MXPA99009706A - Neuronal mort1 isoforms - Google Patents

Abstract

A MORT1 gene initially cloned from HeLa cells and identified as a member of the receptor mediated apoptotic pathway, is expressed in the human neuronal cell line, NTERA2. Isolation of the MORT1 from this cell line revealed a transcript isoform that differed from the known MORT1 sequence by a deletion of 21 base pairs (bp 172-192 of the coding sequence). Cloning of MORT1 from adult human brain revealed two isoforms, one similarly deleted for bp 172-192, the other with a basepair substitution, A for G at position 173. Assessment of MORT1 function in a yeast two hybrid system indicates that the deleted and intact forms of MORT1 differ in their capacity to interact with other membersof the apoptotic pathway.

Description

NEURONAL HORT1 ISOFORMS Field of 9th Invention The present invention relates to apoptosis signaling proteins that contain human intracellular killing domain. In particular, the present invention relates to novel human neuronal MORT1 isoforms.

BACKGROUND OF THE INVENTION Apoptosis, or programmed cell death in multicellular organisms, is one of the fundamental means by which a cell can respond to environmental changes. One of the best-studied systems of mammalian apoptosis involves Fas (also designated APO-1 and CD95), a type I membrane receptor that, when cross-linked by its analogous ligand, induces apoptosis in a wide variety of cells (for reference, see Nagata, 1994). The extracellular interaction of the Fas ligand with the Fas receptor that covers the cell membrane activates a cascade of intracellular signal transduction that eventually activates proteases in the family (ICE) of the I -1 β conversion enzyme (Henkarí , nineteen ninety six). The transduction of an apoptotic signal depends on the interaction between the intracellular "death domain" of Fas with a cytoplasmic protein of 23 kD, MORT1 [(Boldin, et al., 1995), also called FADD (Chinnaiyan et al., nineteen ninety five)]. The events that lead from the production of an activated Fas trimer complex to the cellular destruction mediated by ICE-like proteases have not been determined, but the recruitment of two molecules of MORT1 / FADD into a complex of induction of death signaling with Fas's death domain seems to be a necessary step (Kischkel, et al., 1995). The final result of this route is cell death by a different mechanism characterized by nuclear and cytoplasmic condensation and DNA fragmentation.

The human gene of MORT1 / FADD covers approximately 3.6 kb and contains two exons (286 and 341 base pairs) separated by a 2.0 kb intron. The MORT1 / FADD was mapped to chromosome 11q13.3 by independent PCR screening techniques of hybrid somatic cell mapping panels and fluorescence in situ hybridization (Kim, et al., 1996). The knowledge of the location of the chromosome and the structure of the MORT1 / FADD gene will help in the efforts to determine their involvement in genetic disorders of apoptosis. Defects in apoptosis due to mutations in the Fas receptor have been described (Fisher et al., 1995; Rieux-Laucat, et al, 1995) in patients with a rare autoimmune lymphoproliferative syndrome (ALPS) that includes non-malignant lymphadenopathy, hepatosplenomegaly and extended CD4 CD4 CD8 lymphocyte populations. However, the existence of some patients with clinical findings of ALPS, but without Fas mutation, suggests that defects in other proteins in the Fas pathway can also produce ALPS. The functional involvement of MORT1 / FADD in the Fas route makes it a candidate for mutation analysis in ALPS. The location of ORT1 / FADD at 11q13.3 also makes it a candidate for human diseases associated with this part of the genome. The IDDM4, a predisposition site for insulin-dependent familial diabetes mellitus (IDDM) has been mapped to this region through binding studies (Cordell, et al., 1995, Davies, et al, 1994, Hashimoto, et al., 1995). ). The pathogenesis of IDDM may involve the destruction mediated by autoimmune T lymphocytes of pancreatic β-islet cells that produce insulin (Tisch and McDevitt, 1996). The chromosomal location of MORT1 / FADD, coupled with its known role in lymphocyte apoptosis, makes it a candidate for mutational analysis in patients with familial diabetes linked to IDD 4. In addition, the region of 11q13 is amplified in several human malignancies, including carcinoma of the chest, gallbladder, esophagus, head and neck, and lung (Schuuring, 1995, Szepetowski, et al., 1995). The amplification of this region has been associated with poor prognosis in patients with breast cancer susceptible to surgery (Schuuring, et al., 1992). Mapping of ORT1 / FADD to the breast cancer cell line amplicon MDA-MB-134-VI (Lafaget, et al., 1992) raises the possibility of its involvement in tumor growth. Future analysis by binding and mutation of MORT1 / FADD in other diseases may support the hypothesis that the deregulation of cell death is a fundamental mechanism for the phogenesis of human diseases (Thompson, 1995). A single gene can encode more than one mRNA transcript through transcriptional processing events such as the use of alternative promoters, alternative splicing, and alternative polyadenylation (Farrow, 1997; Lewin, 1994). The resultant variant transcript isoforms may differ in stability, transferability, or the encoded protein sequence, each of which may impact the function of the encoded protein. Transcript variants that result from alternative RNA processing may be tissue-specific, developmentally regulated, endocrine-regulated, or may appear in response to specific exogenous tails. In addition, the transcript variants of a gene may result from differences in genomic sequence (between individuals or between cell lines). To date, only one isoform of transcript has been reported for the MORT1 / FADD gene (Boldin, et al., 1995; Chinnaiyan, et al., 1995). This invention describes the identification of two new isoforms of MORT1.

