Recombinant, active caspases and uses thereof

Abstract

Rev-caspases comprising a primary product in which the small subunit is N-terminal to the large subunit are provided. Rev-caspases are used for screening and identifying caspase inhibitors and enhancers. Rev-caspase genes can be delivered to cells for gene therapy.

Description

TECHNICAL FIELD

The present invention relates generally to regulating apoptosis, and more particularly to the novel aspartate-specific cysteine proteases known as caspases, their coding regions, mutant forms thereof, and their use in screening assays and as pharmaceutical compositions for the controlled death of targeted cells to treat human disease.

BACKGROUND OF THE INVENTION

Tissue homeostasis is maintained by the process of apoptosis—that is, the normal physiological process of programmed cell death. Changes to the apoptotic pathway that prevent or delay normal cell turnover can be just as important in the pathogenesis of diseases as are abnormalities in the regulation of the cell cycle. Like cell division, which is controlled through complex interactions between cell cycle regulatory proteins, apoptosis is similarly regulated under normal circumstances by the interaction of gene products that either prevent or induce cell death.

Since apoptosis functions in maintaining tissue homeostasis in a range of physiological processes such as embryonic development, immune cell regulation and normal cellular turnover, the dysfunction or loss of regulated apoptosis can lead to a variety of pathological disease states. For example, the loss of apoptosis can lead to the pathological accumulation of self-reactive lymphocytes that occurs with many autoimmune diseases. Inappropriate loss or inhibition of apoptosis can also lead to the accumulation of virally infected cells and of hyperproliferative cells such as neoplastic or tumor cells. Similarly, the inappropriate activation of apoptosis can also contribute to a variety of pathological disease states including, for example, acquired immunodeficiency syndrome (AIDS), neurodegenerative diseases and ischemic injury. Treatments which are specifically designed to modulate the apoptotic pathways in these and other pathological conditions can alter the natural progression of many of these diseases.

Although apoptosis is mediated by diverse signals and complex interactions of cellular gene products, the results of these interactions ultimately feed into a cell death pathway that is evolutionarily conserved between humans and invertebrates. The pathway, itself, is a cascade of proteolytic events analogous to that of the blood coagulation cascade.

Several gene families and products that modulate the apoptotic process have now been identified. One family is the aspartate-specific cysteine proteases (“caspases”). The caspase Ced-3, identified in C. elegans , is required for programmed cell death during development of the roundworm C. elegans . Ced-3 homologues as well as other caspases have been characterized. The human caspase family includes, for example, human ICE (interleukin-1-β converting enzyme) (caspase-1), ICE rel II (caspase-4), ICE rel III (caspase-5), Mch5 (caspase-8), Mch4 (caspase-10), ICE-LAP6 (caspase-9), Mch2 (caspase-6), CPP32 (caspase-3), ICE-LAP3 (casepase-7), ICH-1 (caspase-2), Caspase 11-14, and others.

The caspases share many features. In this regard, caspases are cysteine proteases (named for a cysteine residue in the active site) that cleave substrates at Asp-X bonds. Furthermore, the primary caspase product is a zymogen that requires proteolytic cleavage at specific internal aspartate residues for activation. The primary gene product is arranged such that the N-terminal peptide (prodomain) precedes a large subunit domain, which precedes a small subunit domain. Cleavage of a caspase yields two subunits, a large (generally approximately 20 kD) and a small (generally approximately 10 kD) subunit that associate non-covalently: to form a heterodimer, and, in some caspases, an N-terminal peptide of varying length (see FIG. 1 ). The heterodimer may combine non-covalently to form a tetramer.

Caspase zymogens are themselves substrates for caspases. Inspection of the interdomain linkages in each zymogen reveals target sites (i.e. protease sites) that indicate a hierarchical relationship of caspase activation. By analyzing such pathways, it has been demonstrated that caspases are required for apoptosis to occur. Moreover, caspases appear to be necessary for the accurate and limited proteolytic events which are the hallmark of classic apoptosis (see Salvesen and Dixit, Cell, 91: 443-446, 1997). However, when overexpressed in mammalian cells, the short prodomain caspases-3 and -6 cells are unable to undergo autocatalytic processing/activation and do not induce apoptosis. Thus, no cellular model system has been developed in which to test inhibitors of these caspases nor is gene delivery of a caspase commonplace.

Therefore, there exists a need in the art for methods of assaying compounds for their ability to affect caspase activity as well as for methods of regulating caspases in order to treat diseases and syndromes. The present invention provides recombinant caspase constructs that are active in cells, allowing the regulation of apoptosis for the treatment of pathology as well as providing methods and compositions for assaying compounds for caspase inhibitory and, thus, anti-apoptotic effects, while further providing other related advantages.

SUMMARY OF THE INVENTION

The present invention generally provides rev-caspases. In one aspect, the invention provides an isolated nucleic acid molecule encoding a rev-caspase. In certain embodiments, the rev-caspase is selected from the group consisting of rev-caspase-1, rev-caspase-2, rev-caspase-3, rev-caspase-4, rev-caspase-5, rev-caspase-6, rev-caspase-7, rev-caspase-8, rev-caspase-9, rev-caspase-10, rev-caspase-11, rev-caspase-12, rev-caspase-13, and rev-caspase-14. In other preferred embodiments, the rev-caspase is a human rev-caspase. Nucleic acid and amino acid sequences of rev-caspases are provided. The invention also provides rev-caspase proteins.

In another aspect, an expression vector comprising the nucleic acid molecule encoding rev-caspase is provided, wherein the sequence encoding rev-caspase is operatively linked to a promoter. In certain embodiments, the promoter is inducible, such as HIV LTR. Host cells transfected with the expression vectors are also provided.

In the present invention, methods of identifying an inhibitor or enhancer of caspase processing activity are provided, comprising: (a) contacting a sample containing an in vitro translated rev-caspase with a candidate inhibitor or candidate enhancer; and (b) detecting the presence of large and small subunits of rev-caspase, and therefrom determining the level of caspase processing activity, wherein a decrease in processing indicates the presence of a caspase inhibitor, and wherein an increase in processing indicates the presence of a caspase enhancer, wherein processed rev-caspase yields large and small subunits.

In other aspects, methods are provided for identifying an inhibitor or enhancer of caspase processing activity, comprising: (a) contacting a cell transfected with the vector expressing rev-caspase with a candidate inhibitor or candidate enhancer; and (b) detecting the presence of large and small subunits of rev-caspase, and therefrom determining the level of caspase processing activity, wherein a decrease in processing indicates the presence of a caspase inhibitor, and wherein an increase in processing indicates the presence of a caspase enhancer, wherein processed rev-caspase yields large and small subunits.

Methods are also provided for identifying an inhibitor or enhancer of caspase-mediated apoptosis, comprising: (a) contacting a cell transfected with the vector expressing rev-caspase with a candidate inhibitor or candidate enhancer or with a reference compound; and (b) detecting cell viability, wherein viability of cells contacted with a candidate is increased in the presence of an inhibitor and is decreased in the presence of an enhancer compared to cells contacted with a reference compound.

In other aspects, gene delivery vehicles, comprising the nucleic acid molecule encoding a rev-caspase are provided, wherein the rev-caspase sequence is operatively linked to a promoter. In preferred embodiments, the gene delivery vehicle is a retrovirus or adenovirus or the nucleic acid molecule is associated with a polycation. The gene delivery vehicle may further comprise a ligand that binds a cell surface receptor.

The invention also provides methods of treating cancer or autoimmune diseases, comprising administering to a patient the gene delivery vehicles disclosed herein.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, the various references set forth below that describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the processing and folding of human caspase-3 into the mature zymogen represented by bar and ribbon diagrams and is representative of other caspases. α helices are shown as spirals and the β strands are represented by arrows. The N- and the C-termini of the LS are labeled N-LS and C-LS, respectively, and the termini of the SS are similarly labeled N-SS and C-SS.

FIGS. 1B and C are schematic representations of rev-caspase-3 and -6, respectively. In both FIG. 1B and C the N-terminus of the SS and the C-terminus of the LS are labeled as in FIG. 1 A and the linker region between the C-SS and the N-LS which includes the caspase-3 or -6 prodomain is represented by a thin line. Solid arrows indicate the cleavage sites (DEVDG (SEQ ID NO:38), Asp9 and Asp28—for rev-caspase-3 and VEIDA (SEQ ID NO:116), and Asp23—for rev-caspase-6) within the linker region. The hatched boxes represent a 15 residue-long T7-tag on the N-termini of the wild-type and the rev-caspases. All aspartate processing sites are indicated on the bar diagrams. FIG. 1B further depicts a schematic representation of the spontaneous folding of rev-caspase-3 into the mature zymogen represented a ribbon diagram. The ribbon diagram of rev-caspase-3 is based on the published crystal structure of caspase-3.

FIG. 2A is a scanned image of an autoradiogram representing SDS-PAGE analysis of rev-caspase autoprocessing. Caspase-3 and -6 or their rev-versions (Rev) including active site Cys to Ala mutants (Rev C/A) in pRSC-lacZ constructs were in vitro translated in the presence of 35 S-methionine. The translation products were then analyzed by SDS-PAGE and autoradiography. The LS and the SS are indicated.

FIGS. 2B and C are scanned images of autoradiograms representing SDS-PAGE analysis of the autoprocessing of rev-caspase-3 and -6, respectively in the presence of varying levels of selected caspase inhibitors. Rev-caspase-3 ( FIG. 2B ) or rev-caspase-6 ( FIG. 2C ) were in vitro translated in the presence of increasing concentrations of DEVD-CHO (0.04 μM); (SEQ ID NO:52), or zVAD-fmk (0-5 μM). The translation products were then analyzed as in FIG. 2 A. WT, wild-type.

FIGS. 3A and B are scanned images representing the SDS-PAGE analysis of the ability of rev-caspase-3 (imaged by western blot) and -6 (imaged by autoradiogram) to cleave PARP and lamin, respectively. In FIG. 3A purified human PARP was incubated with buffer (lane 1) or BL-21 bacterial extracts prepared from bacteria transformed with caspase-3 (lane 2), rev-caspase-3 (lane 3), caspase-6 (lane 4), rev-caspase-6 (lane 5) constructs or empty pET28a vector (lane 6) for 2 h at 37° C. The reaction products were then analyzed by SDS-PAGE and Western blotting with anti-human PARP antibody. In FIG. 3B a cDNA encoding the C-terminus of lamin A (amino acids) which contain the caspase-6 cleavage site (VEIDA); (SEQ ID NO:116), was amplified by PCR and in vitro translated in the presence of 35 S-methionine. The labeled product was incubated with buffer (lane 1) or the BL-21 bacterial extracts listed above for 2 h at 37° C., and then analyzed by SDS-PAGE and autoradiography. The cleavage products are indicated to the right.

FIGS. 4A and B are bar diagrams representing the ability of rev-caspase-3 and -6 to induce apoptosis in MCF-7 cells. MCF-7 cells were transiently transfected with either rev-caspase-3 (FIG. 4 A), or rev-caspase-6 ( FIG. 4B ) expression constructs in combination with 4-fold of CrmA, p35 or Bcl-2 expression constructs, or 20 μM zVAD-fmk. Cells transfected with an empty vector or the wild-type caspase-3 or -6 were used as controls.

FIG. 5A is a scanned image of an autoradiogram representing SDS-PAGE analysis of the enzymatic activity of uncleavable rev-caspase-3. Uncleavable rev-caspase-3 was in vitro translated in the absence or the presence of increasing concentrations of DEVD-CHO (SEQ ID NO:52),. The translation product contains a cleavable 35 residues-long His6-T7-tag at its N-terminus. The active site mutant rev-caspase-3 (Rev C/A) was used as a control. The p32 cleavage product without the His6-T7-tag is indicated to the right.

FIG. 5B is a plot of an activity assay of bacterially expressed uncleavable rev-caspase-3. The plot measures the ability of the uncleavable rev-caspase-3 to cleave the DEVD-AMC (SEQ ID NO:52), substrate. Rev, rev-caspase-3; Rev-mod, uncleavable rev-caspase-3; Rev-C/A, rev-caspase-3 with an active site mutation.

FIG. 6 is a multiple amino acid sequence alignment of the relatively conserved regions of the caspases (SEQ ID NOs:54-115). In the bottom line, “c” refers to residues involved in catalysis, “b” refers to residues that bind the substrate-carboxylate of P1 Asp, “a” refers to residues adjacent to the substrate P2-P4 recognition responsible amino acids, “DX” indicates known and potential processing sites between the small and large subunits of the caspases. The roman numerals at the left of the figure indicate the caspase subfamilies: Ced-like (I), ICE-like (II), and the Nedd2/Ich-1-like (III). The asterisk represents the non-conservative substitution in the active site pentapeptide sequences of Mch4 (caspase-10), Mch5 (caspase-8), and Mch6 (caspase-9)

FIG. 7 depicts a nucleotide sequence of Rev-caspase-3 (SEQ ID NO:1).

FIG. 8 depicts a nucleotide sequence of uncleavable Rev-caspase-3 (SEQ ID NO:2).

FIG. 9 depicts a nucleotide sequence of Rev-caspase-6 (SEQ ID NO:3).

FIG. 10 depicts a schematic of some possible rev-caspases. 1, intervening sequence; SS, small subunit; P, prodomain; LS, large subunit; X, linker.

FIGS. 11A and 11B depict a nucleotide (SEQ ID NOs:4 and 5) and predicted amino acid sequence of caspase-1 (SEQ ID NO:6).

FIGS. 12A and 12B depict a nucleotide (SEQ ID NOs:7 and 8) and predicted amino acid sequence of caspase-2 (SEQ ID NO:9).

FIGS. 13A and 13B depict a nucleotide (SEQ ID NOs:10 and 11) and predicted amino acid sequence of caspase-3 (SEQ ID NO:12).

FIGS. 14A and 14B depict a nucleotide (SEQ ID NOs:13 and 14) and predicted amino acid sequence of caspase-4 (SEQ ID NO:15).

FIGS. 15A and 15B depict a nucleotide (SEQ ID NOs:16 and 17) and predicted amino acid sequence of caspase-5 (SEQ ID NO:18).

FIGS. 16A and 16B depict a nucleotide (SEQ ID NOs:19 and 20) and predicted amino acid sequence of caspase-6 (SEQ ID NO:21).

FIGS. 17A and 17B depict a nucleotide (SEQ ID NOs:22 and 23) and predicted amino acid sequence of caspase-7 (SEQ ID NO:24).

FIGS. 18A-18C depict a nucleotide (SEQ ID NOs:25 and 26) and predicted amino acid sequence of caspase-8 (SEQ ID NO:27).

FIGS. 19A and 19B depict a nucleotide (SEQ ID NOs:28 and 29) and predicted amino acid sequence of caspase-9 (SEQ ID NO:30).

FIGS. 20A and 20B depict a nucleotide (SEQ ID NOs:31 and 32) and predicted amino acid sequence of caspase-10 (SEQ ID NO:33).

FIGS. 21A , 21 B, and 21 C depict predicted amino acid sequences of Rev-caspase-3 (A; SEQ ID NO:34), uncleavable rev-caspase-3 (B; SEQ ID NO:35), and rev-caspase-6 (C; SEQ ID NO:36).

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms that will be used hereinafter.

As used herein, a caspase refers to a cysteine protease that specifically cleaves proteins after Asp residues. Caspases are initially expressed as zymogens, in which a large subunit is N-terminal to a small subunit. Caspases are generally activated by cleavage at internal Asp residues (FIG. 1 A). These proteins have been identified in many eukaryotes, including C. elegans , Drosophila, mouse, and humans. Currently, there are at least 14 known caspase genes, named caspase-1 through caspase-14. Caspases are found in myriad organisms, including human, mouse, insect (e.g., Drosophila), and other invertebrates (e.g., C. elegans ). In Table 1, ten human caspases are listed along with their alternative names. The nucleotide and amino acid sequences of representative human caspase gene products are presented in SEQ ID NOs:4-33 and FIGS. 11-20 .

Caspase    Alternative name
Caspase-1  ICE
Caspase-2  ICH-1
Caspase-3  CPP32, Yama, apopain
Caspase-4  ICE                                                                        rel                                                                    II; TX, ICH-2
Caspase-5  ICE                                                                        rel                                                                    III; TY
Caspase-6  Mch2
Caspase-7  Mch3, ICE-LAP3, CMH-1
Caspase-8  FLICE; MACH; Mch5
Caspase-9  ICE-LAP6; Mch6
Caspase-10 Mch4, FLICE-2

As used herein, “rev-caspase” refers to a cysteine protease that specifically cleaves proteins after Asp residues and is expressed as a zymogen, in which a small subunit is N-terminal to a large subunit.

Within the context of this invention, it should be understood that a caspase or rev-caspase includes wild-type protein sequences, as well as other variants (including alleles) of the native protein sequence. Briefly, such variants may result from natural polymorphisms or may be synthesized by recombinant methodology, and differ from wild-type protein by one or more amino acid substitutions, insertions, deletions, or the like. Typically, when engineered, amino acid substitutions will be conservative, i.e., substitution of amino acids within groups of polar, non-polar, aromatic, charged, etc. amino acids. In the region of homology to the native sequence, variants should preferably have at least 90% amino acid sequence identity, and within certain embodiments, greater than 92%, 95%, or 97% identity.

As will be appreciated by those skilled in the art, a nucleotide sequence encoding a caspase, rev-caspase or variant may differ from the known native sequences, due to codon degeneracies, nucleotide polymorphisms, or amino acid differences. In other embodiments, variants should preferably hybridize to the native nucleotide sequence at conditions of normal stringency, which is approximately 25-30° C. below Tm of the native duplex (e.g., 5×SSPE, 0.5% SDS, 5×Denhardt's solution, 50% formamide, at 42° C. or equivalent conditions; see generally, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987). Low stringency hybridizations utilize conditions approximately 40° C. below Tm, and high stringency hybridizations utilize conditions approximately 10° C. below Tm. Variants preferably have at least 75% nucleotide identity to native sequence, preferably at least 80%, 85%, and most preferably at least 90% nucleotide identity.

An “isolated nucleic acid molecule” refers to a polynucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid construct, that has been separated from its source cell (including the chromosome it normally resides in) at least once in a substantially pure form. Nucleic acid molecules may be comprised of a wide variety of nucleotides, including DNA, RNA, nucleotide analogues, or some combination of these.

A. Caspase and Rev-caspase Genes and Gene Products

As noted above, the invention provides compositions relating to caspase and rev-caspase genes and gene products, and methods for the use of the genes and gene products. In particular, the invention provides rev-caspase constructs that are active when expressed in cells. Given the disclosure provided herein, a caspase gene can be isolated from a variety of cell types and engineered to produce a rev-caspase.

1. Isolation of Caspase Genes

The present invention, as described herein, provides rev-caspase genes, which are constructed from caspase genes. Caspase genes may be isolated from either genomic DNA or preferably cDNA. Isolation of caspase genes from genomic DNA or cDNA typically can proceed by, first, generating an appropriate DNA library through techniques for constructing libraries that are known in the art (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989) or purchased from commercial sources (e.g., Clontech, Palo Alto, Calif.). Briefly, cDNA libraries can be constructed in bacteriophage vectors (e.g., λZAPII), plasmids, or others, which are suitable for screening, while genomic DNA libraries can be constructed in chromosomal vectors, such as YACs (yeast artificial chromosomes), bacteriophage vectors, such as λEMBL3, λgt10, cosmids, or plasmids.

In one embodiment known caspase sequences may be utilized to design an oligonucleotide hybridization probe suitable for screening genomic or cDNA libraries. Preferably, such oligonucleotide probes are 20-30 bases in length. To facilitate hybridization detection, the oligonucleotide may be conveniently labeled, generally at the 5′ end, with a reporter molecule, such as a radionuclide, (e.g., 32 P), enzymatic label, protein label, fluorescent label, or biotin. Such libraries are then generally plated as phage or colonies, depending upon the vector used. Subsequently, a nitrocellulose or nylon membrane, to which the colonies or phage have been transferred, is probed to identify candidate clones which contain the caspase gene. Such candidates may be verified as containing caspase DNA by any of various means including, for example, DNA sequence analysis or hybridization with a second, non-overlapping probe.

Once a library is identified as containing a caspase gene, the gene can be isolated by amplification. Briefly, when using cDNA library DNA as a template amplification primers are designed based upon known caspase gene sequences (see GenBank Accession Nos. X65019 (caspase-1), U13021 (caspase-2), U13737 (caspase-3), U25804 (caspase-4), U28015 (caspase-5), U20536 (caspase-6), U37448 (caspase-7), U60520 (caspase-8), U56390 (caspase-9), U60519 (caspase-10) Y13089 (caspase-11), Y13090 (caspase-12), AF078533 (caspase-13), AF092997 (caspase-14), and sequences provided herein). Amplification of cDNA libraries made from cells with high caspase activity is preferred. Primers for amplification are preferably derived from sequences in the 5′ and 3′ untranslated region in order to isolate a full-length cDNA. The primers preferably have a GC content of about 50% and contain restriction sites to facilitate cloning and do not have self-complementary sequences nor do they contain complementary sequences at their 3′ end (to prevent primer-dimer formation). The primers are annealed to cDNA or genomic DNA and sufficient amplification cycles are performed to yield a product readily visualized by gel electrophoresis and staining. The amplified fragment is purified and inserted into a vector, such as λgt10 or pBS(M13+), and propagated. Confirmation of the nature of the fragment is obtained by DNA sequence analysis or indirectly through amino acid sequencing of the encoded protein.

