DNA ENCODING PRECURSOR OF INTERLEUKIN-1 beta CONVERTING ENZYME-RELATED CYSTEINE PROTEINASE II (ICErel-II)

Abstract: A complementary DNA (cDNA) encoding full length form of ICErel-II is identified, sequenced and isolated. The cDNA is cloned into expression vectors for expression in recombinant hosts. The cDNA is useful to produce recombinant full length ICErel-II. The cDNA and the recombinant ICErel-II protein derived therefrom are useful in diagnostic kits, laboratory reagents and assays. The cDNA and the recombinant ICErel-II protein may be used to identify compounds that affect ICErel-II function, inflammation and cell apoptosis. ICErel-II function, inflammation and cell apoptosis may also be modulated by ICErel-II antisense or gene therapy.

Description: TITLE OF THE INVENTION DNA ENCODING PRECURSOR OF INTERLEUKIN-lβ CONVERTING ENZYME - RELATED CYSTEINE PROTEINASE II (ICErel-II) CROSS-RELATED TO OTHER APPLICATIONS This is a continuation of U.S. Serial No. 08/225,487, filed April 8, 1994, now pending. BACKGROUND OF THE INVENTION Interleukin-1 β (IL-1 β) is a major mediator of chronic and acute inflammation. Along with IL-1 β, human monocytes produce two additional members of the IL-1 gene family; interleukin-1 α (TL-lo.) and IL-1 receptor antagonist (IL-RA). All three proteins bind to the membrane-anchored forms of the type 1 and type 2 IL-1 receptors (ILIR) on target cells. EL-l and IL-1 β elicit virtually identical biological responses whereas IL-1RA blocks these effects. Both IL-l and ILlβ are synthesized as 31 kDa primary translation products which lack functional hydrophobic signal sequences. The 31 kDa form of IL-lα is fully active without further processing but does not appear to be actively released from cells. IL-lβ, the predominant form of IL-1 released by activated monocytes, is synthesized as an inactive 31 kDa precursor (pIL- lβ) that is processed to its mature 17.5 kDa form (mIL-lβ) by interleukin-1 β converting enzyme (ICE), a novel cysteine proteinase. ICE generates fully active mIL-lβ by cleaving pIL-lβ between Aspl 16 and Alai 17, a unique site for prohormone processing. The sequence around this cleavage site, -Tyr-Val-His-Asp-Ala-, is evolutionarily conserved in all known pIL-lβ polypeptides. Active human ICE as shown by conventional HPLC and affinity purification techniques is a heterodimer consisting of a 1 :1 stoichiometric complex of 19,866 Da (p20) and 10,244 Da (plO) subunits. Cloned cDNAs have revealed that ICE is constituitively expressed as a 45 kDa proenzyme (p45) composed of a 14 kDa prodomain, followed by p20 which contains the active site Cys285- a 19 residue connecting peptide that is not present in the mature enzyme, and plO, a required component of the active enzyme. The mature subunits are flanked by Asp-X sequences. Mutational analysis of these sites and expression in heterologous systems indicates that the generation of active enzyme is autocatalytic. Murine and rat ICE have also been cloned and show a high degree of sequence similarity including these structural motifs. Recently, a family of ICE-like genes has begun to emerge, including the nematode cell death abnormal gene (CED-3) of Caenorhabiditis elegans, Caenorhabiditis briggsae and Caenorhabiditis vulgaris, and the murine neuronal precursor cell embroyonic developmentally downregulated (NEDD-2) gene. The predicted polypeptide sequences of these genes exhibit 29% and 27% sequence identity with human ICE, respectively. The sequence identity of CED-3 with ICE is higher in the regions corresponding to the p20 and plO subunits of mature human ICE. All known sequences for ICE and for CED-3 contain the pentapeptide sequence -Gln-Ala-Cys-Arg-Gly- surrounding the catalytic cysteine of ICE or its equivalent in CED-3. Both CED-3 and murine ICE, when expressed by transfection in fϊbroblast cell lines or by microinjection into neuronal cells, cause programmed cell death (apoptosis) to occur. The pro- apoptotic effects of CED-3 or ICE can be prevented by co-transfection with either bcl-2, a mammalian proto-oncogene which appears to function as a cell death suppressor gene, or with the cytokine response modifier A (crmA) gene product, a serpin-like inhibitor of ICE. SUMMARY OF THE INVENTION A novel human thiol proteinase termed ICErel-II (interleukin-1 β converting enzyme - related cysteine proteinase II) has been isolated and purified. A DNA molecule encoding the full length precursor form of the ICErel-II protein has been isolated, purified and the nucleotide sequence determined. The ICErel-II encoding DNA has been cloned for expression in recombinant hosts. The DNA clones produce recombinant full-length ICE r el-H and the individual subunits of the mature form of the enzyme. Recombinant ICE r el-H is useful for identifying modulators of ICErel-II activity and hence modifiers of pathological conditions related to the pro-inflammatory or pro-apoptotic effects of ICErel-II- ICErel-H antisense molecules are useful for therapeutically reducing or eliminating the pro-inflammatory or pro- apoptotic effects of ICErel-II, whereas gene transplantation or gene therapy with ICE r el-II is useful for enhancing the pro -inflammatory or pro-apoptotic effects of ICErel-H- These therapies are beneficial in the treatment of immune, proliferative and degenerative diseases including, but not limited to, immune deficiency syndromes (such as AIDS), autoimmune diseases, pathogenic infections, cardiovascular and neurological injury, alopecia, aging, cancer, Parkinson's disease and Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Nucleotide sequence of human ICErel-II (cDNA clone PI 01.1.1), its complementary nucleotide sequence, and deduced amino acid sequence. Figure 2. Alignment of the human ICE r el-II amino acid sequence with the amino acid sequence of human ICE. Identical amino acids are indicated by a vertical line between the aligned sequences, whereas highly conservative amino acid differences are indicated by a double dot and conservative amino acid differences are indicated by a single dot between the aligned sequences. Figure 3. Alignment of the human ICErel-II amino acid sequence with the amino acid sequence of Caenorhabiditis elegans CED- 3. Identical amino acids are indicated by a vertical line between the aligned sequences, whereas highly conservative amino acid differences are indicated by a double dot and conservative amino acid differences are indicated by a single dot between the aligned sequences. Figure 4. Alignment of the human ICErel-II amino acid sequence with the amino acid sequence of murine NEDD-2. Identical amino acids are indicated by a vertical line between the aligned sequences, whereas highly conservative amino acid differences are indicated by a double dot and conservative amino acid differences are indicated by a single dot between the aligned sequences. DETAILED DESCRIPTION OF THE INVENTION A complementary DNA (cDNA) which