above) that recognize and bind to survivin ia. When purifying an antibody-based vaccine, survivin produced recombinantly would bind to the antibody, while other proteins and cellular debris would be washed out. The survivin polypeptides can then be isolated and concentrated to a desired concentration. Alternatively, the survivin polypeptides are created by dividing the natural survivin with one or more proteases (e.g., trypsin). The proteolytic fragments are then separated and recovered using SDS-PAGE, high resolution / high pressure separation techniques, or reverse phase HPLC. See R. J: Beynon and J. S. Bond, Proteolytic Enzymes: A Practical Approach, (Oxford University Press, New York, 1989). These isolated peptides are then concentrated to a desired final concentration. Once purified, survivin polypeptides or full-length survivin molecules can be placed in an emulsion containing an adjuvant. Adjuvants contemplated for use with Survivin include aluminum adjuvants, Freund's adjuvant, oil emulsions in water containing tubercle bacilli and interleukin-2 (IL-2). Additional preparations include combining survivin polypeptides with other appropriate antigens, associated with tumor and, optionally, with other immunomodulatory agents, such as cytokines. Other suitable carriers or excipients can be used, including bovine serum albumin, coupling survivin polypeptide hapten, keyhole limpet keyhole, ovalbumin and purified protein derivative of tubercuiina. Peptides can be coupled to carriers using techniques such as those described in Ed Harlow and David Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, 1988). Vaccines can be administered to human subjects in the form of an injected emulsion subcutaneously, intradermally or intramuscularly (IM); Appropriately formulated vaccines can be taken orally. With vaccines containing adjuvants, the vaccine is usually administered, preferably intramuscularly, for example, in the deltoid. The amount of survivin vaccine or survivin peptide vaccine, which will be administered to a patient, will correspond to values typically used for other cancer vaccines. Dose concentrations will vary approximately from 0.25 μg to 1,000 μg per day. The most preferred scales will be around 10 μg to 500 μg per day.

EXAMPLE 16 USE IN DIAGNOSIS OF ANTI-SURVIVAL ANTIBODIES Frequently the antigens associated with the tumor (TAA) are detached from the tumor cells towards the surrounding plasma or towards the blood. As a result, TAA is often found in the blood, and blood samples obtained from patients can be used to detect the presence of cancer, as well as in the manner of a factor in the stage classification of cancers (eg, stage). I, II, II or IV). Survivin is one of said TAA and normal individuals do not express survivin. The results of studies of various cancers have indicated that the presence of survivin (or survivin fragments) correlates with, and is predictive of which the disease may be aggressive or may have metastatic size. A similar strategy of detecting and quantifying survivin levels or 5 survivin fragments can be used to determine residual tumor burden in patients undergoing chemotherapy or irradiation therapy for cancer treatment. Elevated or increasing levels of survivin may reflect an advanced stage of neoplastic disease. ~? ^ For use in diagnostics, it is extracted, using well techniques known, blood from patients who have known loads of cancer or from patients suspected of having cancer. The blood sample is prepared by known techniques and tested for binding to survivin antibodies, which are prepared and, optionally, labeled as discussed further below. Said general antibody detection protocols and the associated reagents are well established in the art. You can also monitor other samples of biological fluid, such as semen, urine or saliva, for the presence of survivin. This diagnostic technique can also be used to monitor the progress of the disease and the response to individualized therapies. This method offers a relatively non-invasive means for monitor the progression or regression of cancer.

EXAMPLE 17 DETECTION OF SURVIVINA BY IMMUNOBIOANALYSIS An illustrative example of immunobioassay to test the The presence of survivin in a patient's blood is based on the ability of monoclonal antibodies to survivin to bind to survivin and remove the detectable survivin from the solution by immunoprecipitation. Said immunobioanalysis is used to detect survivin in patients who are '? ^ cancer suspects, and fractions eluted from fractionation columns.

An aliquot of each patient sample is incubated for two hours at 4 ° C, with a monoclonal antibody that specifically recognizes and binds to survivin, such as the Mab8E2 described above. The monoclonal antibody or agarose beads with anti-mouse IgG are insolubilized, which can be purchased from Sigma Chemical Co., St. Louis, MO, USA The agarose bead complex is prepared (IgG (H + L)) - anti-mouse survivin by first washing the agarose beads with binding buffer containing 0.01 M phosphate buffer (pH 7.2), and 0.25 M NaCl, and then incubating the beads with the monoclonal survivin antibody for 18 hours at 4 ° C , in the same regulator. The agarose beads are then sedimented by centrifugation for 30 seconds at 16,000 x g in a microcentrifuge and the non-specific sites can be blocked by incubation with 2% defatted dry milk in 0.5M NaCl-TMK for 30 minutes at 4 ° C. After blocking, the beads can be washed three times with 0.5 M NaCl-TMK and resuspended in an equal volume of the same regulator. The 20: 1 complex of agarose bead: monoclonal antibody can then be incubated with every 250: 1 of the patient's test sample for two hours at 4 ° C. Any survivin present in the patient's test sample will be found by the survivin monoclonal antibody, which is located on the perias. The pearl complex, now with bound survivin, can be removed by centrifugation for 30 seconds at 16,000 x g. The supernatant is then analyzed for the survivin activity in the bioassay described below. Control samples are treated with blocked beads lacking the survivin monoclonal antibody, and tested for survivin activity in the bioassay.

EXAMPLE 16 DETECTION OF SURVIVINA USING A DIRECT ELISA TEST Normal plasma (control) and plasma samples from cancer patients are diluted 1: 1 with phosphate buffered saline (PBS). One volume of each mixture is added to a centricon-10 filter having a molecular weight limit of 10 kDa, and centrifuged at 5,000 x g (7.00 r.p.m.) for one hour. A volume of PBS is added to the retention product and centrifuged for 30 minutes. The final dilution is around 1: 3. Then you concavities ELISA plate is coated with the product retention to 1: 6, 1: 12, 1: 24, 1: 48 and 1: 96 final dilution in buffer coating bicarbonate having a pH of 9.6 , overnight, at 4 ° C. The plates are then washed twice with a washing buffer containing 5% Tween 20 in phosphate-buffered saline. The residual binding sites are blocked with 4% bovine serum albumin, 300 μ / concavity for two hours. The plates are then washed twice with washing buffer. Then 100 ul of a monoclonal antibody which specifically recognizes and binds to Survivin, as Mab 8E2, at a dilution of 1 is used: 200 in 1% BSA, and added to the concavities and incubated for one hour with stirring . The plates are washed five times with a washing regulator. Next, 100 μl of horseradish peroxidase-conjugated secondary antibody is added, typically at a dilution of 1: 2,000 at each concavity, and incubated for one hour. The plates are washed again 5 times with the washing regulator. each concavity is then added 100 .mu.l / concavity substrate containing 5 ug of survivin and 5 ul of H2O2 / 10 ml of phosphate-citrate buffer, and incubated for 5 minutes. The enzyme reaction is stopped by adding 50 μl / concavity of H2S? 4 2 molar. The absorbance of light at 492 nm is measured in an EIA reader. Patient samples containing survivin will produce a positive reading, while samples that do not contain survivin will be negative.

EXAMPLE 18 FRAGMENTS OF SURVIVINA. PEPTIDES AND ANTAGONISTS OF SMALL MOLECULE As described previously, the key functional residues in survivin, necessary for apoptosis, have been identified. These data provide a template on which synthetic peptides and antagonists and competitive inhibitors of the small molecule survivin function are produced. Preferably the peptides are produced from native survivin or substitutions structure survivin natural peptide, including the functionally important residues Trp67 included - Pro73 - Cys84 can generate peptide fragments of native survivin by common techniques and currents , including protein digests. A determination of which fragments compete with survivin can be easily effected using the apoptosis measurement systems and the apoptosis analysis systems described above. These results provide a unique opportunity to identify a discrete linear sequence in survivin, which is essential for the inhibition of apoptosis. Consistent with the general paradigm of inhibition of apoptosis, dependent on IAP proteins, also predicted that a structural region in the molecule, necessary for anti-apoptotic function, is the primary candidate for a site of interaction with other molecules (such as joint participants). The functionally relevant peptide sequence in survivin, based on the mutagenesis data, is EGWEPDDDPIEEHKKHSSGC (SEQ ID NO: 4). The substitutions with Ala of the underlined residues, results in the complete loss of survivin function in the transfected cells. This linear sequence can be synthesized and used as a more stringent and specific reagent, to isolate associated molecules using common and current biochemical procedures, affinity chromatography; or as a bait for the yeast two-hybrid system. It is also preferred that the ßCOOH coiled helix region of the Survivin is included in survivin fragments and peptides. Recent data indicate that this survivin domain is important for the anti-apoptosis function of survivin. A truncated, recombinant form of survivin lacking the 40 amino acids of the βCOOH end, comprising the coiled helix domain, has been generated. This truncated shape > ^ P was transfected with a lacZ plasmid in NIH3T3 cells, side-by-side with wild-type survivin and XIAP, another member of the IAP gene family. The results, shown in Figure 17, indicate that truncated survivin lost most (about 80%) of the cytoprotective effect in the prevention of apoptosis in transfected cells, induced by hydrogen peroxide.

Incidentally, in this system, survivin was more potent than NAIP to prevent apoptosis. Survivin agonists and antagonists can also be easily identified by conventional techniques. The designed synthetic peptides, based on the natural linear sequence, also function as competitive inhibitors of the interaction of survivin with previously unidentified participating molecules. However, this inhibition must be sufficient to block the anti-apoptosis function of survivin. A similar strategy, based on peptide, has been successful in blocking in vitro and in vivo activation of caspase, which protects cells from apoptosis. See, for example, Milligan, C. E, and co-authors (1995) Neuron. : 385-393.