Other methods may also be used to obtain a caspase encoding nucleic acid molecule. For example, a nucleic acid molecule encoding caspase may be obtained from an expression library by screening with an antibody or antibodies reactive to caspase (see, Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, NY, 1987; Ausubel, et al. Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, NY, 1995).

Caspase genes from a variety of species may be isolated using the compositions provided herein. For closely related species, the human sequence or portion thereof may be utilized as a probe on a genomic or cDNA library. For example, a fragment of caspase that encompasses the catalytic site may be labeled and used as a probe on a library constructed from mouse, primate, rat, dog, or other vertebrate, warm-blooded or mammalian species. An initial hybridization at normal stringency may yield candidate clones or fragments. If no hybridization is initially observed, varying degrees of stringency may be used. (see Sambrook et al. supra, and other well-known sources for stringency conditions) While such probes may also be used to probe libraries from evolutionarily diverse species, such as Drosophila, hybridization conditions will likely be more relaxed.

While relaxed hybridization conditions using probes designed from human sequences may identify caspase genes of evolutionarily diverse species it may be more beneficial to attempt to directly isolate these genes from a library using methods which do not require the human sequence per se. These methods include, but are not limited to, amplification using primers derived from conserved areas, amplification using degenerate primers from various regions, antibody probing of expression libraries, and the like. For example, random-primed amplification (e.g., polymerase chain reaction) may be employed (see, e.g., Methods Enzymol. 254: 275, 1995; Trends Genet. 11: 242, 1995; Liang and Pardee, Science 257: 967, 1992; Welsh et al., Nucl. Acids Res. 20: 4965, 1992). In addition, variations of random-primed PCR may also be used, especially when a particular gene or gene family is desired. In such a method, one of the amplification primers is an “anchored oligo(dT) (oligo(dT)dN)” and the other primer is a degenerate primer based upon amino acid or nucleotide sequence of a related gene. A gene sequence is identified as a caspase by amino acid similarity and/or nucleic acid similarity. Generally, amino acid similarity is preferred. Candidate caspase genes are examined for enzyme activity by one of the functional assays described herein or other equivalent assays.

Variants of caspase and rev-caspase genes provided herein may be engineered from natural variants (e.g., polymorphisms, splice variants, mutants), synthesized or constructed. Many methods have been developed for generating mutants (see, generally, Sambrook et al., supra; Ausubel, et al., supra, and the discussion above). Briefly, preferred methods for generating a few nucleotide substitutions utilize an oligonucleotide that spans the base or bases to be mutated and contains the mutated base or bases. The oligonucleotide is hybridized to complementary single stranded nucleic acid and second strand synthesis is primed from the oligonucleotide. The double-stranded nucleic acid is prepared for transformation into host cells, typically E. coli, but alternatively, other prokaryotes, yeast or other eukaryotes. Standard screening and vector growth protocols are used to identify mutant sequences and obtain high yields.

Similarly, deletions and/or insertions of the caspase or rev-caspase genes may be constructed by any of a variety of known methods as discussed supra. For example, the gene can be digested with restriction enzymes and religated such that a sequence is deleted or religated with additional sequences such that an insertion or large substitution is made. Other means of generating variant sequences may be employed with methods known in the art, for example those described in Sambrook et al. (supra) and Ausubel et al. (supra). Verification of variant sequences is typically accomplished by restriction enzyme mapping, sequence analysis, or probe hybridization. Variants which catalyze Asp-specific cleavages are useful in the context of this invention.

B. Rev-Caspases

The caspases of the present invention are generated by rearranging the gene sequence of the caspase gene such that the nucleic acid sequence encoding the small subunit precedes (is 5′ to) the nucleic acid sequence encoding the large subunit. These rearranged caspases are called rev-caspases.

1. Structure of Rev-caspases

The rev-caspases of the present invention comprise at least a portion of the small subunit and at least a portion of the large subunit. In preferred embodiments, the prodomain or a portion thereof (see FIGS. 1 , 10 ) and/or an intervening sequence or a portion thereof (see FIG. 10 ) are also present in rev-caspase. In other preferred embodiments, a “linker” region is located between the small and large subunits.

The boundaries of the small subunit and large subunit are identified either experimentally by amino acid sequence analysis of the mature caspase or by inspection of structural homology (e.g., the conserved Asp-X cleavage site). For exemplary purposes, the Table below presents the boundaries of the prodomain (P), large subunit (LS), intervening sequence (I), and small subunit (SS) of human caspase-1 through -10. The nucleotide numbers refer to the nucleotides in SEQ ID NOs:4-33 and in FIGS. 11-20 .

			Intervening
Caspase	Prodomain	Large Subunit	sequence	Small Subunit
Caspase-1	1-357	358-891	892-948	949-1212
Caspase-2	1-456	457-948	949-990	991-1305
Caspase-3	1-84	85-525		526-831
Caspase-4	1-240	241-810	811-867	868-1131
Caspase-5	1-363	364-933		934-1254
Caspase-6	1-69	70-537	538-579	580-879
Caspase-7	1-69	70-594		595-909
Caspase-8	1-681	682-1173		1174-1488
Caspase-9	1-390	391-945	946-990	991-1248
Caspase-10	1-657	658-1116		1117-1437

As noted above, a portion of the large subunit and small subunit may be used in rev-caspase constructs. When designing rev-caspases that contain a portion of these subunits, the active site (e.g., QACXG, where X is Arg, Gln, or Gly); (SEQ ID NO:51), which is located near the C-terminus of the large subunit should not be deleted if protease activity is desired. Preferably, the 3-dimensional structure as determined by X-ray crystallography (see Mittl et al., J. Biol. Chem., 272: 6539-6547, 1997; Rotondu et al., Nat. Struct. Biol., 3: 619-625, 1996; Walker et al., Cell, 78: 343-352, 1994; Wilson et al., Nature, 370: 270-275, 1994) is maintained. For example, from the x-ray crystallographic structures of caspases, the amino acids, that are important in binding substrates have been identified. Likewise, substitutions of amino acids in the active site may be detrimental to maintaining activity. Although it is preferred that both subunits are derived from the same caspase, combinations of subunits from different caspases and/or from different species may be used.

The prodomain (sometimes called an N-terminal peptide) is generally not required for enzyme activity and is normally released in vivo. Rev-caspases of the present invention optionally have a prodomain or portion thereof. Similarly, the intervening sequence, which is present in certain caspases, is optional for inclusion in rev-caspases.

In certain embodiments, a linker region is engineered between the small and large subunits. A “linker region”, as used herein, refers to a peptide of from about 5 to about 50 amino acids. In preferred embodiments, the linker may contain a protease sensitive or cleavage site. Any site recognized by an intracellular protease may be used. In addition, multiple protease sensitive sites may be tandemly arranged in the same linker. Preferred protease sensitive sites are susceptible to cleavage by caspases or by viral proteases. Preferred caspase sensitive sites include, but are not limited to DXXDG (wherein X is any amino acid; SEQ ID NO:37); DEVDG (SEQ ID NO:38), IETDG (SEQ ID NO:39), YVADG (SEQ ID NO:40), YVHDG (SEQ ID NO:41), and WEHDG (SEQ ID NO:42). Furthermore, the Gly residue may be Ala or another small amino acid. The latter three sites are specifically cleaved by caspases-1, -4, and -5. Other sites specifically cleaved by only one or a few caspases are preferred in certain embodiments. Viral proteases cleavage sites include, but are not limited to, those recognized and cleaved by HIV protease, HCV (hepatitis C virus) protease, HBV (hepatitis B virus) protease, and rhinovirus protease.

2. Construction of Rev-caspases

Rev-caspases may be constructed from caspase sequences by a variety of methods known in the art. A preferred method is amplification (e.g., polymerase chain reaction (PCR)) to selectively amplify the individual subunits and place these in cloning vectors such as pUC such as described in Example 1. Moreover, such PCR reactions can be performed in a variety of ways such that the primers used for amplification contain specific restriction endonuclease sites to facilitate insertion into a vector.

Further, a variety of other methodologies besides PCR may be used to attain the desired rearrangement. For example, one skilled in the art may employ isothermal methods to amplify the nucleotide sequence of interest, using existing restriction endonuclease sites present in the nucleotide sequence to excise and insert sequences, or by the introduction of distinct restriction endonuclease sites by site-directed mutagenesis followed by excision and insertion. These and other methods are described in Sambrook et al., supra; Ausubel, et al., supra. Briefly, one methodology is to generate single-stranded cDNA of the caspase of interest, followed by annealing a primer, which is complementary except for the desired alteration (e.g., a small insertion, deletion, or mutation such that a unique restriction site is created between the large and small subunits and/or at the 5′ and 3′ ends of both subunits). Bacterial cells are transformed and screened for those which contain desired construct. This construct is then digested to liberate the subunit sequences, which can then be purified and religated into the appropriate orientation.

As indicated above, rev-caspase genes may be manipulated to contain insertions, deletions or substitutions. Moreover, such variant rev-caspase genes useful in the context of this invention include those which facilitate Asp-specific cleavages indicative of caspase activity. Further, variants which are incapable of being cleaved into separate subunits are encompassed within the context of this invention, if those variants are able to facilitate Asp-specific cleavages by way of a cysteine-containing active site. By way of guidance, amino acids involved in catalysis, Asp recognition in substrate, and P2-P4 substrate recognition are provided in FIG. 6 .

C. Vectors, Host Cells and Means of Expressing and Producing Protein

Caspase may be expressed in a variety of host organisms. In certain embodiments, caspase is produced in bacteria, such as E. coli, or mammalian cells (e.g., CHO and COS-7), for which many expression vectors have been developed and are available. Other suitable host organisms include other bacterial species, and eukaryotes, such as yeast (e.g., Saccharomyces cerevisiae ), and insect cells (e.g., Sf9).

A DNA sequence encoding rev-caspase is introduced into an expression vector appropriate for the host. In certain embodiments, rev-caspase is inserted into a vector such that a fusion protein is produced. The rev-caspase sequence is derived as described herein. As discussed above, the sequence may contain alternative codons for each amino acid with multiple codons. The alternative codons can be chosen as “optimal” for the host species. Restriction sites are typically incorporated into the primer sequences and are chosen with regard to the cloning site of the vector. If necessary, translational initiation and termination codons can be engineered into the primer sequences.

At minimum, the vector must contain a promoter sequence. As used herein, a “promoter” refers to a nucleotide sequence that contains elements that direct the transcription of a linked gene. At minimum, a promoter contains an RNA polymerase binding site. More typically, in eukaryotes, promoter sequences contain binding sites for other transcriptional factors that control the rate and timing of gene expression. Such sites include TATA box, CAAT box, POU box, AP1 binding site, and the like. Promoter regions may also contain enhancer elements. When a promoter is linked to a gene so as to enable transcription of the gene, it is “operatively linked”.

Other regulatory sequences may be included. Such sequences include a transcription termination signal sequence, secretion signal sequence, origin of replication, selectable marker, and the like. The regulatory sequences are operationally associated with one another to allow transcription or translation.

The expression vectors used herein include a promoter designed for expression of the proteins in a host cell (e.g., bacterial). Suitable promoters are widely available and are well known in the art. Inducible or constitutive promoters are preferred. Such promoters for expression in bacteria include promoters from the T7 phage and other phages, such as T3, T5, and SP6, and the trp, lpp, and lac operons. Hybrid promoters (see, U.S. Pat. No. 4,551,433), such as tac and trc, may also be used. Promoters for expression in eukaryotic cells include the P10 or polyhedron gene promoter of baculovirus/insect cell expression systems (see, e.g., U.S. Pat. Nos. 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784), MMTV LTR, CMV IE promoter, RSV LTR, SV40, metallothionein promoter (see, e.g., U.S. Pat. No. 4,870,009) and the like.

The promoter controlling transcription of rev-caspase may itself be controlled by a repressor. In some systems, the promoter can be derepressed by altering the physiological conditions of the cell, for example, by the addition of a molecule that competitively binds the repressor, or by altering the temperature of the growth media. Preferred repressor proteins include, but are not limited to the E. coli lacI repressor responsive to IPTG induction, the temperature sensitive λc1857 repressor, and the like. The E. coli lacI repressor is preferred.

In other preferred embodiments, the vector also includes a transcription terminator sequence. A “transcription terminator region” has either a sequence that provides a signal that terminates transcription by the polymerase that recognizes the selected promoter and/or a signal sequence for polyadenylation.

Preferably, the vector is capable of replication in the host cells. Thus, when the host cell is a bacterium, the vector preferably contains a bacterial origin of replication. Preferred bacterial origins of replication include the fl-ori and col E1 origins of replication, especially the ori derived from pUC plasmids. In yeast, ARS or CEN sequences can be used to assure replication. A well-used system in mammalian cells is SV40 ori.

The plasmids also preferably include at least one selectable marker that is functional in the host. A selectable marker gene includes any gene that confers a phenotype on the host that allows transformed cells to be identified and selectively grown. Suitable selectable marker genes for bacterial hosts include the ampicillin resistance gene (Amp r ), tetracycline resistance gene (Tc r ) and the kanamycin resistance gene (Kan r ). The kanamycin resistance gene is presently preferred. Suitable markers for eukaryotes usually require a complementary deficiency in the host (e.g., thymidine kinase (tk) in tk-hosts). However, drug markers are also available (e.g., G418 resistance and hygromycin resistance).

The sequence of nucleotides encoding rev-caspase may also include a secretion signal, whereby the resulting peptide is a precursor protein processed and secreted. The resulting processed protein may be recovered from the periplasmic space or the fermentation medium. Secretion signals suitable for use are widely available and are well known in the art (von Heijne, J. Mol. Biol. 184: 99-105, 1985). Prokaryotic and eukaryotic secretion signals that are functional in E. coli (or other host) may be employed. The presently preferred secretion signals include, but are not limited to, those encoded by the following E. coli genes: pelB (Lei et al., J. Bacteriol. 169: 4379, 1987), phoA, ompA, ompT, ompF, ompC, beta-lactamase, and alkaline phosphatase.

One skilled in the art appreciates that there are a wide variety of suitable vectors for expression in bacterial cells and which are readily obtainable. Vectors such as the pET series (Novagen, Madison, Wis.), the tac and trc series (Pharmacia, Uppsala, Sweden), pTTQ18 (Amersham International plc, England), pACYC 177, pGEX series, and the like are suitable for expression of a rev-caspase. Baculovirus vectors, such as pBlueBac (see, e.g., U.S. Pat. Nos. 5,278,050, 5,244,805, 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784; available from Invitrogen, San Diego) may be used for expression in insect cells, such as Spodoptera frugiperda sf9 cells (see, U.S. Pat. No. 4,745,051). The choice of a bacterial host for the expression of a rev-caspase is dictated in part by the vector. Commercially available vectors are paired with suitable hosts.

A wide variety of suitable vectors for expression in eukaryotic cells are available. Such vectors include pCMVLacI, pXT1 (Stratagene Cloning Systems, La Jolla, Calif.); pCDNA series, pREP series, pEBVHis (Invitrogen, Carlsbad, Calif.). In certain embodiments, rev-caspase gene is cloned into a gene targeting vector, such as pMClneo, a pOG series vector (Stratagene Cloning Systems).

Rev-caspase is isolated by standard methods, such as affinity chromatography, size exclusion chromatography, metal ion chromatography, ionic exchange chromatography, HPLC, and other known protein isolation methods. (see generally Ausubel et al. supra; Sambrook et al. supra). An isolated purified protein gives a single band on SDS-PAGE when stained with Coomassie blue.

Rev-caspase may be expressed as a hexa-his fusion protein and isolated by metal-containing chromatography, such as nickel-coupled beads. Briefly, a sequence encoding His 6 is linked to a DNA sequence encoding a rev-caspase. Although the His 6 sequence can be positioned anywhere in the molecule, preferably it is linked at the 3′ end immediately preceding the termination codon. The fusion may be constructed by any of a variety of methods. A convenient method is amplification of the rev-caspase gene using a downstream primer that contains the codons for His 6 .

Purified rev-caspase protein may be used in assays to screen for inhibitory drugs. These assays may be performed in vitro or in vivo and utilize any of the methods described herein or that are known in the art. The protein may also be crystallized and subjected to X-ray analysis to determine its 3-dimensional structure or used to raise antibodies.

D. Uses of Rev-caspase Gene and Gene Product

1. Inhibitors and Enhancers of Caspase Activity

Candidate inhibitors and enhancers may be isolated or procured from a variety of sources, such as bacteria, fungi, plants, parasites, libraries of chemicals, peptides or peptide derivatives and the like. Inhibitors and enhancers may be also be rationally designed, based on the protein structure determined from X-ray crystallography (see, Mittl et al., J. Biol. Chem., 272: 6539-6547, 1997). In certain preferred embodiments, the inhibitor targets a specific caspase (e.g., caspase-3 and not any other caspases).

Without being held to a particular mechanism, the inhibitor may act by preventing processing of caspase or by preventing enzymatic activity, or by other mechanism. The inhibitor may act directly or indirectly. In preferred embodiments, inhibitors interfere in the processing of the caspase protein. In other preferred embodiments, the inhibitors are small molecules. In a most preferred embodiment, the inhibitors prevent apoptosis. Inhibitors should have a minimum of side effects and are preferably non-toxic. Inhibitors that can penetrate cells are preferred.

In addition, enhancers of caspase activity or expression are desirable in certain circumstances. At times, increasing apoptosis will have a therapeutic effect. For example, tumors or cells that mediate autoimmune diseases are appropriate cells for destruction. Enhancers may increase the rate or efficiency of caspase processing, increase transcription or translation, or act through other mechanisms. As is apparent to one skilled in the art, many of the guidelines presented above apply to the design of enhancers as well.

Screening assays for inhibitors and enhancers will vary according to the type of inhibitor or enhancer and the nature of the activity that is being affected. Assays may be performed in vitro or in vivo. In general, in vitro assays are designed to evaluate caspase protein processing or caspase enzymatic activity, and in vivo assays are designed to evaluate caspase protein processing, caspase enzymatic activity, apoptosis, or caspase cleavage of substrate. In any of the assays, a statistically significant increase or decrease compared to a proper control is indicative of enhancement or inhibition.

One in vitro assay can be performed by examining the effect of a candidate compound on processing of rev-caspase into two subunits. Briefly, a cleavable form of rev-caspase, that is a primary translation product, is obtained from an in vitro translation system. The cleavable form of rev-caspase is preferably constructed to be auto-cleaved, but can be constructed to be cleaved by other protease components present or added to the reaction. This primary product is contacted with or without or translated in the presence or absence of a candidate compound and assessed for appearance of the two subunits to facilitate detection, typically, the primary product of rev-caspase is labeled during translation, cell viability, and the like. The two subunits may be readily detected by autoradiography after gel electrophoresis. One skilled in the art will recognize that other methods of labeling and detection may be used alternatively.

An alternative in vitro assay is designed to measure cleavage of a caspase substrate (e.g., Acetyl DEVD-aminomethyl coumarin (amc) (SEQ ID NO:52), lamin, PRPP, and the like). Substrate turnover may be assayed using either cleavable or noncleavable rev-caspase. Briefly, in this method, rev-caspase is translated and allowed sufficient time to be processed, if a cleavable rev-caspase is being used. The caspase substrate along with the candidate compound is added to the reaction. Detection of cleaved substrate is performed by any one of a variety of standard methods. Generally, the substrate will be labeled and followed by an appropriate detection means.

Moreover, any known enzymatic analysis can be used to follow the inhibitory or enhancing ability of a candidate compound with regard to a rev-caspase of this invention. For example, one could express the rev-caspase of interest in a cell line be it bacterial, insect, mammalian or other, either in cleavable or noncleavable form and purify the rev-caspase. The purified rev-caspase could then be used in a variety of assays to follow its catalytic ability in the presence of candidate compounds, as noted above. Such methods of expressing and purifying recombinant proteins are known in the art and examples can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989 as well as in a number of other sources.

In vivo assays are typically performed in cells transfected either transiently or stably with an expression vector containing a rev-caspase gene, such as those described herein. These cells are used to measure rev-caspase processing, substrate turnover, or apoptosis in the presence or absence of a candidate compound. When assaying apoptosis, a variety of cell analyses may be used including, for example, dye staining and microscopy to examine nucleic acid fragmentation and porosity of the cells. Further, in vivo assaying for the ability of the transfected rev-caspase to cleave known substrates that are co-transfected or placed in the cell culture media in the presence of the candidate compound can be performed thereby allowing for the detection and determination of substrate turnover.