encodes the full length form of ICErel-II is identified, sequenced and isolated. The cDNA is cloned into expression vectors for expression in a recombinant host. The cDNA is useful to produce recombinant full length ICE r el-II. The cDNA and the recombinant ICErel-II protein derived therefrom are useful in the production of antibodies, diagnostic kits, laboratory reagents and assays. The cDNA and the recombinant ICErel-II protein may be used to identify compounds that affect ICErel-II function, inflammation and cell apoptosis. ICErel-II antisense oligonucleotides or antisense mimetics may be clinically useful for reducing the expression of ICErel- II protein and thereby reducing the pro-inflammatory or pro-apoptotic effects of ICErel-II. Similarly, the ICE r el-II coding sequence can be used for gene therapy to introduce ICErel-II into target cells thereby enhancing the pro-inflammatory or pro-apoptotic effects of ICErel-II. A variety of cells and cell lines may be suitable for use to isolate ICErel-H cDNA. Selection of suitable cells may be done by screening for ICErel-II activity in cell extracts or conditioned medium using conventional techniques. Cells which possess ICErel-II activity in this assay may be suitable for the isolation of ICErel-II cDNA. A variety of procedures may be used to molecularly clone ICErel-II cDNA. These methods include, but are not limited to, direct functional expression of the ICErel-II gene following the construction of an ICErel-II-containing cDNA library in an appropriate expression vector system. Another method is to screen an ICErel-II -containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labelled oligonucleotide probe designed from the amino acid sequence of ICErel-π . A variety of libraries constructed from cells may be useful for isolating ICErel-D -encoding DNA. Suitable libraries may be prepared from cells or cell lines which have ICE r el-II activity. Preparation of cDNA libraries can be performed by standard techniques known in the art. Such cDNA library construction techniques as well as other standard molecular biology techniques can be found, for example, in Maniatis, T., Fritsch, E.F., Sambrook, J., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982) or in Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., Current Protocols in Molecular Biology (John Wiley & Sons, New York, New York, 1989). DNA encoding ICErel-II may also be isolated from a suitable genomic DNA library. Construction of genomic DNA libraries can be performed by standard techniques known in the art. Known genomic DNA library construction techiques can be found in Maniatis, T. et al., (supra), Ausubel et ah, (supra). The cloned ICErel-H cDNA may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant ICErel-II. Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, yeast, bluegreen algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector may contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses. A variety of mammalian expression vectors may be used to express recombinant ICErel-H in mammalian cells. Commercially- available mammalian expression vectors which may be suitable for recombinant ICErel-II expression, include but are not limited to, pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO- pSV2-neo (ATCC 37593) pBPV-l(8-2) (ATCC 37110), pdBPV- MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and LZD35 (ATCC 37565). DNA encoding ICErel-II may also be cloned into an expression vector for expression in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria, yeast, mammalian cells and insect cells. Cell lines derived from mammalian species which may be suitable and which are commercially available, include but are not limited to, CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NTH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171). The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, infection, protoplast fusion, and electroporation. The expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce ICErel-II protein. Identification of ICErel-II expressing host cell clones may be done by several means, including but not limited to immuno- logical reactivity with anti-ICErel-H antibodies, and the presence of host cell-associated ICErel-π activity. Expression of ICErel-II cDNA may also be performed using in vitro produced synthetic mRNA. Synthetic mRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes. To determine the ICErel-H cDNA sequence(s) that yields optimal levels of enzymatic activity and/or ICErel-II protein, modified ICErel-II cDNA molecules are constructed. Host cells are transformed with the cDNA molecules and the levels of ICErel-II RNA and protein are measured. Levels of ICErel-II protein in host cells are quantitated by a variety of methods such as immunoaffinity and/or ligand affinity techniques. ICErel-H -specific affinity beads or ICErel-II-specific antibodies are used to isolate 35s-methionine labelled or unlabelled ICErel-II protein. Labelled lCE re l-II protein is analyzed by SDS-PAGE. Unlabelled ICE r el-H protein is detected by Western blotting, ELISA or RIA assays employing ICErel-II specific antibodies. Following expression of ICErel-II in a recombinant host cell, ICErel-II protein may be recovered to provide ICErel-H in active form. Several ICE r e]-H purification procedures are available and suitable for use. Recombinant ICErel-II may be purified from cell lysates or from conditioned culture media, by various combinations of, or individual application of fractionation, or chromatography steps that are known in the art. In addition, recombinant ICE r el-H can be separated from other cellular proteins by use of an immuno-affinity column made with monoclonal or polyclonal antibodies specific for full length nascent ICErel-II or polypeptide fragments of ICErel-H- The recombinant protein may be used to generate antibodies. The term "antibody" as used herein includes both polyclonal and monoclonal antibodies, as well as fragments thereof, such as, Fv, Fab and F(ab)2 fragments that are capable of binding antigen or hapten. Monospecific antibodies to ICErel-H are purified from mammalian antisera containing antibodies reactive against ICErel-H or are prepared as monoclonal antibodies reactive with ICErel-H using standard techniques. Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for ICErel-H. Homogenous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the ICErel-H, as described above. Enzyme-specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with rabbits being preferred, with an appropriate concentration of ICErel- H either with or without an immune adjuvant. Monoclonal antibodies (mAb) reactive with ICErel-H may be prepared by conventional methods, such as by immunizing inbred mice with ICErel-H. The mice are immunized with about 0.1 mg to about 10 mg, preferably about 1 mg, of ICE r el-H in about 0.5 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant. Freund's complete adjuvant is preferred. The mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 0.1 to about 10 mg of ICE r el-H in a buffer solution such as phosphate buffered saline (PBS) by the intravenous (IV) route. Lymphocytes from antibody-positive mice are obtained by removing spleens from immunized mice by standard procedures known in the art. Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner under conditions which will allow the formation of stable hybridomas. Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids are collected form growth positive wells on about days 14, 18, and 21 and are screened for antibody produciton by an immunoassay such as solid phase immunoradioassay (SPIRA) using ICErel-H as the antigen. The culture fluids are also tested in the Ouchterlony precipitation assay to determine the isotype of the mAb. Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973. In vitro production of anti-ICE r el-H is carried out by growing the hydridoma in DMEM containing about 2% fetal calf serum to obtain sufficient quantities of the specific mAb. The mAb are purified by techniques known in the art. Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of ICE r el-H in body fluids or tissue and cell extracts. Methods such as those described above may be used to produce monospecific antibodies may be utilized to produce antibodies specific for ICErel-H polypeptide fragments or full-length nascent ICErel-H polypeptide. ICErel-H antibody affinity columns are made by adding the antibodies to a gel support, such as Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1M ethanolamine HCl (pH 8). The column is washed with water followed by 0.23 M glycine HCl (pH 2.6) to remove any non-conjugated antibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supernatants or cell extracts containing ICE r el-H or ICE r el-H fragments are slowly passed through the column. The column is then washed, and the protein is eluted. The purified ICErel-H protein is then dialyzed against phosphate buffered saline. Kits containing ICE r el-H cDNA, antibodies to ICErel-H or ICErel-H protein may be prepared. Such kits are used to detect DNA which hybridizes to ICErel-H DNA or to detect the presence of ICErel-H protein or peptide fragments in a sample. Such characterization is useful for a variety of purposes including but not limited to forensic analyses and epidemiological studies. The DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of ICErel-H DNA, ICErel-H RNA or ICE re l-II protein. The recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of ICErel-H. Such a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container. The carrier would further comprise reagents such as recombinant ICErel-H protein or anti-ICErel-H antibodies suitable for detecting ICErel-H. The carrier may also contain means for detection such as labeled antigen or enzyme substrates or the like. Nucleotide sequences that are complementary to the ICErel- H encoding cDNA sequence can be synthesized for antisense therapy. These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2'-0-alkylRNA, or other ICErel-H antisense oligonucleotide mimetics. ICErel-H antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harbouring the antisense sequence. ICErel-H antisense therapy may be particularly useful for the treatment of diseases where it is beneficial to reduce ICErel-H acitivy. ICErel-H gene therapy may be used to introduce ICErel-H into the cells of target organs. The ICErel-H gene can be ligated into viral vectors which mediate transfer of the ICErel-H DNA by infection of recipient host cells. Suitable viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus and the like. Alternatively, ICErel-H DNA can be transferred into cells for gene therapy by non-viral techniques including receptor-mediated targetted DNA transfer using ligand-DNA conjugates or adenovirus-ligand-DNA conjugates, lipofection membrane fusion or direct microinjection. These procedures and variations of them are suitable for ex vivo as well as in vivo ICErel-H gene therapy. ICErel-H gene therapy may be particularly useful for the treatment of diseases where it is beneficial to elevate ICErel-H activity. Pharmaceutically useful compositions comprising ICErel-H DNA or ICErel-H protein may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein or DNA. Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose ICErel-H related disorders. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration. The pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Only one member of the set will be identical to the ICErel-H sequence but will be capable of hybridizing to ICErel-H DNA even in the presence of DNA oligonucleotides with mismatches. The mismatched DNA oligonucleotides may still hybridize to the ICErel- HDNA to permit identification and isolation of ICErel-H encoding DNA. DNA encoding ICErel-H from a particular organism may be used to isolate and purify homologues of ICErel-π from other organisms. To accomplish this, the first ICErel-H DNA may be mixed with a sample containing DNA encoding homologues of ICErel-H under appropriate hybridization conditions. The hybridized DNA complex may be isolated and the DNA encoding the homologous DNA may be purified therefrom. It is known that there is a substantial amount of redundancy in the various codons which code for specific amino acids. Therefore, this invention is also directed to those DNA sequences which contain alternative codons which code for the eventual translation of the identical amino acid. For purposes of this specification, a sequence bearing one or more replaced codons will be defined as a degenerate variation. Also included within the scope of this invention are mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide. It is known that DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally-occurring peptide. Methods of altering the DNA sequences include, but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate. As used herein, a "functional derivative" of ICErel-H is a compound that possesses a biological activity (either functional or structural) that is substantially similar to the biological activity of ICErel- H. The term "functional derivatives" is intended to include the "fragments," "variants," "degenerate variants," "analogs" and "homologs" or to "chemical derivatives" of ICErel-H. The term "fragment" is meant to refer to any polypeptide subset of ICErel-H. The term "variant" is meant to refer to a molecule substantially similar in structure and function to either the entire ICErel-H molecule or to a fragment thereof. A molecule is "substantially similar" to ICErel-H if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical. The term "analog" refers to a molecule substantially similar in function to either the entire ICErel-π molecule or to a fragment thereof. The term "chemical derivative" describes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solublity, half-life, absorption, etc of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences. The present invention is also directed to methods for screening for compounds which modulate that expression of DNA or RNA encoding ICErel-H as well as the function of ICE r el-H protein in vivo. Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules. Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding ICE r el-H or the function of ICErel-H protein. Compounds that modulate the expression of DNA or RNA encoding ICErel-H or the function of ICE r el-H protein may be detected by a variety of assays. The assay may be a simple "yes/no" assay to determine whether there is a change in expression or function. The assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample. The following examples illustrate the present invention without, however, limiting the same thereto. EXAMPLE 1 Molecular Cloning Of ICE l-H A partial-length cDNA clone for ICErel-H was identified by replica-filter screening of a THP-1 cell (acute monocytic leukemia cell line; ATCC TIB 202) cDNA library in bacteriophage λgtlO using the [3_2p]-labelled S y n thetic oligonucleotide probe: (5 ATGAAGGATGAGGTGGCCCTGCTGGCT GCTGTGACCCTGCTGGGCGTGCTGCTGCAGGCTGGCTTC TCCCTGCAGGTGATCTCTGCCCGGATTGCCTTCCGG GT(3')(SEQIDNO:l). This initial 748 bp clone, designated Tl 0.3.1, corresponded to bases 544-1291 of the full-length ICErel-H clone described herein. The clone was retrieved from an individual λgtlO plaque by DNA amplification using the polymerase chain reaction (PCR) with the synthetic oligonucleotide primers (5')TGA GCA AGT TCA GCC TGG TTA AGT(3') (SEQ ID NO 2; forward primer) and (5')GGC TTA TGA GTA TTT CTT CCA GGG(3') (SEQ ID NO: 3; reverse primer) which correspond to the sequence of λgtlO on the left and right side, respectively, of the Eco RI restriction enzyme site containing the cDNA insert. The resulting amplification product was ligated into the plasmid vector pCR-H (Invitrogen), transformed into competent Escherichia coli cells, colony-purified and propagated by growth of the resulting transformed cells in liquid culture. The plasmid DNA was purified from the cells and the nucleotide sequence of the clone T10.3.1 insert was determined by dideoxy DNA sequencing. Based on the sequence of this clone, synthetic oligonucleotides specific for the clone Tl 0.3.1 sequence were prepared and after [32p] labelling were used to re-screen the THP-1 cell λgtlO cDNA library. The probes used for replica filter screening were (5')AGG CCA CTT CCA AGG ATG CTG GA(3') SEQ ID NO:4 (one replica filter) and (5')CTG GAA GAT GGT GTC ATA AAG CAG C(3') SEQ ID NO:5 (other paired replica filter). The largest clone identified, designated T15.1.1, was 1207 bp and corresponded to bases 64-1270 of the full-length ICErel-H clone described herein. The clone was retrieved from λgtlO following expansion of the λ bacteriophage by culture on E. co/i- ' -containing agarose plates, then purifying the λ bacteriophage DNA by a combination of polyethylene glycol precipitation, macroporous silica-gel chromato- graphy and alcohol precipitation. The T15.1.1 DNA fragment was excised by partial Eco RI restriction digestion (partial digestion was necessary owing to an internal Eco RI site in the Tl 5.1.1 sequence) followed by agarose gel purification of the 1.2 Kb fragment then ligation of the purified fragment into the Eco RI site of the plasmid vector pBluescript H SK+ (Stratagene). Following transformation into competent E. coli cells, colony purification and propagation of the resulting transformed cells in liquid culture, the plasmid DNA was purified and the nucleotide sequence of the clone T15.1.1 fragment was determined. The full-length ICE re l-H clone (designated Plθl.1.1) was identified by replica filter screening of a human peripheral blood monocyte cDNA library in bacteriophage λgtl 1. The filters were probed with a [32p]-iabelled PCR fragment that had been generated by amplification of the T15.1.1 ICE r el-H clone using the primers (5')GAA CTG GAA GGA AGA GGA AA(3') (SEQ ID NO: 6; forward primer) and (5')TCA TGC ACA GTT CCG CAG ATT CCC T(3') (SEQ ID NO:7; reverse primer) which produced a fragment corresponding to bases 142-702 of the full- length ICErel-H sequence. (This probe was chosen since it was in a region of ICE r el-H with low sequence identity to ICE and would therefore minimize the number of ICE clones identified.) The 1297 bp full-length ICErel-H clone (Plθl.1.1) was retrieved by Eco RI excision from purified λ bacteriophage DNA as described above for clone T15.1.1. The DNA sequence of the Plθl.1.1 ICErel-H clone was identical to four other clones that were isolated during the same series of replica filter screens. The complete cDNA sequence of ICE re l-π (clone P101.1.1) and corresponding amino acid sequence is shown in Figure 1. The longest open reading frame of ICErel-H clone Plθl .1.1 (bases 38 to 1168) encodes a 43.3 kDa polypeptide which has 53% sequence identity (68% sequence similarity) with human interleukin-1 β converting enzyme (Figure 2), 26% sequence identity (52% sequence similarity) with the Caenorhabiditis elegans CED-3 polypeptide (Figure 3) and 23% sequence identity (41 % sequence similarity) with the murine NEDD-2 polypeptide (Figure 4). The particularly high degree of sequence conservation surrounding the catalytic cysteine residue (Cys258 of ICErel-H, Cys285 of human interleukin-1 β converting enzyme, Cys358 of CED-3) as well as other structural motifs throughout the polypeptide is consistent with ICErel-H being a thiol proteinase. EXAMPLE 2 Sub-Cloning Of The ICErel-H cDNA Into Expression Vectors The cDNA encoding ICErel-H was sub-cloned into several vectors for expression of the ICErel-H protein in transfected host cells and for in vitro transcription/translation. These vectors include pBluescript H SK+ (where expression is driven by T7 or T3 promoters), pcDNA I/Amp (where expression is driven by the cytomegalovirus (CMV) promoter), pSZ9016-l (where expression is driven by the HIV long terminal repeat (LTR) promoter) and the baculovirus transfer vector pVL1393 (where expression is driven by the polyhedrin (PH) promoter) for producing recombinant baculovirus containing the ICErel-H encoding DNA sequence. Using techniques such as those described in Maniatis (supra) and Ausubel (supra), ICErel-H may be subcloned into vectors suitable for expression in bacteria or yeast or other expression systems. The predicted/actual amino acid sequence of ICErel-H is shown in Figure 1. a) pBluescript H SK+:ICErgl-H. The full length ICE re l-II cDNA clone was retrieved from λ bacteriophage by limited Eco RI digestion and ligated into Eco Rl-cut, CIP-treated pBluescript H SK+ as described above under "molecular cloning of ICErel-H". Separate subclones were recovered in which the sense orientation of ICErel-H followed