EXAMPLE 19 THERAPEUTIC USES OF SURVIVAL DNA OF OPPOSITE SENSE As described above, transcription of a sequence of opposite sense of survivin altered the equilibrium of EPR-1 / survivin gene. This was demonstrated in HeLa cell transfectants, in which the metallothionein-induced transcription of the "normal-sense" filament of EPR-1, suppressed survivin expression and profoundly influenced cell apoptosis / proliferation. Additionally, transiently cotransfecting a construct of opposite sense of survivin with a plasmid reported by lacZ, decreased the viability of the transfectants of sense of survivin, after a transfection of 48 hours in cells expressing β-galactosidase. Consequently, the level of expression of survivin in a cell or tissue expressing survivin, as in a tumor, decreases when transfecting the cell or tissue with the normal sense filament of EPR-1, in the DNA. Alternatively, the DNA encoding the opposite sense of survivin is used to transfect a target cell or tissue. Said therapy effectively decreases the translation of the mRNA that encodes survivin, to the survivin protein.

EXAMPLE 20 USE OF SURVIVINE AS A PROTECTIVE AGENT AGAINST THE APOPTOSIS Survivin has been shown to protect cells against apoptosis when administered to cells that have been exposed to hydrogen peroxide or other agents that typically induce apoptosis. It is contemplated that it may be necessary to increase the cell permeability, preferably transiently, in order to facilitate the delivery of survivin or its effective fragments to reduce apoptosis. Certain conditions involving transient metabolic inhibition or transient hypoxia are probably adequate to increase cellular permeability, without the need for additional external agents. Agents that may be appropriate include metabolic inhibitors, such as 2-deoxyglucose and sodium azide. However, the ability of survivin to mediate cytoprotection during the transient increase in cellular permeability, offers the possibility of using therapeutic infusion of recombinant survivin to reduce reperfusion damage and cellular damage during myocardial infarction and stroke. It is contemplated that said procedures are mediated by increased tissue damage, due to apoptosis. Survivin treatment may reduce the degree and extent of damaged tissue. The use of survivin or allelic variants of survivin in subjects to modulate or prevent cell death related to apoptosis would be beneficial to treat or ameliorate the effects of a variety of indications related to apoptosis. These indications include, but are not limited to: the dermatological effects of aging (for example, baldness that is caused by apoptosis of hair follicle cells), disorders and diseases, such as immunosuppression, gastrointestinal disturbances (e.g. , damage to the lining of the intestines, ulcers and damage induced by radiation or chemotherapy), cardiovascular disorders, apoptosis related to reperfusion damage (eg, coronary artery obstruction, cerebral infarction, spinal / cranial trauma and severe concomitant paralysis; damage due to aggressions, such as freezing or burns, and any indication that was previously believed to be treated by superoxide dismutase), rejection of tissue transplantation (for example, graft disease against the recipient) and Alzheimer's disease. The administration of survivin can also be cytoprotective against apoptosis induced by chemotherapy or radiation. The survivin protein can be produced for administration as described above, for example, using the cDNA described herein. The protein may require purification for pharmaceutical administration purposes and said purification steps preferably utilize monoclonal antibody separation and purification techniques, which were also described further back. In a clinical application, survivin was administered to patients at pharmaceutically effective doses, i.e., effective doses to reduce the level or degree of apoptosis present otherwise, through various routes. For example, to treat dermatological diseases in which it was involved '- apoptosis, Survivin can be administered in an ointment, a cream, an ointment or in the form of powder. Topical formulations may contain additional pharmaceutical or cosmetic compositions, such as humectants, humectants, odor modifiers, regulators, pigments, preservatives, vitamins (such as A, C or E), emulsifiers, dispersing agents, wetting agents, stabilizers, propellants, agents antimicrobials, sunscreens, enzymes and the like. Typical doses of survivin that can be administered to patients will be from 0.01% to 1.0% by weight. Additional topical pharmaceutical compositions are described in S. Nakai and co-inventors, U.S. Patent No. 5,672,603. Survivin can also be administered, when it can be appropriate for the condition to be treated, in the form of pills, solutions, suspensions, emulsions, granules or capsules. Survivin can be administered orally; injected into solutions administered intravenously, either alone or in admixture with conventional fluids for parenteral infusion (e.g., fluids containing glucose, amino acids, etc.); injected intramuscularly, intradermally, subcutaneously or intraperitoneally; using suppositories, and in the form of ophthalmic solutions, for example, eye drops. Survivin can also be administered using delayed release carriers, such as liposomes, microsponges, microspheres or microcapsules, which are deposited very close to the tissue being treated, for the prevention of cell death, related to apoptosis. Survivin concentrations or functional allelic variants of survivin, administered by routes other than topical administration, will typically vary in dosage from about 10 μg per day to about 25 μg per day, depending on the route of administration. Of course, it would be expected that experts in the field, such as physicians, may alter those values, on a case-by-case basis, as necessary for the particular patient.