OBJECTIVES OF THE INVENTION The present invention is based on the discovery of new isoforms of transcripts of the human neuronal MORT1 gene. The transcripto isoforms and the proteins encoded by them are useful as screening agents in the diagnosis of CNS diseases and in the discovery of CNS-specific anti-apoptotic compounds. The DNA, RNA and proteins encoded by them are useful in a scenario of "gene therapy", either as therapeutic agents in vivo in humans, or as experimental tools in the manipulation of neuronal apoptosis in cell cultures and in systems of animal model Accordingly, in one embodiment, the invention is directed to a neuronal protein encoded by a MORT1 gene, wherein the gene is isolated from NTERA2 or adult human brain tissue. The cDNA encoding the neuronal protein differs from the MORT1 gene known by the 21-base pair deletion and by base pair substitutions.

Brief Description of the Drawings Figure 1 illustrates the investigation of transcript isoforms of MORT1 with C360S of ACHal in a hybrid system of two yeasts: Activation of the reporter gene HIS3. Each of the transcript isoforms of ORT1 was expressed as a fusion protein with Gal4 DNA binding domain. The C360S from MACHal was expressed as a fusion protein with the Gal4 activation domain. Yeast strains were plated at a cell density of 5 x 101, 5 x 102, 5? 103, 5? 104y 5 x 105, (from left to right inside a plate). Fraternal isolates from yeast strains expressing a transcript isoform of MORT1 with C36OS fusion proteins of MACHal were plated on histidine deficient medium and containing 0, 20 or 40 mM of 3-aminotriazole (3-AT ). The upper line plates (1) contained yeast strains expressing two fusion proteins as follows: YCB5: MORT1-pAS1 / C36OS from MACHal-pACTII; YCB9: MORTl? 21-pAS1 / C36OS from MACHal-pACTII; YCB16: G173A from MORT1-pAS1 / C360S from MACHal -pACTI I. The lower line of plates (3) contains negative control strains expressing only the fusion protein of the MORT1 transcript isoform with a recombinant vector containing heterologous DNA not related, as follows: YCB5.1: MORT1-pAS1 / SNF4-pACTII; YCB9.1: G173A from MORT1 / SNF4-pACTU; YCB16.1: MORT1? 21-pAS1 / SNF4-pACTIl. The functional interaction of the fusion protein of the MORT1 transcript isoform with the C36OS fusion protein of MACHal reconstitutes the function of the Gal4 protein and boosts the activity of the reporter gene HIS3 thereby binding the MORT1 isoform / interaction of C36OS from MACHal to the prototropia of histidine and yeast cell growth.

Figure 2 illustrates the investigation of the transcript isoforms of MORT1 with C360S from MACHal in a hybrid system of two yeasts: Activation of the reporter gene CYH2. Each of the transcript isoforms of MORT1 was expressed as a fusion protein with Gal4 DNA binding domain. The C360S from MACHal was expressed as a fusion protein with the Gal4 activation domain. Yeast strains were plated at a cell density of 5? 101, 5 x 102, 5 x 103, 5 x 104 and 5? 105, (from left to right inside a plate). Fraternal isolates from yeast strains expressing a transcript isoform of MORT1 with C360S fusion proteins from MACHal were plated on selective media containing 0, 8 or 12 ug of cyclohexamide / mL of medium. The upper line plates (1) contained yeast strains expressing two fusion proteins as follows: YCB5: MORT1-pAS1 / C360S from MACHal-pACTI I; YCB9: MORTl? 21-pAS1 / C36OS from MACHal -pACTII; YCB16: G173A from MORT1-pAS1 / C36OS from MACHal -pACTI I. The lower line of plates (3) contains negative control strains that express only the fusion protein of the MORT1 transcript isoform with a recombinant vector containing unrelated heterologous DNA, as follows: YCB5.1: MORT1-pAS1 / SNF4 -pACTII; YCB9.1: G173A from MORT1 / SNF4-pACTII: YCB16.1: MORT1? 21-pAS1 / SNF4-pACTII. The functional interaction of the fusion protein of the MORT1 transcript isoform with the C360S fusion protein of MACHal reconstitutes the function of the Gal4 protein and boosts the activity of the reporter gene CYH2 thereby binding the MORT1 isoform / interaction of C360S from MACHal to the sensitivity to cyclohexamide and the abrogation of yeast cell growth.