either the T7 or T3 promoters. b) pcDNA I/Amp:ICErel-H. In order to enable directional cloning, ICErel-H was excised from a purified plasmid preparation of pBluescript H SK+:ICE re l-H ( above) in which the ICErel-H DNA sequence was downstream of the T7 promoter using Eco RV and Xba I. The resulting Eco RV, Xba I ICE r el-H fragment was purified and ligated into Eco RV-cut, Xba I-cut, CIP-treated pcDNA I/Amp such that the ICErel-H encoding DNA was downstream of the CMV promoter. c) pSZ9016-l :ICErpl-II. ICE re l-H was excised from pBluescript H SK+:ICE r el-H ('a'above) by limited Eco RI digestion and subsequent purification of the 1.3 Kb fragment from agarose gels. The resulting Eco RI ICErel-H fragment was ligated into Eco Rl-cut, CIP- treated pSZ9016-l . Subclones were selected in which the sense orientation of ICE r el-H was downstream of the HIV LTR promoter. d) pVL1393:ICEτ l-π and pVL1393:T7 ICEt l-π HA Directional cloning of the ICErel-II encoding DNA into the baculovirus transfer vector pVL1393 was mediated by excising ICErel-H from pcDNA I/Amp:ICE re l-II ('b' above) with Bam HI and Xba I then ligating the resulting 1.3 Kb fragment into Bam Hi-cut, Xba I-cut, CIP- treated pVL1393 producing pVL1393:ICE re l-H. Similarly, ICE re l-H was epitope tagged by engineering a T7 tag at the 5' amino terminus of the ICErel-H open reading frame and a FluHA epitope at the 3' carboxy terminus. The ICErel-H DNA modified in this manner was ligated into the Bam HlfXba I sites of pVL1393 to produce pVL1393:T7 ICErel-H HA. EXAMPLE 3 Expression Of The ICErel-H Polypeptide By In Vitro Transcription/ Translation And Bv Transfection Into Host Cells Vectors containing the ICErel-H encoding DNA sequence were used to drive the translation of the ICErel-H polypeptide in rabbit reticulocyte lysates, mammalian host cells, and in baculovirus infected insect cells. The experimental procedures were essentially those outlined in the manufacturers' instructions. a) In vitro Transcription/Translation. pBluescript H SK+:ICErel-H plasmid DNA (with ICErel-H in the T7 orientation) was linearized by Bam HI digestion downstream of the ICE r el-π insert. The linearized plasmid was purified and used as a template for run-off transcription using T7 RNA polymerase in the presence of m7G(5')ppp(5')G. The resulting capped ICErel-H transcripts were purified by lithium chloride precipitation and used to drive the translation of ICErel-H in nuclease-pretreated rabbit reticulocyte lysate in the presence of L-[35s]methionine. The resulting translation mixtures contained radiolabelled ICErel-H protein which migrated on SDS/polyacrylamide gels with an apparent molecular mass of 45 ± 2 kDa. b) Expression in Mammalian Cells. The ICE r el-H protein was expressed in mammalian host cells following transfection with either pcDNA I/Amp:ICErel-H (under control of the CMV promoter) or pSZ9016-l :ICE re l-H (under control of the HTV LTR promoter). In the latter case (pSZ9016-l :ICE re l-H), cells were co-transfected with the TAT expressing plasmid pSZ90161 :TAT. For both ICErel-π expression plasmids, COS-7 cells were transfected using either DEAE-dextran or lipofection with Lipofectamine (BRL). c) Expression in Insect Cells. The ICErel-H - containing baculovirus transfer vector pVL1393:T7 ICErel-π HA was used to produce recombinant baculovirus (Autographa calif or nica) by in vivo homologous recombination. Epitope tagged ICErel-H was then expressed in Sf9 (Spodoptera frugiperda) insect cells grown in suspension culture following infection with the ICErel-π - containing recombinant baculovirus. EXAMPLE 4 Cloning Of ICErel-H For Expression Of The ICErel-H Polypeptide In Other Host Cell Systems a) Cloning of ICEml-π cDNA into a bacterial expression vector. Recombinant ICErel-H is produced in a bacterium such as E. coli following the insertion of the optimal ICErel-H cDNA sequence into expression vectors designed to direct the expression of heterologous proteins. These vectors are constructed such that recombinant ICErel-H is synthesized alone or as a fusion protein for subsequent manipulation. Expression may be controlled such that recombinant ICE r el-H is recovered as a soluble protein or within insoluble inclusion bodies. Vectors such as pBR322, pSKF, pUR, pATH, pGEX, pT7-5, pT7-6, pT7- 7, pET, pIBI (H3I), pSP6 T7-19 (Gibco/BRL), pBluescript H (Stratagene), pTZ18R, pTZ19R (USB), pSE420 (Invitrogen) or the like are suitable for these purposes. b) Cloning of ICErel-II cDNA into a veast expression vector. Recombinant ICErel-H is produced in a yeast such as Saccharomyces cerevisiae following the insertion of the optimal ICErel- π cDNA cistron into expression vectors designed to direct the intracellular or extracellular expression of heterologous proteins. In the case of intracellular expression, vectors such as EmBLyex4 or the like are ligated to the ICErel-H cistron [Rinas, U. et al, Biotechnology 8: 543-545 (1990); Horowitz B. et al, J. Biol. Chem. 265: 4189-4192 (1989)]. For extracellular expression, the ICErel-H cistron is ligated into yeast expression vectors which fuse a secretion signal (a yeast or mammalian peptide) to the amino terminus of the ICErel-H protein [Jacobson, M. A., Gene 85: 511-516 (1989); Riett L. and Bellon N. Biochem. 28: 2941- 2949 (1989)]. c) Cloning of ICErgl-II cDN A into a viral expression vector. Recombinant ICErel-H is produced in mammalian host cells, such as HeLa S3 cells, after infection with vaccinia virus containing the ICE r el-H cDNA sequence. To produce ICErel-H :vaccinia virus, the ICE r el-H cDNA is first ligated into a transfer vector, such as pSCl 1, pTKgptFls, pMJ601 or other suitable vector, then transferred to vaccinia virus by homologous recombination. After plaque purification and virus amplification, ICE r el-H:vaccinia virus is used to infect mammalian host cells and produce recombinant ICErel-H protein. EXAMPLE 5 Process For The Production Of A Interleukin- 1 β Converting Enzyme - Related Cysteine Proteinase II Polypeptide Recombinant ICErel-H is produced by a) transforming a host cell with DNA encoding ICErel-H protein to produce a recombinant host cell; b) culturing the recombinant host cell under conditions which allow the production of interleukin- lβ converting enzyme - related cysteine proteinase H; and c) recovering the interleukin- lβ converting enzyme - related cysteine proteinase π. The recombinant interleukin- lβ converting enzyme - related cysteine proteinase π is purified and characterized by standard methods. EXAMPLE 6 Compounds that modulate interleukin-1 β converting enzyme - related cysteine proteinase II activity may be detected by a variety of methods. A method of identifying compounds that affect interleukin-1 β converting enzyme - related cysteine proteinase π comprises: (a) mixing a test compound with a solution containing interleukin-1 β converting enzyme - related cysteine proteinase II to form a