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Yale University (B) ADDRESS: 451 College Street (C) CITY: New Haven (D) STATE: CT (E) COUNTRY: USA (F) POSTAL CODE : 06510 (ii) TITLE OF THE INVENTION: SURVIVINE, A PROTEIN THAT INHIBITS CELLULAR APOPTOSIS, AND ITS MODULATION, (iii) NUMBER OF SEQUENCES: 35 (iv) HOW TO READ IN COMPUTER: (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) APPLICATION PROGRAM: Patentln, edition # 1.0, version # 1.30 (v) CURRENT REQUEST DATA: ( A) NUMBER OF APPLICATION: PCT / US97 / 21880 (B) DATE OF SUBMISSION: November 20, 1997 (vi) DATA OF PREVIOUS APPLICATION: (A) NUMBER OF APPLICATION: US 60/031, 435 (B) DATE OF PERSECUTION November 20, 1996 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) NUMBER OF FILAMENTS: one only (D) TOPOLOGY: linear . (ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide". (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 1: TGCTGGCCGC TCCTCCCTC 19 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) NUMBER OF FILAMENTS: one only (D) TOPOLOGY: linear . (I) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide". (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 2: ATGACCTCCA GAGGTTTC 18 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear, (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 3: Wing Pro Thr Leu Pro Pro Wing Trp Gln Pro Phe Leu Lys Asp His Arg 1 5 10 15 Me (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 4: Glu Gly Trp Giu Pro Asp Asp Asp Pro lie Glu Glu His Lys Lys His 1 5 10 15 Ser Ser Gly Cys 20 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) NUMBER OF FILAMENTS: one only (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 5: GCGGGTGAGC TGTCCCTTGC AGATGGC 27 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) NUMBER OF FILAMENTS: one only (D) TOPOLOGY: linear . (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO 6: CCATGTAAGT TGATTTTTCT AGAGAGG 27 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) NUMBER OF FILAMENTS: one only (D) TOPOLOGY: linear , (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 7: AATTGTATGT CTTTATTTCC AGGCAAA 27 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (xi) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 8: Glu Glu Wing Arg Leu Val Thr Phe Gln Asn Trp Pro Asp Wing Phe Leu 1 5 10 15 Thr Pro G n Glu Leu Wing Lys Wing Gly Phe Tyr Tyr Leu Gly Arg Gly 20 25 30 Asp Gln Val Gln Cys Phe Ala Cys Gly Gly Lys Leu Ala 35 40 45 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: ( D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 9: Glu Glu Wing Arg Phe Leu Thr Tyr Ser Met Trp Pro Leu Ser Phe Leu 1 5 10 15 Ser Pro Ala Glu Leu Ala Arg Ala Gly Phe Tyr Tyr He Gly Pro Gly 20 25 30 Asp Arg Val Ala Cys Phe Ala Cys Gly Gly Lys Leu Ser 35 40 45 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 10: Glu Ala Asn Arg Leu Val Thr Phe Lys Asp Trp Pro Asn Pro Asn He 1 5 10 15 Thr Pro Gln Ala Leu Ala Lys Ala Gly Phe Tyr Tyr Leu Asn Arg Leu 20 25 30 Asp His Val Lys Cys Val Trp Cys Asn Gly Val He Wing 35 40 45 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) ) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 11: Glu Glu Val Arg Leu Asn Thr Phe Glu Lys Trp Pro Val Ser Phe Leu 1 5 10 15 Ser Pro Glu Thr Met Ala Lys Asn Gly Phe Tyr Tyr Leu Gly Arg Ser 20 25 30 Asp Glu Val Arg Cys Ala Phe Cys Lys Val Glu He Met 35 40 45 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 12: Lys Ala Ala Arg Leu Gly Thr Tyr Thr Asn Trp Pro Val Gln Phe Leu 1. 5 10 15 Glu Pro Ser Arg Met Wing Wing Ser Gly Phe Tyr Tyr Leu Gly Arg Gly 20 25 30 Asp Glu Val Arg Cys Wing Phe Cys Lys Val Glu He Thr 35 40 45 (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 47 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 13: Glu Glu Wing Arg Leu Wing Being Phe Arg Asn Trp Pro Phe Tyr Val Gln 1 5 10 15 Gly He Ser Pro Cys Val Leu Ser Glu Wing Gly Phe Val Phe Thr Gly 20 25 30 Lys Gln Asp Thr Val Gln Cys Phe Ser Cys Gly Gly Cys Leu Gly 35 4o 45 (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 14: Glu Ala Asn Arg Leu Val Thr Phe Lys Asp Trp Pro Asn Pro Asn He 1 5 10 15 Thr Pro Gln Ala Leu Ala Lys Ala Gly Phe Tyr Tyr Leu Asn Arg Leu 20 25 30 Asp His Val Lys Cys Val Trp Cys Asn Gly Val He Ala 35 40 45 (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 46 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear . (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 15: Glu Glu Wing Arg Leu Lys Ser Phe Gln Asn Trp Pro Asp Tyr Ala His 1 5 10 15 Leu Thr Pro Arg Glu Leu Wing Be Wing Gly Leu Tyr Tyr Thr Gly He 20 25 3o Gly Asp Gln Val Gln Cys Phe Cys Cys Gly Gly Lys Leu Lys 35 40 45 < 2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 46 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 16: Glu Glu Wing Arg Leu Lys Ser Phe Gln Asn Trp Pro Asp Tyr Wing His 1 5 10 15 Leu Thr Pro Arg Glu Leu Wing Be Wing Gly Leu Tyr Tyr Thr Gly Wing 20 25 30 Asp Asp Gln Val Gln Cys Phe Cys Cys Gly Gly Lys Leu Glu 35 40 45 (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 17: Glu Asn Wing Arg Leu Leu Thr Phe Gln Thr Trp Pro Leu Thr Phe Leu 1 5 10 15 Ser Pro Thr Asp Leu Wing Arg Wing Gly Phe Tyr Tyr Thr Gly Pro Gly 20 25 30 Asp Arg Val Wing Cys Phe Wing Cys Gly Gly Lys Leu Ser 35 40 45 (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 18: Glu Glu Wing Arg Phe Leu Thr Tyr His Met Trp Pro Leu Thr Phe Leu 1 5 10 15 Ser Pro Glu Leu Wing Arg Wing Gly Phe Tyr Tyr He Gly Pro Gly 20 25 30 Asp Arg Val Wing Cys Phe Wing Cys Gly Gly Lys Leu Ser - 35 40 45 (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 46 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 19: Glu Glu Wing Arg Leu Lys Ser Phe Gln Asn Trp Pro Asp Tyr Ala His 1 5 10 15 Leu Thr Pro Arg Glu Leu Wing Be Wing Gly Leu Tyr Tyr Thr Gly He 20 25 30 Gly Asp Gln Val Gln Cys Phe Cys Cys Gly Gly Lys