Survivin, a protein that inhibits cellular apoptosis, and its modulation

The present invention provides the amio 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 agents that block Survivin mediated inhibition of cellular apoptosis, methods of using agents that block Survivin mediated inhibition or Survivin expression to modulate biological and pathological processes, and methods of assaying Survivin activity.

FIELD OF THE INVENTION
 The present invention relates to the field of modulating cell apoptosis,
 particularly agents useful to inhibit apoptosis, as well as to diagnostic
 and prognostic assays involving conditions in mediated by the expression
 of inhibitors of apoptosis. The invention specifically relates to the
 identification of a novel human gene, tentatively named Survivin. Survivin
 encodes a protein, Survivin, that inhibits cellular apoptosis,
 particularly in cancer cells and embryonic cells.
 BACKGROUND OF THE INVENTION
 Regulation of cell proliferation by programmed cell death (apoptosis)
 maintains tissue homeostasis during development and differentiation (Raff,
 M. D., Nature (1992) 356:397-400; Vaux, D. L. et al., Cell (1994)
 76:777-779). This process involves an evolutionarily conserved multi-step
 cascade (Oltvai, Z. et al., 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. et al., Cell (1994) 76,
 959-962), and associated signal transducers (Tartaglia, L. A. et al.,
 Immunol Today (1992) 13:151-153), protease gene families (Martin, S. J. et
 al., Cell (1995) 82:349-352), intracellular second messengers (Kroemer, G.
 et al., FASEB J (1995) 9:1277-1287), tumor suppressor genes (Hafifer, R.
 et al., Curr Op Gen Dev (1995) 5:84-90), and negative regulatory proteins
 that counteract apoptotic cell death (Hockenbery, D. et al., Nature (1990)
 348:334-336). Aberrantly increased apoptosis or abnormally prolonged cell
 survival (Oltvai, Z. N. et al., Cell (1994) 79:189-192) may both
 contribute to the pathogenesis of human diseases, including autoimmune
 disorders, neurodegenerative processes, and cancer (Steller, H., Science
 (1995) 267:1445-1449; Thompson, C. B., Science (1995) 267:1456-1462).
 Specifically, for example, inhibitors of apoptosis, most notably of the
 bcl-2 family (Reed, J, J Cell Biol (1994) 124:1-6, and Yang, E, et al.,
 Blood (1996) 88:386-401), maintain lymphoid homeostasis and morphogenesis
 in adult (Hockenbery, D et al, Proc Natl Acad Sci USA (1991) 88:6961-6965)
 and fetal (LeBrun, D. et al (1993) 142:743-753) tissues. Deregulated
 expression of bcl-2 has also been implicated in cancer, by aberrantly
 prolonging cell survival and facilitating the insurgence of transforming
 mutations.
 In addition to bcl-2, several members of a new gene family of inhibitors of
 @ apoptosis related to the baculovirus IAP gene (Birnbaum, M. J. et al., J
 Virology (1994) 68:2521-2528; Clem, R. J. et al., Mol Cell Biol (1994)
 14:5212-5222) have been identified in Drosophila and mammalian cells
 (Duckett, C. S. et al., EMBO J (1996) 15:2685-2694; Hay, B. A. et al.,
 Cell (1995) 83:1253-1262; Liston, P. et al., Nature (1996) 379:349-353;
 Rothe, M. et al., Cell (1995) 83:1243-1252; Roy, N. et al., Cell (1995)
 80:167-178). These molecules are highly conserved evolutionarily; they
 share a similar architecture organized in two or three approximately 70
 amino acid amino terminus Cys/His baculovirus IAP repeats (BIR) and by a
 carboxy terminus zinc-binding domain, designated RING finger (Duckett, C.
 S. et al., EMBO J (1996) 15:2685-2694; Hay, B. A et al., Cell (1995)
 83:1253-1262; Liston, P. et al., Nature (1996) 379:349-353; Rothe, M. et
 al., Cell (1995) 83:1243-1252; Roy, N. et al., Cell (1995) 80:167-178).
 Recombinant expression of IAP proteins blocks apoptosis induced by various
 stimuli in vitro (Duckett, C. S. et al., EMBO J (1996) 15:2685-2694;
 Liston, P. et al., Nature (1996) 379:349-353), and promotes abnormally
 prolonged cell survival in the developmentally-regulated model of the
 Drosophila eye, in vivo (Hay, B. A. et al., Cell (1995) 83:1253-1262).
 Finally, deletions in a IAP neuronal inhibitor of apoptosis, NAIP, were
 reported in 75% of patients with spinal muscular atrophy, thus suggesting
 a potential role of this gene family in human diseases (Roy, N. et al.,
 Cell (1995) 80:167-178).
 Therapeutic and diagnostic uses of nucleic acids that encode various
 inhibitors of apoptosis relating to a member of the LAP 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 such genes and gene products are contemplated for the novel
 protein and its encoding nucleic acid discusssed below.
 Recently, a novel gene encoding a structurally unique IAP apoptosis
 inhibitor, designated Survivin has been identified. Survivin is a -16.5 kD
 cytoplasmic protein containing a single BIR, and a highly charged
 carboxyl-terminus coiled-coil region instead of a RING finger, which
 inhibits apoptosis induced by growth factor (IL-3) withdrawal when
 transferred in B cell precursors (Ambrosini, G. et al., Nature Med. (1997)
 3:917-921). At variance with bcl-2 or other IAP proteins, Survivin is
 undetectable in adult tissues, but becomes prominently expressed in all
 the most common human cancers of lung, colon, breast, pancreas, and
 prostate, and in -50% of high-grade non-Hodgkin's lymphomas, in vivo.
 Intriguingly, the coding strand of the Survivin gene was highly homologous
 to the sequence of Effector cell Protease Receptor-1 (EPR-1) (Altieri, D.
 C., FASEB J (1995) 9:860-865), but oriented in the opposite direction,
 thus 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
 which is nearly identical to EPR-1, but oriented in the opposite
 direction. The antisense EPR-1 gene product, designated Survivin, is a
 distantly related member of the LAP family of inhibitors of apoptosis
 (Duckett, C. S. et al., EMBO J (1996) 15:2685-2694; Hay, B. A. et al.,
 Cell (1995) 83:1253-1262; Liston, P. et al., Nature (1996) 379:349-353;
 Rothe, M. et al., Cell (1995) 83:1243-1252; Roy, N. et al., 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, inhibition of Survivin expression by
 up-regulating its natural antisense EPR-1 transcript resulted in massive
 apoptosis and decreased cell growth.
 SUMMARY 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 Survivin or the Survivin protein.
 Based on this observation, the present invention provides purified
 Survivin protein.
 The present invention further provides nucleic acid molecules that encode
 the Survivin protein. Such nucleic acid molecules can be in an isolated
 form, or can be operably linked to expression control elements or vector
 sequences.
 The present invention further provides methods of identifying other members
 of the Survivin family of proteins. Specifically, the nucleic acid
 sequence of Survivin can be used as a probe, or to generate primers,
 in methods to identify nucleic acid molecules that encode other members of
 the Survivin family of proteins.
 The present invention further provides antibodies that bind to Survivin.
 Such antibodies can be either 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 methods for isolating Survivin
 binding partners. Survivin binding partners are isolated using the
 Survivin protein as a capture probe. Alternatively, Survivin can be used
 as bait in the yeast two-hybrid system to screen an expression library and
 identify genes that encode proteins that bind to the Survivin protein.
 Binding partners isolated by these methods are useful in preparing
 antibodies and also serve as targets for drug development.
 The present invention further provides methods to identify agents that can
 block or modulate the association of Survivin with a binding partner.
 Specifically, an agent can be tested for the 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
 test agent 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 utilize an agent that binds to Survivin or to the binding
 partner.
 The present invention further provides methods of regulating the expression
 of Survivin within a cell. Expression of Survivin within a cell can be
 regulated so as to produce or inhibit the production of Survivin.
 Blocking Survivin/binding partner associations or Survivin expression can
 be used to modulate biological and pathological processes that require
 Survivin. For example, methods that reduce Survivin production induce
 apoptosis of tumor cells. Stimulation of Survivin production can be used
 as a means of extending the culturability of cells or tissues.
 The biological and pathological processes that require Survivin or
 Survivin/binding partner interactions can firther be modulated using gene
 therapy methods. Additional genetic manipulation within an 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, a Survivin gene can be
 altered to correct a genetic deficiency; peptide modulators of Survivin
 activity can be produced within a target cell using genetic transformation
 methods to introduce a modulator encoding nucleic acid molecules into a
 target cell; etc. The use of nucleic acids for antisense and triple helix
 therapies and interventions are expressly contemplated.
 The present invention further provides methods of reducing the severity of
 pathological processes that require Survivin. Since expression of Survivin
 or association of Survivin with a binding partner is required for
 Survivin-mediated biological processes, agents that block Survivin
 expression, Survivin activity or the association of Survivin with a
 binding partner, can be used in therapeutic methods.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
 I. General Description
 The present invention is based in part on identifying a novel protein that
 is expressed in tumor cells and inhibits cellular apoptosis, hereinafter
 the Survivin protein or Survivin. Survivin is also found to be expressed
 in embryonic tissues.
 The Survivin protein can be used as an agent, or serve as a target for
 agents, that can be used to inhibit or stimulate Survivin mediated
 inhibition of cellular apoptosis, for example to block abnormal cell
 growth or to extend cell growth in culture.
 As used herein, modulation of apoptosis means increasing or decreasing the
 number of cells that would otherwise undergo apoptosis in a given cell
 population. This can be effected by increasing or decreasing the amount of
 Survivin present in a cell or by increasing or decreasing the activity of
 the Survivin. Preferably, the given cell population in which apoptosis is
 to be modulated is found in a tumor or other tissue or group of cells in
 which beneficial effect results from the modulation. Also, preferably, the
 increase or decrease in number of cells that would otherwise undergo
 apoptosis in a given cell population is at least about 10%, 20%, 40% or
 more preferably at least about 50% of the cells in 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 fragments of Survivin as discussed below are used as capture
 probes to isolate Survivin binding proteins. Dominant negative proteins,
 DNAs encoding these proteins, antibodies to these binding proteins,
 peptide fragments of these proteins or mimics of these proteins may be
 introduced into cells to affect Survivin function. Additionally, these
 proteins provide novel targets for screening of synthetic small molecules
 and combinatorial or naturally occurring compound libraries to discover
 novel therapeutics to regulate Survivin function.
 II. Identification, General Characterization and Tissue Distribution of
 Survivin
 The present invention is based on the identification on chromosome 17q25 of
 a novel member of the LAP family of inhibitors of apoptosis, designated
 Survivin, which may confer a selective advantage for cancer cell growth.
 Relevant features of the Survivin gene include its developmentally- and
 differentiation-regulated expression, its nearly identical and
 complementary DNA sequence with the factor Xa receptor EPR-1, and its
 abundant in vivo expression in common human malignancies, but not in the
 adjacent non-neoplastic population. As described below, targeting Survivin
 expression by metallothionein-induction of EPR-1 mRNA resulted in
 apoptosis and inhibition of proliferation of HeLa cell transfectants.
 In addition to their contribution to bemostasis, cellular receptors for
 blood proteases have recently emerged as pleiotropic signaling molecules,
 playing a crucial role in embryologic development (Connolly, A. J. et al.,
 Nature (1996) 381:516-519), and vasculogenesis (Carmeliet, P. et al.,
 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
 contributing to procoagulant activity (Altieri, D.C., FASEB J(1995)
 9:860-865), and T cell activation (Duchosal, M. A. et al., Nature (1996)
 380:352-356). Although the Survivin coding sequence was found to be nearly
 identical to the EPR-1 cDNA, its orientation was unambiguously assigned to
 the antisense EPR-1 strand for the position of the consensus splice sites
 at intron-exon boundaries (Padgett, R. A. et al, Ann Rev Biochem (1986)
 55:1119-1150). On the other hand, the authenticity of the EPR-1 "sense"
 strand was demonstrated in previous studies, when mammalian cells
 transfected with the EPR-1 cDNA or with chimeric EPR-1 constructs
 (Ambrosini, G. et al., J Biol Chem (1996) 271:1243-1248 and Altieri, D.
 C., FASEB J(1995) 9:860-865), were recognized by anti-EPR-1 mAbs and bound
 factor Xa in a specific and saturable reaction.
 These findings could be reconciled by the existence of multiple, highly
 homologous, EPR-1 transcripts oriented in opposite directions. The
 heterogeneity of EPR-1 mRNA and the complex pattern of Southern
 hybridization support this hypothesis. Previously, double stand EPR-1
 probes detected three strongly hybridizing bands of 1.9, 3.4 and
 .about.1.5 kb in mRNA of EPR-1.sup.+ cells (Altieri, D. C., FASEB J(1995)
 9:860-865). Here, single strand-specific probes confirmed the presence of
 multiple mature and polyadenylated EPR-1-related messages, and revealed
 that the 1.9 and 3.4 kb bands corresponded to two highly regulated,
 antisense EPR-1 transcripts, while the 1.5 kb band, more accurately
 defined as 1.2 kb, coincided with a genuine EPR-1-encoding message. While
 the 1.9 kb antisense transcript clearly originated from the Survivin gene
 described here, a gene encoding the 1.2 kb "sense" 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
 EPR-1 sequences in various species, and (iii) the numerous expressed
 sequence tag database entries matching (P=0.018-7.times.10.sup.-11) the
 positive (accession n. W46267), or the negative (accession n. W34764,
 W83810, T29149) EPR-1 strand, altogether suggest the existence of at least
 a second, highly-related, EPR-1 gene oriented in the opposite direction to
 that described here, and encoding the previously characterized factor Xa
 receptor (Altieri, D. C., FASEB J(1995) 9:860-865).
 A similar situation could arise from gene duplication event(s) involving
 EPR-1 sequences. Interestingly, the single hybridization signal detected
 on chromosome 17q25, and the single hybridizing bands identified in a
 Southern blot of high molecular weight genomic DNA, suggest that
 EPR-1-related sequences potentially oriented in opposite directions may be
 adjacent in close proximity, within a physical interval of 75-130 kb.
 The presence of multiple EPR1 transcripts oriented in opposite directions
 implies a reciprocal regulatory mechanism by naturally occurring
 antisense. This is consistent with the predominantly discordant and
 mutually exclusive distribution of sense and antisense EPR1 messages in
 developing or adult tissues in vivo, and during HL-60 cell terminal
 differentiation. While antisense regulation is common in prokaryotes
 (Green, P. J. et al., Annu Rev Biochem (1986) 55:569-597), a growing
 number of eukaryotic gene products have been recently characterized for
 the occurrence of functional antisense transcripts potentially
 participating in gene regulation, including basic fibroblast growth factor
 (Kimmelman, D. et al., Cell (1989) 59:687-696; Murphy, P. R. et al.,
 Molecular Endocrinology (1994) 8:852-859), a1(I) collagen (Farrell, C. M.
 et al., J Biol Chem (1995) 270:3400-3408 and Lukens, 1995), n-myc
 (Krystal, G. W. et al., Mol Cell Biol (1990) 10:4180-4191), c-myc (Celano,
 P. et al., J Biol Chem (1992) 267:15092-15096), p53 (Khochbin, S. et al.,
 EMBO J (1989) 8:4107-4114), c-erbAa (Lazar, M. A. et al., Mol Cell Biol
 (1989) 9:1128-1136), and CD3 .xi./.eta./.theta. locus (Lerer, A. et al, J
 Immunol (1993) 151:3152-3162).
 As described below, the existence of a EPR-1/Survivin gene balance
 regulated by functional antisense was demonstrated in HeLa cell
 transfectants, when metallothionein-induced transcription of the EPR-1
 "sense" strand suppressed the expression of Survivin 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
 translational initiation codon. Additional experiments have evaluated the
 ability of a Survivin antisense to inhibit cell growth. This was done by
 transiently co-transfecting the Survivin antisense with a lacZ reported
 plasmid and making a determination of cell viability after a 48-h
 transfection in .beta.-galactosidase expressing cells. The results
 indicated that the viability of Survivin antisense transfectants was &lt;20%
 of control cells transfected with the empty vector. A control antisense of
 ICAM-1 (intercellular adhesion molecule-1) similarly co-transfected in
 HeLa cells was ineffective.
 Survivin was found to be a small protein of 142 amino acids (.about.16.5
 kDa) with no amino acid sequence homology to EPR-1, and designated
 Survivin for the presence of a BIR-homologous domain (Birnbaum, M. J. et
 al., J Virology (1994) 68:2521-2528; Clem, R. J. et al., Mol Cell Biol
 (1994) 14:5212-5222) found in IMP inhibitors of apoptosis (Duckett, C. S.
 et al., EMBO J (1996) 15:2685-2694; Hay, B. A. et al., Cell (1995)
 83:1253-1262; Liston, P. et al., Nature (1996) 379:349-353; Rothe, M. et
 al, Cell (1995) 83:1243-1252; Roy, N. et al., Cell (1995) 80:167-178).
 Based on overall sequence conservation, the absence of a carboxy terminus
 RING finger 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. et al., Cell (1995)
 80:167-178). Thus, unlike bcl-2 or other IAP proteins, Survivin is
 undetectable in adult tissues, but becomes prominetnly expressed in all
 the most common human cancers of lung, colon, breast, pancreas, and
 prostate, and in .about.50% of high-grade non-Hodgkin's lymphomas, in
 vivo. Additionally, unlike other IAP proteins (Deveraux, Q. et al., Nature
 (1997) 388:300-304), Survivin does not bind caspases in a cell-free system
 (Roy, N. et al., Blood (1997) 595:2645.