mixture; (b) measuring interleukin- lβ converting enzyme - related cysteine proteinase II activity in the mixture; and (c) comparing the interleukin-1 β converting enzyme - related cysteine proteinase II activity of the mixture to a standard. Compounds that modulate interleukin- lβ converting enzyme - related cysteine proteinase II activity may be formulated into pharmaceutical compositions. Such pharmaceutical compositions may be useful for treating diseases or conditions that are characterized by altered interleukin- lβ converting enzyme - related cysteine proteinase II activity. Examples of diseases wherein the interleukin-1 β converting enzyme - related cysteine proteinase II activity is increased include immune deficiency syndromes, pathogenic infections, cardiovascular and neurological injury, alopecia, aging, Parkinson's disease and Alzheimers disease. For these diseases, therapeutic treatment comprises treatment with compounds that decrease the interleukin-1 β converting enzyme - related cysteine proteinase II activity. Examples of diseases wherein the interleukin- lβ converting enzyme - related cysteine proteinase II activity is decreased include autoimmune diseases, leukemias, lymphomas and other cancers. For these diseases, therapeutic treatment comprises treatment with compounds that increase interleukin- lβ converting enzyme - related cysteine proteinase II activity. EXAMPLE 7 DNA which is structurally related to DNA encoding interleukin-1 β converting enzyme - related cysteine proteinase II is detected with a probe. A suitable probe may be derived from DNA having all or a portion of the nucleotide sequence of Figure 1 , RNA encoded by DNA having all or a portion of the nucleotide sequence of Figure 1 , degenerate oligonucleotides derived from a portion of the amino acid sequence of Figure 1 or an antibody directed against interleukin- lβ converting enzyme - related cysteine proteinase II. EXAMPLE 8 A kit useful for the detection and characterization of DNA or RNA encoding interleukin-1 β converting enzyme - related cysteine proteinase II or interleukin- lβ converting enzyme - related cysteine proteinase II is prepared by conventional methods. The kit may contain DNA encoding interleukin- lβ converting enzyme - related cysteine proteinase H, recombinant interleukin- lβ converting enzyme - related cysteine proteinase H, RNA corresponding to the DNA encoding interleukin-lβ converting enzyme - related cysteine proteinase II or antibodies to interleukin- l β converting enzyme - related cysteine proteinase II. The kit may be used to characterize test samples, such as forensic samples or epidemiological samples. EXAMPLE 9 Cloning Of Other ICErel-H Genes Using Human ICErH-H Gene The cross hybridization of the DNA representing portions of the ICErel-II gene to genomic DNA isolated from other organisms makes it possible to clone the homologous genes from the parent organisms. To do this, a genomic library from another primate such as a monkey is constructed from genomic DNA according to conventional methods. Using, for example, an EMBL vector, an EMBL genomic library is prepared, plated and screened by hybridization with a 32p-labeled DNA probe. Positive plaques are selected and subjected to additional screening until a purified cross-reacting plaque is selected. The DNA contained in the positive clone is further characterized by physical methods such as restriction mapping, Southern hybridization and DNA sequencing. For example, purified nucleic acid encoding a functional interleukin- lβ converting enzyme - related cysteine proteinase II from such an animal may be isolated by hybridizing an appropriate sample with nucleic acid encoding interleukin- lβ converting enzyme - related cysteine proteinase H under low stringency conditions. EXAMPLE 10 Use of mutagenized ICErel-H DNA encoding ICErel-H is mutagenized using standard methods to produce an altered ICErel-H gene. Host cells are transformed with the altered ICErel-H to produce altered ICErel-H protein. The altered ICErel-H protein may be isolated, purified and used to characterize the function of ICErel-H protein. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: MERCK FROSST CANADA, INC. 5 NICHOLSON, DONALD W. ALI, AMBERDEEN VAILLANCOURT, JOHN P. MUNDAY, NEIL A. (ii) TITLE OF INVENTION: DNA ENCODING PRECURSOR OF INTERLEUKIN-IBETA CONVERTING ENZYME -RELATED CYSTEINE PROTEINASE II (ICE rel-II) 10 (iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: KEVIN P. MURPHY (B) STREET: 1001 DEMAISONNEUVE BLVD. WEST, SUITE 800 (C) CITY: MONTREAL (D) STATE: QUEBEC 15 (E) COUNTRY: CANADA (F) ZIP: H3A 3C8 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 20 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US Case 19038 (B) FILING DATE: 08-APR-1994 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: MURPHY, KEVIN P. (B) REGISTRATION NUMBER: SWABEY, OGILVY, RENAULT 25 (C) REFERENCE/DOCKET NUMBER: 19038 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 157 base pairs (B) TYPE: nucleic acid 30 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ATGAAGGATG AGGTGGCCCT GCTGGCTGCT GTGACCCTGC TGGGCGTGCT GCTGCAGGCT 60 GGCTTCTCCC TGCAGGTGAT CTCTGCCCGG ATTGCCTTCC GGGTTCCATC TAAAAAAGGT 120 A^GTAGGTAA AAGAATTATA TTATCAAGTA TTTATTT 157 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 82 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single 10 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: TGAGCAAGTT CAGCCTGGTT AAGTCTTTTT CCATCTAAAA AAGGTAGGTA GGTAAAAGAA 60 TTATATTATC AAGTATTTAT TT 82 (2) INFORMATION FOR SEQ ID NO:3 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single 20 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGCTTATGAG TATTTCTTCC AGGGAGCAAA AACCCCATCT AAAAAAGGTA GGTAGGTAAA 60 25 AGAATTATAT TATCAAGTAT TTATTT 86 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 75 base pairs (B) TYPE: nucleic acid 30 (C) STRANDEDNESS: single (D) TOPOLOGY: linear . (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION.: SEQ ID NO:4: AGGCCACTTC CAAGGATGCT GGACCATCTA AAAAAGGTAG GTAGGTAAAA GAATTATATT 60 ATCAAGTATT TATTT 75 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 81 base pairs 5 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 10 CTGGAAGATG GTGTCATAAA GCAGCAAATC CATCTAAAAA AGGTAGGTAG GTAAAAGAAT 60 TATATTATCA AGTATTTATT T 81 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: ..