Leu Lys 35 40 45 (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 20: Glu Ala Asn Arg Leu Val Thr Phe Lys Asp Trp Pro Asn Pro Asn He 1 5 10 15 Thr Pro Gln Ala Leu Ala Lys Ala Gly Phe Tyr Tyr Leu Asn Arg Leu 20 25 30 Asp His Val Lys Cys Val Trp Cys Asn Gly Val He Wing 35 40 45 (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) ) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 21: Tyr Val Gly He Gly Asp Lys Val Lys Cys Phe His Cys Asp Gly Gly 1 5 10 15 Leu Arg Asp Trp Glu Pro Gly Asp Asp Pro Trp Glu Glu His Wing Lys 20 25 30 Trp Phe Pro Arg Cys Glu Phe Leu Leu Leu Ala Lys Gly Gln Glu Tyr 35 40 45 Val Ser 50 (2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 22: Tyr Val Asp Arg Asn Asp Asp Val Lys Cys Phe Cys Cys Asp Gly Gly 1 5 10 15 Leu Arg Cys Trp Glu Pro Gly Asp Asp Pro Trp He Glu His Wing Lys 20 25 30 Trp Phe Pro Arg Cys Glu Phe Leu He Arg Met Lys Gly Gln Glu Phe 35 40 45 Val Asp 50 (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 23: Tyr Gln Lys He Gly Asp Gln Val Arg Cys Phe His Cys Asn He Gly 1 5 10 1 Leu Arg Ser Trp Gln Lys Glu Asp Glu Pro Trp Phe Glu His Wing Lys 20 25 30 Trp Ser Pro Lys Cys Gln Phe Val Leu Leu Ala Lys Gly Pro Ala Tyr 35 40 45"Val Ser 50 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 49 amino acids (B) TYPE: amino acid (C) ) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 24: Tyr Thr Gly Tyr Gly Asp Asn Thr Lys Cys Phe Tyr Cys Asp Gly Gly 1 5 10 15 Leu Lys Asp Trp Glu Pro Glu Asp Val Pro Trp Glu Gln His Val Arg 20 25 30 Trp Phe Asp Arg Cys Ala Tyr Val Gln Leu Val Lys Gly Arg Asp Tyr 35 40 45 Val (2) INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 49 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) ) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 25: Tyr Thr Gly Gln Gly Asp Lys Thr Arg Cys Phe Cys Cys Asp Gly Gly 1 5 10 15 Leu Lys Asp Trp Glu Pro Asp Asp Pro Wing Trp Gln Gln His Wing Arg 20 25 30 Trp Tyr Asp Arg Cys Glu Tyr Val Leu Leu Val Lys Gly Arg Asp Phe 35 40 45 Val (2) INFORMATION FOR SEQ ID NO: 26: (") CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (Ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 26: Tyr Thr Gly He Lys Asp He Val Gln Cys Phe Ser Cys Gly Gly Cys 1 5 10 15 Leu Glu Lys Trp Gln Glu Gly Asp Asp Pro Leu Asp Asp His Thr Arg 20 25 30 Cys Phe Pro Asn Cys Pro Phe Leu Gln Asn Met Lys Ser Ser Glu Wing 35 40 45 Val Thr 50 (2) INFORMATION FOR SEQ ID NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 27: Tyr Gln Lys He Gly Asp Gln Val Arg Cys Phe His Cys Asn He Gly 1 5 10 15 Leu Arg Ser Trp Gln Lys Glu Asp Glu Pro Trp Phe Glu His Wing Lys 20 25 30 Trp Ser Pro Lys Cys Gln Phe Val Leu Leu Wing Lys Gly Pro Ser Tyr 35 40 45 Val Ser 50 (2) INFORMATION FOR SEQ ID NO: 28: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 28: Wing Leu Gly Glu Gly Asp Lys Val Lys Cys Phe His Cys Gly Gly Gly 1 5 10 15 Leu Thr Asp Trp Lys Pro Ser Glu Asp Pro Trp Glu Gln His Wing Lys 20 25 30 Trp Tyr Pro Gly Cys Lys Tyr Leu Leu Glu Gln Lys Gly Gln Glu Tyr 35 40 45 He Asn 50 (2) INFORMATION FOR SEQ ID NO: 29: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 29: Wing Leu Gly Glu Gly Asp Lys Val Lys Cys Phe His Cys Gly Gly Gly 1 5 10 15 Leu Thr Asp Trp Lys Pro Ser Glu Asp Pro Trp Glu Gln His Wing Lys 20 25 30 Trp Tyr Pro Gly Cys Lys Tyr Leu Leu Asp Glu Lys Gly Gln Glu Tyr 35 40 45 He Asn 50 (2) INFORMATION FOR SEQ ID NO: 30: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear, (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 30: Tyr Val Gly Asn As Asp Asp Val Lys Cys Phe Cys Cys Asp Gly Gly 1 5 10 15 Leu Arg Cys Trp Glu Ser Gly Asp Asp Pro Trp Val Gln His Wing Lys 20 25 30 Trp Phe Pro Arg Cys Glu Tyr Leu He Arg He Lys Gly Gln Glu Phe 35 40 45 He Arg 50 (2) INFORMATION FOR SEQ ID NO: 31: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 31: Tyr Val Gly Arg Asn Asp Asp Val Lys Cys Phe Gly Cys Asp Gly Gly 1 5 10 15 Leu Arg Cys Trp Glu Ser Gly Asp Asp Pro Trp Val Glu His Wing Lys 20 25 30 Trp Phe Pro Arg Cys Glu Phe Leu He Arg Met Lys Gly Gln Glu Phe 35 40 45 Val Asp 50 (2) INFORMATION FOR SEQ ID NO: 32: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 32: Wing Leu Gly Glu Gly Asp Lys Val Lys Cys Phe His Cys Gly Gly Gly 1 5 10 15 Leu Thr Asp Trp Lys Pro Ser Glu Asp Pro Trp Glu Gln His Wing Lys 20 25 30 Trp Tyr Pro Gly Cys Lys Tyr Leu Leu Glu Glti Lys Gly Gln Glu Tyr 35 40 5 He Asn 50 (2) INFORMATION FOR SEQ ID NO: 33: (¡) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 33: Tyr Gln Lys He Gly Asp Gln Val Arg Cys Phe His Cys Asn He Gly 1 5 10 15 Leu Arg Ser Trp Gln Lys Glu Asp Glu Pro Trp Phe Glu His Wing Lys 20 25 30 Trp Ser Pro Lys Cys Gln Phe Val Leu Leu Wing Lys Gly Pro Ala Tyr 35 40 45 Val Ser 50 (2) INFORMATION FOR SEQ ID NO: 34: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 142 amino acids (B) TYPE: amino acid (C) NUMBER OF FILAMENTS: (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 34: Met Gly Wing Pro Thr Leu Pro Pro Wing Trp Gln Pro Phe Leu Lys Asp 1 5 10 15 His Arg He Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Cys Wing 20 25 30 Cys Thr Pro Glu Arg Met Wing Glu Ala Gly Phe He His Cys Pro Thr 35 40 45 Glu Asn Glu Pro Asp Leu Ala Gln Cys Phe Phe Cys Phe Lys Glu Leu 50 55 60 Glu Gly Trp Glu Pro Asp Asp Asp Pro He Glu Glu His Lys His 65 70 75 80 Being Ser Gly Cys Wing Phe Leu Being Val Lys Lys Gln Phe Glu Glu Leu 85 90 95 Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Wing Lys Asn Lys 100 105 110 Wing Wing Lys Glu Thr Asn Lys Lys Lys Glu Phe Glu Glu Thr Ala 115 120 125 Lys Lys Val Arg Arg Ala He Glu Gln Leu Ala Ala Asp 130 135 140 (2) INFORMATION FOR SEQ ID NO: 35: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25,796 pairs of bases (B) TYPE: nucleic acid (C) NUMBER OF FILAMENTS: one only (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: genomic DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID. NO: 