 Consistent with the anti-apoptosis properties of IAP proteins in vitro
 (Duckett, C. S. et al., EMBO J (1996) 15:2685-2694; Liston, P. et al.,
 Nature (1996) 379:349-353), and in vivo (Hay, B. A. et al., Cell (1995)
 83:1253-1262), inhibition of Survivin expression by the EPR-1 transcript
 (which naturally is antisense to Survivin) resulted in increased
 apoptosis, as determined by in situ internucleosomal DNA fragmentation in
 HeLa cell transfectants. The ability of a RING finger-less IAP protein to
 counteract apoptosis is not without a precedent, as demonstrated by the
 suppression of apoptosis mediated by NAIP (Liston, P. et al., Nature
 (1996) 379:349-353), and by the in vivo gain-of-function of a Drosophila
 IAP protein following deletion of the RING finger (Hay, B. A. et al., Cell
 (1995) 83:1253-1262). Although anti-apoptosis genes are thought to play an
 indirect role in cell growth, by favoring the accumulation of oncogenic
 mutations(s) in aberrantly long-living cells (Reed, J. C., J Cell Biol
 (1994) 124:1-6), down-regulation of Survivin resulted in a profound
 inhibition of HeLa cell proliferation. While this may derive from rapid
 disappearance of HeLa cells expressing the highest levels of antisense
 transcripts by apoptosis, a similar decrease in tumor cell proliferation
 has been reported in vivo after antisense inhibition of bcl-2 (Reed, J. C.
 et al., Proc Natl Acad Sci USA (1990) 87:3660-3664).
 The possibility that IAP proteins may play a more general role in cell
 proliferation, not exclusively restricted to apoptosis inhibition, has
 been proposed earlier. Rothe et al., have recently demonstrated that the
 amino terminus BIR in two IAP proteins (cLAPs) physically interacts with
 the signal transducers associated with the 75 kDa TNF receptor (Rothe, M.
 et al., Cell (1995) 83:1243-1252), a molecule primarily implicated in cell
 proliferation and survival rather than apoptotic signaling (Tartaglia, L.
 A. et al., Immunol Today (1992) 13:151-153). While it is not known if
 Survivin is physically linked to signaling molecules (Rothe, M. et al.,
 Cell (1995) 83:1243-1252), the structural divergence of its BIR as
 compared with other IAP proteins (Duckett, C. S. et al., EMBO J (1996)
 15:2685-2694; Hay, B. A. et al., Cell (1995) 83:1253-1262; Liston, P. et
 al., Nature (1996) 379:349-353; Rothe, M. et al., Cell (1995)
 83:1243-1252; Roy, N. et al., Cell (1995) 80:167-178), may confer
 specificity for supramolecular interaction(s) potentially relevant to its
 particular mechanism of apoptosis inhibition/cell growth.
 Dysregulation of programmed cell death (apoptosis) has recently emerged as
 a primary mechanism contributing to the pathogenesis of various human
 diseases, including cancer (Steller, H., Science (1995) 267:1445-1449;
 Thompson, C. B., Science (1995)267:1456-1462). While the impact of
 anti-apoptosis gene(s) in neoplasia is highlighted by the role of bcl-2 in
 follicular lymphoma (Korsmeyer, S. J., Blood (1992) 80:879-886), a
 potential distribution of LAP proteins in cancer had not been previously
 investigated. In this context, one of the most striking characteristics of
 Survivin was its abundant expression in actively proliferating transformed
 cell lines, and in all the most common human malignancies of lung, colon,
 pancreas, and breast, in vivo, but not in the non-neoplastic adjacent cell
 population. This distribution in multiple human cancers may signal a
 fundamental role of this molecule in apoptosis/cell proliferation
 mechanisms in neoplasia. By analogy with the paradigm of bcd-2,
 over-expression of Survivin in cancer may lead to aberrantly prolonged
 cell viability (Veis, D. J. et al., Cell (1993) 75:229-240), increased
 resistance to chemotherapy-induced apoptosis (Miyashita, T. et al., Blood
 (1993) 81:151-157), and, as suggested by the in vitro studies reported
 above, in a direct advantage for transformed cell proliferation.
 On the other hand, for its presence in normal PBMC and benign breast
 adenomas, in vivo (unpublished observations), Survivin expression cannot
 be interpreted per se as a marker of malignant transformation but may
 reflect a more general, developmental--or cell type-specific response to
 certain stimuli. This is consistent with the presence of Survivin during
 normal embryonic (our unpublished observations) and fetal development, and
 its rapid disappearance in growth-arrested cell types (i.e. vitamin
 D.sub.3 -treated HL-60), and terminally-differentiated tissues, in vivo.
 At variance with other LAP proteins which are constitutively found in
 adult mature tissues (Duckett, C. S. et al., EMBO J (1996) 15:2685-2694;
 Liston, P. et al., Nature (1996) 379:349-353; Rothe, M. et al., Cell
 (1995) 83:1243-1252), this pattern of expression is reminiscent of the
 distribution of bcl-2 in fetal tissues (LeBrun, D. P. et al., Am J Pathol
 (1993) 142:743-753), and its more restricted presence in differentiated
 cells, correlating with susceptibility to apoptosis (Hockenbery, D. M. et
 al, Proc Natl 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 to balance the effect of this potent
 anti-apoptosis gene by manipulating a normal cell regulatory mechanism,
 centered on the expression of EPR-1 (Altieri, D. C., FASEB J (1995)
 9:860-865). Targeting Survivin may then remove a selective advantage for
 transformed cell growth and be therapeutically beneficial to increase the
 susceptibility of cancer cells to chemotherapy-induced apoptosis. Along
 the same line, identification of polymorphic markers and construction of
 extended aplotypes within and around the EPR1 /Survivin locus may provide
 new insights on the population genetics of susceptibility to chemotherapy.
 III. SPECIFIC EMBODIMENTS
 A. Survivin 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 that has the amino acid sequence
 of human Survivin depicted in FIG. 4. The term "Survivin protein" also
 includes naturally occurring allelic variants of Survivin, naturally
 occurring proteins that have a slightly different amino acid sequence than
 that specifically recited above. Allelic variants, though possessing a
 slightly different amino acid sequence than those recited above, will
 still have the requisite ability to inhibit cellular apoptosis.
 As used herein, the Survivin family of proteins refers to Survivin proteins
 that have been isolated from organisms in addition to 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 (inhibitory apoptosis proteins) family of
 protein. However, Survivin is the first member of a unique subfamily of
 IAP proteins that differ from other LAP proteins in significant ways.
 Despite homology and sequence conservation in the BIR module between
 Survivin and other members of this gene family, there are important
 structural differences that are unique to members of the Survivin family
 of proteins. First unlike any other LAP protein, Survivin has only one BIR
 module (most of the other molecules have 2-3). Further, Survivin does not
 contain a carboxy-terminal RING finger but has a predicted coiled-coil
 instead. Only the Neuronal Apoptosis Inhibitory Protein (NAIP) in the IAP
 family lacks a RING finger, but does not contain a carboxy-terminus coiled
 coil. Finally there is no DNA sequence similarity between Survivin and
 other LAP proteins ( primers designed on Survivin are unlikely to
 detect other LAP proteins and vice-versa).
 The Survivin proteins of the present invention are preferably in isolated
 from. As used herein, a protein is said to be isolated when physical,
 mechanical or chemical methods are employed to remove the Survivin protein
 from cellular constituents that are normally associated with the Survivin
 protein. A skilled artisan can readily employ standard purification
 methods to obtain an isolated Survivin protein.
 The Survivin proteins of the present invention further include conservative
 variants of the Survivin proteins herein described. As used herein, a
 conservative variant refers to alterations in the 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 deletion is said to adversely affect the
 Survivin protein when the altered sequence prevents the Survivin protein
 from associating with a Survivin binding partner and/or prevents the
 Survivin protein from inhibiting cellular apoptosis. For example, the
 overall 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.
 The allelic variants, the conservative substitution variants and the
 members of the Survivin family of proteins, will have the ability to
 inhibit cellular apoptosis. Such proteins will ordinarily have an amino
 acid sequence having at least about 75% amino acid sequence identity with
 the human Survivin sequence, more preferably at least about 80%, even more
 preferably at least about 90%, and most preferably at least about 95%.
 Identity or homology with respect to such sequences is defined herein as
 the percentage of amino acid residues in the candidate sequence that are
 identical with the known peptides, after aligning the sequences and
 introducing gaps, if necessary, to achieve the maximum percent homology,
 and including any conservative substitutions as being homologous.
 N-terminal, C-terminal or internal extensions, deletions, or insertions
 into the peptide sequence shall not be construed as affecting homology.
 Thus, the Survivin proteins of the present invention include molecules
 having the amino acid sequences disclosed in FIG. 1; fragments thereof
 having a consecutive sequence of at least about 3, 5, 10 or 15 amino acid
 residues of the Survivin protein; amino acid sequence variants of such
 sequence wherein an amino acid residue has been inserted N- or C-terminal
 to, or within, the disclosed Survivin sequence; amino acid sequence
 variants of the disclosed Survivin sequence, or their fragments as defined
 above, that have been substituted by another residue. Contemplated
 variants further include those containing predetermined mutations by,
 e.g., homologous recombination, site-directed or PCR mutagenesis, and the
 corresponding Survivin proteins of other animal species, including but not
 limited to rabbit, rat, murine, porcine, bovine, ovine, equine and
 non-human primate species, and the alleles or other naturally occurring
 variants of the Survivin family of proteins; and derivatives wherein the
 Survivin protein has been covalently modified by substitution, chemical,
 enzymatic, or other appropriate means with a moiety other than a naturally
 occurring amino acid (for example a detectable moiety such as an enzyme or
 radioisotope). The recombinant Survivin protein also can 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 specific small molecule inhibitors.
 As described below, members of the Survivin family of proteins can be used:
 1) as a target to block Survivin mediated inhibition of cellular
 apoptosis, 2) to identify and isolate binding partners that bind Survivin,
 3) in methods to identify agents that block the association of Survivin
 with a Survivin binding partner, 4) as a target to assay for Survivin
 mediated inhibition of cellular apoptosis, 5) as an agent to block
 cellular apoptosis, administered alone or as part of a combination
 therapy, 6) as a binding partner in an assay to quantitate circulating
 levels of anti-Survivin antibodies, 7) as an antigen to elicit production
 of anti-Survivin antibodies that in turn can be used in an an assay to
 quantitate circulating levels of Survivin and or can be used for
 immunohistochemical purposes, and 8) as a therapeutic anti-cancer vaccine,
 or component of a polyvalent vaccine.
 B. Anti-Survivin Antibodies
 The present invention further provides antibodies that selectively bind to
 a Survivin protein. The anti-Survivin antibodies particularly contemplated
 include monoclonal and polyclonal antibodies as well as fragments
 containing 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, or fragment, in isolated or immunoconjugated
 form (Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). FIG. 9
 provides a Jameson-Wolf plot of the antigenic index of various regions of
 Survivin. Such regions, in combination with the other structural analysis
 provided in FIG. 9, provide suitable fragments for use in generating
 Survivin specific antibodies. Methods for preparing 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
 using, for example, carbodiimide reagents may be used; in other instances
 linking reagents such as those supplied by Pierce Chemical Co., Rockford,
 Ill., may be effective.
 Administration of the Survivin immunogen is conducted generally by
 injection over a suitable time period and with use of a suitable adjuvant,
 as is generally understood in the art. During the immunization schedule,
 titers of antibodies can be taken to determine adequacy of antibody
 formation.
 While the polyclonal antisera produced in this way may be satisfactory for
 some applications, for pharmaceutical compositions, monoclonal antibody
 preparations are preferred. Immortalized cell lines which secrete a
 desired monoclonal antibody may be prepared using the standard method of
 Kohler and Milstein or modifications which effect immortalization of
 lymphocytes or spleen cells, as is generally known. The immortalized cell
 lines secreting the desired antibodies are screened by immunoassay in
 which the antigen is the Survivin peptide. When the appropriate
 immortalized cell culture secreting the desired antibody is identified,
 the cells can be cultured either in vitro or by production in ascites
 fluid.
 The desired monoclonal antibodies are then recovered from the culture
 supernatant or from the ascites supernatant. Fragments of the monoclonals
 or the polyclonal antisera which contain the immunologically significant
 portion can be used as antagonists, as well as the intact antibodies. Use
 of immunologically reactive fragments, such as the Fab, Fab', of
 F(ab').sub.2 fragments is often preferable, especially in a therapeutic
 context, as these fragments are generally less immunogenic than the whole
 immunoglobulin.
 The antibodies or fragments may also be produced, using current technology,
 by recombinant means. Regions that bind specifically to the desired
 regions of receptor can also be produced in the context of chimeras or CDR
 grafted antibodies of multiple species origin.
 The antibodies thus produced are useful not only as modulators of the
 association of Survivin with a Survivin binding partner, but are also
 useful in immunoassays for detecting Survivin expression/activity and for
 the purification of Survivin and associated binding partners.
 C. Survivin Encoding Nucleic Acid Molecules
 The present invention further provides nucleic acid molecules that encode
 Survivin, and the related Survivin proteins herein described, preferably
 in isolated form. For convenience, all Survivin encoding nucleic acid
 molecules will be referred to as the Survivin encoding nucleic acid
 molecule, the Survivin gene, or Survivin. As used herein, "nucleic acid"
 is defined as RNA or DNA that encodes a peptide as defined above, or is
 complementary to a nucleic acid sequence encoding such peptides, or
 hybridizes to such a nucleic acid and remains stably bound to it under
 stringent conditions, or encodes a polypeptide sharing at least 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 antisense molecules, as well as nucleic acids based on
 an alternative backbone or including alternative bases whether derived
 from natural sources or synthesized. Such hybridizing or complementary
 nucleic acid, however, is defined further as being novel and unobvious
 over any prior art nucleic acid including that which encodes, hybridizes
 under appropriate stringency conditions, or is complementary to a nucleic
 acid encoding a Survivin protein according to the present invention.
 As used herein, "stringent conditions" are conditions in which
 hybridization yields a clear and detectable sequence. Stringent conditions
 are those that (1) employ low ionic strength and high temperature for
 washing, for example, 0.015 M NaCl,0.0015 M sodium titrate, 0.1% SDS at
 50.degree. C., or (2) employ during hybridization a denaturing agent such
 as formamide, for example, 50% (vol/vol) formamide 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.degree. C.
 Another example is use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M
 sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium
 pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50
 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree. C., with washes
 at 42.degree. C. in 0.2 x SSC and 0.1% SDS. A skilled artisan can readily
 determine and vary the stringency 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 contaminant nucleic
 acid encoding other polypeptides from the source of nucleic acid.
 The present invention further provides fragments of the Survivin encoding
 nucleic acid molecule. As used herein, a fragment of a Survivin encoding
 nucleic acid molecule refers to a small portion of the entire protein
 encoding sequence. The size of the fragment will be determined by the
 intended use. For example, if the fragment is chosen so as to encode an
 active portion of the Survivin protein, such as the C-terminal .beta.
 coils or the IAP motif, the fragment will need to be large enough to
 encode the functional region(s) of the Survivin protein. If the fragment
 is to be used as a nucleic acid probe or PCR primer, then the fragment
 length is chosen so as to obtain a relatively small number of false
 positives during probing/priming. FIG. 1 identifies fragments of the
 Survivin gene that are particularly useful as selective hybridization
 probes or PCR primers.
 Fragments of the Survivin encoding nucleic acid molecules of the present
 invention (i.e., synthetic oligonucleotides) that are used as probes or
 specific primers for the polymerase chain reaction (PCR), or to synthesize
 gene sequences encoding Survivin proteins can easily be synthesized by
 chemical techniques, for example, the phosphotriester method of Matteucci,
 et al., J Am Chem Soc (1981) 103:3185-3191 or using automated synthesis
 methods. In addition, larger DNA segments can readily be prepared by well
 known methods, such as synthesis of a group of oligonucleotides that
 define various modular segments of the Survivin gene, followed by ligation
 of oligonucleotides to build the complete modified Survivin gene.
 The Survivin encoding nucleic acid molecules of the present invention may
 further be modified so as to contain a detectable label for diagnostic and
 probe purposes. 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 Survivin expression and tumor growth
 potential. A variety of such labels are known in the art and can readily
 be employed with the Survivin encoding molecules herein described.
 Suitable labels include, but are not limited to, biotin, radiolabeled
 nucleotides and the like. A skilled artisan can employ any of the art
 known labels to obtain a labeled Survivin encoding nucleic acid molecule.
 Since the Survivin gene is an antisense or reverse orientation of the EPR-1
 gene, particularly preferred are single-stranded probes for use in
 diagnostic purposes.
 Specifically, single-stranded diagnostic probes can be used to selectively
 hybridize to mRNA that encodes Survival Single-stranded probes can be
 generated using known methods in which one strand of a double-stranded
 probe is isolated or in which a single stranded RNA probe is generated.
 Modifications to the primary structure itself by deletion, addition, or
 alteration of the amino acids incorporated into the protein sequence
 during translation can be made without destroying the activity of the
 protein. Such substitutions or other alterations result in proteins having
 an amino acid sequence encoded by DNA falling within the contemplated
 scope of the present invention.
 D. Isolation of Other Survivin Encoding Nucleic Acid Molecules
 As described above, the identification of the human Survivin encoding
 nucleic acid molecule allows a skilled artisan to isolate nucleic acid
 molecules that encode other members of the Survivin family of proteins in
 addition to the human sequence herein described.