The assays briefly described herein may be used to identify an enhance or inhibitor that is specific for an individual caspase. In a preferred embodiment candidate compounds would be analyzed using a variety of rev-caspases (e.g., rev-caspase-1 through rev-caspase-14) to identify specific for individual caspases.

A variety of methodologies exist can be used to investigate the effect of a candidate compound. Such methodologies are those commonly used to analyze enzymatic reactions and include, for example, SDS-PAGE, spectroscopy, HPLC analysis, autoradiography, chemiluminescence, chromogenic reactions, and immunochemistry (e.g., blotting, precipitating, etc.).

Inhibitors and enhancers may be used in the context of this invention to exert control over the cell death process or cytokine activation (e.g., IL-1, which is activated by caspase-1). Thus, these inhibitors and enhancers will have utility in diseases characterized by either excessive or insufficient levels of apoptosis. Inhibitors of proteases have potential to treat the major neurodegenerative diseases: stroke, Parkinson's Disease, Alzheimer's Disease, and ALS. As well, caspase protease inhibitors may be used to inhibit apoptosis in the heart following myocardial infarction, in the kidney following acute ischemia, and in diseases of the liver. In other embodiments, inhibitors of caspase-1 can be used to inhibit the release of the pro-inflammatory IL-1β, and thus may provide therapeutic benefit in treating inflammation and/or autoimmune disorders. Enhancers of caspase activity may be used in contexts when apoptosis or cytokine activation are desired. For example, inducing or increasing apoptosis in cancer cells or aberrantly proliferating cells may be effected by delivery of a caspase enhancer.

The inhibitors and enhancers may be further coupled with a targeting moiety that binds a cell surface receptor specific to the cells. Administration of inhibitors or enhancers will generally follow established protocols. The compounds of the present invention may be administered either alone, or as a pharmaceutical composition. Briefly, pharmaceutical compositions of the present invention may comprise one or more of the inhibitors or enhancers as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like, carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and preservatives. In addition, pharmaceutical compositions of the present invention may also contain one or more additional active ingredients.

Compositions of the present invention may be formulated for the manner of administration indicated, including for example, for oral, nasal, venous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration. Within other embodiments of the invention, the compositions described herein may be administered as part of a sustained release implant. Within yet other embodiments, compositions of the present invention may be formulized as a lyophilizate, utilizing appropriate excipients which provide stability as a lyophilizate, and subsequent to rehydration.

2. Gene Therapy

As noted above, rev-caspases may be delivered to cells as part of gene delivery vehicles. In many diseases and syndromes, too little apoptosis is an important feature in their development. Treatment of many autoimmune diseases and tumors would benefit from increased apoptosis. One means to increase apoptosis is to provide target cells with caspase genes in an expressible form. This may be accomplished by delivery of DNA or cDNA capable of in vivo transcription of the rev-caspase. More specifically, in order to produce rev-caspases in vivo, a nucleic acid sequence coding for the rev-caspase is placed under the control of a eukaryotic promoter (e.g., a pol III promoter, CMV or SV40 promoter). Where it is desired to more specifically control transcription, the rev-caspase may be placed under the control of a tissue or cell specific promoter (e.g., to target cells in the liver), or an inducible promoter, such as metallothionein.

Many techniques for introduction of nucleic acids into cells are known. Such methods include retroviral vectors and subsequent retrovirus infection, adenoviral or adeno-associated viral vectors and subsequent infection, and complexes of nucleic acid with a condensing agent (e.g., poly-lysine). These complexes or viral vectors may be targeted to particular cell types by way of a ligand incorporated into the vehicle. Many ligands specific for tumor cells and other cells are well known in the art.

A wide variety of vectors may be utilized within the context of the present invention, including for example, plasmids, viruses, retrotransposons and cosmids. Representative examples include adenoviral vectors (e.g., WO 94/26914, WO 93/9191; Yei et al., Gene Therapy 1: 192-200, 1994; Kolls et al., PNAS 91(1):215-219, 1994; Kass-Eisler et al., PNAS 90(24):11498-502, 1993; Guzman et al., Circulation 88(6):2838-48, 1993; Guzman et al., Cir. Res. 73(6):1202-1207, 1993; Zabner et al., Cell 75(2):207-216, 1993; Li et al., Hum Gene Ther. 4(4):403-409, 1993; Caillaud et al., Eur. J Neurosci. 5(10):1287-1291, 1993), adeno-associated type 1 (“AAV-1”) or adeno-associated type 2 (“AAV-2”) vectors (see WO 95/13365; Flotte et al., PNAS 90(22):10613-10617, 1993), hepatitis delta vectors, live, attenuated delta viruses and herpes viral vectors (e.g., U.S. Pat. No. 5,288,641), as well as vectors which are disclosed within U.S. Pat. No. 5,166,320. Other representative vectors include retroviral vectors (e.g., EP 0 415 731; WO 90/07936; WO 91/02805; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218.

Within certain aspects of the invention, nucleic acid molecules that encode the rev-caspase may be introduced into a host cell utilizing a vehicle, or by various physical methods. Representative examples of such methods include transformation using calcium phosphate precipitation (Dubensky et al., PNAS 81: 7529-7533, 1984), direct microinjection of such nucleic acid molecules into intact target cells (Acsadi et al., Nature 352: 815-818, 1991), and electroporation whereby cells suspended in a conducting solution are subjected to an intense electric field in order to transiently polarize the membrane, allowing entry of the nucleic acid molecules. Other procedures include the use of nucleic acid molecules linked to an inactive adenovirus (Cotton et al., PNAS 89: 6094, 1990), lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7417, 1989), microprojectile bombardment (Williams et al., PNAS 88: 2726-2730, 1991), polyeation compounds such as polylysine, receptor specific ligands, liposomes entrapping the nucleic acid molecules, spheroplast fusion whereby E. coli containing the nucleic acid molecules are stripped of their outer cell walls and fused to animal cells using polyethylene glycol, viral transduction, (Cline et al., Pharmac. Ther. 29: 69, 1985; and Friedmann et al., Science 244: 1275, 1989), and DNA ligand (Wu et al, J. of Biol. Chem. 264: 16985-16987, 1989), as well as psoralen inactivated viruses such as Sendai or Adenovirus. In one embodiment, the rev-caspase construct is introduced into the host cell using a liposome.

As noted above, pharmaceutical compositions also are provided by this invention. These compositions may contain any of the above described inhibitors, enhancers, DNA molecules, vectors or host cells, along with a pharmaceutically or physiologically acceptable carrier, excipients or diluents. Generally, such carriers should be nontoxic to recipients at the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails combining the therapeutic agent with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents.

In addition, the pharmaceutical compositions of the present invention may be prepared for administration by a variety of different routes, including for example intraarticularly, intracranially, intradermally, intrahepatically, intramuscularly, intraocularly, intraperitoneally, intrathecally, intravenously, subcutaneously or even directly into a tumor. In addition, pharmaceutical compositions of the present invention may be placed within containers, along with packaging material which provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions will include a tangible expression describing the reagent concentration, as well as within certain embodiments, relative amounts of excipient ingredients or diluents (e.g., water, saline or PBS) which may be necessary to reconstitute the pharmaceutical composition. Pharmaceutical compositions are useful for both diagnostic or therapeutic purposes.

Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease. Dosages may be determined most accurately during clinical trials. Patients may be monitored for therapeutic effectiveness by appropriate technology, including signs of clinical exacerbation, imaging and the like.

The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES

Example 1

Generation of cDNAs Expressing Rev-caspase-3 and 6 Precursors

Generation of cDNAs encoding rev-caspase-3 and 6 precursors were generated by PCR. The large (LS) and small subunits (SS) of caspase-3 were amplified with the following primers using the caspase-3 cDNA as a template:

LS-forward, ATGGAGAACACTGAAAACTCAG (SEQ ID NO:43);
LS-reverse, GTCATCATCAACACCTCAGTCT (SEQ ID NO:44);
SS-forward, GGATCCATGATTGAGACAGACAGTGG (SEQ ID NO:45);
SS-reverse, ATCAACTTCATCGTGATAAAAATAGAGTTC (SEQ ID NO:46).

The PCR products were cloned separately into the Sma I site of pBluescript KS + . The small subunit was then excised from KS + -vector with Bam HI and inserted into the Bam HI site of the second KS + -vector which contains the large subunit. This places the small subunit in-frame 5′ to the large subunit. Rev-caspase-6 was amplified and cloned in the KS + -vector in a similar way. The following PCR primers were used with caspase-6-His6 cDNA as a template:

LS-forward, ATGAGCTCGGCCTCGGGG   (SEQ ID NO:47);
LS-reverse, TTAATCTACTACATCCAAAGG (SEQ ID NO:48);
SS-forward, GGATCCATGGTAGAAATAGATGCAGCCTCCGTTTAC (SEQ ID NO:49)
SS-reverse, ATCAATTTCAACGTGGTGGTGGTGGTGGTGC (SEQ ID NO:50).

The resulting nucleotide sequences were such that the wild type subunit order was reversed thus creating a contiguous nucleotide sequence wherein the coding region for the small subunit preceded that of the large subunit (See FIG. 1 ). The engineered contiguous caspase-3 and 6 molecules (i.e., rev-caspase molecules) in which the SS was fused in frame N-terminal to the LS, and a cleavage site (DEVDG in the case of caspase-3; VEIDS in the case of caspase-6 (these internal cleavage sites were designed to be specific for the caspase in which it was introduced in order to investigate the autocatalytic activity of the particular caspase)) was introduced between the two subunits and is depicted in FIG. 1B and C .

To express the rev-caspases in bacteria, their cDNAs were excised with Bam HI/Xho I and subcloned into the bacterial expression vector pET28a (Novagen, Inc.) in-frame with the T7-tag of this vector.

Example 2

Expression of Rev-caspase in Mammlian Cells and Assay for Apoptosis

To express the rev-caspases in mammalian cells and assay their apoptotic activity, they were amplified with the T7-tag primer and the LS-reverse primers using the pET28a constructs as templates, and subcloned into the mammalian double expression vector pRSC-LacZ (MacFarlane et al., J. Biol. Chem., 272: 25417-25420, 1997; Tsang et al., Bio/Technology, 22: 68, 1997). This vector allows the expression of lacZ under the Rous Sarcoma virus promoter, and the test cDNA under the CMV promoter. To assay for apoptosis, MCF-7 or 293 cells were transfected, using the method commercially available as the Lipofect Amine method (Life Technologies, Inc.), with the pRSC-LacZ constructs in the presence or absence of different apoptosis-inhibitors. 30 h after transfection cells were stained with β-galactosidase and examined for morphological signs of apoptosis. The percentage of round blue apoptotic cells (mean±SD) were represented as a function of total blue cells under each condition (n≧3).

Example 3

In Vitro Translation of Caspases

35 S-labeled caspases (wild-type and rev-caspases) were obtained by in vitro translation in the presence of 35 S-methionine using a coupled transcription/translation system in rabbit reticulocyte lysate using TNT Kit (Promega) according to the manufacturer's recommendations. Unlike the wild-type caspase-3 and 6, FIG. 2A demonstrates that rev-caspase-3 and 6 were able to undergo autocatalytic processing in the in vitro translation reaction. Further, this processing was completely inhibited by mutation of the active site Cys of rev-caspase-3 and 6 ( FIG. 2A , lanes 3 and 6) and by selected caspase inhibitors (See Example 4). Because the in vitro translated products are present at very low concentration in the reaction mixture, the observed cleavage must be attributed to an intramolecular processing within the caspase heterotetramer.

Example 4

Effects of Inhibitors on Rev-caspase-3 and 6 Activity

To test the effect of selected caspase inhibitors on the autocatalytic activity of rev-caspase-3 and 6, the rev-caspases were translated as in Example 3, but in the presence of varying amounts of inhibitors. As demonstrated by FIG. 2B , in the presence of increasing amounts of DEVD-CHO (SEQ ID NO:52), a decrease in the amount of cleavage products and a corresponding increase in the amount of the revcaspase-3 precursor was observed. This corresponded to nearly 50-90% inhibition of the autocatalytic activity of rev-caspase-3 at 40-400 nM concentration. However, the same concentrations of this inhibitor had little effect on the autocatalytic activity of rev-caspase-6 (FIG. 2 C). This is consistent with earlier observations that caspase-6 is poorly inhibited by DEVD-CHO (SEQ ID NO:52); see Srinivasula et al., J. Biol. Chem., 271: 27099-27106, 1996. On the other hand, as is apparent from inspection of FIGS. 2B and 2C, z-VAD-fmk had nearly an equal inhibitory effect on rev-caspase-3 and 6 autocatalytic activity at the concentration used in this experiment. Nevertheless, nearly 10-fold more of z-VAD-fink than DEVD-CHO (SEQ ID NO:52) was required to obtain complete inhibition of caspase-3 activity. Similarly, baculovirus p35 had nearly an equal inhibitory effect on rev-caspase-3 and 6 autocatalytic activity (data not shown).

Example 5

Caspase-3 and 6 Specificity Retention of Bacterially Expressed Rev-caspase-3 and -6

At limited caspase concentrations, poly(ADP) ribose polymerase (PARP) is specifically cleaved by caspase-3 and 7 but not other caspases. Similarly, lamin is specifically cleaved by caspase-6 but not other caspases. To compare the activity of the wild-type and rev-caspase-3 and 6 towards PARP and lamin, rev-caspase-3 and 6 were expressed in bacteria and then incubated with the two substrates PARP and lamin. As shown in FIGS. 3A and B , the activity of the rev-caspases towards these two substrates were indistinguishable from their wild-type counterparts. Both caspase-3 variants (rev and WT), but not caspase-6 variants efficiently cleaved PARP. In contrast, both caspase-6 variants, but not caspase-3 variants efficiently cleaved lamin. These results demonstrate that the mature caspases generated from the rev and the wild type constructs have identical substrate specificity.

Example 6

Induction of Apoptosis in Mammalian Cells by Rev-caspase-3 and -6

To determine the apoptotic activity of rev-caspase-3 and 6 in vivo, the rev-caspases were expressed in human MCF-7 cells, transfected as explained above in Example 2. As evidenced by FIGS. 4A and B , unlike the wild type caspase-3 and 6, the rev-caspases potently induced apoptosis in nearly 90% of the transfected cells. Overexpression of Bc1-2 or CrmA, which protect against different forms of apoptosis, did not significantly reduce their apoptotic activity. Nevertheless, overexpression of the baculovirus p35, which inhibits the activity of most caspases, partially protected against their apoptotic activity. Also, incubation of the transfected cells in the presence of 100 μM z-VAD-fmk, dramatically reduced their apoptotic activity to nearly 30%. These data demonstrate directly that the activity of caspase-3 and 6 are downstream of the CrmA and Bcl-2 block in the apoptotic cascade, and can only be inhibited by high concentration of the pancaspase-inhibitor z-VAD-fmk.

Example 7

Activity of Noncleavable Rev-caspase-3

To demonstrate that the rev-caspase molecules are inherently active and do not require separation of the two subunits and that the two subunits are derived from the same contiguous molecule, the DEVD (SEQ ID NO:52) site was removed and Asp9 and 28, that are present between the two subunits of rev-caspase-3, were mutated (see FIG. 2 A). However, to follow the activity of this molecule a cleavable 35 residue long His6-T7-tag N-terminal to the IETD (SEQ ID NO:53) site was introduced (see FIG. 1 B). FIG. 5 demonstrates that upon in vitro translation of this molecule, as described in Example 3 above, there was no evidence of cleavage between the two subunits. Nevertheless, the translated molecule was active as evident from its ability to cleave its T7-tag to form the p32 species (FIG. 5 ). In the presence of 400 nM DEVD-CHO (SEQ ID NO:52), processing of the T7-tag was inhibited and only the full length p34 species can be seen. Furthermore, expression of this molecule into MCF-7 cells potently induced apoptosis in these cells. These data demonstrate that when the two subunits of a caspase are rearranged in the reverse order, it is not necessary to separate them from each other to generate an active caspase. Thus by mimicking the mature caspase structure, it is possible to design a contiguous active caspase molecules.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