-. (A) LENGTH: 74 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 20 GAACTGGAAG GAAGAGGAAA TTCCATCTAA AAAAGGTAGG TAGGTAAAAG AATTATATTA 60 TCAAGTATTT ATTT 74 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: 25 (A) LENGTH: 78 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 30 ( i) SEQUENCE DESCRIPTION: SEQ ID NO:7: TCATGCACAG TTCCGCAGAT TCCCTACCAT CTAAAAAAGG TAGGTAGGTA AAAGAATTAT 60 ATTATCAAGT ATTTATTT 78 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1299 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TTTCCAACGC TGTAAAAAAG GACAGAGGCT GTTCCCTATG GCAGAAGGCA ACCACAGAAA 60 AAAGCCACTT AAGGTGTTGG AATCCCTGGG CAAAGATTTC CTCACTGGTG TTTTGGATAA 120 CJTg-GGTGGAA CAAAATGTAC TGAACTGGAA GGAAGAGGAA AAAAAGAAAT ATTACGATGC 180 TAAAACTGAA GACAAAGTTC GGGTCATGGC AGACTCTATG CAAGAGAAGC AACGTATGGC 240 AGGACAAATG CTTCTTCAAA CCTTTTTTAA CATAGACCAA ATATCCCCCA ATAAAAAAGC 300 TCATCCGAAT ATGGAGGCTG GACCACCTGA GTCAGGAGAA TCTACAGATG CCCTCAAGCT 360 JTCTCCTCAT GAAGAATTCC TGAGACTATG TAAAGAAAGA GCTGAAGAGA TCTATCCAAT 420 AAAGGAGAGA AACAACCGCA CACGCCTGGC TCTCATCATA TGCAATACAG AGTTTGACCA 480 TCTGCCTCCG AGGAATGGAG CTGACTTTGA CATCACAGGG ATGAAGGAGC TACTTGAGGG 540 TCTGGACTAT AGTGTAGATG TAGAAGAGAA TCTGACAGCC AGGGATATGG AGTCAGCGCT 600 GAGGGCATTT GCTACCAGAC CAGAGCACAA GTCCTCTGAC AGCACATTCT TGGTACTCAT 660 GTCTCATGGC ATCCTGGAGG GAATCTGCGG AACTGTGCAT GATGAGAAAA AACCAGATGT 720 GCTGCTTTAT GACACCATCT TCCAGATATT CAACAACCGC AACTGCCTCA GTCTGAAGGA 780 CAAACCCAAG GTCATCATTG TCCAGGCCTG CAGAGGTGCA AACCGTGGGG AACTGTGGGT 840 CAGAGACTCT CCAGCATCCT TGGAAGTGGC CTCTTCACAG TCATCTGAGA ACCTGGAGGA 900 25 AGATGCTGTT TACAAGACCC ACGTGGAGAA GGACTTCATT GCTTTCTGCT CTTCAACGCC 960 ACACAACGTG TCCTGGAGAG ACAGCACAAT GGGCTCTATC TTCATCACAC AACTCATCAC 1020 ATGCTTCCAG AAATATTCTT GGTGCTGCCA CCTAGAGGAA GTATTTCGGA AGGTACAGCA 1080 ATCATTTGAA ACTCCAAGGG CCAAAGCTCA AATGCCCACC ATAGAACGAC TGTCCATGAC 1140 30 AAGATATTTC TACCTCTTTC CTGGCAATTG AAAATGGAAG CCACAAGCAG CCCAGCCCTC 1200 CTTAATCAAC TTCAAGGAGC ACCTTCATTA GTACAGCTTG CATATTTAAC ATTTTGTATT 1260 TCAATAAAAG TGAAGACAAA CAAAAAAAAA AAAAAAAAA 1299 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 812 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9 : Xaa Met Ala Glu Gly Asn His Arg Lys Lys Pro Leu Lys Val Leu Glu 1 5 10 15 Q Ser Leu Gly Lys Asp Phe Leu Thr Gly Val Leu Asp Asn Leu Val Glu 20 25 30 Gin Asn Val Leu Asn Trp Lys Glu Glu Glu Lys Lys Lys Tyr Tyr Asp 35 40 45 Ala Lys Thr Glu Asp Lys Val Arg Val Met Ala Asp Ser Met Gin Glu 50 55 60 5 Lys Gin Arg Met Ala Gly Gin Met Leu Leu Gin Thr Phe Phe Asn He 65 70 75 80 Asp Gin He Ser Pro Asn Lys Lys Ala His Pro Asn Met Glu Ala Gly 85 90 95 Pro Pro Glu Ser Gly Glu Ser Thr Asp Ala Leu Lys Leu Cys Pro His 100 105 110 0 Glu Glu Phe Leu Arg Leu Cys Lys Glu Arg Ala Glu Glu He Tyr Pro 115 120 125 He Lys Glu Arg Asn Asn Arg Thr Arg Leu Ala Leu He He Cys Asn 130 135 140 Thr Glu Phe Asp His Leu Pro Pro Arg Asn Gly Ala Asp Phe Asp He 5 145 150 155 160 Thr Gly Met Lys Glu Leu Leu Glu Gly Leu Asp Tyr Ser Val Asp Val 165 170 175 Glu Glu Asn Leu Thr Ala Arg Asp Met Glu Ser Ala Leu Arg Ala Phe 180 185 190 0 Ala Thr Arg Pro Glu His Lys Ser Ser Asp Ser Thr Phe Leu Val Leu 195 200 205 Met Ser His Gly He Leu Glu Gly He Cys Gly Thr Val His Asp Glu 210 215 220 Lys Lys Pro Asp Val Leu Leu Tyr Asp Thr He Phe Gin He Phe Asn 225 230 235 240 Asn Arg Asn Cys Leu Ser Leu Lys Asp Lys Pro Lys Val He He Val 245 250 255 Gin Ala Cys Arg Gly Ala Asn Arg Gly Glu Leu Trp Val Arg Asp Ser 260 265 270 Pro Ala Ser Leu Glu Val Ala Ser Ser Gin Ser Ser Glu Asn Leu Glu 275 280 285 Glu Asp Ala Val Tyr Lys Thr His Val Glu Lys Asp Phe He Ala Phe 290 295 300 Cys Ser Ser Thr Pro His Asn Val Ser Trp Arg Asp Ser Thr Met Gly 305 310 315 320 Ser He Phe He Thr Gin Leu He Thr Cys Phe Gin Lys Tyr Ser Trp 325 330 335 Cys Cys His Leu Glu Glu Val Phe Arg Lys Val Gin Gin Ser Phe Glu 340 345 350 Thr Pro Arg Ala Lys Ala Gin Met Pro Thr He Glu Arg Leu Ser Met 355 360 365 Thr Arg Tyr Phe Tyr Leu Phe Pro Gly Asn Thr Thr Thr Cys Cys Ala 370 375 380 Ala Cys Gly Cys Thr Gly Thr Ala Ala Ala Ala Ala Ala Gly Gly Ala 385 390 395 400 Cys Ala Gly Ala Gly Gly Cys Thr Gly Thr Thr Cys Cys Cys Thr Ala 405 410 415 Thr Gly Gly Cys Ala Gly Ala Ala Gly Gly Cys Ala Ala Cys Cys Ala 420 425 430 Cys Ala Gly Ala Ala Ala Ala Ala Ala Gly Cys Cys Ala Cys Thr Thr 435 440 445 Ala Ala Gly Gly Thr Gly Thr Thr Gly Gly Ala Ala Thr Cys Cys Cys 450 455 460 Thr Gly Gly Gly Cys Ala Ala Ala Gly Ala Thr Thr Thr Cys Cys Thr 465 470 475 480 Cys Ala Cys Thr Gly Gly Thr Gly Thr Thr Thr Thr Gly Gly Ala Thr 485 490 495 Ala Ala Cys Thr Thr Gly Gly Thr Gly Gly Ala Ala Cys Ala Ala Ala 500 505 510 Ala Thr Gly Thr Ala Cys Thr Gly Ala Ala Cys Thr Gly Gly Ala Ala 515 520 525 Gly Gly Ala Ala Gly Ala Gly Gly Ala Ala Ala Ala Ala Ala Ala Gly 530 535 540 Ala Ala Ala Thr Ala Thr Thr Ala Cys Gly Ala Thr Gly Cys Thr Ala 545 550 555 560 Ala Ala Ala Cys Thr Gly Ala Ala Gly Ala Cys Ala Ala Ala Gly Thr 565 570 575 Thr Cys Gly Gly Gly Thr Cys Ala Thr Gly Gly Cys Ala Gly Ala Cys 580 585 590 Thr Cys Thr Ala Thr Gly Cys Ala Ala Gly Ala Gly Ala Ala Gly Cys 595 600 605 Ala Ala Cys Cys His Glu Met He Cys Ala Leu Ser Thr Arg Cys Thr 610 615 620 Arg Glu Ser Glu Xaa Ala Met Pro Leu Glu Glu Xaa Ala Met Pro Leu 625 630 635 640 Glu His Glu Ala Asp Glu Xaa Ala Met Pro Leu Glu Ser Asx His Glu 645 650 655 Ala Asp His Ala Asn Gly He Asn Gly He Asn Asp Glu Asn Thr His 660 665 670 Ala Asn Gly He Asn Gly He Asn Asp Glu Asn Thr His Ala Asn Gly 675 680 685 He Asn Gly He Asn Asp Glu Asn Thr Met Ala He Asn His Glu Ala 690 695 700 Asp Ser Asn Met Asx Glu Arg Glu Asp Pro Ala Arg Ala Gly Ala Pro 705 710 715 720 His Ser Pro Ala Thr Glu Asn Thr His Glu Ala Asp Glu Arg Ser Thr 725 730 735 Glu Pro Ser Asx His Glu Ala Asp Thr Glu Xaa Thr Trp His Ala Thr 740 745 750 He Ser Cys Leu Ala He Met Glu Asp Ser Asx His Glu Ala Asp His 755 760 765 Pro Pro Xaa His His His Phe Gly His His Asp Pro Pro Arg Thr He 770 775 780 Met Glu Ser Cys Arg He Glu Arg Pro Ala Thr Glu Asn Thr Asp Glu 785 790 795 800 Pro Thr Pro Ala Thr Glu Asn Thr Asp Glu Pro Thr 805 810 WHAT IS CLAIMED IS: 1. Isolated DNA encoding precursor interleukin- 1 β converting enzyme - related cysteine proteinase II or a functional derivative thereof. 2. The isolated DNA of Claim 1 having the nucleotide sequence of Figure 1 or a functional derivative thereof. 3. The isolated DNA of Claim 1 having the nucleotide sequence: TTTCCAACGC TGTAAAAAAG GACAGAGGCT GTTCCCTATG GCAGAAGGCA ACCACAGAAA AAAGCCACTT AAGGTGTTGG AATCCCTGGG CAAAGATTTC CTCACTGGTG TTTTGGATAA CTTGGTGGAA CAAAATGTAC TGAACTGGAA GGAAGAGGAA AAAAAGAAAT ATTACGATGC TAAAACTGAA GACAAAGTTC GGGTCATGGC AGACTCTATG CAAGAGAAGC AACGTATGGC AGGACAAATG CTTCTTCAAA CCTTTTTTAA CATAGACCAA ATATCCCCCA ATAAAAAAGC TCATCCGAAT ATGGAGGCTG GACCACCTGA GTCAGGAGAA TCTACAGATG CCCTCAAGCT TTGTCCTCAT GAAGAATTCC TGAGACTATG TAAAGAAAGA GCTGAAGAGA TCTATCCAAT AAAGGAGAGA AACAACCGCA CACGCCTGGC TCTCATCATA TGCAATACAG AGTTTGACCA TCTGCCTCCG AGGAATGGAG CTGACTTTGA CATCACAGGG ATGAAGGAGC TACTTGAGGG TCTGGACTAT AGTGTAGATG TAGAAGAGAA TCTGACAGCC AGGGATATGG AGTCAGCGCT GAGGGCATTT GCTACCAGAC CAGAGCACAA GTCCTCTGAC AGCACATTCT TGGTACTCAT GTCTCATGGC ATCCTGGAGG GAATCTGCGG AACTGTGCAT GATGAGAAAA AACCAGATGT GCTGCTTTAT GACACCATCT TCCAGATATT CAACAACCGC AACTGCCTCA GTCTGAAGGA CAAACCCAAG GTCATCATTG TCCAGGCCTG CAGAGGTGCA AACCGTGGGG AACTGTGGGT CAGAGACTCT CCAGCATCCT TGGAAGTGGC CTCTTCACAG TCATCTGAGA ACCTGGAGGA AGATGCTGTT TACAAGACCC ACGTGGAGAA GGACTTCATT GCTTTCTGCT CTTCAACGCC ACACAACGTG TCCTGGAGAG ACAGCACAAT GGGCTCTATC TTCATCACAC AACTCATCAC ATGCTTCCAG AAATATTCTT GGTGCTGCCA CCTAGAGGAA GTATTTCGGA AGGTACAGCA ATCATTTGAA ACTCCAAGGG CCAAAGCTCA AATGCCCACC ATAGAACGAC TGTCCATGAC AAGATATTTC