DETAILED DESCRIPTION OF THE INVENTION The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and yeast biology and genetics, which are within the ordinary knowledge of the art. technique. Such techniques are fully explained in the literature (eg, Ausubel, et al., 1993, Coico, 1994, Freshney, 1987, Glover, 1985, Griffin and Griffin, 1994, Hanes and Higgins, 1984, Perbal, 1988, Rose et al. al., 1990; Sambrook, et al., 1989). All patents, patent applications and publications cited herein, whether supra or infra, are incorporated herein by reference in their entirety. In describing the present invention, the following terms will be employed and are intended to be defined as indicated below. By "MORT1 transcript isoform" is meant a nucleic acid molecule, including DNA, RNA, mRNA, cDNA derived from mRNA, or even synthetic DNA, which is derived either directly or indirectly from a genomic sequence of MORT1. As used herein, the term specifically includes the transcript described in Chinnaiyan, et al., (1995), Boldin, et al., (1995), and Genbank deposit x84709, and as MORT1 by Genbank x84709. The term MORT1? 21 denotes an isolated cDNA sophoris from NTERA2 cells (SEQ ID NO: 1) or adult human brain (SEQ ID NO: 3) having a specific deletion of 21 base pairs (base pairs 172 -192 of the coding sequence) when compared to the published MORT1. The term MORT1G173A (SEQ ID NO: 5) denotes an isoform of cDNA isolated from human brain having a nucleotide substitution (A by G) at position bp 173 of the coding sequence of MORT1. Here, the A of the transcriptional initiation codon of MORT1 is designated position 1, and corresponds to bp 145 of the Genbank sequence x84709. The above terms (transcript isoform of MORT1, MORT1? 21 and MORT1G173A) encompass the sequence of genomic MORT1 encoding them, including introns and eons. Alternatively, a transcript isoform may include transcripts of genomic sequences lacking one or more inines or exons, or transcripts that incorporate non-coding or coding sequence different from those found in the wild-type, full-length sequence. Transcript isoforms can arise from any one of a number of natural occurrence processes, including but not limited to mutation, alternative initiation, alternative splicing, and alternative polyadenylation, each of which can affect the primary structure of some other aspect of the function of the encoded protein. The protein product of a transcript isoform may contain amino acids that differ from the wild-type protein due to insertion, deletion or change of coding nucleotide framework. For example, NTERA2 and MORT1 adult human brain isoforms are described in SEQ ID NOs: 1 and 3. The cDNA encoding intact MORT1 was generated using standard PCR techniques (Finney, 1993; Griffin and Griffin, 1994). ). Hela cell cDNA prepared by reverse transcriptase / standard PCR was the source of the DNA model. Additionally, MORT1 cDNA was prepared using a human placental cDNA library as the source of the model. In each case, a fragment of MORT1 was amplified by PCR, cloned and sequenced. The DNA sequences obtained from placental tissue and HeLa were compared to the MORT1 sequence in the Genbank x84709 reservoir. The existence of a human neuronal MORT1 transcript was determined by RT / PCR. The human neuronal NTERA2 cells were grown under conditions that promoted terminal differentiation for the neuronal phenotype and induced the experimentation of apoptosis by incubation with staurosporine. RNA extracted from these cells was subjected to RT / PCR using specific primers of MORT1. The resulting fragment of MORT1 was cloned and its sequence compared to the MORT1 sequence of the Genbank deposit x84709. MORT1 was also cloned from fetal and adult human brain cDNA libraries by PCR. Fragments of MORT1 were amplified, cloned and their sequences compared to the MORT1 sequence in the Genbank deposit x84709. In summary, MORT1 clones derived from HeLa cells, the human placental library, and human fetal brain coincided with the MORT1 sequence of Genbank. The clones of MORT1 from NTERA2 cells and three of the five clones derived from adult human brain were subjected to deletion for base pairs 172-192 of the MORT1 coding sequence. The other two of the five clones derived from the adult human brain had a single substitution of base pairs (G173A) relative to the MORT1 sequence of the Genbank. The deletion of 21 base pairs and the substitution of G173A both generated a Glu-Pro-Glu amino acid sequence at positions 56-58 of the protein sequence, versus the corresponding Glu-Pro-Gly sequence of the protein type. wild. In addition, the human neuronal MORT1 isoforms include some of the base pair substitutions relative to the Genbank sequence, as described in SEQ ID NO: 1 and 3. The human neuronal MORT1 transcript isoforms are also cloned from phage libraries of human cDNA by probing with a radiolabelled MORT1 probe using published DNA hybridization methods (Ausubel et al., 1993; Sambrook, et al., 1989). Many human brain sub-region libraries are commercially available (Clontech; Stratagene) for screening. For tissue or neuronal cell lines for which there are no commercially available libraries, the regular synthesis of a cDNA library is performed from poly (A) + RNA (service available at Clontech). The selection of poly (A) and total RNA are carried out according to published methods (Glover, 1985; Sambrook, et al., 1989). The genomic sequence of human MORT1 is cloned using standard PCR techniques (Griffin and Griffin, 1994) from human genomic DNA prepared by standard methods (Glover, 1985, Sambrook, et al., 1989). The relative abundance of neuronal MORT1 transcript isoforms within a given tissue or cell line is evaluated by Rnasa protection and S1 nuclease mapping, performed according to published methods (Ausubel, et al., 1993; Berk and Sharp, 1997). Lee and Costlow, 1987). Specifically, oligonucleotide probes are designed to distinguish 21 nucleotide differences between the deletion and intact MORT1 isoforms. A hybrid system of two yeasts as described in Young and Ozenberger, WO 95/34646, published on December 21, 1995, all of which is incorporated herein, was used to functionally characterize the capacity of the isoforms of the transcript. of MORT1 to interact with C360S of MACHal, a protein component of the cytoplasmic apparatus of the Fas / AP01 and TNF receptors. Expression vectors were constructed by fusing the DNA binding domain of GAL4 to the isoforms of the MORT1 transcript. A second expression vector was constructed by fusing the activation domain of GAL4 to C360S of MACHal. To determine the ability of the MORT1 transcript isoforms to interact with MACHal C360S, yeast strains were generated that expressed a single isoform of the MORT1 C360S MORT1 transcript, or a single recombinant plasmid that encoded a fusion protein with its accompanying vector containing unrelated heterologous DNA. Strains were tested for the productive interaction of protein-protein through reporter gene activity and a change in the yeast cell phenotype. These data suggest that the fusion protein of the MORT1? 21 isoform is damaged in its ability to functionally interact with the C36OS fusion protein of MACHal. Yeast strains expressing MORT1? 21 with C36OS from MACHal demonstrated diminished histidine prototopia, and decreased cell growth, as well as decreased sensitivity to cycloheximide and increased cell growth. This was observed in comparison with the growth characteristics of the yeast strain expressing the fusion proteins for MORT1 with C360S from MACHal, or MORTG173A with C360S from MACHal. The present nucleic acids find a wide variety of applications including the use as translatable translatable, hybridization probes, PCR primaries, therapeutic nucleic acids, etc .; use in the detection of the presence of genes and transcripts of genes involved in apoptosis, in the detection or ification of nucleic acids encoding additional MORT1 homologs and structural analogs, and in gene therapy applications. The product genes encoded by these transcripts are useful in their use as target proteins in the development of therapeutic products for the manipulation of the apoptotic route.