35: TC AGACATG CGGATATATT CAAGCTGGGC ACAGCACAGC AGCCCCACCC CAGGCAGCTT 60 GAAATCAGAG CTGGGGTCCA AAGGGACCAC ACCCCGAGGG ACTGTGTGGG GGTCGGGGCA 120 CACAGGCCAC TGCTTCCCCC CGTCTTTCTC AGCCATTCCT GAAGTCAGCC TCACTCTGCT 180 TCTCAGGGAT TTCAAATGTG CAGAGACTCT GGCACTTTTG TAGAAGCCCC TTCTGGTCCT 240 AACTTACACC TGGATGCTGT GGGGCTGCAG CTGCTGCTCG GGCTCGGGAG GATGCTGGGG 300 GCCCGGTGCC CATGAGCTTT TGAAGCTCCT GGAACTCGGT TTTGAGGGTG TTCAGGTCCA 360 GGTGGACACC TGGGCTGTCC TTGTCCATGC ATTTGATGAC ATTGTGTGCA GAAGTGAAAA 420 GGAGTTAGGC CGGGCATGCT GGCTTATGCC TGTAATCCCA GCACTTTGGG AGGCTGAGGC 480 GGGTGGATCA CGAGGTCAGG AGTTCAATAC CAGCCTGGCC AAGATGGTGA AACCCCGTCT 540 CTACTAAAAA TACAAAAAAA TTAGCCGGGC ATGGTGGCGG GCGCATGTAA TCCCAGCTAC 600 TGGGGGGGCT GAGGCAGAGA ATTGCTGGAA CCCAGGAGAT GGAGGTTGCA GTGAGCCAAG 660 ATTGTGCCAC TGCACTGCAC TCCAGCCTGG CGACAGAGCA AGACTCTGTC TCAAAAAAAA 720 AAAAAAAAAG TGAAAAGGAG TTGTTCCTTT CCTCCCTCCT GAGGGCAGGC AACTGCTGCG 780 GTTGCCAGTG GAGGTGGTGC GTCCTTGGTC TGTGCCTGGG GGCCACCCCA GCAGAGGCCA 840 TGGTGGTGCC AGGGCCCGGT TAGCGAGCCA ATCAGCAGGA CCCAGGGGCG ACCTGCCAAA 900 GTCAACTGGA TTTGATAACT GCAGCGAAGT TAAGTTTCCT GATTTTGATG ATTGTGTTGT 960 GGTTGTGTAA GAGAATGAAG TATTTCGGGG TAGTATGGTA ATGCCTTCAA CTTACAAACG 1020 GTTCAGGTAA ACCACCCATA TACATACATA TACATGCATG TGATATATAC ACATACAGGG 1080 ATGTGTGTGT GTTCACATAT ATGAGGGGAG AGAGACTAGG GGAGAGAAAG TAGGTTGGGG 1140 AGAGGGAGAG AGAAAGGAAA ACAGGAGACA GAGAGAGAGC GGGGAGTAGA GAGAGGGAAG 1200 GGGTAAGAGA GGGAGAGGAG GAGAGAAAGG GAGGAAGAAG CAGAGAGTGA ATGTTAAAGG 1260 AAACAGGCAA AACATAAACA GAAAATCTGG GTGAAGGGTA TATGAGTATT CTTTGTACTA 1320 TTCTTGCAAT TATCTTTTAT TTAAATTGAC ATCGGGCCCC GCGCAGTGGC TCACATCTGT 1380 AATCCCAGCA CTTTGGGAGG CCGAGGCAGG CAGATCACTT GAGGTCAGGA GTTTGAGACC 1440 AGCCTGGCAA ACATGGTGAA ACCCCATCTC TACTAAAAAT ACAAAAATTA GCCTGGTGTG 1500 GTGGTGCATG CCTTTAATCT CAGCTACTCG GGAGGCTGAG GCAGGAGAAT CGCTTGAACC 1560 CGTGGCGGGG AGGAGGTTGC AGTGAGCTGA GATCATGCCA CTGCACTCCA GCCTGGGCGA 1620 TAGAGCGAGA CTCAGTTTCA AATAAATAAA TAAACATCAA AATAAAAAGT TACTGTATTA 1680 AAGAATGGGG GCGGGGTGGG AGGGGTGGGG AGAGGTTGCA AAAATAAATA AATAAATAAA 1740 TAAACCCCAA AATGAAAAAG ACAGTGGAGG CACCAGGCCT GCGTGGGGCT GGAGGGCTAA 1800 TAAGGCCAGG CCTCTTATCT CTGGCCATAG AACCAGAGAA GTGAGTGGAT GTGATGCCCA 1860 GCTCCAGAAG TGACTCCAGA ACACCCTGTT CCAAAGCAGA GGACACACTG ATTTTTTTTT 1920 TAATAGGCTG CAGGACTTAC TGTTGGTGGG ACGCCCTGCT TTGCGAAGGG AAAGGAGGAG 1980 TTTGCCCTGA GCACAGGCCC CCACCCTCCA CTGGGCTTTC CCCAGCTCCC TTGTCTTCTT 2040 ATCACGGTAG TGGCCCAGTC CCTGGCCCCT GACTCCAGAA GGTGGCCCTC CTGGAAACCC 2100 AGGTCGTGCA GTCAACGATG TACTCGCCGG GACAGCGATG TCTGCTGCAC TCCATCCCTC 2160 CCCTGTTCAT TTGTCCTTCA TGCCCGTCTG GAGTAGATGC TTTTTGCAGA GGTGGCACCC 2220 TGTAAAGCTC TCCTGTCTGA CTTTTTTTTTT TTTTTTAGAC TGAGTTTTGC TCTTGTTGCC 2280 TAGGCTGGAG TGCAATGGCA CAATCTCAGC TCACTGCACC CTCTGCCTCC CGGGTTCAAG 2340 CGATTCTCCT GCCTCAGCCT CCCGAGTAGT TGGGATTACA GGCATGCACC ACCACGCCCA 2400 GCTAATTTTT GTATTTTTAG TAGAGACAAG GTTTCACCGT GATGGCCAGG CTGGTCTTGA 2460 ACTCCAGGAC TCAAGTGATG CTCCTGCCTA GGCCTCTCAA AGTGTTGGGA TTACAGGCGT 2520 GAGCCACTGC ACCCGGCCTG CACGCGTTCT TTGAAAGCAG TCGAGGGGGC GCTAGGTGTG 2580 GGCAGGGACG AGCTGGCGCG GCGTCGCTGG GTGCACCGCG ACCACGGGCA GAGCCACGCG 2640 GCGGGAGGAC TACAACTCCC GGCACACCCC GCGCCGCCCC GCCTCTACTC CCAGAAGGCC 2700 GCGGGGGGTG GACCGCCTAA GAGGGCGTGC GCTCCCGACA TGCCCCGCGG CGCGCCATTA 2760 ACCGCCAGAT TTGAATCGCG GGACCCGTTG GCAGAGGTGG CGGCGGCGGC ATGGGTGCCC 2820 CGACGTTGCC CCCTGCCTGG CAGCCCTTTC TCAAGGACCA CCGCATCTCT ACATTCAAGA 2880 ACTGGCCCTT CTTGGAGGGC TGCGCCTGCA CCCCGGAGCG GGTGAGACTG CCCGGCCTCC 2940 TGGGGTCCCC CACGCCCGCC TTGCCCTGTC CCTAGCGAGG CCACTGTGAC TGGGCCTCGG 3000 GGGTACAAGC CGCCCTCCCC TCCCCGTCCT GTCCCCAGCG AGGCCACTGT GGCTGGGCCC 3060 CTTGGGTCCA GGCCGGCCTC CCCTCCCTGC TTTGTCCCCA TCGAGGCCTT TGTGGCTGGG 3120 CCTCGGGGTT CCGGGCTGCC ACGTCCACTC ACGAGCTGTG CTGTCCCTTG CAGATGGCCG 3180 AGGCTGGCTT CATCCACTGC CCCACTGAGA ACGAGCCAGA CTTGGCCCAG TGTTTCTTCT 3240 GCTTCAAGGA GCTGGAAGGC TGGGAGCCAG ATGACGACCC CATGTAAGTC TTCTCTGGCC 3300 AGCCTCGATG GGCTTTGTTT TGAACTGAGT TGTCAAAAGA TTTGAGTTGC AAAGACACTT 3360 AG ATGGGAG GGTTGCTTTC CACCCTCATT GCTTCTTAAA CAGCTGTTGT GAACGGATAC 3420 CTCTCTATAT GCTGGTGCCT TGGTGATGCT TACAACCTAA TTAAATCTCA TTTGACCAAA 3480 ATGCCTTGGG GTGGACGTAA GATGCCTGAT GCCTTTCATG TTCAACAGAA TACATCAGCA 3540 GACCCTGTTG TTGTGAACTC CCAGGAATGT CCAAGTGCTT TTTTTGAGAT TTTTTAAAAA 3600 ACAGTTTAAT TGAAATATAA CCTACACAGC ACAAAAATTA CCCTTTGAAA GTGTGCACTT 3660 CACACTTTCG GAGGCTGAGG CGGGCGGATC ACCTGAGGTC AGGAGTTCAA GACCTGCCTG 3720 GCCAACTTGG CGAAACCCCG TCTCTACTAA AAATACAAAA ATTAGCCGGG CATGGTAGCG 3780 CACGCCCGTA ATCCCAGCTA CTCGGGAGGC TAAGGCAGGA GAATCGCTTG AACCTGGGAG 3840 GCGGAGGTTG CAGTGAGCCG AGATTGTGCC AATGCACTCC AGCCTCGGCG ACAGAGCGAG 3900 ACTCCGTCAT AAAAATAAAA AATTGAAAAA AAAAAAAGAA AGAAAGCATA TACTTCAGTG 3960 TTGTTCTGGA TTTTTTTCTT CAAGATGCCT AGTTAATGAC AATGAAATTC TGTACTCGGA 4020 TGGTATCTGT CTTTCCACAC TGTAATGCCA TATTCTTTTC TCACCTTTTT TTCTGTCGGA 4080 TTCAGTTGCT TCCACAGCTT TAATTTTTTT CCCCTGGAGA ATCACCCCAG TTGTTTTTCT 4140 TTTTGGCCAG AAGAGAGTAG CTGTTTTTTT TCTTAGTATG TTTGCTATGG TGGTTATACT 4200 GCATCCCCGT AATCACTGGG AAAAGATCAG TGGTATTCTT CTTGAAAATG AATAAGTGTT 4260 ATGATATTTT CAGATTAGAG TTACAACTGG CTGTCTTTTT GGACTTTGTG TGGCCATGTT 4320 TTCATTGTAA TGCAGTTCTG GTAACGGTGA TAGTCAGTTA TACAGGGAGA CTCCCCTAGC 4380 AGAAAATGAG AGTGTGAGCT AGGGGGTCCC TTGGGGAACC CGGGGCAATA ATGCCCTTCT 4440 CTGCCCTTAA TCCTTACAGT GGGCCGGGCA CGGTGGCTTA CGCCTGTAAT ACCAGCACTT 4500 TGGGAGGCCG AGGCGGGCGG ATCACGAGGT CAGGAGATCG AGACCATCTT GGCTAATACG 4560 GTGAAACCCC GTCTCCACTA AAAATACAAA AAATTAGCCG GGCGTGGTGG TGGGCGCCTG 4620 TAGTCCCAGC TACTCGGGAG GCTGAGGCAG GAGAATGGCG TGAACCCAGG AGGCGGAGCT 4680 TGCAGTGAGC CGAGATTGCA CCACTGCACT CCAGCCTGGG CGACAGAATG AGACTCCGTC 4740 TCAAAAAAAA AAAAAAAAGA AAAAAATCTT TACAGTGGAT TACATAACAA TTCCAGTGAA 4800 ATGAAATTAC TTCAAACAGT TCCTTGAGAA TGTTGGAGGG ATTTGACATG TAATTCCTTT 4860 GGACATATAC CATGTAACAC TTTTCCAACT AATTGCTAAG GAAGTCCAGA TAAAATAGAT 4920 ACATTAGCCA CACAGATGTG GGGGGAGATG TCCACAGGGA