 Essentially, a skilled artisan can readily use the amino acid sequence of
 Survivin to generate antibody probes to screen expression libraries
 prepared from cells. Typically, polyclonal antiserum from mammals such as
 rabbits immunized with the purified Survivin protein (as described below)
 or monoclonal antibodies can be used to probe a mammalian cDNA or genomic
 expression library, such as lambda gtll library, to obtain the appropriate
 coding sequence for Survivin, or other members of the Survivin family of
 proteins. The cloned cDNA sequence can be expressed as a fusion protein,
 expressed directly using its own control sequences, or expressed by
 constructions using control sequences appropriate to the particular host
 used for expression of the enzyme. FIG. 1 identifies important antigenic
 and/or putative operative domains found in the Survivin protein sequence.
 Such regions are preferred sources of antigenic portions of the Survivin
 protein for the production of probe, diagnostic, and therapeutic
 antibodies.
 Alternatively, a portion of the Survivin encoding sequence herein described
 can be synthesized and used as a probe to retrieve DNA encoding a member
 of the Survivin family of proteins from any mammalian organisms that
 contains such a protein. Oligomers containing approximately 18-20
 nucleotides (encoding about a 6-7 amino acid stretch) are prepared and
 used to screen genomic DNA or cDNA libraries to obtain hybridization under
 stringent conditions or conditions of sufficient stringency to eliminate
 an undue level of false positives.
 Additionally, pairs of oligonucleotide primers can be prepared for use in a
 polymerase chain reaction (PCR) to selectively clone a Survivin-encoding
 nucleic acid molecule. A PCR denature/anneal/extend cycle for using such
 PCR primers is well known in the art and can readily be adapted for use in
 isolating other Survivin encoding nucleic acid molecules. FIG. 1
 identifies regions of the human Survivin gene that are particularly well
 suited for use as a probe or as primers.
 E. rDNA Molecules Containing a Survivin Encoding Nucleic Acid Molecule
 The present invention further provides recombinant DNA molecules (rDNAs)
 that contain a Survivin encoding 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 et al., Molecular Cloning (1989). In the
 preferred rDNA molecules, a Survivin encoding DNA sequence is operably
 linked to expression control sequences and/or vector sequences.
 The choice of vector and/or expression control sequences to which one of
 the Survivin encoding sequences of the present invention is operably
 linked depends directly, as is well known in the art, on the functional
 properties desired, e.g., protein expression, and the host cell to be
 transformed. A vector contemplated by the present invention is at least
 capable of directing the replication or insertion into the host
 chromosome, and preferably also expression, of the Survivin gene included
 in the rDNA molecule.
 Expression control elements that are used for regulating the expression of
 an operably linked protein encoding sequence are known in the art and
 include, but are not limited to, inducible promoters, constitutive
 promoters, secretion signals, and other regulatory elements. Preferably,
 the inducible promoter is readily controlled, such as being responsive to
 a nutrient in the host cell's medium.
 In one embodiment, the vector containing a Survivin encoding nucleic acid
 molecule will include a prokaryotic replicon, i.e., a DNA sequence having
 the ability to direct autonomous replication and maintenance of the
 recombinant DNA molecule extrachromosomally in a prokatyotic host cell,
 such as a bacterial host cell, transformed therewith. Such replicons are
 well known in the art. In addition, vectors that include a prokaryotic
 replicon may also include a gene whose expression confers a detectable
 marker such as a drug resistance. Typical bacterial drug resistance genes
 are those that confer resistance to ampicillin or tetracycline.
 Vectors that include a prokaryotic replicon can further include a
 prokaryotic or viral promoter capable of directing the expression
 (transcription and translation) of the Survivin encoding gene sequences in
 a bacterial host cell, such as E. coli. A promoter is an expression
 control element formed by a DNA sequence that permits binding of RNA
 polymerase and transcription to occur. Promoter sequences compatible with
 bacterial hosts are typically provided in plasmid vectors containing
 convenient restriction sites for insertion of a DNA segment of the present
 invention. Typical of such vector plasmids are pUC8, pUC9, pBR322 and
 pBR329 available from Biorad Laboratories, (Richmond, Calif.), pPL and
 pKK223 available from Pharmacia, Piscataway, N.J.
 Expression vectors compatible with eukaryotic cells, preferably those
 compatible with vertebrate cells, can also be used to form rDNA molecules
 that contain a Survivin encoding sequence. Eukayotic cell expression
 vectors are well known in the art and are available from several
 commercial sources. Typically, such vectors are provided containing
 convenient restriction sites for insertion of the desired DNA segment.
 Typical of such vectors are PSVL and pKSV-10 (Pharmacia), pBPV-1/pML2d
 (International Biotechnologies, Inc.), pTDT1 (ATCC, #31255), the vector
 pCDM8 described herein, and the like eukaryotic expression vectors.
 Eukaryotic cell expression vectors used to construct the rDNA molecules of
 the present invention may further include a selectable marker that is
 effective in an eukaryotic cell, preferably a drug resistance selection
 marker. A preferred drug resistance marker is the gene whose expression
 results in neomycin resistance, i.e., the neomycin phosphotransferase
 (neo) gene. Southern et al., J Mol Anal Genet (1982) 1:327-341.
 Alternatively, the selectable marker can be present on a separate plasmid,
 and the two vectors are introduced by co-trasfection of the host cell, and
 selected by culturing in the appropriate drug for the selectable marker.
 F. Host Cells Containing an Exogenously Supplied Survivin Encoding Nucleic
 Acid Molecule
 The present invention fierier provides host cells transformed with a
 nucleic acid molecule that encodes a Survivin protein of the present
 invention The host cell can be either prokatyotic or eukaryotic.
 Eukaryotic cells useful for expression of a Survivin protein are not
 limited, so long as the cell line is compatible with cell culture methods
 and compatible with the propagation of the expression vector and
 expression of the Survivin gene product. Preferred eukaryotic host cells
 include, but are not limited to, yeast, insect and mammalian cells,
 preferably vertebrate cells such as those from a mouse, rat, monkey or
 human fibroblastic cell line, the most preferred being cells that do not
 naturally express a Survivin protein. Preferred eukaryotic host cells
 include the murine IL-3 dependent cell line BaF3, and the like eukaryotic
 tissue culture cell lines.
 Any prokaryotic host can be used to express a Survivin-encoding rDNA
 molecule. The preferred prokaryotic host is E. coli.
 Transformation of appropriate cell hosts with a rDNA molecule of the
 present invention is accomplished by well known methods that typically
 depend on the type of vector used and host system employed. With regard to
 transformation of prokaryotic host cells, electroporation and salt
 treatment methods are typically employed, see, for example, Cohen et al.,
 Proc Natl Acad Sci USA (1972)69:2110; and Maniatis et al., Molecular
 Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
 Harbor, N.Y. (1982). With regard to transformation of vertebrate cells
 with vectors containing rDNAs, electroporation, cationic lipid or salt
 treatment methods are typically employed, see, for example, Graham et al.,
 Virol (1973) 52:456; Wigler et al., Proc Natl Acad Sci USA (1979)
 76:1373-76.
 Successfully transformed cells, i.e., cells that contain a rDNA molecule of
 the present invention, can be identified by well known techniques. For
 example, cells resulting from the introduction of an rDNA of the present
 invention can be cloned to produce single colonies. Cells from those
 colonies can be harvested, lysed and their DNA content examined for the
 presence of the rDNA using a method such as that described by Southern, J
 Mol Biol (1975) 98:503, or Berent et al., Biotech (1985) 3:208 or the
 proteins produced from the cell assayed via an immunological method.
 G. Production of Survivin Using a rDNA Molecule Encoding a Survivin Protein
 The present invention further provides methods for producing a Survivin
 protein that uses one of the Survivin encoding nucleic acid molecules
 herein described. In general terms, the production of a recombinant form
 of a Survivin protein typically involves the following steps.
 First, a nucleic acid molecule is obtained that encodes a Survivin protein,
 such as the nucleic acid molecule depicted in FIG. 1. If the Survivin
 encoding sequence is uninterrupted by introns, it is directly suitable for
 expression in any host. If not, then a spliced form of the Survivin
 encoding nucleic acid molecule can be generated and used or the intron
 containing nucleic acid molecule can be used in a compatible eukaryotic
 expression system.
 The Survivin encoding nucleic acid molecule is then preferably placed in
 operable linkage with suitable control sequences, as described above, to
 form an expression unit containing the Survivin encoding sequences. The
 expression unit is used to transform a suitable host and the transformed
 host is cultured under conditions that allow the production of the
 Survivin protein. Optionally the Survivin protein is isolated from the
 medium or from the cells; recovery and purification of the protein may not
 be necessary in some instances where some impurities may be tolerated.
 Each of the foregoing steps can be done in a variety of ways. For example,
 the desired coding sequences may be obtained from genomic fragments and
 used directly in appropriate hosts. The construction of expression vectors
 that are operable in a variety of hosts is accomplished using appropriate
 replicons and control sequences, as set forth above. The control
 sequences, expression vectors, and transformation methods are dependent on
 the type of host cell used to express the gene and were discussed in
 detail earlier. Suitable restriction sites can, if not normally available,
 be added to the ends of the coding sequence so as to provide an excisable
 gene to insert into these vectors. A skilled artisan can readily adapt any
 host/expression system known in the art for use with Survivin encoding
 sequences to produce a Survivin protein.
 H. Inhibition of Cell Death Using Survivin
 As provided above, Survivin has been shown to inhibit cellular apoptosis.
 Accordingly, Survivin can be used in methods to extend the life of cells.
 In general, cellular apoptosis can be inhibited by contacting a cell with
 Survivin.
 The are a number of situation in which it is desirable to inhibit cellular
 apoptosis. For example, the death of cells in tissues and organs being
 prepared for transport and transplant can be inhibited using the Survivin
 protein. Alternatively, cells lines can be established for long term
 culture using Survivin encoding nucleic acid molecules expressed in the
 cell line.
 Hence, Survivin protein or Survivin gene expression 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 liposomal,
 Penetrin-1 delivery, or inclusion in the cell growth media, to inhibit
 apoptosis. Alternatively, the Survivin gene can be introduced and
 expressed in cells to increase the longevity of cells in culture. These
 provide means and methods for increasing the ability of cultured cells to
 produce desired compounds as well as provide methods of establishing
 long-term culture of primary explants of cells and tissues.
 In tissue transplant, typically tissues and organs are stored and
 transported prior to transplant. Cell death, by mechanisms similar to
 apoptosis, can lead to the loss of viability of the tissues or organs. In
 this setting, infusion with Survivin protein can be used as a method to
 inhibit cell death in such tissues and organs.
 There are pathological conditions characterized by premature and unwanted
 cellular apoptosis, for example in accelerated aging disorders. It is
 already known that inactivating mutations in a IAP protein may cause human
 diseases. The example is for the NAIP (see above). Studies of patients
 with SMA (Spinal muscular atrophy, a neurodegenrative disease that is
 thought to be caused by aberrantly increased apoptosis) has demonstrated
 that the NAIP gene is inactivated and deleted in 75% of these patients
 (Roy et al. 1995, Cell 80:167). By extension, inactivating mutations in
 Survivin can result in degenerative diseases characterized by aberrantly
 increased cell death. Haplotypic markers within and around the Survivin
 locus on chromosome 17q25 can be used in studies of population genetics to
 determine if that locus has already been implicated in diseases with
 increased apoptosis. In such cases, the Survivin gene or the Survivin
 protein can be used to treat the conditions. Accordingly, the Survivin
 protein, or a Survivin encoding nucleic acid molecule is administered to
 an individual as a means of treating abnormal apoptosis.
 I. Methods to Identify Survivin Binding Partners
 Another embodiment of the present invention provides methods for use in
 isolating and identifying binding partners of Survivin. 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, 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 that expresses Survivin under conditions that allow the association
 of a binding partner with Survivin. After mixing, peptides that have
 become associated with Survivin are separated from the mixture. The
 binding partner that bound 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 cellular extract refers to a preparation or fraction that
 is made from a lysed or disrupted cell. The preferred source of cellular
 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 of a cell. Cells can
 be disrupted using either physical or chemical disruption methods.
 Examples of physical disruption methods include, but are not limited to,
 sonication and mechanical shearing. Examples of chemical lysis methods
 include, but are not limited to, detergent lysis and the enzyme lysis. In
 addition, the cellular extract can be prepared from cells that have been
 freshly isolated from a subject or from cells or cell lines which have
 been cultured. A skilled artisan can readily adapt methods for preparing
 cellular extracts in order to obtain extracts for use in the present
 methods.
 Once an extract of a cell is prepared, the extract is mixed with the
 Survivin protein under conditions in which association of Survivin with
 the binding partner can occur. A variety of conditions can be used, the
 most preferred being conditions that closely resemble conditions found in
 the cytoplasm of a Survivin-expressing cell. Features such as osmolarity,
 pH, temperature, and the concentration of cellular extract used, can be
 varied to optimize the association of the Survivin with the binding
 partner.
 After mixing under appropriate conditions, Survivin is separated from the
 mixture. A variety of techniques can be utilized to separate the mixture.
 For example, antibodies specific to Survivin can be used to
 immunoprecipitate the Survivin and associated binding partner.
 Alternatively, standard chemical separation techniques such as
 chromatography and density/sediment centrifugation can be used.
 After removal of nonassociated cellular constituents found in the extract,
 the binding partner can be dissociated from the Survivin protein using
 conventional methods. For example, dissociation can be accomplished by
 altering the salt concentration or pH of the mixture.
 To aid in separating associated Survivin/binding partner pairs from the
 mixed extract, the Survivin protein can be immobilized on a solid support.
 For example, Survivin can be attached to a nitrocellulose matrix or
 acrylic beads. Attachment of Survivin to a solid support further aids in
 separating peptide/binding partner pair from other constituents found in
 the extract.
 Alteratively, the Survivin-encoding nucleic acid molecule 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 readily be adapted to employ
 the Survivin encoding molecules herein described.
 J. Use of Survivin Binding Partners
 Once isolated, the Survivin binding partners obtained using the above
 described methods 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 a
 Survivin binding partner can be used to assay Survivin activity, 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 below.
 K. Methods to Identify Agents that Block Survivin/Binding Partner
 Interactions
 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 allow association of Survivin with the
 Survivin binding partner, the two mixtures are analyzed and compared to
 determine if 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
 decreasing the amount of association present in the sample containing the
 tested agent.
 As used herein, an agent is said to reduce or block Survivin/Survivin
 binding partner association when the presence of the agent decreases the
 extent to which or prevents the Survivin binding partner from becoming
 associated with Survivin. One class of agents will reduce or block the
 association by binding to the Survivin binding partner while another class
 of agents will reduce or block the association by binding to Survivin.
 The Survivin binding partner used in the above assay can either be an
 isolated and fully characterized protein or can be a partially
 characterized protein that binds to Survivin or a Survivin binding partner
 that has been identified as being present in a cellular extract It will be
 apparent to one of ordinary skill in the art that so long as the Survivin
 binding partner has been characterized by an identifiable property, e.g.,
 molecular weight, the present assay can be used.
 Agents that are assayed in the above method can be randomly selected or
 rationally selected or designed. As used herein, an agent is said to be
 randomly selected when the agent is chosen randomly without considering
 the specific sequences involved in the association of the Survivin with
 the Survivin binding partner. An example of randomly selected agents is
 the use a chemical library or a peptide combinatorial library, or a growth
 broth of an organism.
 As used herein, an agent is said to be rationally selected or designed when
 the agent is chosen on a nonrandom basis which takes into account the
 sequence of the target site and/or its conformation in connection with the
 agent's action. As described above, there are two sites of action for
 agents that block Survivin/Survivin binding partner interaction: the
 binding partner contact site on Survivin and the Survivin contact site on
 the Survivin binding partner. Agents can be rationally selected or
 rationally designed by utilizing the peptide sequences that make up the
 contact sites of the Survivin/Survivin binding partner pair. For example,
 a rationally selected peptide agent can be a peptide whose amino acid
 sequence is identical to the Survivin contact site on the Survivin binding
 partner. Such an agent will reduce or block the association of Survivin
 with the binding partner by binding to the Survivin binding partner.
 The agents of the present invention can be, as examples, peptides, small
 molecules, vitamin derivatives, as well as carbohydrates. A skilled
 artisan can readily recognize that there is no limit as 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 chosen
 based on the amino acid sequence of the Survivin protein.
 The peptide agents of the invention can be prepared using standard solid
 phase (or solution phase) peptide synthesis methods, as is known in the
 art. In addition, the DNA encoding these peptides may be synthesized using
 commercially available oligonucleotide synthesis instrumentation and
 produced recombinantly using standard recombinant production systems. The
 production using solid phase peptide synthesis is necessitated if
 non-gene-encoded amino acids are to be included.
 Another class of agents of the present invention are antibodies
 immunoreactive with critical positions of the Survivin or Survivin binding
 partner. As described above, antibodies are obtained by immunization of
 suitable mammalian subjects with peptides, containing as antigenic
 regions, those portions of the Survivin or binding partner, intended to be
 targeted by the antibodies. Critical regions include the contact sites
 involved in the association of the Survivin with the Survivin binding
 partner.
 As discussed below, the important minimal sequence of residues involved in
 Survivin activity define a functional linear domain that can be
 effectively used as a bait for two-hybrid screening and identification of
 potential Survivin-associated molecules. Use of such Survivin fragments
 will significantly increase the specificity of the screening as opposed to
 using the fall length molecule or the entire BIR domain and is therefore
 preferred. Similarly, this linear sequence can be also used as an affinity
 matrix also to isolate Survivin binding proteins using a biochemical
 affinity purification strategy.