The following experiments and examples are proposed by way of illustration and in no way as a limitation.

Examples Example 1: Cloning of MORT1 from HeLa v cells from a cDNA library of human placenta. As a control, the intact MORT1 gene was cloned as described in Boldin, et al., (1995). The oligonucleotides were prepared in an ABI oligosintetizer, designed according to the published cDNA sequence of human MORT1 (Genbank x84709). A 25-base 5 'sense oligonucleotide containing an Ncol site and the initiation codon ("5' MORT1," 5'-ACC CCG CCA TGG ACC CGT TCC TGG t-3 \ corresponding to bases -8 to + was synthesized 17), and a 3 'antisense oligonucleotide of 24 bases spanning the stop codon ("3" MORT1, "5'-ACG GGC CCA TCA GGA CGC TTC GGA-3', complementary to bases 636 to 613 of the sequence MORT1 coding.) The cDNA encoding intact MORT1 was generated using standard PCR techniques (Finney, 1993) .The thermal cycling of these reactions was carried out under the regime of 1 min at 95C, then 30 cycles (40 seconds). at 95C, 1 min at 60C, 1 min at 72C), and 10 min at 72C, using oligonucleotides "5 'MORT1" and "3" MORT1. "cDNA from HeLa cells and a cDNA library (matchmaker) of human placenta (Clontech) served as the DNA models In each case, a 640 bp fragment was obtained by PCR and subsequently ligated into the cloning vector pCR II (Invitrogen). The MORT1 cDNA sequences of placenta and HeLa tied with the coding sequence of the published MORT1 sequence (Genbank deposit X84709).