Claims: TACCTCTTTC CTGGCAATTG AAAATGGAAG CCACAAGCAG CCCAGCCCTC CTTAATCAAC TTCAAGGAGC ACCTTCATTA GTACAGCTTG CATATTTAAC ATTTTGTATT TCAATAAAAG TGAAGACAAA CAAAAAAAAA AAAAAAA (SEQ ID NO:8) or a functional derivative thereof. 4. Isolated RNA encoded by the isolated DNA of Claim 1 or its complementary sequence. 5. An expression vector containing the isolated DNA of Claim 1. 6. A recombinant host cell containing the expression vector of Claim 5. 7. A process for the production of a recombinant interleukin-1 β converting enzyme - related cysteine proteinase II polypeptide, comprising: a) transforming a host cell with the isolated DNA of Claim 1 to produce a recombinant host cell; b) culturing the recombinant host cell under conditions which allow the production of recombinant interleukin-1 β converting enzyme - related cysteine proteinase II; and c) recovering the recombinant interleukin-1 β converting enzyme - related cysteine proteinase π. 8. The recombinant interleukin-1 β converting enzyme - related cysteine proteinase II produced by the process of Claim 7. 9. An isolated and purified interleukin-1 β converting enzyme - related cysteine proteinase II or a homologue or fragment thereof. 10. The isolated and purified interleukin- 1 β converting enzyme - related cysteine proteinase II of Claim 9 which is encoded by the DNA of Figure 1. 11. The isolated and purified interleukin- 1 β converting enzyme - related cysteine proteinase II of Claim 9 having the amino acid sequence: MAEGNHRKKP LKVLESLGKD FLTGVLDNLV EQNVLNWKEE EKKKYYDAKT EDKVRVMADS MQEKQRMAGQ MLLQTFFNID QISPNKKAHP NMEAGPPESG ESTDALKLCP HEEFLRLCKE RAEEIYPIKE RNNRTRLALI ICNTEFDHLP PRNGADFDIT GMKELLEGLD YSVDVEENLT ARDMESALRA FATRPEHKSS DSTFLVLMSH GILEGICGTV HDEKKPDVLL YDTIFQIFNN RNCLSLKDKP KVIIVQACRG ANRGELWVRD SPASLEVASS QSSENLEEDA VYKTHVEKDF IAFCSSTPHN VSWRDSTMGS IFITQLITCF QKYSWCCHLE EVFRKVQQSF ETPRAKAQMP TIERLSMTRY FYLFPGN (SEQ ID N0.9). 12. A method of identifying compounds that modulate interleukin- lβ converting enzyme - related cysteine proteinase II, comprising: (a) mixing a test compound with a solution containing interleukin-1 β converting enzyme - related cysteine proteinase H to form a mixture; (b) measuring interleukin- lβ converting enzyme - related cysteine proteinase H activity in the mixture; and (c) comparing the interleukin-1 β converting enzyme - related cysteine proteinase II activity of the mixture to a standard. 13. Compounds identified by the method of Claim 12. 14. Pharmaceutical compositions comprising the compound of Claim 13. 15. A kit comprising a reagent selected from the group consisting of isolated DNA encoding recombinant interleukin- lβ converting enzyme - related cysteine proteinase II, recombinant interleukin- lβ converting enzyme - related cysteine proteinase II, antibodies to recombinant interleukin- lβ converting enzyme - related cysteine proteinase H and homologues of recombinant interleukin- lβ converting enzyme - related cysteine proteinase II. 16. An antibody immunologically reactive with the interleukin- lβ converting enzyme - related cysteine proteinase II of Claim 9. 17. A method of altering interleukin- 1 β converting enzyme - related cysteine proteinase II activity in vivo comprising administering compound of Claim 13 to an animal. 18. The method of Claim 17 wherein the compound is an antisense molecule complementary to DNA encoding interleukin-1 β converting enzyme - related cysteine proteinase II of Claim 1. 19. A method of altering interleukin- 1 β converting enzyme - related cysteine proteinase H activity by gene therapy comprising in vivo or ex vivo gene transplantation of the isolated DNA of Claim 1. 20. A method for treating an animal in need of treatment of a condition characterized by altered interleukin- lβ converting enzyme - related cysteine proteinase II activity, comprising increasing or decreasing interleukin-1 β converting enzyme - related cysteine proteinase H activity. 21. The method of Claim 20, wherein the condition is selected from the group consisting of: a) Alzheimer's disease b) Parkinson's disease ' c) cancer d) aging e) alopecia f) cardiovascular or neurological injury g) pathogenic infection h) autoimmune disease; and i) immune deficiency syndrome. 22. A method for treating an animal in need of treatment of a condition characterized by altered interleukin- lβ converting enzyme - related cysteine proteinase II activity, comprising increasing or decreasing DNA encoding interleukin- lβ converting enzyme - related cysteine proteinase H activity. 23. The method of Claim 22, wherein the condition is selected from the group consistmg of: a) Alzheimer's disease b) Parkinson's disease c) cancer d) agkg e) alopecia f) cardiovascular or neurological injury g) pathogenic infection h) autoimmune disease; and i) immune deficiency syndrome. 24. A probe for identifying DNA that is structurally related to the DNA of Claim 1 , the probe being selected from the group consisting of: a) DNA having all or a portion of the nucleotide sequence of Figure 1 ; b) RNA encoded by the DNA of a) ; c) degenerate oligonucleotides derived from a portion of the amino acid sequence of Figure 1 ; and d) an antibody directed against recombinant interleukin-lβ converting enzyme - related cysteine proteinase H. 25. A method of isolating DNA encoding interleukin- 1 β converting enzyme - related cysteine proteinase II protein, comprising: (a) hybridizing the isolated DNA of Claim 1 to a sample, the sample containing DNA encoding interleukin-lβ converting enzyme - related cysteine proteinase H, to form a complex; and (b) purifying the complex. 26. Purified nucleic acid encoding functional interleukin- lβ converting enzyme - related cysteine proteinase H which is capable of hybridizing with nucleic acid encoding interleukin-lβ converting enzyme - related cysteine proteinase II under low stringency conditions. 27. Use of a compound of Claim 13 in the manufacture of a medicament for altering interleukein-lβ converting enzyme-related cysteine proteinase II activity. 28. A compound of Claim 13 for use in altering interluekin-lβ converting enzyme-related cysteine proteinase II activity. 29. DNA of claim 1, 2 or 3 for use in altering interleukin-lβ converting enzyme-related cysteine proteinase II activity.

Inventors: ALI AMBEREEN, MUNDAY NEIL A, NICHOLSON DONALD W, VAILLANCOURT JOHN P, ALI, AMBEREEN, MUNDAY, NEIL, A., NICHOLSON, DONALD, W., VAILLANCOURT, JOHN, P.
Applicants: ALI AMBEREEN, MERCK FROSST CANADA INC, MUNDAY NEIL A, NICHOLSON DONALD W, VAILLANCOURT JOHN P, ALI, AMBEREEN, MERCK FROSST CANADA INC., MUNDAY, NEIL, A., NICHOLSON, DONALD, W., VAILLANCOURT, JOHN, P.
Application year: 1995
Main classification: C12N15
IPC codes: A61K38, A61K48, A61P3, A61P9, A61P25, A61P31, A61P35, A61P43, C07K16, C12N1, C12N5, C12N9, C12N15, C12Q1, C12R1