#             SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS: 116
<210> SEQ ID NO 1
<211> LENGTH: 873
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Rev-Caspase-3  contructed
#from human caspase -3
<400> SEQUENCE: 1
atgattgaga cagacagtgg tgttgatgat gacatggcgt gtcataaaat ac
#cagtggag     60
gccgacttct tgtatgcata ctccacagca cctggttatt attcttggcg aa
#attcaaag    120
gatggctcct ggttcatcca gtcgctttgt gccatgctga aacagtatgc cg
#acaagctt    180
gaatttatgc acattcttac ccgggttaac cgaaaggtgg caacagaatt tg
#agtccttt    240
tcctttgacg ctacttttca tgcaaagaaa cagattccat gtattgtttc ca
#tgctcaca    300
aaagaactct atttttatca cgatgaagtt gatgggggat cccccatgga ga
#acactgaa    360
aactcagtgg attcaaaatc cattaaaaat ttggaaccaa agatcataca tg
#gaagcgaa    420
tcaatggact ctggaatatc cctggacaac agttataaaa tggattatcc tg
#agatgggt    480
ttatgtataa taattaataa taagaatttt cataagagca ctggaatgac at
#ctcggtct    540
ggtacagatg tcgatgcagc aaacctcagg gaaacattca gaaacttgaa at
#atgaagtc    600
aggaataaaa atgatcttac acgtgaagaa attgtggaat tgatgcgtga tg
#tttctaaa    660
gaagatcaca gcaaaaggag cagttttgtt tgtgtgcttc tgagccatgg tg
#aagaagga    720
ataatttttg gaacaaatgg acctgttgac ctgaaaaaaa taacaaactt tt
#tcagaggg    780
gatcgttgta gaagtctaac tggaaaaccc aaacttttca ttattcaggc ct
#gccgtggt    840
acagaactgg actgtggcat tgagacagac tga
#
#        873
<210> SEQ ID NO 2
<211> LENGTH: 858
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Uncleavalbe Rev-Caspase-3
#contructed from
human caspase -3
<400> SEQUENCE: 2
atgattgaga cagacagtgg tgttgatgat gacatggcgt gtcataaaat ac
#cagtggag     60
gccgacttct tgtatgcata ctccacagca cctggttatt attcttggcg aa
#attcaaag    120
gatggctcct ggttcatcca gtcgctttgt gccatgctga aacagtatgc cg
#acaagctt    180
gaatttatgc acattcttac ccgggttaac cgaaaggtgg caacagaatt tg
#agtccttt    240
tcctttgacg ctacttttca tgcaaagaaa cagattccat gtattgtttc ca
#tgctcaca    300
aaagaactct atttttatca cggatccccc atggagaaca ctgaaaactc ag
#tggcttca    360
aaatccatta aaaatttgga accaaagatc atacatggaa gcgaatcaat gg
#cctctgga    420
atatccctgg acaacagtta taaaatggat tatcctgaga tgggtttatg ta
#taataatt    480
aataataaga attttcataa gagcactgga atgacatctc ggtctggtac ag
#atgtcgat    540
gcagcaaacc tcagggaaac attcagaaac ttgaaatatg aagtcaggaa ta
#aaaatgat    600
cttacacgtg aagaaattgt ggaattgatg cgtgatgttt ctaaagaaga tc
#acagcaaa    660
aggagcagtt ttgtttgtgt gcttctgagc catggtgaag aaggaataat tt
#ttggaaca    720
aatggacctg ttgacctgaa aaaaataaca aactttttca gaggggatcg tt
#gtagaagt    780
ctaactggaa aacccaaact tttcattatt caggcctgcc gtggtacaga ac
#tggactgt    840
ggcattgaga cagactga
#
#
# 858
<210> SEQ ID NO 3
<211> LENGTH: 903
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Rev-Caspase-6 contructed fro
#m human caspase -3
<400> SEQUENCE: 3
atggtagaaa tagatgcagc ctccgtttac acgctgcctg ctggagctga ct
#tcctcatg     60
tgttactctg ttgcagaagg atattattct caccgggaaa ctgtgaacgg ct
#catggtac    120
attcaagatt tgtgtgagat gttgggaaaa tatggctcct ccttagagtt ca
#cagaactc    180
ctcacactgg tgaacaggaa agtttctcag cgccgagtgg acttttgcaa ag
#acccaagt    240
gcaattggaa agaagcaggt tccctgtttt gcctcaatgc taactaaaaa gc
#tgcatttc    300
tttccaaaat ctaatctcga gcaccaccac caccaccacg ttgaaattga tg
#ggggatcc    360
cccatgagct cggcctcggg gctccgcagg gggcacccgg caggtgggga ag
#aaaacatg    420
acagaaacag atgccttcta taaaagagaa atgtttgatc cggcagaaaa gt
#acaaaatg    480
gaccacagga ggagaggaat tgctttaatc ttcaatcatg agaggttctt tt
#ggcactta    540
acactgccag aaaggcgggg cacctgcgca gatagagaca atcttacccg ca
#ggttttca    600
gatctaggat ttgaagtgaa atgctttaat gatcttaaag cagaagaact ac
#tgctcaaa    660
attcatgagg tgtcaactgt tagccacgca gatgccgatt gctttgtgtg tg
#tcttcctg    720
agccatggcg aaggcaatca catttatgca tatgatgcta aaatcgaaat tc
#agacatta    780
actggcttgt tcaaaggaga caagtgtcac agcctggttg gaaaacccaa ga
#tatttatc    840
atccaggcat gtcggggaaa ccagcacgat gtgccagtca ttcctttgga tg
#tagtagat    900
taa
#
#
#            903
<210> SEQ ID NO 4
<211> LENGTH: 1215
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 4
atggccgaca aggtcctgaa ggagaagaga aagctgttta tccgttccat gg
#gtgaaggt     60
acaataaatg gcttactgga tgaattatta cagacaaggg tgctgaacaa gg
#aagagatg    120
gagaaagtaa aacgtgaaaa tgctacagtt atggataaga cccgagcttt ga
#ttgactcc    180
gttattccga aaggggcaca ggcatgccaa atttgcatca catacatttg tg
#aagaagac    240
agttacctgg cagggacgct gggactctca gcagatcaaa catctggaaa tt
#accttaat    300
atgcaagact ctcaaggagt actttcttcc tttccagctc ctcaggcagt gc
#aggacaac    360
ccagctatgc ccacatcctc aggctcagaa gggaatgtca agctttgctc cc
#tagaagaa    420
gctcaaagga tatggaaaca aaagtcggca gagatttatc caataatgga ca
#agtcaagc    480
cgcacacgtc ttgctctcat tatctgcaat gaagaatttg acagtattcc ta
#gaagaact    540
ggagctgagg ttgacatcac aggcatgaca atgctgctac aaaatctggg gt
#acagcgta    600
gatgtgaaaa aaaatctcac tgcttcggac atgactacag agctggaggc at
#ttgcacac    660
cgcccagagc acaagacctc tgacagcacg ttcctggtgt tcatgtctca tg
#gtattcgg    720
gaaggcattt gtgggaagaa acactctgag caagtcccag atatactaca ac
#tcaatgca    780
atctttaaca tgttgaatac caagaactgc ccaagtttga aggacaaacc ga
#aggtgatc    840
atcatccagg cctgccgtgg tgacagccct ggtgtggtgt ggtttaaaga tt
#cagtagga    900
gtttctggaa acctatcttt accaactaca gaagagtttg aggatgatgc ta
#ttaagaaa    960
gcccacatag agaaggattt tatcgctttc tgctcttcca caccagataa tg
#tttcttgg   1020
agacatccca caatgggctc tgtttttatt ggaagactca ttgaacatat gc
#aagaatat   1080
gcctgttcct gtgatgtgga ggaaattttc cgcaaggttc gattttcatt tg
#agcagcca   1140
gatggtagag cgcagatgcc caccactgaa agagtgactt tgacaagatg tt
#tctacctc   1200
ttcccaggac attaa
#
#
#  1215
<210> SEQ ID NO 5
<211> LENGTH: 1215
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 5
taccggctgt tccaggactt cctcttctct ttcgacaaat aggcaaggta cc
#cacttcca     60
tgttatttac cgaatgacct acttaataat gtctgttccc acgacttgtt cc
#ttctctac    120
ctctttcatt ttgcactttt acgatgtcaa tacctattct gggctcgaaa ct
#aactgagg    180
caataaggct ttccccgtgt ccgtacggtt taaacgtagt gtatgtaaac ac
#ttcttctg    240
tcaatggacc gtccctgcga ccctgagagt cgtctagttt gtagaccttt aa
#tggaatta    300
tacgttctga gagttcctca tgaaagaagg aaaggtcgag gagtccgtca cg
#tcctgttg    360
ggtcgatacg ggtgtaggag tccgagtctt cccttacagt tcgaaacgag gg
#atcttctt    420
cgagtttcct atacctttgt tttcagccgt ctctaaatag gttattacct gt
#tcagttcg    480
gcgtgtgcag aacgagagta atagacgtta cttcttaaac tgtcataagg at
#cttcttga    540
cctcgactcc aactgtagtg tccgtactgt tacgacgatg ttttagaccc ca
#tgtcgcat    600
ctacactttt ttttagagtg acgaagcctg tactgatgtc tcgacctccg ta
#aacgtgtg    660
gcgggtctcg tgttctggag actgtcgtgc aaggaccaca agtacagagt ac
#cataagcc    720
cttccgtaaa cacccttctt tgtgagactc gttcagggtc tatatgatgt tg
#agttacgt    780
tagaaattgt acaacttatg gttcttgacg ggttcaaact tcctgtttgg ct
#tccactag    840
tagtaggtcc ggacggcacc actgtcggga ccacaccaca ccaaatttct aa
#gtcatcct    900
caaagacctt tggatagaaa tggttgatgt cttctcaaac tcctactacg at
#aattcttt    960
cgggtgtatc tcttcctaaa atagcgaaag acgagaaggt gtggtctatt ac
#aaagaacc   1020
tctgtagggt gttacccgag acaaaaataa ccttctgagt aacttgtata cg
#ttcttata   1080
cggacaagga cactacacct cctttaaaag gcgttccaag ctaaaagtaa ac
#tcgtcggt   1140
ctaccatctc gcgtctacgg gtggtgactt tctcactgaa actgttctac aa
#agatggag   1200
aagggtcctg taatt
#
#
#  1215
<210> SEQ ID NO 6
<211> LENGTH: 404
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 6
Met Ala Asp Lys Val Leu Lys Glu Lys Arg Ly
#s Leu Phe Ile Arg Ser
1               5
#                10
#                15
Met Gly Glu Gly Thr Ile Asn Gly Leu Leu As
#p Glu Leu Leu Gln Thr
20
#            25
#            30
Arg Val Leu Asn Lys Glu Glu Met Glu Lys Va
#l Lys Arg Glu Asn Ala
35
#        40
#        45
Thr Val Met Asp Lys Thr Arg Ala Leu Ile As
#p Ser Val Ile Pro Lys
50
#    55
#    60
Gly Ala Gln Ala Cys Gln Ile Cys Ile Thr Ty
#r Ile Cys Glu Glu Asp
65
#70
#75
#80
Ser Tyr Leu Ala Gly Thr Leu Gly Leu Ser Al
#a Asp Gln Thr Ser Gly
85
#                90
#                95
Asn Tyr Leu Asn Met Gln Asp Ser Gln Gly Va
#l Leu Ser Ser Phe Pro
100
#           105
#           110
Ala Pro Gln Ala Val Gln Asp Asn Pro Ala Me
#t Pro Thr Ser Ser Gly
115
#       120
#       125
Ser Glu Gly Asn Val Lys Leu Cys Ser Leu Gl
#u Glu Ala Gln Arg Ile
130
#   135
#   140
Trp Lys Gln Lys Ser Ala Glu Ile Tyr Pro Il
#e Met Asp Lys Ser Ser
145                 1
#50                 1
#55                 1
#60
Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Gl
#u Glu Phe Asp Ser Ile
165
#               170
#               175
Pro Arg Arg Thr Gly Ala Glu Val Asp Ile Th
#r Gly Met Thr Met Leu
180
#           185
#           190
Leu Gln Asn Leu Gly Tyr Ser Val Asp Val Ly
#s Lys Asn Leu Thr Ala
195
#       200
#       205
Ser Asp Met Thr Thr Glu Leu Glu Ala Phe Al
#a His Arg Pro Glu His
210
#   215
#   220
Lys Thr Ser Asp Ser Thr Phe Leu Val Phe Me
#t Ser His Gly Ile Arg
225                 2
#30                 2
#35                 2
#40
Glu Gly Ile Cys Gly Lys Lys His Ser Glu Gl
#n Val Pro Asp Ile Leu
245
#               250
#               255
Gln Leu Asn Ala Ile Phe Asn Met Leu Asn Th
#r Lys Asn Cys Pro Ser
260
#           265
#           270
Leu Lys Asp Lys Pro Lys Val Ile Ile Ile Gl
#n Ala Cys Arg Gly Asp
275
#       280
#       285
Ser Pro Gly Val Val Trp Phe Lys Asp Ser Va
#l Gly Val Ser Gly Asn
290
#   295
#   300
Leu Ser Leu Pro Thr Thr Glu Glu Phe Glu As
#p Asp Ala Ile Lys Lys
305                 3
#10                 3
#15                 3
#20
Ala His Ile Glu Lys Asp Phe Ile Ala Phe Cy
#s Ser Ser Thr Pro Asp
325
#               330
#               335
Asn Val Ser Trp Arg His Pro Thr Met Gly Se
#r Val Phe Ile Gly Arg
340
#           345
#           350
Leu Ile Glu His Met Gln Glu Tyr Ala Cys Se
#r Cys Asp Val Glu Glu
355
#       360
#       365
Ile Phe Arg Lys Val Arg Phe Ser Phe Glu Gl
#n Pro Asp Gly Arg Ala
370
#   375
#   380
Gln Met Pro Thr Thr Glu Arg Val Thr Leu Th
#r Arg Cys Phe Tyr Leu
385                 3
#90                 3
#95                 4
#00
Phe Pro Gly His
<210> SEQ ID NO 7
<211> LENGTH: 1308
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 7
atggccgctg acaggggacg caggatattg ggagtgtgtg gcatgcatcc tc
#atcatcag     60
gaaactctaa aaaagaaccg agtggtgcta gccaaacagc tgttgttgag cg
#aattgtta    120
gaacatcttc tggagaagga catcatcacc ttggaaatga gggagctcat cc
#aggccaaa    180
gtgggcagtt tcagccagaa tgtggaactc ctcaacttgc tgcctaagag gg
#gtccccaa    240
gcttttgatg ccttctgtga agcactgagg gagaccaagc aaggccacct gg
#aggatatg    300
ttgctcacca ccctttctgg gcttcagcat gtactcccac cgttgagctg tg
#actacgac    360
ttgagtctcc cttttccggt gtgtgagtcc tgtccccttt acaagaagct cc
#gcctgtcg    420
acagatactg tggaacactc cctagacaat aaagatggtc ctgtctgcct tc
#aggtgaag    480
ccttgcactc ctgaatttta tcaaacacac ttccagctgg catataggtt gc
#agtctcgg    540
cctcgtggcc tagcactggt gttgagcaat gtgcacttca ctggagagaa ag
#aactggaa    600
tttcgctctg gaggggatgt ggaccacagt actctagtca ccctcttcaa gc
#ttttgggc    660
tatgacgtcc atgttctatg tgaccagact gcacaggaaa tgcaagagaa ac
#tgcagaat    720
tttgcacagt tacctgcaca ccgagtcacg gactcctgca tcgtggcact cc
#tctcgcat    780
ggtgtggagg gcgccatcta tggtgtggat gggaaactgc tccagctcca ag
#aggttttt    840
cagctctttg acaacgccaa ctgcccaagc ctacagaaca aaccaaaaat gt
#tcttcatc    900
caggcctgcc gtggagatga gactgatcgt ggggttgacc aacaagatgg aa
#agaaccac    960
gcaggatccc ctgggtgcga ggagagtgat gccggtaaag aaaagttgcc ga
#agatgaga   1020
ctgcccacgc gctcagacat gatatgcggc tatgcctgcc tcaaagggac tg
#ccgccatg   1080
cggaacacca aacgaggttc ctggtacatc gaggctcttg ctcaagtgtt tt
#ctgagcgg   1140
gcttgtgata tgcacgtggc cgacatgctg gttaaggtga acgcacttat ca
#aggatcgg   1200
gaaggttatg ctcctggcac agaattccac cggtgcaagg aaatgtctga at
#actgcagc   1260
actctgtgcc gccacctcta cctgttccca ggacaccctc ccacatga
#              1308
<210> SEQ ID NO 8
<211> LENGTH: 1308
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 8
taccggcgac tgtcccctgc gtcctataac cctcacacac cgtacgtagg ag
#tagtagtc     60
ctttgagatt ttttcttggc tcaccacgat cggtttgtcg acaacaactc gc
#ttaacaat    120
cttgtagaag acctcttcct gtagtagtgg aacctttact ccctcgagta gg
#tccggttt    180
cacccgtcaa agtcggtctt acaccttgag gagttgaacg acggattctc cc
#caggggtt    240
cgaaaactac ggaagacact tcgtgactcc ctctggttcg ttccggtgga cc
#tcctatac    300
aacgagtggt gggaaagacc cgaagtcgta catgagggtg gcaactcgac ac
#tgatgctg    360
aactcagagg gaaaaggcca cacactcagg acaggggaaa tgttcttcga gg
#cggacagc    420
tgtctatgac accttgtgag ggatctgtta tttctaccag gacagacgga ag
#tccacttc    480
ggaacgtgag gacttaaaat agtttgtgtg aaggtcgacc gtatatccaa cg
#tcagagcc    540
ggagcaccgg atcgtgacca caactcgtta cacgtgaagt gacctctctt tc
#ttgacctt    600
aaagcgagac ctcccctaca cctggtgtca tgagatcagt gggagaagtt cg
#aaaacccg    660
atactgcagg tacaagatac actggtctga cgtgtccttt acgttctctt tg
#acgtctta    720
aaacgtgtca atggacgtgt ggctcagtgc ctgaggacgt agcaccgtga gg
#agagcgta    780
ccacacctcc cgcggtagat accacaccta ccctttgacg aggtcgaggt tc
#tccaaaaa    840
gtcgagaaac tgttgcggtt gacgggttcg gatgtcttgt ttggttttta ca
#agaagtag    900
gtccggacgg cacctctact ctgactagca ccccaactgg ttgttctacc tt
#tcttggtg    960
cgtcctaggg gacccacgct cctctcacta cggccatttc ttttcaacgg ct
#tctactct   1020
gacgggtgcg cgagtctgta ctatacgccg atacggacgg agtttccctg ac
#ggcggtac   1080
gccttgtggt ttgctccaag gaccatgtag ctccgagaac gagttcacaa aa
#gactcgcc   1140
cgaacactat acgtgcaccg gctgtacgac caattccact tgcgtgaata gt
#tcctagcc   1200
cttccaatac gaggaccgtg tcttaaggtg gccacgttcc tttacagact ta
#tgacgtcg   1260
tgagacacgg cggtggagat ggacaagggt cctgtgggag ggtgtact
#              1308
<210> SEQ ID NO 9
<211> LENGTH: 435
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 9
Met Ala Ala Asp Arg Gly Arg Arg Ile Leu Gl
#y Val Cys Gly Met His
1               5
#                10
#                15
Pro His His Gln Glu Thr Leu Lys Lys Asn Ar
#g Val Val Leu Ala Lys
20
#            25
#            30
Gln Leu Leu Leu Ser Glu Leu Leu Glu His Le
#u Leu Glu Lys Asp Ile
35
#        40
#        45
Ile Thr Leu Glu Met Arg Glu Leu Ile Gln Al
#a Lys Val Gly Ser Phe
50
#    55
#    60
Ser Gln Asn Val Glu Leu Leu Asn Leu Leu Pr
#o Lys Arg Gly Pro Gln
65
#70
#75
#80
Ala Phe Asp Ala Phe Cys Glu Ala Leu Arg Gl
#u Thr Lys Gln Gly His
85
#                90
#                95
Leu Glu Asp Met Leu Leu Thr Thr Leu Ser Gl
#y Leu Gln His Val Leu
100
#           105
#           110
Pro Pro Leu Ser Cys Asp Tyr Asp Leu Ser Le
#u Pro Phe Pro Val Cys
115
#       120
#       125
Glu Ser Cys Pro Leu Tyr Lys Lys Leu Arg Le
#u Ser Thr Asp Thr Val
130
#   135
#   140
Glu His Ser Leu Asp Asn Lys Asp Gly Pro Va
#l Cys Leu Gln Val Lys
145                 1
#50                 1
#55                 1
#60
Pro Cys Thr Pro Glu Phe Tyr Gln Thr His Ph
#e Gln Leu Ala Tyr Arg
165
#               170
#               175
Leu Gln Ser Arg Pro Arg Gly Leu Ala Leu Va
#l Leu Ser Asn Val His
180
#           185
#           190
Phe Thr Gly Glu Lys Glu Leu Glu Phe Arg Se
#r Gly Gly Asp Val Asp
195
#       200
#       205
His Ser Thr Leu Val Thr Leu Phe Lys Leu Le
#u Gly Tyr Asp Val His
210
#   215
#   220
Val Leu Cys Asp Gln Thr Ala Gln Glu Met Gl
#n Glu Lys Leu Gln Asn
225                 2
#30                 2
#35                 2
#40
Phe Ala Gln Leu Pro Ala His Arg Val Thr As
#p Ser Cys Ile Val Ala
245
#               250
#               255
Leu Leu Ser His Gly Val Glu Gly Ala Ile Ty
#r Gly Val Asp Gly Lys
260
#           265
#           270
Leu Leu Gln Leu Gln Glu Val Phe Gln Leu Ph
#e Asp Asn Ala Asn Cys
275
#       280
#       285
Pro Ser Leu Gln Asn Lys Pro Lys Met Phe Ph
#e Ile Gln Ala Cys Arg
290
#   295
#   300
Gly Asp Glu Thr Asp Arg Gly Val Asp Gln Gl
#n Asp Gly Lys Asn His
305                 3
#10                 3
#15                 3
#20
Ala Gly Ser Pro Gly Cys Glu Glu Ser Asp Al
#a Gly Lys Glu Lys Leu
325
#               330
#               335
Pro Lys Met Arg Leu Pro Thr Arg Ser Asp Me
#t Ile Cys Gly Tyr Ala
340
#           345
#           350
Cys Leu Lys Gly Thr Ala Ala Met Arg Asn Th
#r Lys Arg Gly Ser Trp
355
#       360
#       365
Tyr Ile Glu Ala Leu Ala Gln Val Phe Ser Gl
#u Arg Ala Cys Asp Met
370
#   375
#   380
His Val Ala Asp Met Leu Val Lys Val Asn Al
#a Leu Ile Lys Asp Arg
385                 3
#90                 3
#95                 4
#00
Glu Gly Tyr Ala Pro Gly Thr Glu Phe His Ar
#g Cys Lys Glu Met Ser
405
#               410
#               415
Glu Tyr Cys Ser Thr Leu Cys Arg His Leu Ty
#r Leu Phe Pro Gly His
420
#           425
#           430
Pro Pro Thr
435
<210> SEQ ID NO 10
<211> LENGTH: 835
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (1)...(835)
<223> OTHER INFORMATION: n = A,T,C or G
<400> SEQUENCE: 10
atggagaaca ctgaaaactc agtggattca aaatccatta aaaatttgga ac
#caaagatc     60
atacatggaa gcgaatcaat ggactctgga atatccctgg acaacagtta ta
#aaatggat    120
tatcctgaga tgggtttatg tataataatt aataataaga attttcataa aa
#gcactgga    180
atgacatctc ggtctggtac agatgtcgat gcagcaaacc tcagggaaac at
#tcagaaac    240
ttgaaatatg aagtcaggaa taaaaatgat cttacacgtg aagaaattgt gg
#aattgatg    300
cgtgatgttt ctaaagaaga tcacagcaaa aggagcagtt ttgtttgtgt gc
#ttctgagc    360
catggtgaag aaggaataat ttttggaaca aatggacctg ttgacctgaa aa
#aaataaca    420
aactttttca gaggggatcg ttgtagaagt ctaactggaa aacccaaact tt
#tcattatt    480
caggcctgcc gtggtacaga actggactgt ggcattgaga cagacagtgg tg
#ttgatgat    540
gacatggcgt gtcataaaat accagtggat gccgacttct tgtatgcata ct
#ccacagca    600
cctggttatt attcttggcg aaattcaaag gatggctcct ggttcatcca gt
#cgctttgt    660
gccatgctga aacagtatgc cgacaagctt gaatttatgc acattcttac cc
#gggttaac    720
cgaaaggtgg caacagaatt tgagtccttt tcctttgacg ctacttttca tg
#caaagaaa    780
cagattccat gtattgtttc catgctcaca aaagaactct atttttatca ct
#aan         835
<210> SEQ ID NO 11
<211> LENGTH: 835
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (1)...(835)
<223> OTHER INFORMATION: n = A,T,C or G
<400> SEQUENCE: 11
tacctcttgt gacttttgag tcacctaagt tttaggtaat ttttaaacct tg
#gtttctag     60
tatgtacctt cgcttagtta cctgagacct tatagggacc tgttgtcaat at
#tttaccta    120
ataggactct acccaaatac atattattaa ttattattct taaaagtatt tt
#cgtgacct    180
tactgtagag ccagaccatg tctacagcta cgtcgtttgg agtccctttg ta
#agtctttg    240
aactttatac ttcagtcctt atttttacta gaatgtgcac ttctttaaca cc
#ttaactac    300
gcactacaaa gatttcttct agtgtcgttt tcctcgtcaa aacaaacaca cg
#aagactcg    360
gtaccacttc ttccttatta aaaaccttgt ttacctggac aactggactt tt
#tttattgt    420
ttgaaaaagt ctcccctagc aacatcttca gattgacctt ttgggtttga aa
#agtaataa    480
gtccggacgg caccatgtct tgacctgaca ccgtaactct gtctgtcacc ac
#aactacta    540
ctgtaccgca cagtatttta tggtcaccta cggctgaaga acatacgtat ga
#ggtgtcgt    600
ggaccaataa taagaaccgc tttaagtttc ctaccgagga ccaagtaggt ca
#gcgaaaca    660
cggtacgact ttgtcatacg gctgttcgaa cttaaatacg tgtaagaatg gg
#cccaattg    720
gctttccacc gttgtcttaa actcaggaaa aggaaactgc gatgaaaagt ac
#gtttcttt    780
gtctaaggta cataacaaag gtacgagtgt tttcttgaga taaaaatagt ga
#ttn         835
<210> SEQ ID NO 12
<211> LENGTH: 277
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 12
Met Glu Asn Thr Glu Asn Ser Val Asp Ser Ly
#s Ser Ile Lys Asn Leu
1               5
#                10
#                15
Glu Pro Lys Ile Ile His Gly Ser Glu Ser Me
#t Asp Ser Gly Ile Ser
20
#            25
#            30
Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Gl
#u Met Gly Leu Cys Ile
35
#        40
#        45
Ile Ile Asn Asn Lys Asn Phe His Lys Ser Th
#r Gly Met Thr Ser Arg
50
#    55
#    60
Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Ar
#g Glu Thr Phe Arg Asn
65
#70
#75
#80
Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Le
#u Thr Arg Glu Glu Ile
85
#                90
#                95
Val Glu Leu Met Arg Asp Val Ser Lys Glu As
#p His Ser Lys Arg Ser
100
#           105
#           110
Ser Phe Val Cys Val Leu Leu Ser His Gly Gl
#u Glu Gly Ile Ile Phe
115
#       120
#       125
Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Il
#e Thr Asn Phe Phe Arg
130
#   135
#   140
Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pr
#o Lys Leu Phe Ile Ile
145                 1
#50                 1
#55                 1
#60
Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gl
#y Ile Glu Thr Asp Ser
165
#               170
#               175
Gly Val Asp Asp Asp Met Ala Cys His Lys Il
#e Pro Val Asp Ala Asp
180
#           185
#           190
Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Ty
#r Tyr Ser Trp Arg Asn
195
#       200
#       205
Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Le
#u Cys Ala Met Leu Lys