 L. Uses for Agents that Block the Association of Survivin with a Survivin
 Binding Partner
 As provided in the Background section, Survivin inhibits cellular
 apoptosis. Agents that reduce or block the interactions of Survivin with a
 Survivin binding partner can be used to modulate biological and pathologic
 processes associated with Survivin function and activity.
 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, so long as the mammal is in
 need of modulation of a pathological or biological process mediated by
 Survivin. The term "mammal" is meant an individual belonging to the class
 Mammalia. The invention is particularly useful in the treatment of human
 subjects.
 As used herein, a biological or pathological process mediated by Survivin
 or Survivin binding to a Survivin binding partner refers to the wide
 variety of cellular events mediated by Survivin. Pathological processes
 refer to a category of biological processes which produce a deleterious
 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 Survivin/Survivin
 binding partner association or block Survivin expression.
 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 tumor cell proliferation when the agent decrease
 the rate or extent of cell division.
 M. Administration of Survivin or Agents that Affect Survivin Activity
 The agents of the present invention, whether they be agents that block
 Survivin/binding partner association or the Survivin protein, can be
 administered via parenteral, subcutaneous, intravenous, intramuscular,
 intraperitoneal, transdermal, or buccal routes. Alternatively, or
 concurrently, administration may be by the oral route. The dosage
 administered will be dependent upon the age, health, and weight of the
 recipient, kind of concurrent treatment, if any, frequency of treatment,
 and the nature of the effect desired. For example, to treat tumor cells as
 a means of blocking Survivin inhibition of apoptosis, an agent that blocks
 Survivin expression or the interaction of Survivin with a binding partner,
 is administered systemically or locally to the individual being treated.
 As described below, there are many methods that can readily be adapted to
 administer such agents.
 The present invention further provides compositions containing Survivin or
 one or more agents that block Survivin/binding partner association. While
 individual needs vary, a determination of optimal ranges of effective
 amounts of each component is within the skill of the art. Typical dosages
 comprise 0.1 to 100 .mu.g/kg body wt. The preferred dosages comprise 0.1
 to 10 .mu.g/kg body wt. The most preferred dosages comprise 0.1 to 1
 .mu.g/kg body wt.
 In addition to the pharmacologically active agent, the compositions of the
 present invention may contain suitable pharmaceutically acceptable
 carriers comprising excipients and auxiliaries which facilitate processing
 of the active compounds into preparations which can be used
 pharmaceutically for delivery to the site of action. Suitable formulations
 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 as appropriate oily
 injection suspensions may be administered. Suitable lipophilic solvents or
 vehicles include fatty oils, for example, sesame oil, or synthetic fatty
 acid esters, for example, ethyl oleate or triglycerides. Aqueous injection
 suspensions may contain substances which increase the viscosity of the
 suspension 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 for delivery into the
 cell.
 The pharmaceutical formulation for systemic administration according to the
 invention may be formulated for enteral, parenteral or topical
 administration. Indeed, all three types of formulations may be used
 simultaneously to achieve systemic administration of the active
 ingredient.
 Suitable formulations for oral administration include hard or soft gelatin
 capsules, pills, tablets, including coated tablets, elixirs, suspensions,
 syrups or inhalations and controlled release forms thereof.
 In practicing the methods of this invention, the compounds of this
 invention may 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 along with other
 compounds typically prescribed for these conditions according to generally
 accepted medical practice, such as chemotherapeutic agents.
 N. Combination Therapy
 Survivin, as well as agents of the present invention that modulate Survivin
 activity, can be provided alone, or in combination with another agents
 that modulate a particular biological or pathological process. For
 example, an agent of the present invention that reduces Survivin inhibited
 apoptosis can be administered in combination with other anti-cancer agents
 in methods to control cancer cell growth.
 Alternatively, Survivin can be administered with other protective agents as
 a means for reducing cellular apoptosis. As used herein, two agents are
 said to be administered in combination when the two agents are
 administered simultaneously or are administered independently in a fashion
 such that the agents will act at the same time.
 Inhibition of Survivin activity/expression can be used in combination with
 conventional chemotherapies. The timing for using a chemotherapeutic agent
 in combination with inhibiting Survivin activity/expression depends upon
 chemotherapeutic agent used and the tumor cell type treated. Examples of
 chemotherapeutic agents that can be used in combination with agents the
 effect Survivin activity/expression, includes, but is 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 the like alkylating agents; anti-metabolites, such as methotrexate
 (MTX), etoposide (VP16; vepesid) 6-mercaptopuine (6MP), 6-thiocguanie
 (6TG), cytarabine (Ara-C), 5-fluorouracil (5FU), dacarbazine (DTIC), and
 the like anti-metabolites; antibiotics, such as actinomycin D, doxorubicin
 (DXR; adriamycin), daunorubicin (daunomycin), bleomycin, mitbramycin and
 the like antibiotics; alkaloids, such as vinca alkaloids such as
 vincristne (VCR), vinblastine, and the like; and other antitumor agents,
 such as taxol and taxol derivatives, the cytostatic agents glucocorticoids
 such as dexamethasone (DEX; decadron) and corticosteroids such as
 prednisone, nucleoside enzyme inhibitors such as hydroxyurea, amino acid
 depleting enzymes such as asparaginase, and the like diverse antitumor
 agents.
 The use of the cytotoxic agents described above in chemotherapeutic
 regimens is generally well characterized in the cancer therapy arts, and
 their use herein falls under the same considerations for monitoring
 tolerance and effectiveness and for controlling administration routes and
 dosages, with some adjustments. For example, the actual dosages of the
 cytotoxic agents may vary depending upon the patient s cultured cell
 response determined by using the present histoculture methods. Generally,
 the dosage will be reduced compared to the amount used in the absence of
 agents the effect Survivin activity/expression.
 Typical dosages of an effective cytotoxic agent can be in the ranges
 recommended by the manufacturer, and where indicated by in vitro responses
 or responses in animal models, can be reduced by up to about one order of
 magnitude concentration or amount. Thus, the actual dosage will depend
 upon the judgment of the physician, the condition of the patient, and the
 effectiveness of the therapeutic method based on the in vitro
 responsiveness of the primary cultured malignant cells or histocultured
 tissue sample, or the responses observed in the appropriate animal models.
 O. Methods for Identifying Survivin Expression and Survivin-Mediated
 Inhibition of Apoptosis
 The present invention further provides methods for identifying cells
 involved in Survivin-mediated inhibition of apoptosis as well as
 techniques that can be applied to diagnose biological and pathological
 processes associated with Survivin activity, the progression of such
 conditions, the susceptibility of such conditions to treatment and the
 effectiveness of treatment for such conditions. Specifically,
 Survivin-mediated inhibition of apoptosis can be identified by determining
 whether the Survivin protein is expressed in a cell. Cells expressing
 Survivin are considered to be inhibited from natural cellular apoptosis.
 A variety of immunological and nucleic acid techniques can be used to
 determine if the Survivin protein, or a Survivin encoding mRNA, is
 produced in a particular cell. In one example, an extract of cells is
 prepared. The extract is then assayed to determine whether Survivin is
 expressed in the cell. The degree of expression provides a measurement of
 the degree of inhibition of apoptosis. An increase in expression is a
 measurement of an increased inhibition of apoptosis.
 The measurement of Survivin expression can be used as a marker for a
 variety of purposes. In tumors, the present of Survivin expression
 correlates with the proliferative potential of the tumor. In the Examples,
 it is shown that lymphomas display varying levels of Survivin expression;
 lymphomas showing little to no Survivin expression are low grade lymphomas
 that can be effectively treated while lymphomas showing high levels of
 Survivin expression are high grade aggressive lymphomas that typically
 cannot be effectively treated. Accordingly, the level of Survivin
 expression in a lymphoma, or other tumor, can be used as a predictive
 measurement of the aggressiveness and treatability of the tumor: the
 higher the level of Survivin expression, the higher the aggressiveness of
 the tumor and the more difficult the treatment will be.
 For example, to determine a tumor's proliferative potential or
 easy/prognosis of treatment, an extract is made of the tumor cells and the
 extract is then analyzed, for example, by gel electrophoresis, to
 determine whether a Survivin protein is present. The presence and level of
 Survivin correlates with the proliferative potential of the cancer and the
 ease of treatment. Alternatively, as described above, single-strand probes
 can be used to identify Survivin-encoding mRNA in the cellular extracts.
 In addition to being a marker of tumor aggressiveness and treatment
 potential, Survivin expression can be used as a measurement of the
 effectiveness of anti-tumor therapy. In the Examples, it is shown that
 HL-60, a promylocytic cell line, had high levels of Survivin expression.
 Treatment of HL60 cells with retenoic acid, and anti-cancer agent that
 acts by causing the differention of tumor cells, resulted in a reduction
 and elimination of Survivin expression. The reduction in expression
 correlated with the degree of differentiation, the greater the
 differentiation, the lower the level of Survivin expression. Accordingly,
 Survivin expression can be used to measure the effectiveness of anti-tumor
 treatment: if Survivin expression decreases during treatment, the
 treatment protocol is effective and can be continued, whereas if Survivin
 expression remains unaltered, a different therapeutic regime or protocol
 needs to be performed.
 P. Other Methods to Control Survivin Expression
 The present ivention further provides additional methods that can be used
 to control Survivin expression in a cell. As discussed above and below,
 the Survivin promoter has a CPG island upstream from its promoter. CPG
 islands are known targets for DNA methylation. The DNA methylation sites
 in the CPG island serves as a means for regulating Survivin expression:
 methylation of CPG islands results in the suppression of transcription of
 the gene found downstream from the promoter. Accordingly, agents that
 methylate DNA, such as DNA methylase, and agents that stimulate the
 production of endogenous methylases, can be used to control Survivin
 expression. Specifically, Survivin expression in a cell can be reduced or
 eliminated by causing the cell to increase the level of DNA methylation,
 particularly at the CPG island found upstream from the Survivin gene.
 In another method, Survivin expression can be reduced by increasing the
 level of EPR-1 expression. As shown in the Examples, Survivin expression
 and EPR-1 expression are generally mutually exclusive, expression of EPR-1
 results in a decrease or elimination of Survivin expression and
 visa-a-versa. Accordingly, Survivin expression can be reduced by causing a
 cell to increase EPR1 expression.
 Q. Animal Models
 We have isolated almost the complete structure of the mouse Survivin gene.
 The gene is very conserved with its human counterpart including sizes of
 introns, exons and intron-exon boundaries. The coding regions of the mouse
 Survivin gene are 88%, to the extent sequenced, identical to the human
 protein, thereby demonstrating strong evolutionary conservation. We have
 also determined the differential and developmentally-regulated
 distribution of Survivin during both human and mouse development. The
 availability of the complete structure of the mouse Survivin gene and
 protein will allow the preparation of targeting vectors for gene knockout
 experiments and a more rational approach for the generation of transgenic
 mice expressing Survivin under the control of tissue-specific promoters.
 The Survivin gene and the Survivin protein can serve as a target for gene
 therapy in a variety of contexts. For example, in one application,
 Survivin-deficient non-human animals can be generated using standard
 knock-out procedures to inactivate a Survivin gene or, if such animals are
 non-viable, inducible Survivin antisense molecules can be used to regulate
 Survivin activity/expression. Alternatively, an animal can be altered so
 as to contain a Survivin or antisense-Survivin expression unit that
 directs the expression of Survivin or the antisense molecule in a tissue
 specific fashion In such a uses, a non-human mammal, for example a mouse
 or a rat, is generated in which the expression of the Survivin gene is
 altered by inactivated or activation. This can be accomplished using a
 variety of art-known procedures such as targeted recombination. Once
 generated, the Survivin-deficient animal, the animal that expresses
 Survivin in a tissue specific manner, or an animal that expresses an
 antisense molecule can be used to 1) identify biological and pathological
 processes mediated by Survivin, 2) identify proteins and other genes that
 interact with Survivin, 3) identify agents that can be exogenously
 supplied to overcome Survivin deficiency and 4) serve as an appropriate
 screen for identifying 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-fashion and test the
 effect of over-expression of the protein in district that normally do not
 contain Survivin. This strategy has been successfully used for another
 family of apoptosis inhibitors, namely bcl-2 (Veis et al., Cell (1993)
 75:229). Such an approach can readily be applied to the Survivin protein
 and can be used to address the issue of a potential beneficial effect of
 Survivin in a specific tissue area to protect cells from apoptosis
 (transplant).
 R. Survivin Gene Therapy
 In another embodiment, genetic therapy can be used as a means for
 modulating a Survivin-mediated biological or pathological processes. For
 example, in tumor therapy, it may be desirable to introduce into the
 subject being treated a genetic expression unit that encodes a modulator
 of Survivin expression, such as an antisense encoding nucleic acid
 molecule. Such a modulator can either be constitutively produced or
 inducible within a cell or specific target cell. This allows a continual
 or inducible supply of a modulator of Survivin expression within the
 subject. Blocking Survivin expression allows for the control of tumor cell
 growth. Similarly, cells may be genetically engineered to express
 Survivin, e.g., in allograft pancreatic .beta. cells for transplantation.
 The level of Survivin gene expression may correlate with the level of
 resistance to apoptosis. Thus, Survivin genes also find use in
 anti-apoptosis gene therapy. In particular, a functional Survivin gene may
 be used to sustain neuronal cells that undergo apoptosis in the course of
 a neurodegenerative disease, lymphocytes (i.e., T cells and B cells), or
 cells that have been injured by ischemia.
 Retroviral vectors, adenoviral vectors, adeno- associated viral vectors, or
 other viral vectors with the appropriate tropism for cells likely to be
 involved in apoptosis (for example, epithelial cells) may be used as a
 gene transfer delivery system for a therapeutic Survivin gene construct.
 Numerous vectors useful for this purpose are generally known (Miller,
 Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989;
 Eglitis and Anderson, BioTechniques 6:608-614, 1988; Tolstoshev and
 Anderson, current opinion in biotechnology 1:55-61, 1990; Sharp, The
 Lancet 337:1277-1278, 1991; Cornetta et al., i Nucleic Acid Research and
 Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984;
 Moen, blood Cells 17:407-416, 1991; Miller et al., Biotechniques
 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and
 Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly
 well developed and have ben used in clinical settings (Rosenberg et al.,
 N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).
 Non-viral approaches may also be employed for the introduction of
 therapeutic DNA into cells otherwise predicted to undergo apoptosis. For
 example, Survivin may be introduced into a neuron or a T cell by
 lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono
 et al., Neurosci. Lett. 117:259, 190; Brigham et al., Meth. Enz. 101:512,
 1983), asialorosonucoid-polylysine conjugation (Wu et al., J. Biol. Chem.
 263:14621, 1988; Wu et al., J. Biol. Chem. 264:16985, 1989); or, less
 preferably, microinjection under surgical conditions (Wolff et al.,
 Science 247:1465, 1990).
 For any of the methods of application described above, the therapeutic
 Survivin nucleic acid construct is preferably applied to the site of the
 predicted apoptosis event (for example, by injection). However, it may
 also be applied to tissue in the vicinity of the predicted apoptosis event
 or to a blood vessel supplying the cells predicted to undergo apoptosis.
 In the constructs described, Survivin cDNA expression can be directed from
 any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus
 40 (SV40), or metallothionein promoters), and regulated by any appropriate
 mammalian regulatory element. For example, if desired, enhancers known to
 preferentially direct gene expression in neural cells, T cells, or B cells
 may be used to direct Survivin expression. The enhancers used could
 include, without limitation, those that are characterized as tissue- or
 cell-specific in their expression. Alternatively, if a Survivin genomic
 clone is used as a therapeutic construct (for example, following its
 isolation by hybridization with the Survivin cDNA described above),
 regulation may be mediated by the cognate regulatory sequences or, if
 desired, by regulatory sequences derived from a heterologous source,
 including any of the promoters or regulatory elements described above.
 S. Use of the Survivin Promoter to Direct Gene Expression
 The present invention further provides the promoter of the Survivin gene in
 a form that can be used in generating expression vectors. Specifically,
 the Survivin promoter, identified as being 5' from the ATG start codon in
 of Survivin, can be used to direct the expression of an operably linked
 protein encoding DNA sequence. Since the Survivin promoter does not have a
 TATA box, a skilled artisan would use a 5' fragment, such as nucleotides
 2560-2920 (including exon 1). The Survivin promoter is expressed in fetal
 tissues and can therefore be used to target protein expression in specific
 cell types during specific stages of development. As discussed below,
 transfection of 3T3 cells with the c-myc oncogene results in the
 up-regulation of Survivin mRNA as detected by Northem blots. Accordingly,
 DNA encoding anti-tumor polypeptides under the control of the Survivin
 promoter could be used to transfect tumor cell where they would be
 expressed. A skilled artisan can readily use the Survivin promoter in
 expression vectors using methods known in the art.
 T. Preventative Anti-Apoptotic Therapy
 In a patient diagnosed to be heterozygous for a Survivin mutation or to be
 susceptible to Survivin mutations (even if those mutations do not yet
 result in alteration or loss of Survivin biological activity), or a
 patient diagnosed with a degenerative disease (e.g., motor neuron
 degenerative diseases such as SMA or ALS diseases), or diagnosed as HIV
 positive, any of the disclosed therapies may be administered before the
 occurrence of the disease phenotype. For example, the therapies may be
 provided to a patient who is HIV positive but does not yet show a
 diminished T cell count or other overt signs of AIDS. In particular,
 compounds shown to increase Survivin expression or Survivin biological
 activity may be administered by any standard dosage and route of
 administration. Alteratively, gene therapy using a Survivin expression
 construct may be undertaken to reverse or prevent the cell defect prior to
 the development of the degenerative disease.