Example 2: Cloning of a neuronal MORT1 from NTERA2 cells by RT / PCR using a gene-specific RT primer. In order to investigate if MORT1 was present in neuronal cells, NTERA-2 cells, which originated from a human teratocarcinoma and represented a late human embryonic neuronal phenotype, were investigated for the presence of MORT1. NTERA2 cells (Stratagene) were grown under conditions that promoted terminal differentiation to the neuronal phenotype, including long-term incubation in retinoic acid (Andrews, 1984). Eighteen hours before RNA extraction, staurosporine (Calbiochem) was added to the cell culture medium to a final concentration of 100 nM to induce apoptosis. Total RNA extraction was performed by the RNeasy Total RNA Kit (Life Technologies) using reagents and instructions provided by the manufacturer. Reverse transcription of the total RNA was carried out with reagents and instructions for the gene-specific reverse transcription provided in the BRL Preamplification Kit (Life Technologies). Specifically, an antisense oligonucleotide 3 'of 27 bases ("MORT1 downstream," 5'-TAG ATG CCT GTG GTC CAC CAG CGC AAA-3', complementary to bases 663 to 637 of the sequence) was synthesized in an ABI oligosintetizer. of coding of MORT1). The reverse transcription of 1 μg of total RNA was prepared using this oligonucleotide. PCR amplification of the NTERA2 cDNA was carried out using DNA Taq polymerase and other reagents provided by the manufacturer (Life Technologies) and equimolar amounts of oligonucleotides "5 'MORT1" and "31 MORT1" (see above). The thermal cyclization of the PCRs was in the Gene Amp 2400 thermal cycler (Perkin Elmer) under the regime 1 min at 95C, 35 cycles (15 seconds at 95C, 30 seconds at 50C, 1 min at 72C), 7 min at 72C. The resulting PCR product (cDNA corresponding to the MORT1? 21 isoform) was purified on agarose gel using Qiaex II reagents and instructions provided by the manufacturer (Qiagen) and ligated to the cloning vector pCR2.1 (Invitrogen). Recombinant plasmids were obtained by standard methodology, including transformation in E. Coli One Shot cells (Invitrogen), growth of transformed bacterial cells on LB agar plates containing 100 μg / mL of ampicillin (Sigma), growth of bacterial colonies the LB medium (Life Technologies) and preparation of plasmid DNA using the QIAprep Spin Miniprep Kit (Qiagen). The DNA sequence was obtained using reagents (ABI) of dideoxy termination cycle sequencing and synthetic oligonucleotides (see below) reacted on a Gene Amp 9600 thermal cycler (Perkin Elmer) under the manufacturer's instructions. The reactions were carried out and the information was generated in an automated ABI sequencer. The information of the sequence was analyzed using the computer program Lasergene (DNA star). Oligonucleotides were prepared for use in DNA sequencing (on an ABI synthesizer), designed according to the sequence of the polylinker region of pCR2.1 and published MORT1 sequence. The following oligonucleotides were generated: "Advanced TA" (5'-CAG GAA ACA GCT ATG ACC ATG-3 ', corresponding to the sense chain of the lacZ gene in pCR2.1, 67 base pairs upstream of the cloning site of TA), "Inverse TA" (5'-ACG TTG TAA AAC GAC GGC CAG-3 ', corresponding to the antisense chain of the lacZ gene in pCR2.1, 112 base pairs downstream of the TA cloning site "MORT 150"(5'-ACC TCT TCT CCA TGC TGC TG-3 ', sense chain corresponding to bases 131 to 150 of the coding sequence of MORT1)" MORT 230"(5'-TCG AAG TCG TCG ACG CGC CG- 3 ', antisense chain complementary to bases 248 to 229 of the coding sequence of MORT1) "MORT 400" (5'-TCG ACÁ GCA TCG AGG ACÁ GA-3', sense string corresponding to bases 377 to 396 of the coding sequence of MORT1, and "MORT 420" (5'-GAT TCT CAG TGA CTC CCG CA-3 ', antisense chain, complementary to bases 441 to 422 of the coding sequence of M ORT'I).

Example 3: Cloning of MORT1 from NTERA2 cells using primary nested NTERA2 cells (Stratagene) were grown under conditions that promoted terminal differentiation to the neuronal phenotype, including long-term incubation in retinoic acid (Andrews, 1984). Eighteen hours before RNA extraction, staurosporine (Calbiochem) was added to the cell culture medium to a final concentration of 100 nM to induce apoptosis. Total RNA extraction was performed using the BRL Rneasy Total RNA Kit (Life Technologies, Inc.) using reagents and instructions provided by the manufacturer. Reverse transcription of total RNA was carried out with reagents and instructions provided in the BRL Preamplification Kit (Life Technologies, Inc.). Specifically, the reverse transcription of 4 μg of total RNA was primed using oligo (dT). Oligonucleotides for use in PCR were prepared (in an ABI oligosintetizer), designed according to the published MORT1 published cDNA sequence (Genbank X84709). An oligonucleotide of sense 5"of 25 bases (" MORT1 upstream, "5'-AAG CGG CGA GAC CTG GCC ACG GCC A-3 ', corresponding to bases -90 to -66, relative to the initiation site of The PCR amplification of NTERA2 cDNA was carried out using DNA Taq polymerase and other reagents provided by the manufacturer (Life Technologies) and equimolar amounts of oligonucleotides of "MORT1 upstream" and "MORT1 downstream" (see above) The thermal cyclization of the PCRs was carried out in the Gene Amp 2400 thermal cycler (Perkin Elmer, Inc.) under the regime of 1 min at 95C, 35 cycles (15 seconds at 95C, 30 seconds at 50C, 1 min at 72 ° C), 7 min at 72 ° C. An aliquot of 2 μL of the 100 μL reaction served as the DNA model for subsequent amplification by PCR using equimolar amounts of oligonucleotides "5" MORT1"and" 3 'MORT1". The resulting PCR product (The cDNA corresponding to the MORT1? 21 isoform) was purified on agarose gel using Qiaex II reagents and instructions provided by the manufacturer (Qiagen) and ligated into the cloning vector pCR2.1 (Invitrogen). The recombinant plasmids were obtained by standard methodology, including the transformation into E. Coli One Shot cells (Invitrogen), growth of bacterial cells transformed into LB-agar plates containing 100 μg / mL of ampicillin (Sigma), growth of the bacterial colonies in LB medium (Life Technologies), and preparation of plasmid DNA using the QIAprep Spin Miniprep Kit (Qiagen). The DNA sequence was obtained using dideoxy terminating cycle sequencing reagents (ABII) and synthetic oligonucleotides (see above) reacted in a Gene Amp 9600 thermal cycler (Perkin Elmer) under the manufacturer's instructions. The reactions were carried out and data was generated in an automated ABI sequencer. The information of the sequence was analyzed using the computer program Lasergene (DNA Star).