210
#   215
#   220
Gln Tyr Ala Asp Lys Leu Glu Phe Met His Il
#e Leu Thr Arg Val Asn
225                 2
#30                 2
#35                 2
#40
Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Se
#r Phe Asp Ala Thr Phe
245
#               250
#               255
His Ala Lys Lys Gln Ile Pro Cys Ile Val Se
#r Met Leu Thr Lys Glu
260
#           265
#           270
Leu Tyr Phe Tyr His
275
<210> SEQ ID NO 13
<211> LENGTH: 1134
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 13
atggcagaag gcaaccacag aaaaaagcca cttaaggtgt tggaatccct gg
#gcaaagat     60
ttcctcactg gtgttttgga taacttggtg gaacaaaatg tactgaactg ga
#aggaagag    120
gaaaaaaaga aatattacga tgctaaaact gaagacaaag ttcgggtcat gg
#cagactct    180
atgcaagaga agcaacgtat ggcaggacaa atgcttcttc aaaccttttt ta
#acatagac    240
caaatatccc ccaataaaaa agctcatccg aatatggagg ctggaccacc tg
#agtcagga    300
gaatctacag atgccctcaa gctttgtcct catgaagaat tcctgagact at
#gtaaagaa    360
agagctgaag agatctatcc aataaaggag agaaacaacc gcacacgcct gg
#ctctcatc    420
atatgcaata cagagtttga ccatctgcct ccgaggaatg gagctgactt tg
#acatcaca    480
gggatgaagg agctacttga gggtctggac tatagtgtag atgtagaaga ga
#atctgaca    540
gccagggata tggagtcagc gctgagggca tttgctacca gaccagagca ca
#agtcctct    600
gacagcacat tcttggtact catgtctcat ggcatcctgg agggaatctg cg
#gaactgtg    660
catgatgaga aaaaaccaga tgtgctgctt tatgacacca tcttccagat at
#tcaacaac    720
cgcaactgcc tcagtctgaa ggacaaaccc aaggtcatca ttgtccaggc ct
#gcagaggt    780
gcaaaccgtg gggaactgtg ggtcagagac tctccagcat ccttggaagt gg
#cctcttca    840
cagtcatctg agaacctgga ggaagatgct gtttacaaga cccacgtgga ga
#aggacttc    900
attgctttct gctcttcaac gccacacaac gtgtcctgga gagacagcac aa
#tgggctct    960
atcttcatca cacaactcat cacatgcttc cagaaatatt cttggtgctg cc
#acctagag   1020
gaagtatttc ggaaggtaca gcaatcattt gaaactccaa gggccaaagc tc
#aaatgccc   1080
accatagaac gactgtccat gacaagatat ttctacctct ttcctggcaa tt
#ga         1134
<210> SEQ ID NO 14
<211> LENGTH: 1134
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 14
taccgtcttc cgttggtgtc ttttttcggt gaattccaca accttaggga cc
#cgtttcta     60
aaggagtgac cacaaaacct attgaaccac cttgttttac atgacttgac ct
#tccttctc    120
ctttttttct ttataatgct acgattttga cttctgtttc aagcccagta cc
#gtctgaga    180
tacgttctct tcgttgcata ccgtcctgtt tacgaagaag tttggaaaaa at
#tgtatctg    240
gtttataggg ggttattttt tcgagtaggc ttatacctcc gacctggtgg ac
#tcagtcct    300
cttagatgtc tacgggagtt cgaaacagga gtacttctta aggactctga ta
#catttctt    360
tctcgacttc tctagatagg ttatttcctc tctttgttgg cgtgtgcgga cc
#gagagtag    420
tatacgttat gtctcaaact ggtagacgga ggctccttac ctcgactgaa ac
#tgtagtgt    480
ccctacttcc tcgatgaact cccagacctg atatcacatc tacatcttct ct
#tagactgt    540
cggtccctat acctcagtcg cgactcccgt aaacgatggt ctggtctcgt gt
#tcaggaga    600
ctgtcgtgta agaaccatga gtacagagta ccgtaggacc tcccttagac gc
#cttgacac    660
gtactactct tttttggtct acacgacgaa atactgtggt agaaggtcta ta
#agttgttg    720
gcgttgacgg agtcagactt cctgtttggg ttccagtagt aacaggtccg ga
#cgtctcca    780
cgtttggcac cccttgacac ccagtctctg agaggtcgta ggaaccttca cc
#ggagaagt    840
gtcagtagac tcttggacct ccttctacga caaatgttct gggtgcacct ct
#tcctgaag    900
taacgaaaga cgagaagttg cggtgtgttg cacaggacct ctctgtcgtg tt
#acccgaga    960
tagaagtagt gtgttgagta gtgtacgaag gtctttataa gaaccacgac gg
#tggatctc   1020
cttcataaag ccttccatgt cgttagtaaa ctttgaggtt cccggtttcg ag
#tttacggg   1080
tggtatcttg ctgacaggta ctgttctata aagatggaga aaggaccgtt aa
#ct         1134
<210> SEQ ID NO 15
<211> LENGTH: 377
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 15
Met Ala Glu Gly Asn His Arg Lys Lys Pro Le
#u Lys Val Leu Glu Ser
1               5
#                10
#                15
Leu Gly Lys Asp Phe Leu Thr Gly Val Leu As
#p Asn Leu Val Glu Gln
20
#            25
#            30
Asn Val Leu Asn Trp Lys Glu Glu Glu Lys Ly
#s Lys Tyr Tyr Asp Ala
35
#        40
#        45
Lys Thr Glu Asp Lys Val Arg Val Met Ala As
#p Ser Met Gln Glu Lys
50
#    55
#    60
Gln Arg Met Ala Gly Gln Met Leu Leu Gln Th
#r Phe Phe Asn Ile Asp
65
#70
#75
#80
Gln Ile Ser Pro Asn Lys Lys Ala His Pro As
#n Met Glu Ala Gly Pro
85
#                90
#                95
Pro Glu Ser Gly Glu Ser Thr Asp Ala Leu Ly
#s Leu Cys Pro His Glu
100
#           105
#           110
Glu Phe Leu Arg Leu Cys Lys Glu Arg Ala Gl
#u Glu Ile Tyr Pro Ile
115
#       120
#       125
Lys Glu Arg Asn Asn Arg Thr Arg Leu Ala Le
#u Ile Ile Cys Asn Thr
130
#   135
#   140
Glu Phe Asp His Leu Pro Pro Arg Asn Gly Al
#a Asp Phe Asp Ile Thr
145                 1
#50                 1
#55                 1
#60
Gly Met Lys Glu Leu Leu Glu Gly Leu Asp Ty
#r Ser Val Asp Val Glu
165
#               170
#               175
Glu Asn Leu Thr Ala Arg Asp Met Glu Ser Al
#a Leu Arg Ala Phe Ala
180
#           185
#           190
Thr Arg Pro Glu His Lys Ser Ser Asp Ser Th
#r Phe Leu Val Leu Met
195
#       200
#       205
Ser His Gly Ile Leu Glu Gly Ile Cys Gly Th
#r Val His Asp Glu Lys
210
#   215
#   220
Lys Pro Asp Val Leu Leu Tyr Asp Thr Ile Ph
#e Gln Ile Phe Asn Asn
225                 2
#30                 2
#35                 2
#40
Arg Asn Cys Leu Ser Leu Lys Asp Lys Pro Ly
#s Val Ile Ile Val Gln
245
#               250
#               255
Ala Cys Arg Gly Ala Asn Arg Gly Glu Leu Tr
#p Val Arg Asp Ser Pro
260
#           265
#           270
Ala Ser Leu Glu Val Ala Ser Ser Gln Ser Se
#r Glu Asn Leu Glu Glu
275
#       280
#       285
Asp Ala Val Tyr Lys Thr His Val Glu Lys As
#p Phe Ile Ala Phe Cys
290
#   295
#   300
Ser Ser Thr Pro His Asn Val Ser Trp Arg As
#p Ser Thr Met Gly Ser
305                 3
#10                 3
#15                 3
#20
Ile Phe Ile Thr Gln Leu Ile Thr Cys Phe Gl
#n Lys Tyr Ser Trp Cys
325
#               330
#               335
Cys His Leu Glu Glu Val Phe Arg Lys Val Gl
#n Gln Ser Phe Glu Thr
340
#           345
#           350
Pro Arg Ala Lys Ala Gln Met Pro Thr Ile Gl
#u Arg Leu Ser Met Thr
355
#       360
#       365
Arg Tyr Phe Tyr Leu Phe Pro Gly Asn
370
#   375
<210> SEQ ID NO 16
<211> LENGTH: 1258
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (1)...(1258)
<223> OTHER INFORMATION: n = A,T,C or G
<400> SEQUENCE: 16
atgttcaaag gtatccttca gagtggattg gataacttcg tgataaacca ca
#tgctaaag     60
aacaacgtgg ctggacaaac atctatccag accctagtac ctaatacgga tc
#aaaagtcg    120
accagtgtaa aaaaagacaa ccacaaaaaa aaaacagtta agatgttgga at
#acctgggc    180
aaagatgttc ttcatggtgt ttttaattat ttggcaaaac acgatgttct ga
#cattgaag    240
gaagaggaaa agaaaaaata ttatgatgcc aaaattgaag acaaggccct ga
#tcttggta    300
gactctttgc gaaagaatcg cgtggctcat caaatgttta cccaaacact tc
#tcaatatg    360
gaccaaaaga tcaccagtgt aaaacctctt ctgcaaatcg aggctggacc ac
#ctgagtca    420
gcagaatcta caaatatact caaactttgt cctcgtgaag aattcctgag ac
#tgtgtaaa    480
aaaaatcatg atgagatcta tccaataaaa aagagagagg accgcagacg cc
#tggctctc    540
atcatatgca atacaaagtt tgatcacctg cctgcaagga atggggctca ct
#atgacatc    600
gtggggatga aaaggctgct tcaaggcctg ggctacactg tggttgacga aa
#agaatctc    660
acagccaggg atatggagtc agtgctgagg gcatttgctg ccagaccaga gc
#acaagtcc    720
tctgacagca cgttcttggt actcatgtct catggcatcc tagagggaat ct
#gcggaact    780
gcgcataaaa agaaaaaacc ggatgtgctg ctttatgaca ccatcttcca ga
#tattcaac    840
aaccgcaact gcctcagtct aaaggacaaa cccaaggtca tcattgtcca gg
#cctgcaga    900
ggtgaaaaac atggggaact ctgggtcaga gactctccag catccttggc ag
#tcatctct    960
tcacagtcat ctgagaacct ggaggcagat tctgtttgca agatccacga gg
#agaaggac   1020
ttcattgctt tctgttcttc aacaccacat aacgtgtcct ggagagaccg ca
#caaggggc   1080
tccatcttca ttacggaact catcacatgc ttccagaaat attcttgctg ct
#gccaccta   1140
atggaaatat ttcggaaggt acagaaatca tttgaagttc cacaggctaa ag
#cccagatg   1200
cccaccatag aacgagcaac cttgacaaga gatttctacc tctttcctgg ca
#attgan     1258
<210> SEQ ID NO 17
<211> LENGTH: 1258
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (1)...(1258)
<223> OTHER INFORMATION: n = A,T,C or G
<400> SEQUENCE: 17
tacaagtttc cataggaagt ctcacctaac ctattgaagc actatttggt gt
#acgatttc     60
ttgttgcacc gacctgtttg tagataggtc tgggatcatg gattatgcct ag
#ttttcagc    120
tggtcacatt tttttctgtt ggtgtttttt ttttgtcaat tctacaacct ta
#tggacccg    180
tttctacaag aagtaccaca aaaattaata aaccgttttg tgctacaaga ct
#gtaacttc    240
cttctccttt tcttttttat aatactacgg ttttaacttc tgttccggga ct
#agaaccat    300
ctgagaaacg ctttcttagc gcaccgagta gtttacaaat gggtttgtga ag
#agttatac    360
ctggttttct agtggtcaca ttttggagaa gacgtttagc tccgacctgg tg
#gactcagt    420
cgtcttagat gtttatatga gtttgaaaca ggagcacttc ttaaggactc tg
#acacattt    480
tttttagtac tactctagat aggttatttt ttctctctcc tggcgtctgc gg
#accgagag    540
tagtatacgt tatgtttcaa actagtggac ggacgttcct taccccgagt ga
#tactgtag    600
cacccctact tttccgacga agttccggac ccgatgtgac accaactgct tt
#tcttagag    660
tgtcggtccc tatacctcag tcacgactcc cgtaaacgac ggtctggtct cg
#tgttcagg    720
agactgtcgt gcaagaacca tgagtacaga gtaccgtagg atctccctta ga
#cgccttga    780
cgcgtatttt tcttttttgg cctacacgac gaaatactgt ggtagaaggt ct
#ataagttg    840
ttggcgttga cggagtcaga tttcctgttt gggttccagt agtaacaggt cc
#ggacgtct    900
ccactttttg taccccttga gacccagtct ctgagaggtc gtaggaaccg tc
#agtagaga    960
agtgtcagta gactcttgga cctccgtcta agacaaacgt tctaggtgct cc
#tcttcctg   1020
aagtaacgaa agacaagaag ttgtggtgta ttgcacagga cctctctggc gt
#gttccccg   1080
aggtagaagt aatgccttga gtagtgtacg aaggtcttta taagaacgac ga
#cggtggat   1140
tacctttata aagccttcca tgtctttagt aaacttcaag gtgtccgatt tc
#gggtctac   1200
gggtggtatc ttgctcgttg gaactgttct ctaaagatgg agaaaggacc gt
#taactn     1258
<210> SEQ ID NO 18
<211> LENGTH: 418
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 18
Met Phe Lys Gly Ile Leu Gln Ser Gly Leu As
#p Asn Phe Val Ile Asn
1               5
#                10
#                15
His Met Leu Lys Asn Asn Val Ala Gly Gln Th
#r Ser Ile Gln Thr Leu
20
#            25
#            30
Val Pro Asn Thr Asp Gln Lys Ser Thr Ser Va
#l Lys Lys Asp Asn His
35
#        40
#        45
Lys Lys Lys Thr Val Lys Met Leu Glu Tyr Le
#u Gly Lys Asp Val Leu
50
#    55
#    60
His Gly Val Phe Asn Tyr Leu Ala Lys His As
#p Val Leu Thr Leu Lys
65
#70
#75
#80
Glu Glu Glu Lys Lys Lys Tyr Tyr Asp Ala Ly
#s Ile Glu Asp Lys Ala
85
#                90
#                95
Leu Ile Leu Val Asp Ser Leu Arg Lys Asn Ar
#g Val Ala His Gln Met
100
#           105
#           110
Phe Thr Gln Thr Leu Leu Asn Met Asp Gln Ly
#s Ile Thr Ser Val Lys
115
#       120
#       125
Pro Leu Leu Gln Ile Glu Ala Gly Pro Pro Gl
#u Ser Ala Glu Ser Thr
130
#   135
#   140
Asn Ile Leu Lys Leu Cys Pro Arg Glu Glu Ph
#e Leu Arg Leu Cys Lys
145                 1
#50                 1
#55                 1
#60
Lys Asn His Asp Glu Ile Tyr Pro Ile Lys Ly
#s Arg Glu Asp Arg Arg
165
#               170
#               175
Arg Leu Ala Leu Ile Ile Cys Asn Thr Lys Ph
#e Asp His Leu Pro Ala
180
#           185
#           190
Arg Asn Gly Ala His Tyr Asp Ile Val Gly Me
#t Lys Arg Leu Leu Gln
195
#       200
#       205
Gly Leu Gly Tyr Thr Val Val Asp Glu Lys As
#n Leu Thr Ala Arg Asp
210
#   215
#   220
Met Glu Ser Val Leu Arg Ala Phe Ala Ala Ar
#g Pro Glu His Lys Ser
225                 2
#30                 2
#35                 2
#40
Ser Asp Ser Thr Phe Leu Val Leu Met Ser Hi
#s Gly Ile Leu Glu Gly
245
#               250
#               255
Ile Cys Gly Thr Ala His Lys Lys Lys Lys Pr
#o Asp Val Leu Leu Tyr
260
#           265
#           270
Asp Thr Ile Phe Gln Ile Phe Asn Asn Arg As
#n Cys Leu Ser Leu Lys
275
#       280
#       285
Asp Lys Pro Lys Val Ile Ile Val Gln Ala Cy
#s Arg Gly Glu Lys His
290
#   295
#   300
Gly Glu Leu Trp Val Arg Asp Ser Pro Ala Se
#r Leu Ala Val Ile Ser
305                 3
#10                 3
#15                 3
#20
Ser Gln Ser Ser Glu Asn Leu Glu Ala Asp Se
#r Val Cys Lys Ile His
325
#               330
#               335
Glu Glu Lys Asp Phe Ile Ala Phe Cys Ser Se
#r Thr Pro His Asn Val
340
#           345
#           350
Ser Trp Arg Asp Arg Thr Arg Gly Ser Ile Ph
#e Ile Thr Glu Leu Ile
355
#       360
#       365
Thr Cys Phe Gln Lys Tyr Ser Cys Cys Cys Hi
#s Leu Met Glu Ile Phe
370
#   375
#   380
Arg Lys Val Gln Lys Ser Phe Glu Val Pro Gl
#n Ala Lys Ala Gln Met
385                 3
#90                 3
#95                 4
#00
Pro Thr Ile Glu Arg Ala Thr Leu Thr Arg As
#p Phe Tyr Leu Phe Pro
405
#               410
#               415
Gly Asn
<210> SEQ ID NO 19
<211> LENGTH: 883
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (1)...(883)
<223> OTHER INFORMATION: n = A,T,C or G
<400> SEQUENCE: 19
atgagctcgg cctcggggct ccgcaggggg cacccggcag gtggggaaga aa
#acatgaca     60
gaaacagatg ccttctataa aagagaaatg tttgatccgg cagaaaagta ca
#aaatggac    120
cacaggagga gaggaattgc tttaatcttc aatcatgaga ggttcttttg gc
#acttaaca    180
ctgccagaaa ggcggggcac ctgcgcagat agagacaatc ttacccgcag gt
#tttcagat    240
ctaggatttg aagtgaaatg ctttaatgat cttaaagcag aagaactact gc
#tcaaaatt    300
catgaggtgt caactgttag ccacgcagat gccgattgct ttgtgtgtgt ct
#tcctgagc    360
catggcgaag gcaatcacat ttatgcatat gatgctaaaa tcgaaattca ga
#cattaact    420
ggcttgttca aaggagacaa gtgtcacagc ctggttggaa aacccaagat at
#ttatcatc    480
caggcatgtc ggggaaacca gcacgatgtg ccagtcattc ctttggatgt ag
#tagataat    540
cagacagaga agttggacac caacataact gaggtggatg cagcctccgt tt
#acacgctg    600
cctgctggag ctgacttcct catgtgttac tctgttgcag aaggatatta tt
#ctcaccgg    660
gaaactgtga acggctcatg gtacattcaa gatttgtgtg agatgttggg aa
#aatatggc    720
tcctccttag agttcacaga actcctcaca ctggtgaaca ggaaagtttc tc
#agcgccga    780
gtggactttt gcaaagaccc aagtgcaatt ggaaagaagc aggttccctg tt
#ttgcctca    840
atgctaacta aaaagctgca tttctttcca aaatctaatt aan
#
#883
<210> SEQ ID NO 20
<211> LENGTH: 883
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (1)...(883)
<223> OTHER INFORMATION: n = A,T,C or G
<400> SEQUENCE: 20
tactcgagcc ggagccccga ggcgtccccc gtgggccgtc caccccttct tt
#tgtactgt     60
ctttgtctac ggaagatatt ttctctttac aaactaggcc gtcttttcat gt
#tttacctg    120
gtgtcctcct ctccttaacg aaattagaag ttagtactct ccaagaaaac cg
#tgaattgt    180
gacggtcttt ccgccccgtg gacgcgtcta tctctgttag aatgggcgtc ca
#aaagtcta    240
gatcctaaac ttcactttac gaaattacta gaatttcgtc ttcttgatga cg
#agttttaa    300
gtactccaca gttgacaatc ggtgcgtcta cggctaacga aacacacaca ga
#aggactcg    360
gtaccgcttc cgttagtgta aatacgtata ctacgatttt agctttaagt ct
#gtaattga    420
ccgaacaagt ttcctctgtt cacagtgtcg gaccaacctt ttgggttcta ta
#aatagtag    480
gtccgtacag cccctttggt cgtgctacac ggtcagtaag gaaacctaca tc
#atctatta    540
gtctgtctct tcaacctgtg gttgtattga ctccacctac gtcggaggca aa
#tgtgcgac    600
ggacgacctc gactgaagga gtacacaatg agacaacgtc ttcctataat aa
#gagtggcc    660
ctttgacact tgccgagtac catgtaagtt ctaaacacac tctacaaccc tt
#ttataccg    720
aggaggaatc tcaagtgtct tgaggagtgt gaccacttgt cctttcaaag ag
#tcgcggct    780
cacctgaaaa cgtttctggg ttcacgttaa cctttcttcg tccaagggac aa
#aacggagt    840
tacgattgat ttttcgacgt aaagaaaggt tttagattaa ttn
#
#883
<210> SEQ ID NO 21
<211> LENGTH: 293
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 21
Met Ser Ser Ala Ser Gly Leu Arg Arg Gly Hi
#s Pro Ala Gly Gly Glu
1               5
#                10
#                15
Glu Asn Met Thr Glu Thr Asp Ala Phe Tyr Ly
#s Arg Glu Met Phe Asp
20
#            25
#            30
Pro Ala Glu Lys Tyr Lys Met Asp His Arg Ar
#g Arg Gly Ile Ala Leu
35
#        40
#        45
Ile Phe Asn His Glu Arg Phe Phe Trp His Le
#u Thr Leu Pro Glu Arg
50
#    55
#    60
Arg Gly Thr Cys Ala Asp Arg Asp Asn Leu Th
#r Arg Arg Phe Ser Asp
65
#70
#75
#80
Leu Gly Phe Glu Val Lys Cys Phe Asn Asp Le
#u Lys Ala Glu Glu Leu
85
#                90
#                95
Leu Leu Lys Ile His Glu Val Ser Thr Val Se
#r His Ala Asp Ala Asp
100
#           105
#           110
Cys Phe Val Cys Val Phe Leu Ser His Gly Gl
#u Gly Asn His Ile Tyr
115
#       120
#       125
Ala Tyr Asp Ala Lys Ile Glu Ile Gln Thr Le
#u Thr Gly Leu Phe Lys
130
#   135
#   140
Gly Asp Lys Cys His Ser Leu Val Gly Lys Pr
#o Lys Ile Phe Ile Ile
145                 1
#50                 1
#55                 1
#60
Gln Ala Cys Arg Gly Asn Gln His Asp Val Pr
#o Val Ile Pro Leu Asp
165
#               170
#               175
Val Val Asp Asn Gln Thr Glu Lys Leu Asp Th
#r Asn Ile Thr Glu Val
180
#           185
#           190
Asp Ala Ala Ser Val Tyr Thr Leu Pro Ala Gl
#y Ala Asp Phe Leu Met
195
#       200
#       205
Cys Tyr Ser Val Ala Glu Gly Tyr Tyr Ser Hi
#s Arg Glu Thr Val Asn
210
#   215
#   220
Gly Ser Trp Tyr Ile Gln Asp Leu Cys Glu Me
#t Leu Gly Lys Tyr Gly
225                 2
#30                 2
#35                 2
#40
Ser Ser Leu Glu Phe Thr Glu Leu Leu Thr Le
#u Val Asn Arg Lys Val
245
#               250
#               255
Ser Gln Arg Arg Val Asp Phe Cys Lys Asp Pr
#o Ser Ala Ile Gly Lys
260
#           265
#           270
Lys Gln Val Pro Cys Phe Ala Ser Met Leu Th
#r Lys Lys Leu His Phe
275
#       280
#       285
Phe Pro Lys Ser Asn
290
<210> SEQ ID NO 22
<211> LENGTH: 913
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (1)...(913)
<223> OTHER INFORMATION: n = A,T,C or G
<400> SEQUENCE: 22
atggcagatg atcagggctg tattgaagag cagggggttg aggattcagc aa
#atgaagat     60
tcagtggatg ctaagccaga ccggtcctcg tttgtaccgt ccctcttcag ta
#agaagaag    120
aaaaatgtca ccatgcgatc catcaagacc acccgggacc gagtgcctac at
#atcagtac    180
aacatgaatt ttgaaaagct gggcaaatgc atcataataa acaacaagaa ct
#ttgataaa    240
gtgacaggta tgggcgttcg aaacggaaca gacaaagatg ccgaggcgct ct
#tcaagtgc    300
ttccgaagcc tgggttttga cgtgattgtc tataatgact gctcttgtgc ca
#agatgcaa    360
gatctgctta aaaaagcttc tgaagaggac catacaaatg ccgcctgctt cg
#cctgcatc    420
ctcttaagcc atggagaaga aaatgtaatt tatgggaaag atggtgtcac ac
#caataaag    480
gatttgacag cccactttag gggggataga tgcaaaaccc ttttagagaa ac
#ccaaactc    540
ttcttcattc aggcttgccg agggaccgag cttgatgatg gcatccaggc cg
#actcgggg    600
cccatcaatg acacagatgc taatcctcga tacaagatcc cagtggaagc tg
#acttcctc    660
ttcgcctatt ccacggttcc aggctattac tcgtggagga gcccaggaag ag
#gctcctgg    720
tttgtgcaag ccctctgctc catcctggag gagcacggaa aagacctgga aa
#tcatgcag    780
atcctcacca gggtgaatga cagagttgcc aggcactttg agtctcagtc tg
#atgaccca    840
cacttccatg agaagaagca gatcccctgt gtggtctcca tgctcaccaa gg
#aactctac    900
ttcagtcaat agn
#
#
#     913
<210> SEQ ID NO 23
<211> LENGTH: 913
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (1)...(913)
<223> OTHER INFORMATION: n = A,T,C or G
<400> SEQUENCE: 23
taccgtctac tagtcccgac ataacttctc gtcccccaac tcctaagtcg tt
#tacttcta     60
agtcacctac gattcggtct ggccaggagc aaacatggca gggagaagtc at
#tcttcttc    120
tttttacagt ggtacgctag gtagttctgg tgggccctgg ctcacggatg ta
#tagtcatg    180
ttgtacttaa aacttttcga cccgtttacg tagtattatt tgttgttctt ga
#aactattt    240
cactgtccat acccgcaagc tttgccttgt ctgtttctac ggctccgcga ga
#agttcacg    300
aaggcttcgg acccaaaact gcactaacag atattactga cgagaacacg gt
#tctacgtt    360
ctagacgaat tttttcgaag acttctcctg gtatgtttac ggcggacgaa gc
#ggacgtag    420
gagaattcgg tacctcttct tttacattaa ataccctttc taccacagtg tg
#gttatttc    480
ctaaactgtc gggtgaaatc ccccctatct acgttttggg aaaatctctt tg
#ggtttgag    540
aagaagtaag tccgaacggc tccctggctc gaactactac cgtaggtccg gc
#tgagcccc    600
gggtagttac tgtgtctacg attaggagct atgttctagg gtcaccttcg ac
#tgaaggag    660
aagcggataa ggtgccaagg tccgataatg agcacctcct cgggtccttc tc
#cgaggacc    720
aaacacgttc gggagacgag gtaggacctc ctcgtgcctt ttctggacct tt
#agtacgtc    780
taggagtggt cccacttact gtctcaacgg tccgtgaaac tcagagtcag ac
#tactgggt    840
gtgaaggtac tcttcttcgt ctaggggaca caccagaggt acgagtggtt cc
#ttgagatg    900
aagtcagtta tcn
#
#
#     913
<210> SEQ ID NO 24
<211> LENGTH: 303
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 24
Met Ala Asp Asp Gln Gly Cys Ile Glu Glu Gl
#n Gly Val Glu Asp Ser
1               5
#                10
#                15
Ala Asn Glu Asp Ser Val Asp Ala Lys Pro As
#p Arg Ser Ser Phe Val
20
#            25
#            30
Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Va
#l Thr Met Arg Ser Ile
35
#        40
#        45
Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gl
#n Tyr Asn Met Asn Phe
50
#    55
#    60
Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn As
#n Lys Asn Phe Asp Lys
65
#70
#75
#80
Val Thr Gly Met Gly Val Arg Asn Gly Thr As
#p Lys Asp Ala Glu Ala
85
#                90
#                95
Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe As
#p Val Ile Val Tyr Asn
100
#           105
#           110
Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Le