GAGAGAAGGT GCTAAGAGGT 4980 GCCATATGGG AATGTGGCTT GGGCAAAGCA CTGATGCCAT CAACTTCAGA CTTGACGTCT 5040 TACTCCTGAG GCAGAGCAGG GTGTGCCTGT GGAGGGCGTG GGGAGGTGGC CCGTGGGGAG 5100 TGGACTGCCG CTTTAATCCC TTCAGCTGCC TTTCCGCTGT TGTTTTGATT TTTCTAGAGA 5160 GGAACATAAA AAGCATTCGT CCGGTTGCGC TTTCCTTTCT GTCAAGAAGC AGTTTGAAGA 5220 ATTAACCCTT GGTGAATTTT TGAAACTGGA CAGAGAAAGA GCCAAGAACA AAATTGTATG 5280 TATTGGGAAT AAGAACTGCT CAAACCCTGT TCAATGTCTT TAGCACTAAA CTACCTAGTC 5340 CCTCAAAGGG ACTCTGTGTT TTCCTCAGGA AGCATTTTTT TTTTTTTTTCT GAGATAGAGT 5400 TTCACTCTTG TTGCCCAGGC TGGAGTGCAA TGGTGCAATC TTGGCTCACT GCAACCTCTG 5460 CCTCTCGGGT TCAAGTGATT CTCCTGCCTC AGCCTCCCAA GTAACTGGGA TTACAGGGAA 5520 GTGCCACCAC ACCCAGCTAA TTTTTGTATT TTTAGTAGAG ATGGGGTTTC ACCACATTGC 5580 CCAGGCTGGT CTTGAACTCC TGACCTCGTG ATTCGCCCAC CTTGGCCTCC CAAAGTGCTG 5640 GGATTACAGG CGTGAACCAC CACGCCTGGC TTTTTTTTTT TTGTTCTGAG ACACAGTTTC 5700 ACTCTGTTAC CCAGGCTGGA GTAGGGTGGC CTGATCTCGG ATCACTGCAA CCTCCGCCTC 5760 CTGGGCTCAA GTGATTTGCC TGCTTCAGCC TCCCAAGTAG CCGAGATTAC AGGCATGTGC 5820 CACCACACCC AGGTAATTTT TGTATTTTTG GTAGAGACGA GGTTTCACCA TGTTGGCCAG 5880 GCTGGTTTTG AACTCCTGAC CTCAGGTGAT CCACCCGCCT CAGCCTCCCA AAGTGCTGAG 5940 ATTATAGGTG TGAGCCACCA CACCTGGCCT CAGGAAGTAT TTTTATTTTT AAATTTATTT 6000 ATTTATTTGA GATGGAGTCT TGCTCTGTCG CCCAGGCTAG AGTGCAGCGA CGGGATCTCG 6060 GCTCACTGCA AGCTCCGCCC CCCAGGTTCA AGCCATTCTC CTGCCTCAGC CTCCCGAGTA 6120 GCTGGGACTA CAGGCGCCCG CCACCACACC CGGCTAATTT TTTTGTATTT TTAGTAGAGA 6180 CGGGTTTTCA CCGTGTTAGC CAGGAGGGTC TTGATCTCCT GACCTCGTGA TCTGCCTGCC 6240 TCGGCCTCCC AAAGTGCTGG GATTACAGGT GTGAGCCACC ACACCCGGCT ATTTTTATTT 6300 TTTTGAGACA GGGACTCACT CTGTCACCTG GGCTGCAGTG CAGTGGTACA CCATAGCTCA 6360 CTGCAGCCTC GAACTCCTGA GCTCAAGTGA TCCTCCCACC TCATCCTCAC AAGTAATTGG 6420 GACTACAGGT GCACCCCACC ATGCCCACCT AATTTATTTA TTTATTTATT TATTTATTTT 6480 CATAGAGATG AGGGTTCCCT GTGTTGTCCA GGCTGGTCTT GAACTCCTGA GCTCACGGGA 6540 TCCTTTTGCC TGGGCCTCCC AAAGTGCTGA GATTACAGGC ATGAGCCACC GTGCCCAGCT 6600 AGGAATCATT TTTAAAGCCC CTAGGATGTC TGTGTGATTT TAAAGCTCCT GGAGTGTGGC 6660 CGGTATAAGT ATATACCGGT ATAAGTAAAT CCCACATTTT GTGTCAGTAT TTACTAGAAA 6720 CTTAGTCATT TATCTGAAGT TGAAATGTAA CTGGGCTTTA TTTATTTATT TATTTATTTA 6780 TTTATTTTTA ATTTTTTTTTT TTGAGACGAG TCTCACTTTG TCACCCAGGC TGGAGTGCAG 6840 TGGCACGATC TCGGCTCACT GCAACCTCTG CCTCCCGGGG TCAAGCGATT CTCCTGCCTT 6900 AGCCTCCCGA GTAGCTGGGA CTACAGGCAC GCACCACCAT GCCTGGCTAA TTTTTGTATT 6960 TTTAGTAGAC GGGGTTTCAC CATGCTGGCC AAGCTGGTCT CAAACTCCTG ACCTTGTGAT 7020 CTGCCCGCTT TAGCCTCCCA GAGTGCTGGG ATTACAGGCA TGAGCCACCA TGCGTGGTCT 7080 TTTTAAAATT TTTTGATTTT TTTTTTTTTTT GAGACAGAGC CTTGCTCTGT CGCCCAGGCT 7140 GGAGTGCAGT GGCACGATCT CAGCTCACTA CAAGCTCCGC CTCCCGGGTT CACGCCATTC 7200 TTCTGCCTCA GCCTCCTGAG TAGCTGGGAC TACAGGTGCC CACCACCACG CCTGGCTAAT 7260 TTTTTTTTGGT ATTTTTATTA GAGACAAGGT TTCATCATGT TGGCCAGGCT GGTCTCAAAC 7320 TCCTGACCTC AAGTGATCTG CCTGCCTCGG CCTCCCAAAG CGCTGAGATT ACAGGTGTGA 7380 TCTACTGCGC CAGGCCTGGG CGTCATATAT TCTTATTTGC TAAGTCTGGC AGCCCCACAC 7440 AGAATAAGTA CTGGGGGATT CCATATCCTT GTAGCAAAGC CCTGGGTGGA GAGTCAGGAG 7500 ATGTTGTAGT TCTGTCTCTG CCACTTGCAG ACTTTGAGTT TAAGCCAGTC GTGCTCATGC 7560 TTTCCTTGCT AAATAGAGGT TAGACCCCCT ATCCCATGGT TTCTCAGGTT GCTTTTCAGC 7620 TTGAAAATTG TATTCCTTTG TAGAGATCAG CGTAAAATAA TTCTGTCCTT ATATGTGGCT 7680 TTATTTTAAT TTGAGACAGA GTGTCACTCA GTCGCCCAGG CTGGAGTGTG GTGGTGCGAT 7740 CTTGGCTCAC TGCGACCTCC ACCTCCCAGG TTCAAGCGAT TCTCGTGCCT CAGGCTCCCA 7800 AGTAGCTGAG ATTATAGGTG TGTGCCACCA GGCCCAGCTA ACTTTTGTAT TTTTAGTAGA 7860 GACAGGGTTT TGCCATGTTG GCTAAGCTGG TCTCGAACTC CTGGCCTCAA GTGATCTGCC 7920 CGCCTTGGCA TCCCAAAGTG CTGGGATTAC AGGTGTGAAC CACCACACCT GGCCTCAATA 7980 TAGTGGCTTT TAAGTGCTAA GGACTGAGAT TGTGTTTTGT CAGGAAGAGG CCAGTTGTGG 8040 GTGAAGCATG CTGTGAGAGA GCTTGTCACC TGGTTGAGGT TGTGGGAGCT GCAGCGTGGG 8100 AACTGGAAAG TGGGCTGGGG ATCATCTTTT TCCAGGTCAG GGGTCAGCCA GCTTTTCTGC 8160 AGCGTGCCAT AGACCATCTC TTAGCCCTCG TGGGTCAGAG TCTCTGTTGC ATATTGTCTT 8220 TTGTTGTTTT TCACAACCTT TTAGAAACAT AAAAAGCATT CTTAGCCCGT GGGCTGGACA 8280 AAAAAAGGCC ATGACGGGCT GTATGGATTT GGCCCAGCAG GCCCTTGCTT GCCAAGCCCT 8340 GTTTTAGACA AGGAGCAGCT TGTGTGCCTG GAACCATCAT GGGCACAGGG GAGGAGCAGA 8400 GTGGATGTGG AGGTGTGAGC TGGAAACCAG GTCCCAGAGC GCTGAGAAAG ACAGAGGGTT 8460 TTTGCCCTTG CAAGTAGAGC AACTGAAATC TGACACCATC CAGTTCCAGA AAGCCCTGAA 8520 GTGCTGGTGG ACGCTGCGGG GTGCTCCGCT CTAGGGTTAC AGGGATGAAG ATGCAGTCTG 8580 GTAGGGGGAG TCCACTCACC TGTTGGAAGA TGTGATTAAG AAAAGTAGAC TTTCAGGGCC 8640 GGGCATGGTG GCTCACGCCT GTAATCCCAG CACTTTGGGA GGCCGAGGCG GGTGGATCAC 8700 GAGGTCAGGA GATCGAGACC ATCCTGGCTA ACATGGTGAA ACCCCGTCTT TACTAAAAAT 8760 ACAAAAAATT AGCTGGGCGT GGTGGCGGGC GCCTGTAGTC CCAGCTACTC GGGAGGCTGA 8820 GGCAGGAGAA TGGCGTGAAC CTGGGAGGTG GAGCTTGCTG TGAGCCGAGA TCGCGCCACT 8880 GCACTCCAGC CTGGGCGACA GAGCGAGACT CCGTCTCAAA AAAAAAAAAA AAAGTAGGCT 8940 TTCATGATGT GTGAGCTGAA GGCGCAGTAG GCAGAAGTAG AGGCCTCAGT CCCTGCAGGA 9000 GACCCCTCGG TCTCTATCTC CTGATAGTCA GACCCAGCCA CACTGGAAAG AGGGGAGACA 9060 TTACAGCCTG CGAGAAAAGT AGGGAGATTT AAAAACTGCT TGGCTTTTAT TTTGAACTGT 9120 TTTTTTTTGTT TGTTTGTTTT CCCCAATTCA GAATACAGAA TACTTTTATG GATTTGTTTT 9180 TATTACTTTA ATTTTGAAAC AATATAATCT TTTTTTTTGTT GTTTTTTTGA GACAGGGTCT 9240 TACTCTGTCA CCCAGGCTGA GTGCAGTGGT GTGATCTTGG CTCACCTCAG CCTCGACCCC 9300 CTGGGCTCAA ATGATTCTCC CACCTCAGCT TCCCAAGTAG CTGGGACCAC AGGTGCGTGT 9360 GTTGCGCTAT ACAAATCCTG AAGACAAGGA TGCTGTTGCT GGTGATGCTG GGGATTCCCA 9420 AGATCCCAGA TTTGATGGCA GGATGCCCCT GTCTGCTGCC TTGCCAGGGT GCCAGGAGGG 9480 CGCTGCTGTG GAAGCTGAGG CCCGGCCATC CAGGGCGATG CATTGGGCGC TGATTCTTGT 9540 TCCTGCTGCT GCCTCGGTGC TTAGCTTTTG AAACAATGAA ATAAATTAGA ACCAGTGTGA 9600 AAATCGATCA GGGAATAAAT TTAATGTGGA AATAAACTGA ACAACTTAGT TCTTCATAAG 9660 AGTTTACTTG GTAAATACTT GTGATGAGGA CAAAACGAAG CACTAGAAGG AGAGGCGAGT 9720 TGTAGACCTG GGTGGCAGGA GTGTTTTGTT TGTTTTCTTT GGCAGGGTCT TGCTCTGTTG 9780 CTCAGGCTGG AGTACAGTGG CACAATCACA GCTCACTATA GCCTCGACCT CCTGGACTCA 9840 AGCAATCCTC CTGCCTCAGC CTCCCAGTAG CTGGGACTAC AGGCGCATGC CACCATGCCT 9900 