 The methods of the instant invention may be used to reduce or diagnose the
 disorders described herein in any mammal, for example, humans, domestics
 pets, or livestock. Where a non-human mammal is treated or diagnosed, the
 Survivin polypeptide, nucleic acid, or antibody employed is preferably
 specific for that species.
 U. Examples of Additional Apoptosis Assays
 In addition to the foregoing discussion, specific examples of apoptosis
 assays are also provided in the following references. Assays for apoptosis
 in lymphocytes are disclosed by: Li et al., "Induction of apoptosis in
 uninfected lymphocytes by HIV-1 Tat protein", Science 268:429-431, 1995;
 Gibellini et al., "Tat-expressing Jurkat cells show an increased
 resistance to different apoptotic stimuli, including acute human
 immunodeficiency virus-type 1 (HIV-1) infection", Br. J. Haematol.
 89:24-33, 1995; Martin et al., "HIV-1infection of human CD.sup.+ T cells
 in vitro. Differential induction of apoptosis in these cells." J. Immunol.
 152:330-42, 1994; Terai et al., "Apoptosis as a mechanism of cell death in
 cultured T lymphoblasts acutely infected with HIV-1", J. Clin Invest.
 87:1710-5, 1991; Dhein et al., "Autocrine T-cell suicide mediated by
 APO-1/(Fas/CD95)11, Nature 373:438-441, 1995; Katsikis et al., "Fas
 antigen stimulation induces marked apoptosis of T lymphocytes in human
 immunodeficiency virus-infected individuals", J. Exp. Med. 1815:2029-2036,
 1995; Westendorp et al., Sensitization of T cells to CD95-mediated
 apoptosis by HIV-1 Tat and gp120", Nature 375:497, 1995; DeRossi et al.,
 Virology 198:234-44, 1994.
 Assays for apoptosis in fibroblasts are disclosed by: Vossbeck et al.,
 "Direct transforming activity of TGF-beta on rat fibroblasts", Int. J.
 Cancer 61:92-97, 1995; Goruppi et al., "Dissection of c-myc domains
 involved in S phase induction of HIH3T3 fibroblasts", Oncogene 9:1537-44,
 1994; Fernandez et al., "Differential sensitivity of normal and Ha-ras
 transformed C3H mouse embryo fibroblasts tumor necrosis factor; induction
 of bcl-2, c-myc, and manganese superoxide dismutase in resistant cells",
 Oncogene 9:2009-17, 1994; Harrington et al., "c Myc-induced apoptosis in
 fibroblasts is inhibited by specific cytokines", EMBO J., 13:3286-3295,
 1994; Itoh et al., "A novel protein domain required for apoptosis.
 Mutational analysis of human Fas antigen", J. Biol. Chem. 268:10932-7,
 1993.
 Assays for apoptosis in neuronal cells are disclosed by: Melino et al.,
 "Tissue transglutaminase and apoptosis: sense and antisense transfection
 studies with human neuroblastoma cells", Mol. Cell Biol. 14:6584-6596,
 1994; Rosenblaum et al., "Evidence for hypoxia-induced, programmed cell
 death of cultured neurons", Ann. Neurol. 36:864-870, 1994; Sato et al.,
 "Neuronal differentiation of PC12 cells as a result of prevention of cell
 death by bcl-2", J. Neurobiol. 25:1227-1234, 1994; Ferrari et al.,
 "N-acetylcysteine D- and L-stereoisomers prevents apoptotic death of
 neuronal cells", J. Neurosci. 1516:2857-2866, 1995; Talley et al., "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. 1585:2359-2366, 1995; Talley et al., "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; Walkinshaw et al., "Induction of
 apoptosis in catecholaminergic PC12 cells by L-DOPA. Implications for the
 treatment of Parkinson's disease," J. Clin. Invest. 95:2458-2464, 1995.
 Assays for apoptosis in insect cells are disclosed by: Clem et al.,
 "Prevention of apoptosis by a baculovirus gene during infection on insect
 cells", Science 254:1388-90, 1991; Crook et al., "An apoptosis-inbibiting
 baculovirus gene with a zinc finger-like motif", J. Virol: 67:2168-74,
 1993; Rabizadeh et al., "Expression of the baculovirus p35 gene inhibits
 mammalian neural cell death", J. Neurochem. 61:2318-21, 1993; Birnbaum et
 al., "An apoptosis inhibiting gene from a nuclear polyhedrosis virus
 encoding a polypeptide with Cys/His sequence motifs", J. Virol. 68:2521-8,
 1994; Clem et al., Mol. Cell. Biol. 14:5212-5222, 1994.
 V. Use of Survivin in Tissue and Organ Transplantation
 The present invention includes methods of inhibiting or preventing tissue
 or organ transplant rejection in a subject, comprising the local
 administration of a Survivin polypeptide, Survivin polypeptide fragment,
 an apoptosis,,inhibiting peptidomimetic thereof, a transgene encoding a
 Survivin polypeptide or a transgene encoding a Survivin polypeptide
 fragment to the tissue, organ or to a site proximal to the transplant.
 Local delivery of the polypeptides, peptidomimetics to the tissue, organ
 or to a site proximal to the transplant is accomplished by any means
 commonly available, including but not limited to direct local perfusion,
 injection, microsponges, microcapsules, liposomes or time-released
 delivery vehicles.
 Local delivery of a transgene encoding a Survivin polypeptide or a
 transgene encoding a Survivin polypeptide fragment to the tissue, organ or
 to a site proximal to the transplant may be accomplished with any
 available vector, via lipofection or via direct plasmid DNA injection. See
 Qin et al (1995) Transplantation 59(6): 809-816; Le Coultre et al. (1997)
 Eur. J. Pediatr. Surg. 7(4):221-226; Wang et al. (1992) Transplantation
 53(3):703-705; Wang et al. (1996) Transplantation 61(12):1726-1729; Schmid
 et al., (1997) Eur. J Cardiothorac. Surg. 11(6):1023-28; and
 Boasquevisque, C. et al (1997) Ann. Thorac. Surg. 63(6):1556-1561. Vectors
 encoding the transgene include both replicable and replication,,defective
 vectors, such as retroviral vectors, adenovirus vectors or other vectors
 with the appropriate tropism for the cells likely to be involved in
 apoptosis or cells proximal to the site of apoptosis. In the transgene
 constructs, expression can be directed from any suitable promoter,
 including tissue specific promoters which direct gene expression in
 specific cell types, such as the human insulin promoter. Local delivery of
 the transgene to the tissue, organ or to a site proximal to the transplant
 is accomplished by any means commonly available, including but not limited
 to direct local perfusion, injection, microsponges, microcapsules,
 liposomes or time-released delivery vehicles.
 Without further description, it is believed that one of ordinary skill in
 the art can, using the preceding description and the following
 illustrative examples, make and utilize the compounds of the present
 invention and practice the claimed methods. The following working examples
 therefore, specifically point out preferred embodiments of the present
 invention, and are not to be construed as limiting in any way the
 remainder of the disclosure. Other generic configurations will be apparent
 to one skilled in the art. All journal articles and other published
 documents such as patents and patent applications are hereby incorporated
 by reference in their entireties.
 EXAMPLES
 Example 1
 Experimental Procedure and Cloning
 Cells and cell culture. The following cell lines were obtained from
 American Type Culture Collection (ATCC, Rockville, Md.), erythroleukemia
 HEL, B-lymphoma Daudi and JY, monocytic THP-1, T leukemia Jurkat,
 epithelial carcinoma HeLa, promyelocytic HL-60, and non-transformed human
 lung fibroblast Lul 8. The T leukemia cell line MOLT13 was characterized
 previously (Altieri, D.C., FASEB J(1995) 9:860-865). Cells were maintained
 in culture in complete medium RPMI 1640 or DMEM (HeLa, Lu18)
 (BioWhittaker, Walkersville, Md.), supplemented with 10% heat-inactivated
 fetal bovine serum (FBS, Whittaker), 2 mM L-glutamine, and 10 mM HEPES.
 Human umbilical vein endothelial cells HUVEC) were isolated by collagenase
 treatment and maintained in culture in DMEM medium supplemented with 20%
 FBS, 2 mM L-glutamine and endothelial cell growth factor (Biomedical
 Technologies, Stoughton, Mass.).
 Peripheral blood mononuclear cells (PBMC) were isolated from heparinized
 blood collected from normal informed volunteers by differential
 centrifugation on Ficoll-Hypaque (Pharmacia, Piscataway, N.J.) at 400 g
 for 22.degree. C., and washed in phosphate buffered saline (PBS), pH 7.4.
 In some experiments, HL-60 cells were terminally differentiated to a
 mature monocytic phenotype by a 72 h culture in the presence of 0.1 .mu.M
 1,25-dihydroxy-vitamin D.sub.3 and 17.8 .mu.g/ml indomethacin (Sigma
 Chemical Co., St. Louis, Mo.). De novo induction of
 differentiation-dependent markers on vitamin D.sub.3 -treated HL-60 cells,
 including CD11b/CD18 integrin (Hickstein, D. D. et al., J Immunol (1987)
 138:513-519) was determined by flow cytometry with anti-CD11b mAb LM2/1.
 Genomic and cDNA cloning, chromosomal localization and Southern blots. A
 human P1 genomic library (Genome Systems, St. Louis, Mo.) was screened by
 hybridization with a 1.6 kb fragment containing the complete human EPR1
 cDNA (Altieri, D. C., FASEB J(1995) 9:860-865). Three overlapping clones
 were isolated, purified and confirmed by Southern hybridization with the
 EPR-1 cDNA. Hybridizing fragments generated by restriction digest with
 BamHI, HindIII and XbaI (Boehringer Mannheim, Indianapolis, Ind.) were
 cloned in pBluescript (pBSKS.sup.-, Sagene, San Diego, Calif.) for further
 analysis. An overlapping contig spanning 14796 bp from two EPR1
 -hybridizing P1 clones was arrayed, characterized by restriction analysis,
 and completely sequenced on both strands by Taq FS polymerase-based
 automated sequencing using a Applied BioSystem Prism 377 automated
 sequencer (Foster City, Calif.). In some experiments, 10 mg of total RNA
 extracted from HeLa cells by the guanidinium isothiocyanate method was
 primed with EPR-1 forward "sense" oligonucleotide C3/27 (bp 80-102) and
 reverse transcribed in the presence of 200 U of Superscript II (Life
 Science, Grand Island, N.Y.) for 50 min at 42.degree. C.
 The resulting cDNA was amplified by PCR in the presence of 0.5 mg of
 EPR-1-derived primers T5/27 (bp 161-184) and G11/16 (1124-1098, numbering
 from the EPR-1 coding sequence), 200 mM dNTs (New England Biolabs,
 Beverly, Mass.) and 2 U Vent DNA polymerase (New England Biolabs) in a
 total volume of 50 ml. After 35 cycles of amplification with annealing at
 58.degree. C. for 1 min, denaturation at 94.degree. C. for 1 min and
 extension at 72.degree. C. for 1 min, the product was analyzed by agarose
 gel electrophoresis, subeloned in pCRII (Invitrogen Corp., San Diego,
 Calif.), and completely sequenced on both strands. Contig assembly, and
 DNA and protein sequence analyses were performed using Lasergene (DNASTAR,
 Madison, Wis.) and MacVector (Eastman Kodak, Rochester, N.Y.) software
 packages. Chromosomal location of the EPR-1-hybridizing gene was carried
 out by fluorescence in situ hybridization. Purified DNA from a
 EPR-1-hybridizing P1 clone was labeled with digoxigenin dUTP (Amersham
 Corp., Arlington Heights, Ill.) by nick translation.
 The labeled probe was combined with sheared human DNA and hybridized to
 normal metaphase chromosomes derived from phytohemagglutinin-stimulated
 PBMC in a solution containing 50% formamide, 10% dextran sulfate and 2X
 SSC. For two-color staining, biotin-conjugated probe D17Z1, specific for
 the centromere of chromosome 17, was co-hybridized with the
 digoxigenin-labeled P1 clone. Specific staining was detected by incubating
 the hybridized slides with fluoresceinated anti-digoxigenin antibodies and
 Texas red avidin. Slides were counterstained with propidium iodide for one
 color labeling, or with DAPI for two color labeling. A total of 80
 metaphase cells were analyzed with 69 cells exhibiting specific labeling.
 For Southern hybridization, human genomic DNA was extracted from HeLa
 cells according to published protocols, digested with EcoRl, BamHI, XbaI
 or HindIII, separated on a 0.8% agarose gel and transferred to GeneScreen
 nylon membranes (New England Nuclear, Boston, Mass.).
 After UV cross-linking (Stratalinker, Stratagene, San Diego, Calif.), the
 membrane was prehybridized with 100 mg/ml of denatured salmon sperm DNA
 (Promega Corp. Madison, Wis.) in 5X SSC, 0.5% SDS, 5X Denhardt's solution
 and 0.1% sodium pyrophosphate at 65.degree. C. in a roller hybridization
 oven (Hoefer Scientific, San Francisco, Calif.). Hybridization was carried
 out with gel-purified (GeneClean Bio101, Vista, Calif.), .sup.32 P-dCTP
 (Amersham) random-primed labeled (Boehringer-Mannheim, Indianapolis, Ind.)
 1.6 kb EPR-1 cDNA for 16 h at 65.degree. C.
 After two washes in 2X SSC, 1% SDS for 30 min at 65.degree. C., and 0.2X
 SSC at 22.degree. C., radioactive bands were visualized by autoradiography
 using a Kodak X-Omat AR X-ray film and intensifying screens (DuPont de
 Nemours, Wilmington, DE). In other experiments, cultured lymphoblastoid
 cells were embedded in LMP agarose (Bio Rad, Richmond, Calif.) at the
 concentration of 2=10.sup.6 /220 .mu.l block and DNA was extracted by
 standard procedures. After block digestion with MluI or NotI, samples were
 separated by pulsed field gel electrophoresis on a 1% agarose gel for 20 h
 at 200 V with a pulse time of 75 sec using a Bio-Rad CHEF DRII apparatus
 (Hercules, Calif.). After transfer to nylon membranes, and UV
 cross-linking, hybridization with the EPR-1 cDNA and washes were carried
 out as described above.
 In another series of experiments, a blot containing aliquots of genomic DNA
 isolated from several species (Clontech, San Francisco, Calif.) was
 hybridized with a 3' 548 bp fragment of the EPR1 cDNA, as described above.
 Northern blots. Single strand probes specific for sense or antisense EPR-1
 sequences were generated by asymmetric PCR amplification of a 301 bp
 fragment of the EPR1 cDNA. The template, comprising the first 5'226 bp of
 the EPR-1 coding sequence plus 75 bp of the retained regulatory intron
 (Altieri, D. C., FASEB J(1995) 9:860-865), was generated by restriction
 digest of the EPR-1 cDNA with EcoRI (cloning site) and SacII,
 gel-purified, and mixed in a total volume of 10 ml with 15 pmol dNTP (New
 England Biolabs), 7.5 pmol dCTP, and 25 mCi .sup.32 P-dCTP (Amersham), in
 the presence of 20 mM Tris HCl, 50 mM KCl, pH 8.4, 1.5 mM MgCl.sub.2, and
 2.5 U of Taq DNA polymerase (Life Science).
 Generation of a EPR-1-specific antisense probe was carried out by addition
 of 0.2 mg/ml of a "SacII" reverse oligonucleotide
 5'TGCTGGCCGCTCCTCCCTC3'(SEQ ID NO: 1), while extension of the EPR-1
 positive sod and generation of a Survivin-specific probe was performed
 using 0.2 mg/ml of forward F11 oligonucleotide 5'ATGACCTCCAGAGGTTTC3'(SEQ
 ID NO: 2). Twenty-five cycles of amplification were carried with
 denaturation at 94.degree. C. for 1 min, annealing at 52.degree. C. for 1
 min, and extension at 72.degree. C. for 1 min. The EPR-1 sense or
 antisense probes were centrifuged through a Sephadex G-50 spin column
 (Worthington Biochemical Corp., Freehold, N.J.) at 14,000 g for 5 min to
 separate free from incorporated radioactivity, heated at 100.degree. C.
 for 2 min, and immediately added to the hybridization reaction.
 Identical strand-specific probes were used for hybridization of multiple
 tissue blots of adult or fetal human mRNA (Clontech), in 5X SSPE, 10X
 Denhardt's solution, 2% SDS, 100 mg/ml denatured salmon sperm DNA at
 60.degree. C. for 14 h, and washes at 60.degree. C., as described above.
 Aliquots of total RNA extracted from undifferentiated or vitamin D.sub.3
 terminally differentiated HL-60 cells, were processed for Northern
 hybridization with Survivin-specific single strand probe, as described
 above.
 EXAMPLE 2
 Production of Anti-Survivin Antibodies
 A Survivin sequence-specific antibody, called JC700, was produced and
 characterized as follows. A seventeenmer peptide corresponding to the
 Survivin sequence A.sup.3 PTLPPAWQPFLKDHRI.sup.19 (SEQ ID NO: 3), was
 synthesized and characterized by mass spectrometry. One hundred mg of the
 Survivin peptide were coupled in a 1:1 ratio to Keyhole Limpet Hemocyanin
 and injected s. c. into a rabbit in complete Freund's adjuvant. After a
 4-week interval, animals were boosted with s. c. injection of 100 mg of
 peptide in incomplete Freund's adjuvant and sequentially boosted and bled
 at alternate weeks.
 Purification of the anti-Survivin antibody was carried out by affinity
 chromatography on a peptide-Sepharose matrix (5 mg/ml of peptide) with
 elution of the specific IgG fraction in 1 mM glycine, pH 2.5. 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 OD.sub.405.