#u Lys Lys Ala Ser Glu
115
#       120
#       125
Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cy
#s Ile Leu Leu Ser His
130
#   135
#   140
Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gl
#y Val Thr Pro Ile Lys
145                 1
#50                 1
#55                 1
#60
Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cy
#s Lys Thr Leu Leu Glu
165
#               170
#               175
Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Ar
#g Gly Thr Glu Leu Asp
180
#           185
#           190
Asp Gly Ile Gln Ala Asp Ser Gly Pro Ile As
#n Asp Thr Asp Ala Asn
195
#       200
#       205
Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Ph
#e Leu Phe Ala Tyr Ser
210
#   215
#   220
Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pr
#o Gly Arg Gly Ser Trp
225                 2
#30                 2
#35                 2
#40
Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Gl
#u His Gly Lys Asp Leu
245
#               250
#               255
Glu Ile Met Gln Ile Leu Thr Arg Val Asn As
#p Arg Val Ala Arg His
260
#           265
#           270
Phe Glu Ser Gln Ser Asp Asp Pro His Phe Hi
#s Glu Lys Lys Gln Ile
275
#       280
#       285
Pro Cys Val Val Ser Met Leu Thr Lys Glu Le
#u Tyr Phe Ser Gln
290
#   295
#   300
<210> SEQ ID NO 25
<211> LENGTH: 1493
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (1)...(1493)
<223> OTHER INFORMATION: n = A,T,C or G
<400> SEQUENCE: 25
atggacttca gcagaaatct ttatgatatt ggggaacaac tggacagtga ag
#atctggcc     60
tccctcaagt tcctgagcct ggactacatt ccgcaaagga agcaagaacc ca
#tcaaggat    120
gccttgatgt tattccagag actccaggaa aagagaatgt tggaggaaag ca
#atctgtcc    180
ttcctgaagg agctgctctt ccgaattaat agactggatt tgctgattac ct
#acctaaac    240
actagaaagg aggagatgga aagggaactt cagacaccag gcagggctca aa
#tttctgcc    300
tacaggttcc acttctgccg catgagctgg gctgaagcaa acagccagtg cc
#agacacag    360
tctgtacctt tctggcggag ggtcgatcat ctattaataa gggtcatgct ct
#atcagatt    420
tcagaagaag tgagcagatc agaattgagg tcttttaagt ttcttttgca ag
#aggaaatc    480
tccaaatgca aactggatga tgacatgaac ctgctggata ttttcataga ga
#tggagaag    540
agggtcatcc tgggagaagg aaagttggac atcctgaaaa gagtctgtgc cc
#aaatcaac    600
aagagcctgc tgaagataat caacgactat gaagaattca gcaaagggga gg
#agttgtgt    660
ggggtaatga caatctcgga ctctccaaga gaacaggata gtgaatcaca ga
#ctttggac    720
aaagtttacc aaatgaaaag caaacctcgg ggatactgtc tgatcatcaa ca
#atcacaat    780
tttgcaaaag cacgggagaa agtgcccaaa cttcacagca ttagggacag ga
#atggaaca    840
cacttggatg caggggcttt gaccacgacc tttgaagagc ttcattttga ga
#tcaagccc    900
caccatgact gcacagtaga gcaaatctat gagattttga aaatctacca ac
#tcatggac    960
cacagtaaca tggactgctt catctgctgt atcctctccc atggagacaa gg
#gcatcatc   1020
tatggcactg atggacagga ggcccccatc tatgagctga catctcagtt ca
#ctggtttg   1080
aagtgccctt cccttgctgg aaaacccaaa gtgtttttta ttcaggcttg tc
#agggggat   1140
aactaccaga aaggtatacc tgttgagact gattcagagg agcaacccta tt
#tagaaatg   1200
gatttatcat cacctcaaac gagatatatc ccggatgagg ctgactttct gc
#tggggatg   1260
gccactgtga ataactgtgt ttcctaccga aaccctgcag agggaacctg gt
#acatccag   1320
tcactttgcc agagcctgag agagcgatgt cctcgaggcg atgatattct ca
#ccatcctg   1380
actgaagtga actatgaagt aagcaacaag gatgacaaga aaaacatggg ga
#aacagatg   1440
cctcagccta ctttcacact aagaaaaaaa cttgtcttcc cttctgattg an
#n          1493
<210> SEQ ID NO 26
<211> LENGTH: 1493
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (1)...(1493)
<223> OTHER INFORMATION: n = A,T,C or G
<400> SEQUENCE: 26
tacctgaagt cgtctttaga aatactataa ccccttgttg acctgtcact tc
#tagaccgg     60
agggagttca aggactcgga cctgatgtaa ggcgtttcct tcgttcttgg gt
#agttccta    120
cggaactaca ataaggtctc tgaggtcctt ttctcttaca acctcctttc gt
#tagacagg    180
aaggacttcc tcgacgagaa ggcttaatta tctgacctaa acgactaatg ga
#tggatttg    240
tgatctttcc tcctctacct ttcccttgaa gtctgtggtc cgtcccgagt tt
#aaagacgg    300
atgtccaagg tgaagacggc gtactcgacc cgacttcgtt tgtcggtcac gg
#tctgtgtc    360
agacatggaa agaccgcctc ccagctagta gataattatt cccagtacga ga
#tagtctaa    420
agtcttcttc actcgtctag tcttaactcc agaaaattca aagaaaacgt tc
#tcctttag    480
aggtttacgt ttgacctact actgtacttg gacgacctat aaaagtatct ct
#acctcttc    540
tcccagtagg accctcttcc tttcaacctg taggactttt ctcagacacg gg
#tttagttg    600
ttctcggacg acttctatta gttgctgata cttcttaagt cgtttcccct cc
#tcaacaca    660
ccccattact gttagagcct gagaggttct cttgtcctat cacttagtgt ct
#gaaacctg    720
tttcaaatgg tttacttttc gtttggagcc cctatgacag actagtagtt gt
#tagtgtta    780
aaacgttttc gtgccctctt tcacgggttt gaagtgtcgt aatccctgtc ct
#taccttgt    840
gtgaacctac gtccccgaaa ctggtgctgg aaacttctcg aagtaaaact ct
#agttcggg    900
gtggtactga cgtgtcatct cgtttagata ctctaaaact tttagatggt tg
#agtacctg    960
gtgtcattgt acctgacgaa gtagacgaca taggagaggg tacctctgtt cc
#cgtagtag   1020
ataccgtgac tacctgtcct ccgggggtag atactcgact gtagagtcaa gt
#gaccaaac   1080
ttcacgggaa gggaacgacc ttttgggttt cacaaaaaat aagtccgaac ag
#tcccccta   1140
ttgatggtct ttccatatgg acaactctga ctaagtctcc tcgttgggat aa
#atctttac   1200
ctaaatagta gtggagtttg ctctatatag ggcctactcc gactgaaaga cg
#acccctac   1260
cggtgacact tattgacaca aaggatggct ttgggacgtc tcccttggac ca
#tgtaggtc   1320
agtgaaacgg tctcggactc tctcgctaca ggagctccgc tactataaga gt
#ggtaggac   1380
tgacttcact tgatacttca ttcgttgttc ctactgttct ttttgtaccc ct
#ttgtctac   1440
ggagtcggat gaaagtgtga ttcttttttt gaacagaagg gaagactaac tn
#n          1493
<210> SEQ ID NO 27
<211> LENGTH: 476
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 27
Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gl
#y Glu Gln Leu Asp Ser
1               5
#                10
#                15
Glu Asp Leu Ala Ser Leu Lys Phe Leu Ser Le
#u Asp Tyr Ile Pro Gln
20
#            25
#            30
Arg Lys Gln Glu Pro Ile Lys Asp Ala Leu Me
#t Leu Phe Gln Arg Leu
35
#        40
#        45
Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Le
#u Ser Phe Leu Lys Glu
50
#    55
#    60
Leu Leu Phe Arg Ile Asn Arg Leu Asp Leu Le
#u Ile Thr Tyr Leu Asn
65
#70
#75
#80
Thr Arg Lys Glu Glu Met Glu Arg Glu Leu Gl
#n Thr Pro Gly Arg Ala
85
#                90
#                95
Gln Ile Ser Ala Tyr Arg Phe His Phe Cys Ar
#g Met Ser Trp Ala Glu
100
#           105
#           110
Ala Asn Ser Gln Cys Gln Thr Gln Ser Val Pr
#o Phe Trp Arg Arg Val
115
#       120
#       125
Asp His Leu Leu Ile Arg Val Met Leu Tyr Gl
#n Ile Ser Glu Glu Val
130
#   135
#   140
Ser Arg Ser Glu Leu Arg Ser Phe Lys Phe Le
#u Leu Gln Glu Glu Ile
145                 1
#50                 1
#55                 1
#60
Ser Lys Cys Lys Leu Asp Asp Asp Met Asn Le
#u Leu Asp Ile Phe Ile
165
#               170
#               175
Glu Met Glu Lys Arg Val Ile Leu Gly Glu Gl
#y Lys Leu Asp Ile Leu
180
#           185
#           190
Lys Arg Val Cys Ala Gln Ile Asn Lys Ser Le
#u Leu Lys Ile Ile Asn
195
#       200
#       205
Asp Tyr Glu Glu Phe Ser Lys Gly Glu Glu Le
#u Cys Lys Val Tyr Gln
210
#   215
#   220
Met Lys Ser Lys Pro Arg Gly Tyr Cys Leu Il
#e Ile Asn Asn His Asn
225                 2
#30                 2
#35                 2
#40
Phe Ala Lys Ala Arg Glu Lys Val Pro Lys Le
#u His Ser Ile Arg Asp
245
#               250
#               255
Arg Asn Gly Thr His Leu Asp Ala Gly Ala Le
#u Thr Thr Thr Phe Glu
260
#           265
#           270
Glu Leu His Phe Glu Ile Lys Pro His His As
#p Cys Thr Val Glu Gln
275
#       280
#       285
Ile Tyr Glu Ile Leu Lys Ile Tyr Gln Leu Me
#t Asp His Ser Asn Met
290
#   295
#   300
Asp Cys Phe Ile Cys Cys Ile Leu Ser His Gl
#y Asp Lys Gly Ile Ile
305                 3
#10                 3
#15                 3
#20
Tyr Gly Thr Asp Gly Gln Glu Ala Pro Ile Ty
#r Glu Leu Thr Ser Gln
325
#               330
#               335
Phe Thr Gly Leu Lys Cys Pro Ser Leu Ala Gl
#y Lys Pro Lys Val Phe
340
#           345
#           350
Phe Ile Gln Ala Cys Gln Gly Asp Asn Tyr Gl
#n Lys Gly Ile Pro Val
355
#       360
#       365
Glu Thr Asp Ser Glu Glu Gln Pro Tyr Leu Gl
#u Met Asp Leu Ser Ser
370
#   375
#   380
Pro Gln Thr Arg Tyr Ile Pro Asp Glu Ala As
#p Phe Leu Leu Gly Met
385                 3
#90                 3
#95                 4
#00
Ala Thr Val Asn Asn Cys Val Ser Tyr Arg As
#n Pro Ala Glu Gly Thr
405
#               410
#               415
Trp Tyr Ile Gln Ser Leu Cys Gln Ser Leu Ar
#g Glu Arg Cys Pro Arg
420
#           425
#           430
Gly Asp Asp Ile Leu Thr Ile Leu Thr Glu Va
#l Asn Tyr Glu Val Ser
435
#       440
#       445
Asn Lys Asp Asp Lys Lys Asn Met Gly Lys Gl
#n Met Pro Gln Pro Thr
450
#   455
#   460
Phe Thr Leu Arg Lys Lys Leu Val Phe Pro Se
#r Asp
465                 4
#70                 4
#75
<210> SEQ ID NO 28
<211> LENGTH: 1252
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (1)...(1252)
<223> OTHER INFORMATION: n = A,T,C or G
<400> SEQUENCE: 28
atggacgaag cggatcggcg gctcctgcgg cggtgccggc tgcggctggt gg
#aagagctg     60
caggtggacc agctctggga cgccctgctg agcagcgagc tgttcaggcc cc
#atatgatc    120
gaggacatcc agcgggcagg ctctggatct cggcgggatc aggccaggca gc
#tgatcata    180
gatctggaga ctcgagggag tcaggctctt cctttgttca tctcctgctt ag
#aggacaca    240
ggccaggaca tgctggcttc gtttctgcga actaacaggc aagcagcaaa gt
#tgtcgaag    300
ccaaccctag aaaaccttac cccagtggtg ctcagaccag agattcgcaa ac
#cagaggtt    360
ctcagaccgg aaacacccag accagtggac attggttctg gaggatttgg tg
#atgtcggt    420
gctcttgaga gtttgagggg aaatgcagat ttggcttaca tcctgagcat gg
#agccctgt    480
ggccactgcc tcattatcaa caatgtgaac ttctgccgtg agtccgggct cc
#gcacccgc    540
actggctcca acatcgactg tgagaagttg cggcgtcgct tctcctcgcc gc
#atttcatg    600
gtggaggtga agggcgacct gactgccaag aaaatggtgc tggctttgct gg
#agctggcg    660
cggcaggacc acggtgctct ggactgctgc gtggtggtca ttctctctca cg
#gctgtcag    720
gccagccacc tgcagttccc aggggctgtc tacggcacag atggatgccc tg
#tgtcggtc    780
gagaagattg tgaacatctt caatgggacc agctgcccca gcctgggagg aa
#agcccaag    840
ctctttttca tccaggcctg tggtggggag cagaaagacc atgggtttga gg
#tggcctcc    900
acttcccctg aagacgagtc ccctggcagt aaccccgagc cagatgccac cc
#cgttccag    960
gaaggtttga ggaccttcga ccagctggac gccatatcta gtttgcccac ac
#ccagtgac   1020
atctttgtgt cctactctac tttcccaggt tttgtttcct ggagggaccc ca
#agagtggc   1080
tcctggtacg ttgagaccct ggacgacatc tttgagcagt gggctcactc tg
#aagacctg   1140
cagtccctcc tgcttagggt cgctaatgct gtttcggtga aagggattta ta
#aacagatg   1200
cctggttgct ttaatttcct ccggaaaaaa cttttcttta aaacatcata an
#           1252
<210> SEQ ID NO 29
<211> LENGTH: 1252
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (1)...(1252)
<223> OTHER INFORMATION: n = A,T,C or G
<400> SEQUENCE: 29
tacctgcttc gcctagccgc cgaggacgcc gccacggccg acgccgacca cc
#ttctcgac     60
gtccacctgg tcgagaccct gcgggacgac tcgtcgctcg acaagtccgg gg
#tatactag    120
ctcctgtagg tcgcccgtcc gagacctaga gccgccctag tccggtccgt cg
#actagtat    180
ctagacctct gagctccctc agtccgagaa ggaaacaagt agaggacgaa tc
#tcctgtgt    240
ccggtcctgt acgaccgaag caaagacgct tgattgtccg ttcgtcgttt ca
#acagcttc    300
ggttgggatc ttttggaatg gggtcaccac gagtctggtc tctaagcgtt tg
#gtctccaa    360
gagtctggcc tttgtgggtc tggtcacctg taaccaagac ctcctaaacc ac
#tacagcca    420
cgagaactct caaactcccc tttacgtcta aaccgaatgt aggactcgta cc
#tcgggaca    480
ccggtgacgg agtaatagtt gttacacttg aagacggcac tcaggcccga gg
#cgtgggcg    540
tgaccgaggt tgtagctgac actcttcaac gccgcagcga agaggagcgg cg
#taaagtac    600
cacctccact tcccgctgga ctgacggttc ttttaccacg accgaaacga cc
#tcgaccgc    660
gccgtcctgg tgccacgaga cctgacgacg caccaccagt aagagagagt gc
#cgacagtc    720
cggtcggtgg acgtcaaggg tccccgacag atgccgtgtc tacctacggg ac
#acagccag    780
ctcttctaac acttgtagaa gttaccctgg tcgacggggt cggaccctcc tt
#tcgggttc    840
gagaaaaagt aggtccggac accacccctc gtctttctgg tacccaaact cc
#accggagg    900
tgaaggggac ttctgctcag gggaccgtca ttggggctcg gtctacggtg gg
#gcaaggtc    960
cttccaaact cctggaagct ggtcgacctg cggtatagat caaacgggtg tg
#ggtcactg   1020
tagaaacaca ggatgagatg aaagggtcca aaacaaagga cctccctggg gt
#tctcaccg   1080
aggaccatgc aactctggga cctgctgtag aaactcgtca cccgagtgag ac
#ttctggac   1140
gtcagggagg acgaatccca gcgattacga caaagccact ttccctaaat at
#ttgtctac   1200
ggaccaacga aattaaagga ggcctttttt gaaaagaaat tttgtagtat tn
#           1252
<210> SEQ ID NO 30
<211> LENGTH: 416
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 30
Met Asp Glu Ala Asp Arg Arg Leu Leu Arg Ar
#g Cys Arg Leu Arg Leu
1               5
#                10
#                15
Val Glu Glu Leu Gln Val Asp Gln Leu Trp As
#p Ala Leu Leu Ser Ser
20
#            25
#            30
Glu Leu Phe Arg Pro His Met Ile Glu Asp Il
#e Gln Arg Ala Gly Ser
35
#        40
#        45
Gly Ser Arg Arg Asp Gln Ala Arg Gln Leu Il
#e Ile Asp Leu Glu Thr
50
#    55
#    60
Arg Gly Ser Gln Ala Leu Pro Leu Phe Ile Se
#r Cys Leu Glu Asp Thr
65
#70
#75
#80
Gly Gln Asp Met Leu Ala Ser Phe Leu Arg Th
#r Asn Arg Gln Ala Ala
85
#                90
#                95
Lys Leu Ser Lys Pro Thr Leu Glu Asn Leu Th
#r Pro Val Val Leu Arg
100
#           105
#           110
Pro Glu Ile Arg Lys Pro Glu Val Leu Arg Pr
#o Glu Thr Pro Arg Pro
115
#       120
#       125
Val Asp Ile Gly Ser Gly Gly Phe Gly Asp Va
#l Gly Ala Leu Glu Ser
130
#   135
#   140
Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Le
#u Ser Met Glu Pro Cys
145                 1
#50                 1
#55                 1
#60
Gly His Cys Leu Ile Ile Asn Asn Val Asn Ph
#e Cys Arg Glu Ser Gly
165
#               170
#               175
Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cy
#s Glu Lys Leu Arg Arg
180
#           185
#           190
Arg Phe Ser Ser Pro His Phe Met Val Glu Va
#l Lys Gly Asp Leu Thr
195
#       200
#       205
Ala Lys Lys Met Val Leu Ala Leu Leu Glu Le
#u Ala Arg Gln Asp His
210
#   215
#   220
Gly Ala Leu Asp Cys Cys Val Val Val Ile Le
#u Ser His Gly Cys Gln
225                 2
#30                 2
#35                 2
#40
Ala Ser His Leu Gln Phe Pro Gly Ala Val Ty
#r Gly Thr Asp Gly Cys
245
#               250
#               255
Pro Val Ser Val Glu Lys Ile Val Asn Ile Ph
#e Asn Gly Thr Ser Cys
260
#           265
#           270
Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Ph
#e Ile Gln Ala Cys Gly
275
#       280
#       285
Gly Glu Gln Lys Asp His Gly Phe Glu Val Al
#a Ser Thr Ser Pro Glu
290
#   295
#   300
Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro As
#p Ala Thr Pro Phe Gln
305                 3
#10                 3
#15                 3
#20
Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Al
#a Ile Ser Ser Leu Pro
325
#               330
#               335
Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Th
#r Phe Pro Gly Phe Val
340
#           345
#           350
Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Ty
#r Val Glu Thr Leu Asp
355
#       360
#       365
Asp Ile Phe Glu Gln Trp Ala His Ser Glu As
#p Leu Gln Ser Leu Leu
370
#   375
#   380
Leu Arg Val Ala Asn Ala Val Ser Val Lys Gl
#y Ile Tyr Lys Gln Met
385                 3
#90                 3
#95                 4
#00
Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Le
#u Phe Phe Lys Thr Ser
405
#               410
#               415
<210> SEQ ID NO 31
<211> LENGTH: 1440
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 31
atgaaatctc aaggtcaaca ttggtattcc agttcagata aaaactgtaa ag
#tgagcttt     60
cgtgagaagc ttctgattat tgattcaaac ctgggggtcc aagatgtgga ga
#acctcaag    120
tttctctgca taggattggt ccccaacaag aagctggaga agtccagctc ag
#cctcagat    180
gtttttgaac atctcttggc agaggatctg ctgagtgagg aagacccttt ct
#tcctggca    240
gaactcctct atatcatacg gcagaagaag ctgctgcagc acctcaactg ta
#ccaaagag    300
gaagtggagc gactgctgcc cacccgacaa agggtttctc tgtttagaaa cc
#tgctctac    360
gaactgtcag aaggcattga ctcagagaac ttaaaggaca tgatcttcct tc
#tgaaagac    420
tcgcttccca aaactgaaat gacctcccta agtttcctgg catttctaga ga
#aacaaggt    480
aaaatagatg aagataatct gacatgcctg gaggacctct gcaaaacagt tg
#tacctaaa    540
cttttgagaa acatagagaa atacaaaaga gagaaagcta tccagatagt ga
#cacctcct    600
gtagacaagg aagccgagtc gtatcaagga gaggaagaac tagtttccca aa
#cagatgtt    660
aagacattct tggaagcctt accgagggca gctgtgtaca ggatgaatcg ga
#accacaga    720
ggcctctgtg tcattgtcaa caaccacagc tttacctccc tgaaggacag ac
#aaggaacc    780
cataaagatg ctgagatcct gagtcatgtg ttccagtggc ttgggttcac ag
#tgcatata    840
cacaataatg tgacgaaagt ggaaatggag atggtcctgc agaagcagaa gt
#gcaatcca    900
gcccatgccg acggggactg cttcgtgttc tgtattctga cccatgggag at
#ttggagct    960
gtctactctt cggatgaggc cctcattccc attcgggaga tcatgtctca ct
#tcacagcc   1020
ctgcagtgcc ctagactggc tgaaaaacct aaactctttt tcatccaggc ct
#gccaaggt   1080
gaagagatac agccttccgt atccatcgaa gcagatgctc tgaaccctga gc
#aggcaccc   1140
acttccctgc aggacagtat tcctgccgag gctgacttcc tacttggtct gg
#ccactgtc   1200
ccaggctatg tatcctttcg gcatgtggag gaaggcagct ggtatattca gt
#ctctgtgt   1260
aatcatctga agaaattggt cccaagacat gaagacatct tatccatcct ca
#ctgctgtc   1320
aacgatgatg tgagtcgaag agtggacaaa cagggaacaa agaaacagat gc
#cccagcct   1380
gctttcacac taaggaaaaa actagtattc cctgtgcccc tggatgcact tt
#caatatag   1440
<210> SEQ ID NO 32
<211> LENGTH: 1440
<212> TYPE: DNA
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 32
tactttagag ttccagttgt aaccataagg tcaagtctat ttttgacatt tc
#actcgaaa     60
gcactcttcg aagactaata actaagtttg gacccccagg ttctacacct ct
#tggagttc    120
aaagagacgt atcctaacca ggggttgttc ttcgacctct tcaggtcgag tc
#ggagtcta    180
caaaaacttg tagagaaccg tctcctagac gactcactcc ttctgggaaa ga
#aggaccgt    240
cttgaggaga tatagtatgc cgtcttcttc gacgacgtcg tggagttgac at
#ggtttctc    300
cttcacctcg ctgacgacgg gtgggctgtt tcccaaagag acaaatcttt gg
#acgagatg    360
cttgacagtc ttccgtaact gagtctcttg aatttcctgt actagaagga ag
#actttctg    420
agcgaagggt tttgacttta ctggagggat tcaaaggacc gtaaagatct ct
#ttgttcca    480
ttttatctac ttctattaga ctgtacggac ctcctggaga cgttttgtca ac
#atggattt    540
gaaaactctt tgtatctctt tatgttttct ctctttcgat aggtctatca ct
#gtggagga    600
catctgttcc ttcggctcag catagttcct ctccttcttg atcaaagggt tt
#gtctacaa    660
ttctgtaaga accttcggaa tggctcccgt cgacacatgt cctacttagc ct
#tggtgtct    720
ccggagacac agtaacagtt gttggtgtcg aaatggaggg acttcctgtc tg
#ttccttgg    780
gtatttctac gactctagga ctcagtacac aaggtcaccg aacccaagtg tc
#acgtatat    840
gtgttattac actgctttca cctttacctc taccaggacg tcttcgtctt ca
#cgttaggt    900
cgggtacggc tgcccctgac gaagcacaag acataagact gggtaccctc ta
#aacctcga    960
cagatgagaa gcctactccg ggagtaaggg taagccctct agtacagagt ga
#agtgtcgg   1020
gacgtcacgg gatctgaccg actttttgga tttgagaaaa agtaggtccg ga
#cggttcca   1080
cttctctatg tcggaaggca taggtagctt cgtctacgag acttgggact cg
#tccgtggg   1140
tgaagggacg tcctgtcata aggacggctc cgactgaagg atgaaccaga cc
#ggtgacag   1200
ggtccgatac ataggaaagc cgtacacctc cttccgtcga ccatataagt ca
#gagacaca   1260
ttagtagact tctttaacca gggttctgta cttctgtaga ataggtagga gt
#gacgacag   1320
ttgctactac actcagcttc tcacctgttt gtcccttgtt tctttgtcta cg
#gggtcgga   1380
cgaaagtgtg attccttttt tgatcataag ggacacgggg acctacgtga aa
#gttatatc   1440
<210> SEQ ID NO 33
<211> LENGTH: 479
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 33
Met Lys Ser Gln Gly Gln His Trp Tyr Ser Se
#r Ser Asp Lys Asn Cys
1               5
#                10
#                15
Lys Val Ser Phe Arg Glu Lys Leu Leu Ile Il
#e Asp Ser Asn Leu Gly
20
#            25
#            30
Val Gln Asp Val Glu Asn Leu Lys Phe Leu Cy
#s Ile Gly Leu Val Pro
35
#        40
#        45
Asn Lys Lys Leu Glu Lys Ser Ser Ser Ala Se
#r Asp Val Phe Glu His
50
#    55
#    60
Leu Leu Ala Glu Asp Leu Leu Ser Glu Glu As
#p Pro Phe Phe Leu Ala
65
#70
#75
#80
Glu Leu Leu Tyr Ile Ile Arg Gln Lys Lys Le
#u Leu Gln His Leu Asn
85
#                90
#                95
Cys Thr Lys Glu Glu Val Glu Arg Leu Leu Pr
#o Thr Arg Gln Arg Val
100
#           105
#           110
Ser Leu Phe Arg Asn Leu Leu Tyr Glu Leu Se
#r Glu Gly Ile Asp Ser
115
#       120
#       125
Glu Asn Leu Lys Asp Met Ile Phe Leu Leu Ly
#s Asp Ser Leu Pro Lys
130
#   135
#   140
Thr Glu Met Thr Ser Leu Ser Phe Leu Ala Ph
#e Leu Glu Lys Gln Gly
145                 1
#50                 1
#55                 1
#60
Lys Ile Asp Glu Asp Asn Leu Thr Cys Leu Gl
#u Asp Leu Cys Lys Thr
165
#               170
#               175
Val Val Pro Lys Leu Leu Arg Asn Ile Glu Ly
#s Tyr Lys Arg Glu Lys
180
#           185
#           190
Ala Ile Gln Ile Val Thr Pro Pro Val Asp Ly
#s Glu Ala Glu Ser Tyr
195
#       200
#       205
Gln Gly Glu Glu Glu Leu Val Ser Gln Thr As