GGCTAATTTT AAATTTTTTT TTTTCTCTTT TTTGAGATGG AATCTCACTC TGTCGCCCAG 9960 GCTGGAGTGC AGTGGCGTGA TCTCGGCTGA CGGCAAGCTC CGCCTCCCAG GTTCACTCCA 10020 TTCGCCTGCC TCAGCCTCCC AAGTAGCTGG GACTACAGGC GCTGGGATTA CAAACCCAAA 10080 CCCAAAGTGC TGGGATTACA GGCGTGAGCC ACTGCACCCG GCCTGTTTTG TCTTTCAATA 10140 GCAAGAGTTG TGTTTGCTTC GCCCCTACCT TTAGTGGAAA AATGTATAAA ATGGAGATAT 10200 TGACCTCCAC ATTGGGGTGG TTAAATTATA GCATGTATGC AAAGGAGCTT CGCTAATTTA 10260 AGGCTTTTTT GAAAGAGAAG AAACTGAATA ATCCATGTGT GTATATATAT TTTAAAAGCC 10320 ATGGTCATCT TTCCATATCA GTAAAGCTGA GGCTCCCTGG GACTGCAGAG TTGTCCATCA 10380 CAGTCCAT A TAAGTGCGCT GCTGGGCCAG GTGCAGTGGC TTGTGCCTGA ATCCCAGCAC 10440 TTTGGGAGGC CAAGGCAGGA GGATTCATTG AGCCCAGGAG TTTTGAGGCG AGCCTGGGCA 10500 ATGTGGCCAG ACCTCATCTC TTCAAAAAAT ACACAAAAAA TTAGCCAGGC ATGGTGGCAC 10560 GTGCCTGTAG TCTCAGCTAC TCAGGAGGCT GAGGTGGGAG GATCACTTTG AGCCTTGCAG 10620 GTCAAAGCTG CAGTAAGCCA TGATCTTGCC ACTGCATTCC AGCCTGGATG ACAGAGCGAG 10680 ACCCTGTCTC TAAAAAAAAA AAAAACCAAA CGGTGCACTG TTTTCTTTTT TCTTATCAAT 10740 TTATTATTTT TAAATTAAAT TTTCTTTTAA TAATTTATAA ATTATAAATT TATATTAAAA 10800 AATGACAAAT TTTTATTACT TATACATGAG GTAAAACTTA GGATATATAA AGTACATATT 10860 GAAAAGTAAT TTTTTGGCTG GCACAGTGGC TCACACCTGT AATCCCAGCA CTTTGGGAGG 10920 CCGTGGCGGG CAGATCACAT GAGATCATGA GTTCGAGACC AACCTGACCA ACATGGAGAG 10980 ACCCCATCTC TACTAAAAAT ACAAAATTAG CCGGGGTGGT GGCGCATGCC TGTAATCCCA 11040 GCTACTCGGG AGGCTGAGGC AGGAGAATCT CTTGAACCCG GGAGGCAGAG GTTGCGGTGA 11100 GCCAAGATCG TGCCTTTGCA CACCAGCCTA GGCAACAAGA GCGAAAGTCC GTCTCAAAAA 11160 AAAAGTAATT TTTTTTAAGT TAACCTCTGT CAGCAAACAA ATTTAACCCA ATAAAGGTCT 11220 TTGTTTTTTA ATGTAGTAGA GGAGTTAGGG TTTATAAAAA ATATGGTAGG GAAGGGGGTC 11280 CCTGGATTTG CTAATGTGAT TGTCATTTGC CCCTTAGGAG AGAGCTCTGT TAGCAGAATG 11340 AAAAAATTGG AAGCCAGATT CAGGGAGGGA CTGGAAGCAA AAGAATTTCT GTTCGAGGAA 11400 GAGCCTGATG TTTGCCAGGG TCTGTTTAAC TGGACATGAA GAGGAAGGCT CTGGACTTTC 11460 CTCCAGGAGT TTCAGGAGAA AGGTAGGGCA GTGGTTAAGA GCAGAGCTCT GCCTAGACTA 11520 GCTGGGGTGC CTAGACTAGC TGGGGTGCCC AGACTAGCTG GGGTGCCTAG ACTAGCTGGG 11580 TACTTTGAGT GGCTCCTTCA GCCTGGACCT CGGTTTCCTC ACCTGTATAG TAGAGATATG 11640 GGAGCACCCA GCGCAGGATC ACTGTGAACA TAAATCAGTT AATGGAGGAA GCAGGTAGAG 11700 TGGTGCTGGG TGCATACCAA GCACTCCGTC AGTGTTTCCT GTTATTCGAT GATTAGGAGG 11760 CAGCTTAAAC TAGAGGGAGT TGAGCTGAAT CAGGATGTTT GTCCCAGGTA GCTGGGAATC 11820 TGCCTAGCCC AGTGCCCAGT TTATTTAGGT GCTCTCTCAG TGTTCCCTGA TTGTTTTTTC 11880 CTTTGTCATC TTATCTACAG GATGTGACTG GGAAGCTCTG GTTTCAGTGT CATGTGTCTA 11940 TTCTTTATTT CCAGGCAAAG GAAACCAACA ATAAGAAGAA AGAATTTGAG GAAACTGCGA 12000 AGAAAGTGCG CCGTGCCATC GAGCAGCTGG CTGCCATGGA TTGAGGCCTC TGGCCGGAGC 12060 TGCCTGGTCC CAGAGTGGCT GCACCACTTC CAGGGTTTAT TCCCTGGTGC CACCAGCCTT 12120 CCTGTGGGCC CCTTAGCAAT GTCTTAGGAA AGGAGATCAA CATTTTCAAA TTAGATGTTT 12180 CAACTGTGCT CCTGTTTTGT CTTGAAAGTG GCACCAGAGG TGCTTCTGCC TGTGCAGCGG 12240 GTGCTGCTGG TAACAGTGGC TGCTTCTCTC TCTCTCTCTC TTTTTTGGGG GCTCATTTTT 12300 GCTGTTTTGA TTCCCGGGCT TACCAGGTGA GAAGTGAGGG AGGAAGAAGG CAGTGTCCCT 12360 TTTGCTAGAG CTGACAGCTT TGTTCGCGTG GGCAGAGCCT TCCACAGTGA ATGTGTCTGG 12420 ACCTCATGTT GTTGAGGCTG TCACAGTCCT GAGTGTGGAC TTGGCAGGTG CCTGTTGAAT 12480 CTGAGCTGCA GGTTCCTTAT CTGTCACACC TGTGCCTCCT CAGAGGACAG TTTTTTTGTT 12540 GTTGTGTTTT TTTGTTTTTT TTTTTTGGTA GATGCATGAC TTGTGTGTGA TGAGAGAATG 12600 GAGACAGAGT CCCTGGCTCC TCTACTGTTT AACAACATGG CTTTCTTATT TTGTTTGAAT 12660 TGTTAATTCA CAGAATAGCA CAAACTACAA TTAAAACTAA GCACAAAGCC ATTCTAAGTC 12720 ATTGGGGAAA CGGGGTGAAC TTCAGGTGGA TGAGGAGACA GAATAGAGTG ATAGGAAGCG 12780 TCTGGCAGAT ACTCCTTTTG CCACTGCTGT GTGATTAGAC AGGCCCAGTG AGCCGCGGGG 12840 CACATGCTGG CCGCTCCTCC CTCAGAAAAA GGCAGTGGCC TAAATCCTTT TTAAATGACT 12900 TGGCTCGATG CTGTGGGGGA CTGGCTGGGC TGCTGCAGGC CGTGTGTCTG TCAGCCCAAC 12960 CTTCACATCT GTCACGTTCT CCACACGGGG GAGAGACGCA GTCCGCCCAG GTCCCCGCTT 13020 TCTTTGGAGG CAGCAGCTCC CGCAGGGCTG AAGTCTGGCG TAAGATGATG GATTTGATTC 13080 GCCCTCCTCC CTGTCATAGA GCTGCAGGGT GGATTGTTAC AGCTTCGCTG GAAACCTCTG 13140 GAGGTCATCT CGGCTGTTCC TGAGAAATAA AAAGCCTGTC ATTTCAAACA CTGCTGTGGA 13200 CCCTACTGGG TTTTTAAAAT ATTGTCAGTT TTTCATCGTC GTCCCTAGCC TGCCAACAGC 13260 CATCTGCCCA GACAGCCGCA GTGAGGATGA GCGTCCTGGC AGAGACGCAG TTGTCTCTGG 13320 GCGCTTGCCA GAGCCACGAA CCCCAGACCT GTTTGTATCA TCCGGGCTCC TTCCGGGCAG 13380 AAACAACTGA AAATGCACTT CAGACCCACT TATTTATGCC ACATCTGAGT CGGCCTGAGA 13440 TAGACTTTTC CCTCTAAACT GGGAGAATAT CACAGTGGTT TTTGTTAGCA GAAAATGCAC 13500 TCCAGCCTCT GTACTCATCT AAGCTGCTTA TTTTTGATAT TTGTGTCAGT CTGTAAATGG 13560 ATACTTCACT TTAATAACTG TTGCTTAGTA ATTGGCTTTG TAGAGAAGCT GGAAAAAAAT 13620 GGTTTTTGTCT TCAACTCCTT TGCATGCCAG GCGGTGATGT GGATCTCGGC TTCTGTGAGC 13680 CTGTGCTGTG GGCAGGGCTG AGCTGGAGCC GCCCCTCTCA GCCCGCCTGC CACGGCCTTT 13740 CCTTAAAGGC CATCCTTAAA ACCAGACCCT CATGGCTGCC AGCACCTGAA AGCTTCCTCG 13800 ACATCTGTTA ATAAAGCCGT AGGCCCTTGT CTAAGCGCAA CCGCCTAGAC TTTCTTTCAG 13860 ATACATGTCC ACATGTCCAT TTTTCAGGTT CTCTAAGTTG GAGTGGAGTC TGGGAAGGGT 13920 TGTGAATGAG GCTTCTGGGC TATGGGTGAG GTTCCAATGG CAGGTTAGAG CCCCTCGGGC 13980 CAACTGCCAT CCTGGAAAGT AGAGACAGCA GTGCCCGCTG CCCAGAAGAG ACCAGCAAGC 14040 CAAACTGGAG CCCCCATTGC AGGCTGTCGC CATGTGGAAA GAGTAACTCA CAATTGCCAA 14100 TAAAGTCTCA TGTGGTTTTA TCTACTTTTT GAGACAAGGC 14160 CTTGCCCTCC CAGGCTGGAG TGCAGTGGAA TGACCACAGC TCACCGCAAC CTCAAATTCT 14220 TGCGTTCAAG TGAACCTCCC ACTTTAGCCT CCCAAGTAGC TGGGACTACA GGCGCACGCC 14280 ATCACACCCG GCTAATTGAA AAATTTTTTT TTTTGTTTAG ATGGAATCTC ACTTTGTTGC 14340 CCAGGCTGGT CTCAAACTCC TGGGCTCAAG TGATCATCCT GCTTCAGCGT CCGACTTGTT 14400 GGTATTATAG GCGTGAGCCA CTGGGCCTGA CCTAGCTACC ATTTTTTAAT GCAGAAATGA 14460 AGACTTGTAG AAATGAAATA ACTTGTCCAG GATAGTCGAA TAAGTAACTT TTAGAGCTGG 14520 GATTTGAACC CAGGCAATCT GGCTCCAGAG CTGGGCCCTC ACTGCTGAAG GACACTGTCA 14580 GCTTGGGAGG GTGGCTATGG TCGGCTGTCT GATTCTAGGG AGTGAGGGCT GTCTTTAAAG 14640 CAT CACCCCATTC TTTCAGA CAGCTTTGTC AGAAAGGCTG TCATATGGAG CTGACACCTG 14700 CCTCCCCAAG GCTTCCATAG ATCCTCTCTG TACATTGTAA CCTTTTATTT TGAAATGAAA 14760 ATTCACAGGA AGTTGTAAGG CTAGTACAGG GGATCC 14796