 EXAMPLE 3
 Production of a Monoclonal Antibody Against a Survivin Fusion Protein
 The Survivin cDNA was expressed as a GST-fusion protein in E.Coli BL21
 strain and purified to homogeneity with removal of the GST fiame. The
 purified protein was used to inject mice and generate monoclonal
 antibodies using standard 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 the immobilized, purified recombinant Survivin by ELISA and
 bound to Survivin in immunoblots, as shown in FIG. 11.
 EXAMPLE 4
 Immunoblotting and in situ Hybridization
 For immunoblotting, aliquots of SDS-solubilized extracts of various
 transformed cell lines, non-transformed HUVEC, PBMC or Lu18, or
 undifferentiated or vitamin D.sub.3 -differentiated HL-60 cells, were
 normalized for protein content by absorbance at OD.sub.280, separated by
 electrophoresis on a 5-20% SDS polyacrylamide gradient gel under non
 reducing conditions, and electroblotted to hmmobilon membranes (Millipore
 Corp., New Bedford, Mass.) at 1.1 A for 30 min at 22.degree. C. The
 membrane was blocked in TBS, pH 7.4, plus 5% milk, and incubated with 20
 mg/ml of control non-immune rabbit IgG or anti-Survivin antibody JC700 for
 1 h at 22.degree. C., followed by washes in TBS, pH 7.4, and addition of a
 1:7500 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG
 (Promega) for 30 min at 22.degree. C. Binding of the primary antibody was
 revealed by addition of 75 mg/ml nitro blue tetrazolium in 70%
 dimethylformamide (Sigma Chemical Co., St. Louis, Mo.) plus 50 mg/ml
 5-bromo4-chloro-3-indolyl phosphate (Sigma) in 100% dimethylformamide.
 Tissue samples, immunohistochemistry and in situ hybridization. Tissue
 samples from colon adenocarcinoma (6 cases), lung squamous cell carcinoma
 (6cases), lung adenocarcinoma (9 cases), pancreas adenocarcinoma (2
 cases), invasive breast adenocarcinoma (7 cases), were obtained from the
 archives of Yale-New Haven Hospital and used in the present study. Samples
 of 44 high grade lymphoma tissues and 7 low grade lymphoma tissue was also
 obtained. Tissue samples were fixed in formalin, embedded in paraffin, cut
 in 5 .mu.m sections, deparaffinized in xylene, and rehydrated in graded
 alcohol followed by quenching of endogenous peroxidase activity by
 treatment with 2% H.sub.2 O.sub.2 in methanol.
 For immunostaining, the slides were boiled for 5 min in a standard pressure
 cooker, blocked in 10% normal goat serum, and incubated with
 affinity-purified anti-Survivin antibody JC700 (20 .mu.g/ml) for 14 h at
 4.degree. C. After washes in PBS, pH 7.4, slides were further incubated
 with biotin-conjugated goat anti-rabbit IgG (Vector Laboratories,
 Burlingame, Calif.) for 30 min at 22.degree. C., and washed in PBS, pH
 7.4. After addition of streptavidin-biotin conjugated peroxidase
 (Boehringer Mannheim) for 30 min at 22.degree. C., slides were washed, and
 binding of the primary mAbs was revealed by addition of
 3'--3-diamino-benzidine (DAB) and counterstaining with hematoxylin.
 Negative controls were carried out by 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 mg/ml of
 the Survivin 3-19 peptide before tissue staining. For in situ
 hybridization, 1 .mu.g of the Survivin cDNA containing the entire coding
 sequence plus 271 bp of 3' untranslated region in pcDNA3 (nitrogen), was
 completely digested with EcoRI and transcribed in the antisense
 orientation using T7 RNA polymerase in the presence of digoxigenin
 11-uridine-5' triphosphate (Boelringer Mannheim). Tissue slides were
 coated with 1% gelatin, 0.1% chrome-alum, baked at 120.degree. C. for 2 h,
 and stored dust-free at 22.degree. C. Sections were deparaffinized and
 rehydrated through graded alcohol, digested with proteinase K (1 .mu.g/ml
 in 100 mM Tris HCl pH 8.7, 50 mM EDTA) for 30 min at 37.degree. C., and
 acetylated in 0.25% acetic anhydride acid and 100 mM triethanolamine pH
 8.0 for 10 min at 22.degree. C.
 Detection of Survivin mRNA in human tissues was carried out by in situ
 hybridization of the Survivin antisense riboprobe in a buffer containing
 4X SSC, 1X Denhardt's solution, 50% deionized formamide, 250 .mu.g/ml
 yeast tRNA, 500 .mu.g/ml salmon sperm DNA and 5% dextran for 16 h at
 50.degree. C. After washes in 2X SSC for 90 min at 48.degree. C.,
 immobilized digoxigenin was detected using an anti-digoxigenin mAb
 (Boehringer Mannheim) at a 1:3000 dilution, and revealed by alkaline
 phosphatase staining with NBT/BCIP cytochemical stain.
 EXAMPLE 5
 Expression of Survivin in Human Cancers
 Survivin is prominently expressed in human cancer. For its abundant
 distribution in transformed cell types, a potential expression of Survivin
 in neoplasia was investigated, in vivo. Immunohistochemical analysis of
 formalin-fixed, paraffin embedded tissue sections with the
 affinity-purified anti-Survivin JC700 antibody demonstrated abundant
 expression of Survivin in all cases examined of human lung cancer,
 including adenocarcinoma (FIG. 6A), and squamous cell carcinoma (FIG. 6C).
 Consistent with the topography of other IAP proteins (Duckett, C. S. et
 al., EMBO J (1996) 15:2685-2694), expression of the protein was
 exclusively localized to the cytoplasm of tumor cells, while the adjacent
 normal gland epithelium of the lung did not express Survivin (FIG. 6C,
 arrow). No staining was observed when the anti-Survivin antibody was
 substituted with control goat serum (not shown), or after pre-adsorption
 with the immunizing Survivin 3-19 peptide (FIG. 6B), thus confirming the
 specificity of the observed recognition.
 Prominent accumulation of Survivin mRNA in squamous lung cell carcinoma was
 independently demonstrated by in situ hybridization with a
 Survivin-specific single strand riboprobe FIG. 6D). Survivin was also
 abundantly detected in all cases examined of adenocarcinoma of pancreas
 (FIG. 6E), and breast (not shown) by immunohistochemistry, and colon (FIG.
 6G) by in situ hybridization. However, consistent with its absence in
 non-transformed cell types HUVEC and Lu18 (FIG. 4C), in mature tissues
 (FIG. 3), and in terminally-differentiated HL-60 cells (FIG. 5), no
 reactivity of the anti-Survivin JC700 antibody was observed with normal
 exocrine pancreatic epithelial cells by immunohistochemistry (FIG. 6F),
 and no Survivin mRNA was found in the adjacent non-neoplastic colon gland
 epithelium by in situ hybridization (FIG. 6H).
 Expression of Survivin in Lymphoma Tissue. Tissue samples were obtained
 from 44 patients with aggressive, high grade lymphoma and 7 samples were
 obtained from 7 patients with non-aggressive, low grade lymphoma. The
 sample were treated as described above and examined for Survivin
 expression. None of the low grade lymphoma samples displayed Survivin
 expression whereas 27 samples (61%) from patients with high grade lymphoma
 expressed Survivin.
 EXAMPLE 6
 Expression of Survivin in other Cancers
 In addition to the malignant forms of cancer discussed above, the
 expression of Survivin in other types of cancers was investigated in the
 inventors' laboratory or collaboratively with other academic
 investigators. Survivin was found prominently expressed in the most
 aggressive and metastatic forms of malignant thymoma (-100 if cases
 tested), in head and-neck squamous cell carcinoma (-140 cases) and in all
 forms of prostate cancer (15 cases), including the transition lesion of
 benign prostate hyperplasia. The most aggressive forms of neuroblastoma
 are also positive for Survivin as discussed below.
 EXAMPLE 7
 Tissue Specific Expression of Survivin
 Survivin, was recently found in all the most common human cancers but not
 in normal, terminally differentiated adult tissues. The expression of
 Survivin in embryonic and fetal development was investigated.
 Immunohistochemistry and in situ hybridization studies demonstrated strong
 expression of Survivin in several apoptosis-regulated fetal tissues,
 including the stem cell layer of stratified epithelia, endocrine pancreas
 and thymic medulla, with a pattern non-overlapping with that of another
 apoptosis inhibitor, i.e. bcl-2. A sequence-specific antibody to Survivin
 immunoblotted a single -16.5 kd) Survivin band in human fetal lung, liver,
 heart, kidney and gastrointestinal tract. In mouse embryo, prominent and
 nearly ubiquitous distribution of Survivin was found at embryonic date (E)
 11.5, whereas at E15-21, Survivin expression was restricted to the distal
 bronchiolar epithelium of the lung and neural crest-derived cells,
 including dorsal root ganglion neurons, hypophysis and the chorioid
 plexus. These data suggest that expression of Survivin in embryonic and
 fetal development may contribute to tissue homeostasis and differentiation
 independently of bcl-2.
 EXAMPLE 8
 Preparation of Survivin Transfectants
 Inducible Survivin antisense transfectants and apoptosis/proliferation
 experts. A 708 bp SmaI-EcoRI fragment comprising nucleotides 379-1087 of
 the EPR-1 cDNA, was directionally cloned in the sense orientation in the
 mammalian cell expression vector pML1 (generously provided by Dr. R.
 Pytela, Cardiovascular Research Institute, University of California, San
 Francisco). The vector is derived from the episomal mammalian cell
 expression vector pCEP4 by replacing the cytomegalovirus promoter cassette
 with the mMT1 promoter, directing Zn.sup.2+ -dependent expression of
 recombinant proteins in mammalian cells (Lukashev, M. E. et al, J Biol
 Chem (1994) 269:18311-18314).
 Ten million HeLa cells were incubated with 10 mg of pML1 DNA containing the
 Survivin antisense construct plus 50 mg of salmon sperm DNA for 15 min on
 ice, followed by a single electric pulse delivered by a Gene Pulser
 apparatus (Bio-Rad) at 350 V at 960 .mu.F. Forty-eight h after
 transfection, cells were diluted fifteen fold, plated onto 100 mm diameter
 tissue culture dishes and selected for 4 weeks in complete growth medium
 containing 0.4 mg/ml hygromycin. Apoptosis in control cultures or Survivin
 antisense HeLa cell transfectants was evaluated by in situ detection of
 intermucleosomal DNA degradation after Zn.sup.2+ -dependent induction of
 EPR-1 transcription under serum-starving conditions.
 Briefly, control or antisense Survivin transfectants were treated with 200
 mM ZnSO.sub.4 in 0% FBS for 24 h at 37.degree. C. Cells were harvested,
 centrifuged at 800 g for 10 min at 4.degree. C., and the pellet was fixed
 in 10% formalin overnight, dehydrated, embedded in paraffin blocks, and
 sections of 3-5 mm were put on high adhesive slides. Samples were treated
 with 20 mg/ml proteinase K for 15 min at 22.degree. C., washed in
 distilled water, quenched of endogenous peroxidase in 2% H.sub.2 O.sub.2
 in PBS, and subsequently mixed with digoxigenin-labeled dUTP in the
 presence of terminal deoxynucleotidyl transferase (TdT) followed by
 peroxidase conjugated anti-digoxigenin antibody.
 Nuclear staining in apoptotic cells was detected by DAB, according to the
 manufacturer's instructions (AptoTag, Oncor, Gaithersburg, Md.). Control
 experiments were performed by omitting the enzyme incubation step.
 Morphologic features of apoptotic cells (apoptotic bodies) under the
 various conditions tested were detected by hematoxylinieosin staining of
 the same slides.
 For proliferation experiments, vector control HeLa cells or Survivin
 antisense transfectants were plated at 20.times.10.sup.4 /well onto
 24-well tissue culture plates (Costar), induced with 200 mM ZnSO.sub.4 for
 16 h at 37.degree. C., harvested at 24 h intervals, and cell proliferation
 under the various conditions tested was determined microscopically by
 direct cell count. Down-regulation of Survivin expression under these
 experimental conditions was assessed by immunoblotting with JC700
 antibody.
 EXAMPLE 9
 Identification of EPR-1 Complementary Gene
 Three overlapping clones were isolated by hybridization screening of a
 human P1 plasmid genomic library with the EPR-1 cDNA and confirmed by
 Southern blot. This gene was located to the long arm of chromosome 17, to
 band 17q25, by fluorescence in situ hybridization FIGS. 1A, B).
 A contig of P1 fragments spanning 14796 bp was cloned in pBSKS.sup.- and
 completely sequenced on both strands (FIG. 1C). Three putative splice
 sites, matching perfectly the consensus sequences for eukayotic
 intron-exon boundaries (Padgett, R. A. et al., Ann Rev Biochem (1986)
 55:1119-1150), were identified at position 2922, 3284, and 5276 (donor),
 and 3173, 5157, and 11954 (acceptor), thus defining a gene organization in
 four exons and three introns of 252, 1874, and 6678 bp, respectively (FIG.
 1D).
 Sequence analysis of the putative coding regions demonstrated a nearly
 complete identity with the EPR-1 cDNA (Altieri, D. C., FASEB J (1995)
 9:860-865), except for 5 nucleotide changes and 6 nucleotide insertions.
 However, the three splice sites were found on the complementary, antisense
 strand of the EPR1 coding sequence. Consistent with this unexpected
 orientation, the EPR-1 complementary gene revealed a 5' GC rich region,
 comprising nucleotides 2560-2920 and including exon 1 (see below), which
 fulfilled the base composition criteria of a CpG island (Gardiner-Garde,
 M. et al., J Mol Biol (1987) 196:261-282 and Frommer, 1987). Sequencing
 the 2.5 kb upstream the CpG island revealed a TATA-less promoter with
 numerous Sp1 sites (not shown).
 Compex hybridization pattern and evolutionary conservation of EPR-1
 sequences. Probing human genomic DNA with the EPR-1 cDNA revealed several
 hybridizing fragments (FIG. 2A). Of these, a 7.5 kb XbaI, a 7.6 kb BamHI,
 and 4 HindIII fragments of .about.15, 7.5, 6.4, and 3.7 kb, respectively
 (FIG. 2A, arrows), could not be recapitulated by the restriction map of
 the antisense EPR-1 gene (FIG. 1C). In contrast, other bands of comparable
 intensity, including a 5.15 kb XbaI and a 7.1 kb BamHI fragment, genuinely
 originated from the antisense EPR-1 gene and comprised the first two, or
 three exons, respectively (FIG. 2A). At variance with this complex
 hybridization pattern, Southem blot of high molecular weight human genomic
 DNA digested with MluI or NotI and separated by pulsed field gel
 electrophoresis, revealed single EPR-1-hybridizing bands of .about.75 kb
 and 130 kb, respectively (FIG. 2B). Finally, Southem blots of multiple
 species genomic DNA revealed significant evolutionary conservation of
 EPR-1-related sequences FIG. 2C), with numerous strongly hybridizing bands
 in mammalian species and fainter signals in rabbit or chicken genomic DNA,
 under high stringency hybridization conditions (FIG. 2C).
 Discordant tissue distribution of sense/antisense EPR-1 transcripts. The
 potential expression of distinct sense or antisense EPR-1 transcripts was
 investigated Northern blots with single strand-specific probes. Consistent
 with the size of the spliced EPR-1 message (Altieri, D. C., FASEB J (1995)
 9:860-865), an EPR-1 strand-specific probe detected a prominent .about.1.2
 kb band in mRNA extracted from all adult and terminally-differentiated
 human tissues examined (FIG. 3A). In contrast, no specific bands
 hybridized with a EPR-1 antisense-specific single strand probe in adult
 tissues, under the same experimental conditions (FIG. 3B). A similar
 .about.1.2 kb band was detected by the single strand EPR-1-specific probe
 in fetal kidney, and, to a lesser extent, in fetal liver, lung and brain
 (FIG. 3A). At variance with the absence of hybridization in adult tissues,
 the EPR-1 antisense-specific probe recognized a prominent .about.1.9 kb
 band, and a larger 3.2 kb species corresponding to the size of an
 incompletely processed transcript, in fetal liver, while fainter
 hybridization bands were also seen in fetal kidney, lung and brain (FIG.
 3B). A control hybridization with an actin probe confirmed comparable
 loading of mRNA in adult or fetal samples (FIG. 3C).
 Characterization of the antisense EPR-1 gene product. Inspection of the 5'
 CpG island in the antisense EPR-1 gene revealed a putative ATG initiation
 codon at position 2811, surrounded by a sequence (CGGATGCG) that conformed
 well to the consensus for eukaryotic initiation of translation (Kozak, M.,
 Nucleic Acids Res (1984) 12:857-872). Analysis of the antisense EPR-1
 sequence in the 5'.fwdarw.3' direction dictated by the position of
 intron-exon boundaries revealed an open reading frame of 426 bp, spanning
 all four exons, and terminating 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
 primed with EPR-1 "sense" oligonucleotides matched perfectly the genomic
 sequence and confirmed the open reading frame and the predicted
 intron-exon boundaries (not shown).