Example 4: Cloning of MORT1 isoforms from brain tissue MORT1 cDNA from human fetal brain was generated using standard PCR techniques. The thermal cyclization was carried out using the regimen, 1 min at 95 ° C, then 30 cycles (40 seconds at 95 ° C, 1 min at 60 ° C, 1 min at 72 ° C), 10 min at 72 ° C, and using the oligonucleotides "5 'MORT1" and "3 'MORT1" (see above). A cDNA library (matchmaker) of human fetal brain (clontech) and a library (Clontech) of cDNA (matchmaker) of adult full brain, from human, were used as DNA models for PCR. PCR products of approximately 640 bp were obtained, ligated to the pCR II vector (Invitrogen), and recombinant plasmids were obtained by standard methodology, including transformation into E. coli One Shot competent cells, growth of bacterial cells transformed into plates of LB-agar containing 100 μg / mL of ampicillin (Sigma), growth of bacterial colonies in LB Medium (Life Technologies), and preparation of plasmid DNA using Wizard Plus Mipreps (Promega) or by Qiagen Midiprep (Qiagen). The recombinant plasmids were confirmed by digestion with restriction enzyme. The cDNA sequence was determined by dye-dideoxy termination reaction using ABI-Perkin-Elmer reagents and protocols and the ABI 373A automated sequencer. The sequence of each of five isolated clones from human fetal brain cDNA matched the published MORT1 sequence. Five isolated clones derived from human adult brain cDNA were also sequenced. Three out of five clones coded for the isoform containing the 172-192 base pair deletion of the MORT1 coding sequence; the other two clones encoded for a single base pair substitution (G173A) relative to the MORT1 sequence of Genbank (x84709).

Example 5: Cloning of MACHal from cDNA from NTERA2 cells NTERA2 cells (Stratagene) were grown under conditions that promoted terminal differentiation to the neuronal phenotype, including long-term incubation in retinoic acid (Andrews, 1984). Eighteen hours before RNA extraction, staurosporine (Calbiochem) was added to the cell culture medium to a final concentration of 100 nM to induce apoptosis. Total RNA extraction was performed using the BRL RNeasy Total RNA Kit (Life Technologies, Inc.) using reagents and instructions provided by the manufacturer. Reverse transcription of total RNA was carried out with reagents and instructions provided in the BRL Preamplification Kit (Life Technologies, Inc.). Specifically, the reverse transcription of 4 μg of total RNA was primed using oligo (dT). Oligonucleotides for use in PCR were prepared (in an ABI oligosintetizer), designed according to the published MACH sequence (Bolding, et al., 1996). A 24-base molecule (MACH 5 'a, 5'-TTT-AAA-AAG-ATG-GAC-TTC-AGC-AGA-3' was synthesized, encompassing the translation initiation codon of MACHal and other MACH isoforms) . Another 24 base molecule (MACH 3 'a, 5'-ATA-GCA-CCA-TCA-ATC-AGA-AGG-GAA-3', complementary to the coding sequence, and encompassing the stop codon of MACHal and MACHa2, PCR amplification of NTERA2 cDNA was carried out using DNA Taq polymerase and other reagents provided by the manufacturer (Life Technologies) and equimolar quantities of oligonucleotides MACH 5 'a and MACH 3' a (see above). the PCRs was carried out in the Gene Amp 2400 thermal cycler (Perkin Elmer, Inc.) under the regime of 1 min at 95C, 35 cycles (15 seconds at 95C, 30 seconds at 50C, 1 min at 72C), 7 min at 72 C. Two PCR products, both between 1.4 and 1.5 kb, were obtained.The largest product (cDNA corresponding to MACHal) was purified on agarose gel using Qiaex II reagents and instructions provided by the manufacturer (Qiagen) and ligated to the vector of cloning pCR2.1 (Invitrogen) The recombinant plasmids were obtained by methodology Standard, including transformation into E. Coli One Shot cells (Invitrogen), growth of bacterial cells transformed into LB-agar plates containing 100 μg / mL of ampicillin (Sigma), growth of bacterial colonies in LB medium (Life Technologies), and preparation of plasmid DNA using the QIAprep Spin Miniprep Kit (Qiagen). The DNA sequence was obtained using dideoxy cycle sequencing termination reagents (ABII) and synthetic oligonucleotides reacted in a Gene Amp 9600 thermal cycler (Perkin Elmer) under the manufacturer's instructions. The reactions were carried out and data was generated in an automated ABI sequencer. The information of the sequence was analyzed using the computer program Lasergene (DNA Star).