Claims (59)

NOVELTY OF THE INVENTION CLAIMS

1. - The use of an agent that modulates the amount or activity of survivin, for the manufacture of a drug to modulate apoptosis in a cell.

2. The use according to claim 1, further characterized in that the level of apoptosis is increased by decreasing the amount or activity of survivin in the cell.

3. The use according to claim 1, further characterized in that the level of apoptosis is decreased by increasing the amount or activity of survivin in the cell.

4. The use of the survivin polypeptide, a survivin polypeptide fragment or an apoptosis inhibiting peptidomimetic, for the manufacture of a medicament for inhibiting apoptosis in a cell.

5. The use of a transgene encoding a survivin polypeptide or a survivin polypeptide fragment, for the manufacture of a medicament for inhibiting apoptosis in a cell, wherein the transgene is effective to elicit the expression of the survivin polypeptide or the fragment of it.

6. The use of an agent that decreases the inhibitory activity of apoptosis of the survivin polypeptide, for the manufacture of a drug to increase apoptosis in a cell.

7. The use according to claim 6, further characterized in that the agent is a polypeptide comprising the sequence EGWEPDDDPIEEHKKHSSGC, its conservatively substituted homologs or small molecule peptidomimetics thereof.

8. The use of an agent that increases the transcription of the normal sense filament of EPR-1 for the manufacture of a drug to increase the level of apoptosis of a cell; wherein said agent inhibits the translation of the mRNA that encodes a survivin polypeptide, thereby decreasing the transcription of the survivin polypeptide.

9. An isolated nucleic acid molecule, which encodes the amino acid sequence illustrated in SEQ ID NO. 34, the allelic variants of the amino acid sequence of SEQ ID NO. 34, and its fragments, which are effective in inhibiting apoptosis.

10. The isolated nucleic acid molecule according to claim 9, further characterized in that the nucleic acid molecule is operably linked to one or more expression controlling elements.

11. The isolated nucleic acid molecule according to claim 9, further characterized in that the nucleic acid molecule or is included in a vector.

12. An isolated nucleic acid molecule, which encodes a member of the survivin family of proteins, characterized in that the nucleic acid molecule is hybridized to a nucleic acid molecule of claim 9, under sufficiently stringent conditions to produce a clear signal .

13. A host cell transformed to contain the nucleic acid molecule of claim 9.

14. The host cell according to claim 13, further characterized in that the host is selected from the group consisting of prokaryotic hosts and eukaryotic hosts.

15. A method for producing a survivin protein, characterized in that it comprises the step of culturing a transformed host with the nucleic acid molecule of claim 11, under conditions in which the survivin protein is expressed.

16. The method according to claim 15, further characterized in that the host of the group consisting of prokaryotic hosts and eukaryotic hosts is selected.

17. An isolated polypeptide, characterized in that it comprises the amino acid sequence illustrated in SEQ ID NO: 34, its allelic variants and their fragments that retain the ability to inhibit cellular apoptosis.

18. A polypeptide, characterized in that it comprises the sequence EGWEPDDDPIEEHKKHSSGC, its conservatively substituted homologs and its small molecule peptidomimetics.

19. A monoclonal antibody that binds to the polypeptide, its allelic variants and its fragments, which retain the ability to inhibit cellular apoptosis, according to claim 17.

20. - The monoclonal antibody according to claim 19, further characterized in that it has been humanized.

21. The use of an agent that reduces the expression or activity of survivin, for the manufacture of a drug to reduce the severity of a pathological condition mediated by survivin.

22. The use according to claim 21, further characterized in that the pathological condition is caused by abnormal cell growth.

23. The use according to claim 21, further characterized in that the expression of survivin is reduced by contacting the affected cells with an RNA molecule that is complementary to a mRNA molecule encoding survivin.

24. The use according to claim 21, further characterized in that the activity of survivin is reduced by blocking the intracellular interaction of survivin with a survivin binding partner, which comprises the step of contacting the survivin with a blocking agent. the union of survivin with the binding partner.

25. The use according to claim 24, further characterized in that said agent blocks the binding of survivin to the binding partner, selectively binding to survivin.

26. The use according to claim 25, further characterized in that the agent blocks the binding of survivin to the binding partner by selectively binding to the binding partner.

27. - The use according to claim 26, further characterized in that said agent is a polypeptide fragment of survivin.

28. The use according to claim 26, further characterized in that the agent comprises the polypeptide sequence EGWEPDDDPIEEHKKHSSGC, its conservatively substituted homologs and its small molecule peptidomimetics.

29. A method for identifying agents that block the interaction of survivin with a survivin binding partner, characterized in that it comprises the steps of: a) incubating survivin or a fragment thereof, or a polypeptide comprising the sequence EGWEPDDDPIEEHKKHSSGC, and a member of a union or a fragment thereof, with an agent to be tested; and b) determining whether said agent blocks the binding of survivin to the survivin binding partner.

30. A method for analyzing the presence of survivin, characterized in that it comprises the step of determining if a survivin protein is expressed by a sample.

31.- The method according to claim 30, further characterized in that the sample is selected from the group consisting of a tissue biopsy, evacuation, blood, urine and saliva.

32. The method according to claim 30, further characterized in that it further comprises the steps of: a) preparing an extract of the cells in said sample; and b) examine the proteins of the cell extract, to determine the presence of the survivin protein.

33. - The method according to claim 30, characterized in that it additionally comprises the steps of: a) preparing an extract of the cells in the sample; and b) examining the mRNA of the cell extract to determine the presence of a mRNA that encodes survivin.

34. The method according to claim 30, further characterized in that the method for determining the growth potential of a tumor cell is used, correlating the level of expression of survivin with the control samples, to indicate the growth potential of the tumor. tumor.

35.- A method for detecting or monitoring the state or progress of a cancer, characterized in that it comprises the steps of: obtaining a sample of biological fluid from a subject; contact the sample with a monoclonal antibody that recognizes and binds to survivin; and to determine if the monoclonal antibody recognizes and binds survivin present in the sample; so the presence of survivin predicts the presence of cancer.

36.- The method according to claim 35, characterized also because the presence of survivin predicts a neoplastic disease in advanced stage.

37. A method for preserving the growth of cells in culture, characterized in that it comprises the step of contacting the cells with an amount of survivin that is effective to reduce apoptosis.

38.- A device for detecting the presence of survivin in a sample, characterized in that it comprises an antibody that specifically binds survivin, and reagents to detect the antibody-survivin binding pair.

39.- The use of components consisting of: (1) survivin protein or fragments of it, sufficient to evoke a cellular response mediated; (2) a DNA molecule that is opposite in direction to the mRNA of survivin or portions thereof, sufficient to inhibit the translation of survivin; (3) the normal sense DNA strand, EPR-1 acid or portions thereof, for the manufacture of a vaccine to decrease the activity of survivin in a host.

40. The use according to claim 39, further characterized in that the vaccine modulates the level of apoptosis mediated by survivin, in a host.

41.- The use of a survivin polypeptide, a survivin polypeptide fragment, a peptidomimetic thereof, apoptosis inhibitor, a transgene encoding a survivin polypeptide or a transgene encoding a survivin polypeptide fragment, for the manufacture of a medicament antiapoptotic for the prophylaxis or prevention of a disease phenotype associated with cellular apoptosis.

42. The use according to claim 41, further characterized in that the disease phenotype is a decreased T cell count in a subject infected with HIV.

43. The use according to claim 41, further characterized in that the phenotype of the disease is a degenerative disease.

44. - The use according to any of claims 6 and 20 to 22, further characterized in that it additionally comprises the administration of one or more additional agents to control the growth of the cell.

45.- The use of an agent that decreases the amount or activity of survivin, for the manufacture of a drug to treat cancer in a patient, increasing the level of apoptosis in one or more cancer cells.

46. The use according to claim 45, further characterized in that said agent is an agent that decreases the translation of the survivin mRNA in said one or more cancer cells.

47. The use according to claim 45, further characterized in that the agent is an agent that decreases the apoptosis inhibitory activity of the survivin polypeptide present in said one or more cancer cells.

48. The use according to claim 47, further characterized in that said agent that decreases the apoptosis inhibitory activity of survivin is an agent that blocks the intracellular interaction of survivin with a survivin binding partner.

49. The use according to claim 48, further characterized in that the agent blocks the binding of survivin to the binding partner by selectively binding to survivin.

50. The use according to claim 48, further characterized in that the agent blocks the binding of survivin to the binding partner by selectively binding to the binding partner.

51. The use according to claim 50, further characterized in that the agent is a survivin fragment.

52. The use according to claim 50, further characterized in that said agent comprises the polypeptide sequence EGWEPDDDPIEEHKKHSSGC, its conservatively substituted homologs and its small molecule peptidomimetics.

53. The use of a survivin polypeptide, a survivin inhibitor polypeptide fragment of apoptosis, or a peptidomimetic apoptosis inhibitor thereof, for the manufacture of a medicament for inhibiting or reversing reperfusion damage in a subject.

54. The use according to claim 53, further characterized in that said medicament is administered locally to the site of the damage.

55.- The use of a survivin polypeptide, a survivin-inhibiting polypeptide fragment of apoptosis, a peptidomimetic inhibitor of apoptosis, thereof; a transgene encoding a survivin polypeptide or a transgene encoding a survivin-inhibiting apoptosis polypeptide fragment, for the manufacture of a medicament for inhibiting or preventing rejection of tissue or organ transplantation.

56.- The use of a survivin polypeptide, a survivin polypeptide fragment, apoptosis inhibitor, or a peptidomimetic thereof, inhibitor of apoptosis, for the manufacture of a drug to increase the viability of organs and tissues before transplantation to a subject.

57. The use according to claim 56, further characterized in that the transgene is supplied by means of a viral vector.

58. The use according to claim 57, further characterized in that said vector lacks reproduction.

59. The use according to claim 56, further characterized in that the transgene is supplied as a naked nucleic acid.