 Two .lambda.gt11 cDNA clones isolated by hybridization of a HEL library
 with the EPR-1 cDNA, also matched the consensus genomic sequence and
 revealed a homopolymeric A tail on the antisense EPR-1 stand at position
 13186, 14 bp downstream the polyadenylation signal, generating a 3'
 untranslated region of 1144 bp. In these clones, the 5' untranslated
 region upstream from the initiating ATG was of 49 bp, beginning at
 position 2762 in the genomic sequence, and contained an in-frame
 termination codon. Translation of the antisense EPR-1 open reading frame
 predicted a new protein of 142 amino acids, with an estimated molecular
 weight of 16,389 and an acidic pI of 5.74, lacking an amino-tenninus
 signal peptide or a carboxy terminus hydrophobic stretch for membrane
 insertion (FIG. 4A).
 A coiled coil was predicted for the last 40 carboxy terminus residues
 (Lupas, A. et al., Science (1991) 252:1162-1164). BLAST database searches
 revealed a significant degree of similarity between residues 18-88 of the
 antisense EPR-1 gene product and the BIR module in the IAP family of
 inhibitors of apoptosis (Birnbaum, M. J. et al., J Virology (1994)
 68:2521-2528; Clem, R. J. et al., Mol Cell Biol (1994) 14:5212-5222). For
 this analogy, the antisense EPR-1 gene product was designated Survivin. At
 variance with other IAP proteins, Survivin contained only one BIR, encoded
 by the first three exons of the gene, and lacked a carboxy terminus RING
 finger, without additional/alternative exon(s) potentially encoding this
 domain (FIG. 1C).
 An alignment by the Clustal method between the Survivin BIR and that of
 other known IAP proteins is shown in FIG. 4B. Despite the overall match of
 the consensus and several conservative substitutions, phylogenetic
 analysis suggested that Survivin is a distantly related member of the IAP
 family, most closely related to NAIP, which also lacked a RING finger
 (FIG. 4B, shaded boxes) (Roy, N. et al., Cell (1995) 80:167-178).
 A rabbit polyclonal antiserum designated JC700, was raised against residues
 A.sup.3 PTLPPAWQPFLKDHRI.sup.19 (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, JC700
 antibody immunoblotted a single band of .about.16.5 kDa from
 detergent-solubilized extracts of all transformed cell lines examined,
 including B lymphoma Daudi and JY, T leukemia Jurkat and MOLT13, monocytic
 THP-1, and erthroleukemia HEL (FIG. 4C).
 Survivin was also found in isolated peripheral blood mononuclear cells
 (PBMC). In contrast, no expression of Survivin was detected in
 non-transformed Lu-18 human lung fibroblasts or human umbilical vein
 endothelial cells (HUVEC) (FIG. 4C). No specific bands were immunoblotted
 by control non-immune rabbit IgG, under the same experimental conditions
 (FIG. 4C).
 Identification of agents that modulate transcription of the EPR-1 gene.
 Agents that increase the transcription of the EPR-1 gene may be identified
 by conventional techniques. Preferably, a candidate agent is brought into
 contact with a cell that expresses the EPR-1 gene product and the level of
 expression of this product or the level of transcription are determined
 and agents that increase or decrease EPR-1 gene transcripts may readily be
 identified. Alternatively, the EPR-1 transcriptional regulatory elements
 may be placed upstream of a reporter gene such as CAT or
 .beta.-galactosidase.
 EXAMPLE 10
 Regulation of Survivin Expression by Cell Growth/Differentiation
 Consistent with the expression of Survivin in transformed cell lines (FIG.
 4C), undifferentiated and actively proliferating promyelocytic HL-60 cells
 constitutively expressed high levels of Survivin, as demonstrated by
 immunoblotting of a single .about.16.5 kDa band with JC700 antibody, and
 Northern hybridization of a .about.1.9 kb transcript with a single
 strand-specific probe (FIG. 5). In contrast, no specific bands were
 recognized by control non-immune rabbit IgG under the same experimental
 conditions (FIG. 5).
 Vitamin D.sub.3 -induced terninal differentiation of HL-60 cells to a
 mature monocytic phenotype resulted in growth arrest of these cells and de
 novo induction of differentiation-specific markers, including a
 .about.200-fold increased expression of leukocyte CD11b/CD18 integrin
 detected by flow cytometry (not shown), and in agreement with previous
 observations (Hickstein, D.D. et al., J Immunol (1987) 138:513-519). Under
 these experimental conditions, the anti-Survivin JC700 antibody failed to
 immunoblot specific bands from vitamin D.sub.3 -treated HL-60 extracts,
 and no Survivin transcript(s) were detected by Northern hybridization with
 a single strand-specific probe (FIG. 5).
 In contrast, an anti-EPR-1 polyclonal antibody immunoblotted a single
 .about.62 kDa band corresponding to EPR-1 in vitamin D.sub.3
 -differentiated HL-60 extracts under the same experimental conditions (not
 shown). Moreover, down-regulation of Survivin in vitamin D.sub.3
 differentiated HL-60 cells was accompanied by a 5- to 10-fold increased
 surface expression of EPR-1 in these cells, as detected by flow cytometry
 with anti-EPR-1 monoclonal antibodies B6 or 12H1 (FIG. 8).
 As shown in FIG. 16, Survivin is down regulated by the combination of
 cytokines .gamma. interferon and tumor necrosis factor .alpha., but not by
 either cytokine alone. Similarly, the transfection of 3T3 cells with the
 c-myc oncogene results in the up-regulation of Survivin mRNA as detected
 by Northern blots.
 EXAMPLE 11
 Promoting Apoptsis with Survivin
 Targeting Survivin promotes apoptsis and inhibits cell proiferation.
 Transfection of the Survivin cDNA in mouse or hamster cell lines (NIH 3T3,
 CHO) was not suitable for the presence of immunochemically
 indistinguishable endogenous homologues in these cells (not shown).
 Similarly, initial attempts to target the Survivin gene in stable
 antisense transfectants were unsuccessful for slow cell growth and rapid
 loss of viability (not shown). Therefore, Survivin.sup.+ HeLa cells were
 transfected with the 3' end of the EPR-1 cDNA (Survivin antisense) under
 the control of a metallothionein-inducible promoter (Lukashev, M. E. et
 al., J Biol Chem (1994) 269:18311-18314), selected in hygromycin, and
 analyzed for apoptosis and cell proliferation after Zn.sup.2+ -dependent
 activation of transcription.
 Consistent with the expression of Survivin in transformed cell lines (FIG.
 4C), the JC700 antibody immunoblotted 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 bands were recognized by
 JC700 antibody in metallothionein-induced HeLa cells transfected with the
 EPR-1 cDNA (Survivin antisense) (FIG. 7A). Under these experimental
 conditions, in situ analysis of internucleosomal DNA fragmentation by
 AptoTag staining revealed only a few apoptotic cells in serum-starved,
 Zn.sup.2+ -induced, vector control HeLa cells (FIG. 7B).
 In contrast, as discussed above, inhibition of Survivin expression in
 Zn.sup.2+ -induced antisense HeLa cell transfectants was associated with
 prominent nuclear staining in the vast majority of cells examined FIG.
 7B). No nuclear staining was detected in the absence of TdT tagging of the
 digoxigenin-labeled dUTP (not shown).
 Typical morphologic features of apoptosis, including numerous apoptotic
 bodies, were also demonstrated in induced antisense HeLa cell
 transfectants by hematoxylin/eosin staining, while only occasional
 apoptotic bodies were observed in vector control HeLa cultures, under the
 same experimental conditions FIG. 7B).
 A potential effect of Survivin on cell growth was also investigated. In
 these experiments, metallothionein-controlled, EPR-1-dependent, inhibition
 of Survivin expression caused a profound reduction of serum-dependent HeLA
 cell proliferation (FIG. 7C). Three days after Zn.sup.2+ induction, the
 cell count in vector control HeLa cultures increased by 288%, as opposed
 to only a 20% increase in Survivin antisense transfectants, under the same
 experimental conditions (FIG. 7C).
 EXAMPLE 12
 Structure--Function Relationship of Survivin
 The minimal structural requirements involved in Survivin-mediated
 inhibition of apoptosis have been identified through a mutagenesis
 strategy of Ala substitutions of the most evolutionarily conserved
 residues in the single Survivin BIR (baculovirus IAP repeat) module. These
 residues included in the amino-terminal half of the Survivin BIR,
 Arg.sup.18, Phe.sup.22, Trp.sup.25, Pro.sup.26, Pro.sup.35, Ala.sup.39,
 Ala.sup.41, Gly.sup.42, and Cys.sup.46. In the carboxyl-terminal half of
 the Survivin BIR, Ala mutants were first targeted at the Cys.sup.57
 X.sub.2 Cys.sup.60 X.sub.16 His.sup.77 X.sub.6 Cys.sup.84 putative zinc
 binding motif. Additional conserved residues targeted by mutagenesis
 include Asp.sup.53, Leu.sup.64, Trp.sup.67, Pro.sup.69, Asp.sup.71,
 ASP.sup.72 and Pro.sup.73.
 The Survivin mutants are characterized in stable and transiently
 transfected cells, IL-3-dependent BaF3 cells and NIH3T3, respectively. In
 addition to these point mutants, a Survivin chimeric molecule containing a
 caiboxyl-terminal RING finger has also been generated and screened for
 apoptosis inhibition (the RING finger 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 carboxylterminus residues,
 containing a predicted coiled-coil structure, have been deleted. As shown
 in FIG. 12, Ala mutagenesis of key conserved residues in Survivin
 Tr.sup.67 -Pro.sup.73 -Cys.sup.84 produced a recombinant molecule which
 lacked the ability to protect BaF3 cells from apoptosis induced by IL-3
 withdrawal.
 EXAMPLE 13
 Cytoprotective Effects of Survivin
 Classical examples of cell damage to stable cell populations mediated by
 apoptosis include allograft rejection by infiltrating lymphocytes,
 Alzheimer's disease and reperfusion injury following myocardial
 infarction. In addition to being expressed in cancer, thereby functioning
 as a growth-advantage factor for cancer cells, the targeted expression of
 Survivin is useful to protect stable cell populations from apoptosis and
 other cellular insults. This application of Survivin was tested by adding
 increasing concentrations of purified recombinant Survivin to monolayers
 of human endothelial cells injured with hydrogen peroxide (H.sub.2
 O.sub.2), a classical apoptosis-inducing stimulus. The results are
 summarized in FIG. 13. Increasing concentrations of added Survivin
 resulted in a significant increased viability of the treated cells as
 opposed to control cultures treated with control protein myoglobin.
 Similarly, Survivin protected NIH3T3 cells from apoptosis induced by
 hydrogen peroxide after transient co-transfection with a lacZ reporter
 gene as shown in FIG. 17.
 EXAMPLE 14
 Survivin as a Predictive Prognostic Factor
 The presence of Survivin can be utilized as predictive-prognostic negative
 factor in neuroblastoma and non-Hodgkin's lymphoma, and in other cancers.
 Neuroblastoma. A large series of neuroblastoma cases (72) was screened for
 Survivin expression in a multicentric study. As shown in FIG. 14, Survivin
 expression increased dramatically when patients contained at least one
 negative prognostic factor for aggressive and rapidly progressing disease.
 Secondly, expression of Survivin strongly correlated with a more
 aggressive disease and unfavorable histology. Importantly, expression of
 Survivin was a more sensitive prognostic index than simple histology.
 Survivin-positive cases with early diagnosis of favorable histology were
 found to contain at least one negative prognostic factor for disease
 progression and dissemination.
 Hodgkin's Lymphoma. A similar multicentric study has been recently
 completed on analysis of Survivin expression in high grade non-Hodgkin's
 lymphoma (n=48). The results are similar to those observed for
 neuroblastoma. As shown in FIG. 15, expression of Survivin strongly
 correlated with a more widespread disease predominantly in stage IV.
 Clinically, Survivin-expressing patients had fewer episodes of complete
 remission and more episodes of incomplete remission, no remission or
 relapses as compared with Survivin-negative patients.
 Potential implications. The demonstrated role of Survivin as a negative
 predictive prognostic factor in these two embryologically different types
 of cancer iterates the potential use of this molecules a dinnostic tool to
 monitor disease progression and response to the therapy. It can also be
 used for staging purposes and to identify populations of patients
 potentially susceptible to multi-drug resistance (groups with no
 remissions or incomplete remissions). Also, Survivin derived primers
 easily designed from the complete sequence of the Survivin gene can be
 used as a screening tool to identify potential cases of cancer in which
 the Survivin gene has been deleted or mutated. These cases will be very
 important to identify because targeted inactivation of the Survivin gene
 would confer a favorable prognostic factor to cancer patients, removing a
 potential drug-resistance gene. Inactivating mutations in the Survivin
 gene can target the same key residues identified in our initial screening
 of Ala-based mutagenesis or result in an abortive or truncated protein for
 premature termination of translation.
 EXAMPLE 15
 Survivin Cancer Vaccine
 Vaccines directed against Survivin, as found in various types of cancer,
 may be developed as with other disease-related intracellular protein
 targets. These techniques are commonly available and representative
 approaches are described by the references cited below. Vaccines may also
 include the systemic administration of peptide fragments of Survivin and
 the use of vectors to deliver mini-genes encoding Survivin peptides to
 tumor cell targets are contemplated. As mentioned above, Survivin is not
 expressed in normal cells, even in proliferating stem cells in the bone
 marrow. This ensures that the immune response mounted against Survivin
 will be highly selective and specific and will not involve normal cells.
 Devel and Administration of Polypeptide-based vaccines
 Methods of the use of peptide components in a monovalent or a polyvalent
 cancer immunotherapy-vaccine product are described by Nardi, N. et al.,
 Mol. Med. (1995) 1(5):563-567. Additional references that discuss the
 different cancer vaccine and cancer immunotherapies currently being used
 include: N. P. Restifo and M. Sznol "Cancer Vaccines," in DeVita's Cancer.
 Principles & Practice of Oncology 3023-3043 (Lippincott-Raven,
 Philadelphia; 1997); J. Galea-Lauri et al., Cancer Gene Ther. (1996) 3(3):
 202-214; D. C. Linehan et al., Ann. Surg. Oncol. (1996) 3(2): 219-228; and
 J. Vieweg et al., Cancer Invest. (1995) 13(2): 193-201.
 Consistent with the foregoing approach, Survivin polypeptides or full
 length Survivin are synthesized either chemically by known techniques or
 recombinantly by expressing appropriate cDNAs in prokaryotic or eukaryotic
 cells. Survivin proteins so 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 Survivin, such as the monoclonal antibody JC700 or the antibody 8E2
 (both described above) which recognize and bind to Survivin. In purifying
 an antibody-based vaccine, the recombinantly produced Survivin would bind
 to the antibodies while other proteins and cellular debris would be washed
 out. Survivin polypeptides are then be isolated and concentrated to a
 desired strength.
 Alternatively Survivin polypeptides are created by cleaving the native
 Survivin with one or more proteases (e.g., trypsin). Proteolytic fragments
 are then be 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 be concentrated to a desired final concentration.
 Once purified, Survivin polypeptides or full length Survivin molecules may
 then placed in an emulsion containing an adjuvant Adjuvants contemplated
 for use with Survivin include aluminum adjuvants, Freund's adjuvant,
 oil-in-water emulsions containing tubercle bacilli, and interleukin-2
 (IL-2). Additional preparations include combining the Survivin
 polypeptides with other appropriate tumor-associated antigens and,
 optionally, other immunomodulatory agents such as cytokines. Other
 suitable carriers or excipients can be used including bovine serum
 albumin, coupling the Survivin polypeptide with haptens, keyhole limpet
 hemocyanin, ovalbumin, and purified protein derivative of tuberculin.
 Peptides may be coupled to carniers using techniques such as those
 described in ED HARLOW AND DAVID LANE, ANTIBODIES: A LABORATORY MANUAL
 (Cold Spring Harbor Laboratory, 1988).
 Vaccines in human subjects may be anministered in the form of an emulsion
 injected subcutaneously, intradermally or intramuscularly (IM); vaccines
 appropriately formulated can be taken orally. With vaccines containing
 adjuvants, the vaccine is generally preferably be given IM, e.g., in the
 deltoid.
 The amount of Survivin vaccine or Survivin peptide vaccine to be
 administered to a patient will correspond to values typically used in for
 other cancer vaccines. Dosage concentrations will range from about 0.25 g
 to about 1000 g per day. More preferred ranges will be from about 10 .mu.g
 to about 500 .mu.g per day.
 EXAMPLE 16
 Diagnostic use of Anti-Survivin Antibodies
 Frequently, tumor associated antigens (TAA) are shed from tumor cells into
 the surrounding plasma or into the blood. As a result, TAA often are found
 in the blood, and blood samples obtained from patients may be used in
 detecting the presence of cancer, as well as used as a factor is staging
 cancers (e.g., stage I, II, III, or IV). Survivin is one such TAA, and
 healthy, normal individuals do not express Survivin. Results from studies
 of several cancers have indicated that the presence of Survivin (or
 Survivin fragments) correlates with and is predictive that the disease may
 be aggressive or may have metastasized. A similar strategy of detecting
 and quantifying the levels of Survivin or Survivin fragments can be used
 to determine residual tumor burden in patients undergoing chemotherapy or
 radiation therapy for cancer treatment. Elevated or increasing levels of
 Survivin may reflect late stage neoplastic disease.
 For diagnostic uses, blood is drawn from patients, by well known
 techniques, who have known cancer loads or from patients suspected of
 having cancer. The blood sample is prepared by known techniques and is
 tested for binding with antibodies to Survivin that are prepared and,
 optionally, labeled, as discussed above. Such general antibody detection
 protocols and associated reagents are well established in the art. Other
 biological fluid samples such as semen, urine, or saliva can also be
 monitored for the presence of Survivin. This diagnostic technique also can
 be used to monitor disease progression and response to individualized
 therapies. This method offers a relatively non-invasive means of tracking
 cancer progression or regression.