Example 6: Interaction of transcript isoforms of MQRT1 with C36QS of MACHal To investigate the interaction of MORT1 transcript isoforms, a two-hybrid interaction paradigm with MORT1 and C360S of MACHal was first established. The genes coding for the fusion proteins were generated by cloning MORT1 and C360S cDNA sequences from MACHal into plasmids containing the coding regions for the domains of the yeast transcriptional activator proteins. The fusion of DNA binding domain (Gal4) is constructed in pAS1 having a 2m origin of replication that is described in Wade-Harper et al., (1993). Gene domain (Gal4) fusions are constructed in pACTII (described in Durfee et al., 1993). The yeast system used in this study is described by Young and Ozenberger (1995). The MORT1 cDNA is obtained as a Ncol-BamH! Fragment. from plasmid of MORT1-pCRII and directionally cloned in pAS1 via the restriction sites Ncol and BamHI to generate plasmid of MORT1-pAS1. The wild type protein of MACHal is lethal in yeast. Mutation of the cisine at amino acid position 360 to a serine prevents the lethal phenotype and does not interfere with the ability of the protein to interact with MORT1 (Boldin, et al., 1996). The amino acid substitution to generate C360S from MACHal was obtained using standard PCR techniques (Finney, 1993). The thermal cyclization of these reactions was carried out under the regime, 1 min at 95C, then 30 cycles (40 seconds at 95C, 1 min at 60C, 1 min at 72C) and 10 min at 72C, and using specific oligonucleotides. All oligonucleotides were prepared in an ABI Oligosintetizer and were designated according to the cDNA published for human MACH (Boldin et al., 1996); Genbank X98172-X98178). Four oligonucleotides were prepared. A mutagenic oligonucleotide 5 'of 36 bases (oCB1) contained the substitution C360S (TGT to TCT) and silent mutations in Ala 359 (GCT to GCA) to encrypt a diagnostic restriction enzyme site for Sfil [5'-GTG TTT TTT ATT CAG GCA TCT CAG GGG GAT AAC TAC-31], a mutagenic oligonucleotide 3 'of 36 bases (oCB2) [5'-GTA GTT ATC CCC CTG AGA TGC CTG AAT AAA AAA CAC-3'] contained the sequence complementary to oCB1 . An oligonucleotide (oCB3) of 34 bases containing the BamHI site [5'-CGG GAT CCG TAT GGA CTT CAG CAG AAA TCT TTA T-3 '], a 3' oligonucleotide (oCB4) of 38 bases contained restriction enzyme sites Sali and BamHI [5-CGG GAT CCG ACG TCG ACT CAA TCA GAA GGG AAG ACA AG-3 ']. The plasmid MACHal -pCR2.1 (see example 5, above) was used as a model with pair of nucleotides oCB2 and oCB3 to generate a fragment of 1100 base pairs, and as a model for the pair of oligonucleotides oCB1 and oCB4 to generate a fragment of 360 base pairs. A subsequent PCR reaction was carried out using the 1100 and 360 base pair fragments with the oligonucleotide pair oCB3 and oCB4 to generate a 1460 base pair fragment that was ligated to the pCRI1 vector (Invitrogen) to generate C360S-pCRII of MACHal. The recombinant plasmids were obtained by standard methodology, including transformation into E. Coli One Shot cells (Invitrogen). Growth of transformed bacterial cells on LB-agar plates (Gibco, Inc.) containing either 100 mg / mL ampicillin (Sigma or 50 mg / mL Kanamycin (Sigma) as described in the manufacturer's standard protocol. Growth of the bacterial colonies was in LB medium and the plasmid DNA preparation was carried out using Qiagen Midiprep (Qiagen) or Wizard Plus Minipreps (Promega) The recombinant plasmids were confirmed by restriction enzyme digestion The cDNA sequence was obtained by dye-deoxy terminator reactions using ABI-Perkin-Elmer reagents and protocols and the ABI 373A automated sequencer.The cDNA coding for MACHal C360S was obtained as a BamHI-Sall fragment and was directionally cloned into pACTII via BamHi-Xhol sites to generate C360S-pACTII from MACHal DNA from final recombinant vectors was transformed into yeast strain (s) by the lithium acetate method (Rose et al. al., 1990). A host strain of yeast (CY770) as described in Young and Qzenberger (1995) was transformed with the fusion constructs of both MORT1 and C360S of MACHal (YCB5) or a single fusion construct plus the opposing vector containing heterologous DNA. unrelated (YCB5.1) or YCB2.1, respectively). It was found that all strains showed the same growth in non-selective medium. The strains were then tested for growth on a selective medium (ie, growth medium lacking histidine). Only cells expressing fusions of both MORT1 and C360S of MACHal were able to grow in selective medium, while strains containing either the MORT1 fusion or the C360S fusion of MACHal with an unrelated fusion could not grow ( information not shown). The SEQ of G173A of MORT1 (ID NO.5) was obtained as a fragment of Ncol-BamHI from G173A-pCRH of MORT1 and directionally cloned into pAS1 to generate plasmid G173A-pAS1 of MORT1. MORT1? 21 was obtained as a fragment of Ncol-BamHI from MORTl? 21-pCRII and directionally cloned in pAS1 to the plasmid MORT1? 21-pAS1 generated. These plasmids and the C3dOS-pACTII plasmid from MACHal were used to transform the yeast host strain, CY770. Strains were generated expressing fusion constructs (YCB16) of C360S from MACHal G173A of MORT1 or the fusion construct of G173A from MORT1 plus the opposing vector containing unrelated heterologous DNA (YCB16.1). Strains expressing fusion constructs (YCB9) of C360S from MACHal and MORT1? 21 or the fusion construct of MORT1? 21 plus the opposing vector containing unrelated heterologous DNA (YCB9.1) were generated. Two independent samples of each strain were scored on a standard synthetic medium containing 0, 20 or 40 mM of 3-aminotriazole (Figure 2). Plates were incubated at 30C for 3 days. It was found that all strains showed the same growth in non-selective medium. The strains were then tested for growth on selective media (this is growth in medium lacking histidine). The cells expressing the fusion of both MACHal C360S and MORT1, and the cells expressing the C360S fusions of MACHal and the G173A isoform of MORT1, were able to grow in selective medium while the strains containing the fusions of C360S from MACHal and MORT1? 21 were not able to grow (Figure 2). Negative control strains expressing either the MORT1 isoform fusions or the C360S fusion of MACHal with an unrelated fusion could not grow. These data suggest that the fusion protein of the MORT1? 21 isoform is damaged in its ability to functionally interact with the MACHal C360S fusion protein.