#p Val Lys Thr Phe Leu
210
#   215
#   220
Glu Ala Leu Pro Arg Ala Ala Val Tyr Arg Me
#t Asn Arg Asn His Arg
225                 2
#30                 2
#35                 2
#40
Gly Leu Cys Val Ile Val Asn Asn His Ser Ph
#e Thr Ser Leu Lys Asp
245
#               250
#               255
Arg Gln Gly Thr His Lys Asp Ala Glu Ile Le
#u Ser His Val Phe Gln
260
#           265
#           270
Trp Leu Gly Phe Thr Val His Ile His Asn As
#n Val Thr Lys Val Glu
275
#       280
#       285
Met Glu Met Val Leu Gln Lys Gln Lys Cys As
#n Pro Ala His Ala Asp
290
#   295
#   300
Gly Asp Cys Phe Val Phe Cys Ile Leu Thr Hi
#s Gly Arg Phe Gly Ala
305                 3
#10                 3
#15                 3
#20
Val Tyr Ser Ser Asp Glu Ala Leu Ile Pro Il
#e Arg Glu Ile Met Ser
325
#               330
#               335
His Phe Thr Ala Leu Gln Cys Pro Arg Leu Al
#a Glu Lys Pro Lys Leu
340
#           345
#           350
Phe Phe Ile Gln Ala Cys Gln Gly Glu Glu Il
#e Gln Pro Ser Val Ser
355
#       360
#       365
Ile Glu Ala Asp Ala Leu Asn Pro Glu Gln Al
#a Pro Thr Ser Leu Gln
370
#   375
#   380
Asp Ser Ile Pro Ala Glu Ala Asp Phe Leu Le
#u Gly Leu Ala Thr Val
385                 3
#90                 3
#95                 4
#00
Pro Gly Tyr Val Ser Phe Arg His Val Glu Gl
#u Gly Ser Trp Tyr Ile
405
#               410
#               415
Gln Ser Leu Cys Asn His Leu Lys Lys Leu Va
#l Pro Arg His Glu Asp
420
#           425
#           430
Ile Leu Ser Ile Leu Thr Ala Val Asn Asp As
#p Val Ser Arg Arg Val
435
#       440
#       445
Asp Lys Gln Gly Thr Lys Lys Gln Met Pro Gl
#n Pro Ala Phe Thr Leu
450
#   455
#   460
Arg Lys Lys Leu Val Phe Pro Val Pro Leu As
#p Ala Leu Ser Ile
465                 4
#70                 4
#75
<210> SEQ ID NO 34
<211> LENGTH: 290
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Rev-Caspase-3 contructed fro
#m human caspase -3
<400> SEQUENCE: 34
Met Ile Glu Thr Asp Ser Gly Val Asp Asp As
#p Met Ala Cys His Lys
1               5
#                10
#                15
Ile Pro Val Glu Ala Asp Phe Leu Tyr Ala Ty
#r Ser Thr Ala Pro Gly
20
#            25
#            30
Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Se
#r Trp Phe Ile Gln Ser
35
#        40
#        45
Leu Cys Ala Met Leu Lys Gln Tyr Ala Asp Ly
#s Leu Glu Phe Met His
50
#    55
#    60
Ile Leu Thr Arg Val Asn Arg Lys Val Ala Th
#r Glu Phe Glu Ser Phe
65
#70
#75
#80
Ser Phe Asp Ala Thr Phe His Ala Lys Lys Gl
#n Ile Pro Cys Ile Val
85
#                90
#                95
Ser Met Leu Thr Lys Glu Leu Tyr Phe Tyr Hi
#s Asp Glu Val Asp Gly
100
#           105
#           110
Gly Ser Pro Met Glu Asn Thr Glu Asn Ser Va
#l Asp Ser Lys Ser Ile
115
#       120
#       125
Lys Asn Leu Glu Pro Lys Ile Ile His Gly Se
#r Glu Ser Met Asp Ser
130
#   135
#   140
Gly Ile Ser Leu Asp Asn Ser Tyr Lys Met As
#p Tyr Pro Glu Met Gly
145                 1
#50                 1
#55                 1
#60
Leu Cys Ile Ile Ile Asn Asn Lys Asn Phe Hi
#s Lys Ser Thr Gly Met
165
#               170
#               175
Thr Ser Arg Ser Gly Thr Asp Val Asp Ala Al
#a Asn Leu Arg Glu Thr
180
#           185
#           190
Phe Arg Asn Leu Lys Tyr Glu Val Arg Asn Ly
#s Asn Asp Leu Thr Arg
195
#       200
#       205
Glu Glu Ile Val Glu Leu Met Arg Asp Val Se
#r Lys Glu Asp His Ser
210
#   215
#   220
Lys Arg Ser Ser Phe Val Cys Val Leu Leu Se
#r His Gly Glu Glu Gly
225                 2
#30                 2
#35                 2
#40
Ile Ile Phe Gly Thr Asn Gly Pro Val Asp Le
#u Lys Lys Ile Thr Asn
245
#               250
#               255
Phe Phe Arg Gly Asp Arg Cys Arg Ser Leu Th
#r Gly Lys Pro Lys Leu
260
#           265
#           270
Phe Ile Ile Gln Ala Cys Arg Gly Thr Glu Le
#u Asp Cys Gly Ile Glu
275
#       280
#       285
Thr Asp
290
<210> SEQ ID NO 35
<211> LENGTH: 285
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Uncleavable Rev-Caspase-3 co
#ntructed from human
caspase -3
<400> SEQUENCE: 35
Met Ile Glu Thr Asp Ser Gly Val Asp Asp As
#p Met Ala Cys His Lys
1               5
#                10
#                15
Ile Pro Val Glu Ala Asp Phe Leu Tyr Ala Ty
#r Ser Thr Ala Pro Gly
20
#            25
#            30
Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Se
#r Trp Phe Ile Gln Ser
35
#        40
#        45
Leu Cys Ala Met Leu Lys Gln Tyr Ala Asp Ly
#s Leu Glu Phe Met His
50
#    55
#    60
Ile Leu Thr Arg Val Asn Arg Lys Val Ala Th
#r Glu Phe Glu Ser Phe
65
#70
#75
#80
Ser Phe Asp Ala Thr Phe His Ala Lys Lys Gl
#n Ile Pro Cys Ile Val
85
#                90
#                95
Ser Met Leu Thr Lys Glu Leu Tyr Phe Tyr Hi
#s Gly Ser Pro Met Glu
100
#           105
#           110
Asn Thr Glu Asn Ser Val Ala Ser Lys Ser Il
#e Lys Asn Leu Glu Pro
115
#       120
#       125
Lys Ile Ile His Gly Ser Glu Ser Met Ala Se
#r Gly Ile Ser Leu Asp
130
#   135
#   140
Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gl
#y Leu Cys Ile Ile Ile
145                 1
#50                 1
#55                 1
#60
Asn Asn Lys Asn Phe His Lys Ser Thr Gly Me
#t Thr Ser Arg Ser Gly
165
#               170
#               175
Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Th
#r Phe Arg Asn Leu Lys
180
#           185
#           190
Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Ar
#g Glu Glu Ile Val Glu
195
#       200
#       205
Leu Met Arg Asp Val Ser Lys Glu Asp His Se
#r Lys Arg Ser Ser Phe
210
#   215
#   220
Val Cys Val Leu Leu Ser His Gly Glu Glu Gl
#y Ile Ile Phe Gly Thr
225                 2
#30                 2
#35                 2
#40
Asn Gly Pro Val Asp Leu Lys Lys Ile Thr As
#n Phe Phe Arg Gly Asp
245
#               250
#               255
Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Le
#u Phe Ile Ile Gln Ala
260
#           265
#           270
Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Gl
#u Thr Asp
275
#       280
#       285
<210> SEQ ID NO 36
<211> LENGTH: 300
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Rev-Caspase-6 contructed fro
#m human caspase-6
<400> SEQUENCE: 36
Met Val Glu Ile Asp Ala Ala Ser Val Tyr Th
#r Leu Pro Ala Gly Ala
1               5
#                10
#                15
Asp Phe Leu Met Cys Tyr Ser Val Ala Glu Gl
#y Tyr Tyr Ser His Arg
20
#            25
#            30
Glu Thr Val Asn Gly Ser Trp Tyr Ile Gln As
#p Leu Cys Glu Met Leu
35
#        40
#        45
Gly Lys Tyr Gly Ser Ser Leu Glu Phe Thr Gl
#u Leu Leu Thr Leu Val
50
#    55
#    60
Asn Arg Lys Val Ser Gln Arg Arg Val Asp Ph
#e Cys Lys Asp Pro Ser
65
#70
#75
#80
Ala Ile Gly Lys Lys Gln Val Pro Cys Phe Al
#a Ser Met Leu Thr Lys
85
#                90
#                95
Lys Leu His Phe Phe Pro Lys Ser Asn Leu Gl
#u His His His His His
100
#           105
#           110
His Val Glu Ile Asp Gly Gly Ser Pro Met Se
#r Ser Ala Ser Gly Leu
115
#       120
#       125
Arg Arg Gly His Pro Ala Gly Gly Glu Glu As
#n Met Thr Glu Thr Asp
130
#   135
#   140
Ala Phe Tyr Lys Arg Glu Met Phe Asp Pro Al
#a Glu Lys Tyr Lys Met
145                 1
#50                 1
#55                 1
#60
Asp His Arg Arg Arg Gly Ile Ala Leu Ile Ph
#e Asn His Glu Arg Phe
165
#               170
#               175
Phe Trp His Leu Thr Leu Pro Glu Arg Arg Gl
#y Thr Cys Ala Asp Arg
180
#           185
#           190
Asp Asn Leu Thr Arg Arg Phe Ser Asp Leu Gl
#y Phe Glu Val Lys Cys
195
#       200
#       205
Phe Asn Asp Leu Lys Ala Glu Glu Leu Leu Le
#u Lys Ile His Glu Val
210
#   215
#   220
Ser Thr Val Ser His Ala Asp Ala Asp Cys Ph
#e Val Cys Val Phe Leu
225                 2
#30                 2
#35                 2
#40
Ser His Gly Glu Gly Asn His Ile Tyr Ala Ty
#r Asp Ala Lys Ile Glu
245
#               250
#               255
Ile Gln Thr Leu Thr Gly Leu Phe Lys Gly As
#p Lys Cys His Ser Leu
260
#           265
#           270
Val Gly Lys Pro Lys Ile Phe Ile Ile Gln Al
#a Cys Arg Gly Asn Gln
275
#       280
#       285
His Asp Val Pro Val Ile Pro Leu Asp Val Va
#l Asp
290
#   295
#   300
<210> SEQ ID NO 37
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Protease sensitive linker
#region between the
small and large subunits of a
#Rev-caspase
<221> NAME/KEY: VARIANT
<222> LOCATION: (1)...(5)
<223> OTHER INFORMATION: Xaa = Any Amino Aci
#d
<400> SEQUENCE: 37
Asp Xaa Xaa Asp Gly
1               5
<210> SEQ ID NO 38
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Protease sensitive linker
#region between the
small and large subunits of a
#Rev-caspase
<400> SEQUENCE: 38
Asp Glu Val Asp Gly
1               5
<210> SEQ ID NO 39
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Protease sensitive linker
#region between the
small and large subunits of a
#Rev-caspase
<400> SEQUENCE: 39
Ile Glu Thr Asp Gly
1               5
<210> SEQ ID NO 40
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Protease sensitive linker
#region between the
small and large subunits of a
#Rev-caspase
<400> SEQUENCE: 40
Tyr Val Ala Asp Gly
1               5
<210> SEQ ID NO 41
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Protease sensitive linker
#region between the
small and large subunits of a
#Rev-caspase
<400> SEQUENCE: 41
Tyr Val His Asp Gly
1               5
<210> SEQ ID NO 42
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Protease sensitive linker
#region between the
small and large subunits of a
#Rev-caspase
<400> SEQUENCE: 42
Trp Glu His Asp Gly
1               5
<210> SEQ ID NO 43
<211> LENGTH: 22
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR primer for large s
#ubunit of caspase-3
<400> SEQUENCE: 43
atggagaaca ctgaaaactc ag
#
#                 22
<210> SEQ ID NO 44
<211> LENGTH: 22
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR primer for large s
#ubunit of caspase-3
<400> SEQUENCE: 44
gtcatcatca acacctcagt ct
#
#                 22
<210> SEQ ID NO 45
<211> LENGTH: 26
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR primer for small s
#ubunit of caspase-3
<400> SEQUENCE: 45
ggatccatga ttgagacaga cagtgg
#
#              26
<210> SEQ ID NO 46
<211> LENGTH: 30
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR primer for small s
#ubunit of caspase-3
<400> SEQUENCE: 46
atcaacttca tcgtgataaa aatagagttc
#
#           30
<210> SEQ ID NO 47
<211> LENGTH: 18
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR primer for large s
#ubunit of caspase-6
<400> SEQUENCE: 47
atgagctcgg cctcgggg
#
#
#  18
<210> SEQ ID NO 48
<211> LENGTH: 21
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR primer for large s
#ubunit of caspase-6
<400> SEQUENCE: 48
ttaatctact acatccaaag g
#
#
#21
<210> SEQ ID NO 49
<211> LENGTH: 36
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR primer for small s
#ubunit of caspase-6
<400> SEQUENCE: 49
ggatccatgg tagaaataga tgcagcctcc gtttac
#
#       36
<210> SEQ ID NO 50
<211> LENGTH: 30
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: PCR primer for small s
#ubunit of caspase-6
<400> SEQUENCE: 50
atcaattcaa cgtggtggtg gtggtggtgc
#
#           30
<210> SEQ ID NO 51
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<220> FEATURE:
<221> NAME/KEY: VARIANT
<222> LOCATION: (1)...(5)
<223> OTHER INFORMATION: Xaa = Any Amino Aci
#d
<400> SEQUENCE: 51
Gln Ala Cys Xaa Gly
1               5
<210> SEQ ID NO 52
<211> LENGTH: 4
<212> TYPE: PRT
<213> ORGANISM: Homo Sapien
<400> SEQUENCE: 52
Asp Glu Val Asp
1
<210> SEQ ID NO 53
<211> LENGTH: 4
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 53
Ile Glu Thr Asp
1
<210> SEQ ID NO 54
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 54
Arg Thr Arg Thr Gly Ser
1               5
<210> SEQ ID NO 55
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 55
Leu Ser His Gly Cys Gln
1               5
<210> SEQ ID NO 56
<211> LENGTH: 9
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 56
Phe Ile Gln Ala Cys Gly Gly Glu Gln
1               5
<210> SEQ ID NO 57
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 57
Pro Glu Pro Asp Ala
1               5
<210> SEQ ID NO 58
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 58
Asp Gln Leu Asp Ala
1               5
<210> SEQ ID NO 59
<211> LENGTH: 15
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 59
Gly Phe Val Ser Trp Arg Asp Pro Lys Ser Gl
#y Ser Trp Tyr Val
1               5
#                10
#                15
<210> SEQ ID NO 60
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 60
Arg Asp Arg Asn Gly Thr
1               5
<210> SEQ ID NO 61
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 61
Leu Ser His Gly Asp Lys
1               5
<210> SEQ ID NO 62
<211> LENGTH: 9
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 62
Phe Ile Gln Ala Cys Gln Gly Asp Asn
1               5
<210> SEQ ID NO 63
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 63
Val Glu Thr Asp Ser
1               5
<210> SEQ ID NO 64
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 64
Leu Glu Met Asp Leu
1               5
<210> SEQ ID NO 65
<211> LENGTH: 15
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 65
Asn Cys Val Ser Tyr Arg Asn Pro Ala Glu Gl
#y Thr Trp Tyr Ile
1               5
#                10
#                15
<210> SEQ ID NO 66
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 66
Lys Asp Arg Gln Gly Thr
1               5
<210> SEQ ID NO 67
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 67
Leu Thr His Gly Arg Phe
1               5
<210> SEQ ID NO 68
<211> LENGTH: 9
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 68
Phe Ile Gln Ala Cys Gln Gly Glu Glu
1               5
<210> SEQ ID NO 69
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 69
Ile Glu Ala Asp Ala
1               5
<210> SEQ ID NO 70
<211> LENGTH: 15
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 70
Gly Tyr Val Ser Phe Arg His Val Glu Glu Gl
#y Ser Trp Tyr Ile
1               5
#                10
#                15
<210> SEQ ID NO 71
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 71
Gly Val Arg Asn Gly Thr
1               5
<210> SEQ ID NO 72
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 72
Leu Ser His Gly Glu Glu
1               5
<210> SEQ ID NO 73
<211> LENGTH: 9
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 73
Phe Ile Gln Ala Cys Arg Gly Thr Glu
1               5
<210> SEQ ID NO 74
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 74
Ile Gln Ala Asp Ser
1               5
<210> SEQ ID NO 75
<211> LENGTH: 15
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 75
Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gl
#y Ser Trp Phe Val
1               5
#                10
#                15
<210> SEQ ID NO 76
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 76
Pro Glu Arg Arg Gly Thr
1               5
<210> SEQ ID NO 77
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 77
Leu Ser His Gly Glu Gly
1               5
<210> SEQ ID NO 78
<211> LENGTH: 9
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 78
Ile Ile Gln Ala Cys Arg Gly Asn Gln
1               5
<210> SEQ ID NO 79
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 79
Asp Val Val Asp Asn
1               5
<210> SEQ ID NO 80
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 80
Thr Glu Val Asp Ala
1               5
<210> SEQ ID NO 81
<211> LENGTH: 15
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 81
Gly Tyr Tyr Ser His Arg Glu Thr Val Asn Gl
#y Ser Trp Tyr Ile
1               5
#                10
#                15
<210> SEQ ID NO 82
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 82
Thr Ser Arg Ser Gly Thr
1               5
<210> SEQ ID NO 83
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 83
Leu Ser His Gly Glu Glu
1               5
<210> SEQ ID NO 84
<211> LENGTH: 9
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 84
Ile Ile Gln Ala Cys Arg Gly Thr Glu
1               5
<210> SEQ ID NO 85
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 85
Ile Glu Thr Asp Ser
1               5
<210> SEQ ID NO 86
<211> LENGTH: 15
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 86
Gly Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gl
#y Ser Trp Phe Ile
1               5
#                10
#                15
<210> SEQ ID NO 87
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 87
Pro Thr Arg Asn Gly Thr
1               5
<210> SEQ ID NO 88
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 88
Leu Ser His Gly Glu Glu
1               5
<210> SEQ ID NO 89
<211> LENGTH: 9
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 89
Phe Val Gln Ala Cys Arg Gly Glu Arg
1               5
<210> SEQ ID NO 90
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 90
Asp Ser Val Asp Gly
1               5
<210> SEQ ID NO 91
<211> LENGTH: 15
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 91
Gln Tyr Val Ser Trp Arg Asn Ser Ala Arg Gl
#y Ser Trp Phe Ile
1               5
#                10
#                15
<210> SEQ ID NO 92
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 92
Pro Arg Arg Thr Gly Ala
1               5
<210> SEQ ID NO 93
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 93
Met Ser His Gly Ile Arg
1               5
<210> SEQ ID NO 94
<211> LENGTH: 9
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 94
Ile Ile Gln Ala Cys Arg Gly Asp Ser
1               5
<210> SEQ ID NO 95
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 95
Trp Phe Lys Asp Ser
1               5
<210> SEQ ID NO 96
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 96
Phe Glu Asp Asp Ala
1               5
<210> SEQ ID NO 97
<211> LENGTH: 15
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 97
Asp Asn Val Ser Trp Arg His Pro Thr Met Gl
#y Ser Val Phe Ile
1               5
#                10
#                15
<210> SEQ ID NO 98
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 98
Pro Pro Arg Asn Gly Ala
1               5
<210> SEQ ID NO 99
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 99
Met Ser His Gly Ile Leu
1               5
<210> SEQ ID NO 100
<211> LENGTH: 9
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 100
Ile Val Gln Ala Cys Arg Gly Ala Asn
1               5
<210> SEQ ID NO 101
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 101
Trp Val Lys Asp Ser
1               5
<210> SEQ ID NO 102
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 102
Leu Glu Glu Asp Ala
1               5
<210> SEQ ID NO 103
<211> LENGTH: 15
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 103
His Asn Val Ser Trp Arg Asp Ser Thr Met Gl
#y Ser Ile Phe Ile
1               5
#                10
#                15
<210> SEQ ID NO 104
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 104
Pro Ala Arg Asn Gly Ala
1               5
<210> SEQ ID NO 105
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 105
Met Ser His Gly Ile Leu
1               5
<210> SEQ ID NO 106
<211> LENGTH: 9
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 106
Ile Val Gln Ala Cys Arg Gly Glu Lys
1               5
<210> SEQ ID NO 107
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 107
Trp Val Arg Asp Ser
1               5
<210> SEQ ID NO 108
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 108
Leu Glu Ala Asp Ser
1               5
<210> SEQ ID NO 109
<211> LENGTH: 15
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 109
His Asn Val Ser Trp Arg Asp Arg Thr Arg Gl
#y Ser Ile Phe Ile
1               5
#                10
#                15
<210> SEQ ID NO 110
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 110
Glu Phe Arg Ser Gly Gly
1               5
<210> SEQ ID NO 111
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 111
Leu Ser His Gly Val Glu
1               5
<210> SEQ ID NO 112
<211> LENGTH: 9
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 112
Phe Ile Gln Ala Cys Arg Gly Asp Glu
1               5
<210> SEQ ID NO 113
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 113
Asp Gln Gln Asp Gly
1               5
<210> SEQ ID NO 114
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 114
Glu Glu Ser Asp Ala
1               5
<210> SEQ ID NO 115
<211> LENGTH: 15
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQUENCE: 115
Gly Thr Ala Ala Met Arg Asn Thr Lys Arg Gl
#y Ser Trp Tyr Ile
1               5
#                10
#                15
<210> SEQ ID NO 116
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Protease sensitive linker
#region between the
small and large subunits of a
#Rev-caspase
<400> SEQUENCE: 116
Val Glu Ile Asp Ser
1               5