 EXAMPLE 17
 Detection of Survivin by Immunobioassay
 An illustrative example of an immunobioassay to test for the presence of
 Survivin in the blood of patient relies on the ability of the monoclonal
 antibodies to Survivin to bind Survivin and remove the detectable Survivin
 from solution by immunoprecipitation. Such an immunobioassay is used to
 detect Survivin in suspected cancer patients and in fractions eluted from
 fractionation columns. An aliquot of each patient sample is incubated for
 2 hours at 4.degree. C. with a monoclonal antibody that specifically
 recognizes and binds Survivin, such as the Mab 8E2, described above. The
 monoclonal antibody is insolubilized on anti-mouse IgG agarose beads,
 which can be acquired from Sigma Chemical Co., St Louis, Mo.
 The agarose bead anti-mouse (IgG(H+L))Survivin complex is prepared 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
 Survivin monoclonal antibody for 18 hours at 4.degree. C. in the same
 buffer. The agarose beads may then be sedimented by centrifugation for 30
 seconds at 16,000 x g in a microcentrifuge and non-specific sites may be
 blocked by incubation with 2% non-fat dry milk in 0.5 M NaCl-TMK for 30
 minutes at 4.degree. C. After blocking, the beads may be washed 3 times
 with 0.5 M NaCl-TMK and resuspended in an equal volume of the same buffer.
 20:1 of the agarose bead-monoclonal antibody complex may then incubated
 with each 250:1 of the patient test sample for 2 hours at 4.degree. C. Any
 Survivin present in the patient test sample will be found by the Survivin
 monoclonal antibody on the beads. The bead complex, now with Survivin
 bound, may be removed by centrifugation for 30 seconds at 16,000 x g. The
 supernatant is then assayed for Survivin activity in the bioassay as
 described below. Control samples are treated with blocked beads that
 lacked the Survivin monoclonal antibody and tested for Survivin activity
 in the bioassay.
 EXAMPLE 16
 Detecting Survivin using a Direct Elisa Test
 Samples of normal plasma (control) and cancer patient-plasma are diluted
 1:1 with phosphate buffered saline (PBS). One volume of each mixture is
 added to centricon-10 filter having a 10 kD molecular weight limit and
 centrifuged at 5000 x g (7000 rpm) for 1 hour. One volume of PBS is added
 to the retentate and centrifuged for 30 min. The final dilution is about
 1:3. The ELISA plate wells are then coated with retentate at 1:6, 1:12,
 1:24, 1:48 and 1:96 final dilution in bicarbonate coating buffer, having a
 pH 9.6 overnight at 4.degree. C. C. The plates are then washed 2 times
 with wash buffer containing 5% Tween 20 in phosphate buffered saline.
 Residual binding sites are blocked with 4% bovine serum albumin (BSA), 30
 .mu./well for 2 hours. Plates are then washed 2 times with wash buffer.
 Next, 100 .mu.l of a monoclonal antibody that specifically recognizes and
 binds to Survivin, such as Mab 8E2, is used at 1:200 dilution in 1% BSA is
 added to the wells and incubated for 1 hour with agitation. Plates are
 washed 5 times with wash buffer. Next, 100 .mu.l horseradish peroxidase
 conjugated secondary antibody is added, typically at a 1:2,000 dilution to
 each well, and incubated for 1 hour. Plates are again washed 5 times with
 wash buffer. Next, 100 .mu.l/well of substrate containing 5 .mu.g of
 Survivin and 5 .mu.l H.sub.2 O.sub.2 /10 ml citrate-phosphate buffer is
 added to each well and incubated for 5 minutes. The enzyme reaction is
 stopped by adding 50 .mu.l/well 2 M H.sub.2 SO.sub.4. The absorbance of
 light is measured at 492 nm in an EIA reader. Patient samples that contain
 Survivin will produce a positive reading, whereas those samples that do
 not contain Survivin will be negative.
 EXAMPLE 18
 Survivin Fragments, Peptides and Small Molecule Antagonists
 As described above, key functional residues in Survivin required for
 apoptosis have been identified. These data provide a template upon which
 to produce synthetic peptides and small molecule antagonists and
 competitive inhibitors of Survivin function. Preferably, the peptides are
 produced from native Survivin or include substitutions from the native
 Survivin peptide backbone that include the functionally relevant residues
 Trp.sup.67 -Pro.sup.73 -Cys.sup.84. Peptide figments of native Survivin
 can be generated by standard techniques, including protein digests. A
 determination of which fragments compete with Survivin can readily be made
 by using the apoptosis measurement systems and apoptosis assay systems
 described above. These results provide a unique opportunity to identify a
 discrete linear sequence in Survivin, that is essential for inhibition of
 apoptosis.
 Consistent with the general paradigm of IAP proteins-dependent inhibition
 of apoptosis, it also was predicted that a structural region in the
 molecule required for the anti-apoptotic function is the primary candidate
 for being a site of interaction with other molecules (such as binding
 partners). The functionally relevant peptide sequence in Survivin, based
 on the mutagenesis data, is: EGWEPDDDPIEEHKKHSSGC(SEQ ID NO:4). Ala
 substitutions of the underlined residues results in a complete loss of
 function of Survivin in transfected cells. This linear sequence can be
 synthesized and used as a much more stringent and specific reagent to
 isolate associated molecules using standard biochemical procedures of
 affinity chromatography or as a bait for the yeast two-hybrid system.
 Also, preferably, the .beta.COOH coiled-coil region of Survivin is included
 in Survivin fragments and peptides. Recent data indicates that this
 Survivin domain is important for the anti-apoptosis function of Survivin.
 We have generated a recombinant truncated form of Survivin lacking the
 last 40 OCOOH terminus amino acids comprising the coiled-coil domain. This
 truncated form was co-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 FIG. 17, indicate that the truncated
 Survivin had lost most (.about.80%) of the cytoprotective effect at
 preventing apoptosis in transfected cells induced by hydrogen peroxide.
 Incidentally, in this system, Survivin was more potent than NAIP at
 preventing apoptosis.
 Agonists and antagonists of Survivin also can readily be identified through
 conventional techniques. Designed, synthetic peptides based on the native
 linear sequence also function as competitive inhibitors of Survivin's
 interaction with as yet unidentified partner molecules. However, this
 inhibition should be sufficient to block the anti-apoptosis function of
 Survivin.
 A similar peptide-based strategy has been successful to block caspase
 activation in vitro and in vivo, protecting cells from apoptosis. See,
 e.g., Milligan, C. E. et al., (1995) Neuron 15:385-393.
 EXAMPLE 19
 Therapeutic uses of Antisense Survivin DNA
 As described above, the transcription of a Survivin antisense sequence
 altered the EPR-1 /Survivin gene balance. This was demonstrated in HeLa
 cell transfectants, in which metallothionein-induced transcription of the
 EPR-1 "sense" strand suppressed the expression of Survivin and profoundly
 influenced apoptosis/cell proliferation. Additionally, transiently
 co-transfecting a Survivin antisense construct with a lacZ reported
 plasmid decreased the viability of Survivin antisense transfectants after
 a 48-h transfection in .beta.-galactosidase expressing cells. Accordingly,
 the level of expression of Survivin in a Survivin expressing cell or
 tissue, such as a tumor, is decreased by transfecting the cell or tissue
 with the EPR-1 sense strand of DNA. Alternatively, a Survivin
 antisense-encoding DNA is used to transfect a target cell or tissue. Such
 therapy effectively decreases the tlation of Survivin-encoding mRNA into
 Survivin protein.
 EXAMPLE 20
 Use of Survivin as a Protective Agent Against Apoptosis
 Survivin has been shown to protect cells from apoptosis when administered
 to cells that have been exposed to hydrogen peroxide or other agents that
 typically induce apoptosis. It is contemplated that cellular permeability
 may need to be increased, preferably in a transient manner in order to
 facilitate delivery of Survivin, or fragments thereof effective to reduce
 apoptosis. Certain conditions involving transient metabolic inhibition or
 transient hypoxia are likely to increase cellular permeability without the
 need for additional, external agents. Agents that may be appropriate
 include, metabolic inhibitors like 2-deoxygluocose and sodium azide.
 However, the ability of Survivin to mediate cytoprotection during a
 transient increase in cellular permeability offers the possibility of
 using therapeutic infusion of recombinant Survivin to decrease reperfusion
 injury and cellular damage during myocardial infarction and stroke. It is
 contemplated that such processes are mediated by increased tissue damage
 due to apoptosis. Treatment with Survivin could reduce the extent and
 magnitude of the injured tissue.
 The use of Survivin or allelic varients of Survivin in subjects to modulate
 or prevent apoptosis related cell death would be beneficial in treating or
 ameliorating the effects of a variety of apoptosis-related indications.
 These indications include, but are not limited to, dermatological effects
 of aging (e.g., baldness that is caused by apoptosis of cells of hair
 follicle cells), disorders and diseases such as immunosuppression,
 gastrointestinal perturbations (e.g., damage of lining of the gut, ulcers,
 and radiation or chemotherapy induced damage), cardiovascular disorders,
 apoptosis related to reperfusion damage (e.g., coronary artery
 obstruction, cerebral infarction, spinal/head trauma and concomitant
 severe paralysis, damage due to insults such as frostbite or burns, and
 any indication previously thought to be treatable by superoxide
 dismutase), rejection of tissue transplantation (e.g., graft versus host
 disease), and Alzheimer's disease. The administration of Survivin also may
 be cytoprotective against chemotherapy or radiation-induced apoptosis.
 Survivin protein for administration can be produced as described above,
 e.g., using the cDNA described herein. The protein may require
 purification for purposes of pharmaceutical administration and such
 purification steps preferably utilize monoclonal antibody separation and
 purification techniques as also described above.
 In a clinical setting, Survivin is administered to patients in
 pharmaceutically effective dosages, i.e., in dosages effective to reduce
 the level or extent of apoptosis otherwise present, via several routes.
 For example, to treat dermatological ailments that involve apoptosis,
 Survivin can be administered in a salve, cream, ointment or powder form.
 Topical formulations may contain additional pharmaceutical or cosmetic
 compositions such as moisturizers, humectants, odor modifiers, buffer,
 pigment, preservatives, vitamins (such as A, C or E), emulsifiers,
 dispersing agents, wetting agents, stabilizers, propellants, antimicrobial
 agents, sunscreen, enzymes and the like. Typical dosages of Survivin that
 may be administered to patients will be 0.01% to 1.0% by weight.
 Additional topical pharmaceutical compositions are described in S. Nakai
 et al., U.S. Pat. No. 5,672,603.
 Survivin may also be administered, as may be appropriate for the condition
 being treated, in the form of pills, solutions, suspensions, emulsions,
 granules or capsules. Survivin can be administered orally; injected in
 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 such as eye drops. Survivin can also be
 administered using delayed release carriers, such as liposomes,
 rnicrosponges, rnicrospheres or microcapsules that are deposited in close
 proximity to the tissue being treated for prevention of apoptosis related
 cell death.
 Concentrations of Survivin or functional allelic variants of Survivin
 administered via routes other than topical administration typically would
 range in dose from about 10 .mu.g per day to about 25 mg per day depending
 on the route of administration. Of course, it would be expected that
 skilled artisans, such as physicians, may alter these values on a case by
 case basis as required for the particular patient.
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 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18
 Glu Glu Ala Arg Phe Leu Thr Tyr His Met Trp Pro Leu Thr Phe Leu
 1 5 10 15
 Ser Pro Ser Glu Leu Ala Arg Ala Gly Phe Tyr Tyr Ile 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: 19:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 46 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS:
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19
 Glu Glu Ala Arg Leu Lys Ser Phe Gln Asn Trp Pro Asp Tyr Ala His
 1 5 10 15
 Leu Thr Pro Arg Glu Leu Ala Ser Ala Gly Leu Tyr Tyr Thr Gly Ile
 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) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 45 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS:
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20
 Glu Ala Asn Arg Leu Val Thr Phe Lys Asp Trp Pro Asn Pro Asn Ile
 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 Ile Ala
 35 40 45
 (2) INFORMATION FOR SEQ ID NO: 21:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 50 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS:
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21
 Tyr Val Gly Ile 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 Ala 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) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 50 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS:
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: 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 Ile Glu His Ala Lys
 20 25 30
 Trp Phe Pro Arg Cys Glu Phe Leu Ile Arg Met Lys Gly Gln Glu Phe
 35 40 45
 Val Asp
 50
 (2) INFORMATION FOR SEQ ID NO: 23:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 50 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS:
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23
 Tyr Gln Lys Ile Gly Asp Gln Val Arg Cys Phe His Cys Asn Ile Gly
 1 5 10 15
 Leu Arg Ser Trp Gln Lys Glu Asp Glu Pro Trp Phe Glu His Ala 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) STRANDEDNESS:
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: 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) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 49 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS:
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: 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 Ala Pro Trp Gln Gln His Ala 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:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 50 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS:
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26
 Tyr Thr Gly Ile Lys Asp Ile 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 Ala Glu
 35 40 45
 Val Thr
 50
 (2) INFORMATION FOR SEQ ID NO: 27:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 50 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS:
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27
 Tyr Gln Lys Ile Gly Asp Gln Val Arg Cys Phe His Cys Asn Ile Gly
 1 5 10 15
 Leu Arg Ser Trp Gln Lys Glu Asp Glu Pro Trp Phe Glu His Ala Lys
 20 25 30
 Trp Ser Pro Lys Cys Gln Phe Val Leu Leu Ala Lys Gly Pro Ser Tyr
 35 40 45
 Val Ser
 50
 (2) INFORMATION FOR SEQ ID NO: 28:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 50 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS:
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28
 Ala 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 Ala Lys
 20 25 30
 Trp Tyr Pro Gly Cys Lys Tyr Leu Leu Glu Gln Lys Gly Gln Glu Tyr
 35 40 45
 Ile Asn
 50
 (2) INFORMATION FOR SEQ ID NO: 29:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 50 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS:
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29
 Ala 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 Ala Lys
 20 25 30
 Trp Tyr Pro Gly Cys Lys Tyr Leu Leu Asp Glu Lys Gly Gln Glu Tyr
 35 40 45
 Ile Asn
 50
 (2) INFORMATION FOR SEQ ID NO: 30:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 50 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS:
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30
 Tyr Val Gly Asn Ser 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 Ala Lys
 20 25 30
 Trp Phe Pro Arg Cys Glu Tyr Leu Ile Arg Ile Lys Gly Gln Glu Phe
 35 40 45
 Ile Arg
 50
 (2) INFORMATION FOR SEQ ID NO: 31:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 50 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS:
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: 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 Ala Lys
 20 25 30
 Trp Phe Pro Arg Cys Glu Phe Leu Ile Arg Met Lys Gly Gln Glu Phe
 35 40 45
 Val Asp
 50
 (2) INFORMATION FOR SEQ ID NO: 32:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 50 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS:
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32
 Ala 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 Ala Lys
 20 25 30
 Trp Tyr Pro Gly Cys Lys Tyr Leu Leu Glu Gln Lys Gly Gln Glu Tyr
 35 40 45
 Ile Asn
 50
 (2) INFORMATION FOR SEQ ID NO: 33:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 50 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS:
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33
 Tyr Gln Lys Ile Gly Asp Gln Val Arg Cys Phe His Cys Asn Ile Gly
 1 5 10 15
 Leu Arg Ser Trp Gln Lys Glu Asp Glu Pro Trp Phe Glu His Ala 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: 34:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 142 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS:
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34
 Met Gly Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp
 1 5 10 15
 His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Cys Ala
 20 25 30
 Cys Thr Pro Glu Arg Met Ala Glu Ala Gly Phe Ile 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 Ile Glu Glu His Lys Lys His
 65 70 75 80
 Ser Ser Gly Cys Ala Phe Leu Ser Val Lys Lys Gln Phe Glu Glu Leu
 85 90 95
 Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn Lys
 100 105 110
 Ile Ala Lys Glu Thr Asn Asn Lys Lys Lys Glu Phe Glu Glu Thr Ala
 115 120 125
 Lys Lys Val Arg Arg Ala Ile Glu Gln Leu Ala Ala Met Asp
 130 135 140
 (2) INFORMATION FOR SEQ ID NO: 35:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 14796 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: unknown
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35
 TCTAGACATG 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 ATCGGGCCGG 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 CTTTTTTTTT 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
 AGTATGGGAG 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 TTTTTTTTCT 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 ATTTTTTTTT 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 TTTTTTTTTT GAGACAGAGC CTTGCTCTGT CGCCCAGGCT 7140
 GGAGTGCAGT GGCACGATCT CAGCTCACTA CAAGCTCCGC CTCCCGGGTT CACGCCATTC 7200
 TTCTGCCTCA GCCTCCTGAG TAGCTGGGAC TACAGGTGCC CACCACCACG CCTGGCTAAT 7260
 TTTTTTTGGT 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
 TTTTTTTGTT TGTTTGTTTT CCCCAATTCA GAATACAGAA TACTTTTATG GATTTGTTTT 9180
 TATTACTTTA ATTTTGAAAC AATATAATCT TTTTTTTGTT 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
 CAGTCCATTA 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
 GGTTTTGTCT 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 TTTTCTTTTT CTTTTTTTTT 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
 CACCCCATTC CATTTTCAGA CAGCTTTGTC AGAAAGGCTG TCATATGGAG CTGACACCTG 14700
 CCTCCCCAAG GCTTCCATAG ATCCTCTCTG TACATTGTAA CCTTTTATTT TGAAATGAAA 14760
 ATTCACAGGA AGTTGTAAGG CTAGTACAGG GGATCC 14796