Claims

1. A method of identifying an inhibitor or enhancer of caspase processing activity, comprising:(a) contacting a sample containing an in vitro translated rev-caspase with a candidate inhibitor or candidate enhancer; and (b) detecting the presence of large and small subunits of rev-caspase, and therefrom determining the level of caspase processing activity, wherein a decrease in processing indicates the presence of a caspase inhibitor, and wherein an increase in processing indicates the presence of a caspase enhancer, wherein processed rev-caspase yields large and small subunits.

2. A method of identifying an inhibitor or enhancer of caspase processing activity, comprising:(a) contacting a cell transfected with a vector expressing rev-caspase with a candidate inhibitor or candidate enhancer; and (b) detecting the presence of large and small subunits of rev-caspase, and therefrom determining the level of caspase processing activity, wherein a decrease in processing indicates the presence of a caspase inhibitor, and wherein an increase in processing indicates the presence of a caspase enhancer, wherein processed rev-caspase yields large and small subunits.

3. A method of identifying an inhibitor or enhancer of caspase-mediated apoptosis, comprising:(a) contacting a cell transfected with a vector expressing rev-caspase with a candidate inhibitor or candidate enhancer or with a reference compound; and (b) detecting cell viability, wherein viability of cells contacted with a candidate is increased in the presence of an inhibitor and is decreased in the presence of an enhancer compared to cells contacted with a reference compound.

4. The method of any one of claims 1-3, wherein the rev-caspase is selected from the group consisting of rev-caspase-1, rev-caspase-2, rev-caspase-3, rev-caspase-4, rev-caspase-5, rev-caspase-6, rev-caspase-7, rev-caspase-8, rev-caspase-9, rev-caspase-10, rev-caspase-11, rev-caspase-12, rev-caspase-13, and rev-caspase-14.

5. The method of either of claims 1 or 2, wherein detecting comprises gel electrophoresis.

6. The method of claim 1, wherein the in vitro translated rev-caspase is labeled.

7. The method of claim 6, wherein the label is a radioactive label, a peptide tag, an enzyme or biotin.