Example 7: Tamizaie for compounds that affect the interaction of transcript isoforms of MORT1 with other interaction proteins (members of the MACH family). Screening methods as described in Young and Ozenberger, WO 95/34646 published December 21, 1995, the entirety of which is incorporated herein by reference, are employed to identify compounds that affect the interaction of MORT1 transcript isoforms with proteins such as MACHal's C360S.

Low copy number plasmids expressing transcript isoforms of MORT1 (G173A of MORT1 or MORT1? 21) and C360S of MACHal as fusion proteins of GAL4 are constructed to reduce the expression of these proteins. CDNA inserts coding for MORT1 transcript isoforms are directly subcloned from the vector (s) of recombinant pAS1 (s) to pUN30AS via a fragment of Ncol-BamHI to generate either G173-pUN30AS of MORT1 or D21-pUN30AS of MORT1. MACHal's C360S is subcloned directly from the MACHal C360S-pCRII as a BamHI-Sal1 fragment in pUNIOOACT via BamHI-Xhol sites to generate C36OS-pUN100ACT from MACHal. These plasmids are transformed into yeast strains CY770 (Young and Ozenberger, 1995) with reporter plasmid pOZ146 (Young and Ozenberger, 1995) to generate YCB6 yeast strains. The presence of three plasmids is necessary to confer the necessary phenotype to make possible a rescue screening method as described in Young and Ozenberger (1995). The yeast strain (YCB18) containing the fusion plasmids of MORT1 G173A and MACHal C360S plus the reporter plasmid, or the yeast strain (YCB17) containing MORT1? 21 and MACHal C360S plus the reporter plasmid, form the basis of a simple primary screening for compounds that interrupt the interaction of the gene products of the isoform of the MORT1 and C360S transcript of MACHal. Example 8: Tamizaie for compounds that affect the interaction of MORT1 transcript isoforms and protein that contains a death domain motif (TRAPO, Fas / APOi receptor, TNFR). Screening methods as described in Young and Ozenberger, WO 95/34646 published December 21, 1995, all of which is incorporated herein by reference, are employed to identify compounds that affect the interaction of transcript isoforms. of MORT1 with proteins that contain a motif of death domain. Low copy number plasmids expressing MORT1 transcript isoforms (MORT1 G173A or MORT1? 21) and the cytoplasmic domain of the TNF receptor containing a motif of death domain as the GAL4 fusion proteins are constructed to reduce the expression of these proteins. CDNA inserts coding for MORT1 transcript isoforms are directly subcloned from G173A-pAS1 from MORT1 or the recombinant from MORTl? 21-pAS1 to pUN30AS via a Ncol-BamHI fragment to generate either G173-pUN30AS from MORT1 or MORTl? 21-pUN30AS. The cytoplasmic domain of TNFR is subcloned directly from TNFcito-pCRIl as an EcoRI fragment in pUNIOOACT via EcoRI sites to generate TNFRcito-pUNIOOACT. The plasmids are transformed into the yeast strains CY770 (Young and Ozenberger, 1995) with the reporter plasmid pOZ146 (Young and Ozenberger, 1995). The presence of three plasmids is necessary to confer the necessary phenotype to make possible a rescue screening method as described in Young and Ozenberger (1995). The yeast strain (YCB21) containing the fusion plasmids of G173A of MORT1 and TNFRciio plus the reporter plasmid, or the yeast strain (YCB20) containing the MORT1? 21 and TNFRcito plus the plasmid. reporter, forms the basis of a simple primary screening for compounds that interrupt the interaction of the gene products of the MORT1 transcript isoform and a protein containing the death domain, cytoplasmic domain of TNFR.

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LISTING D? SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANTWood, Andre T Bingham, Brendan W Young, Kathleen H Birsan, Camellia (ii) TITLE OF THE INVENTION: Neuronal Isoforms of M0RT1 (iii) NUMBER OF SEQUENCES: 6 (iv) ADDRESS FOR CORRESPONDENCE: (A) DISCRIMINATORY: Andrea C. Walsh (B) STREET: One Campus Drive (C) CITY: Parsippany (D) STATE: New Jersey (E) COUNTRY: USA (F) POSTAL CODE: 07054 (V) COMPUTER LEADABLE FORM: (A) TYPE OF MEDIUM: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patentln Relay # 1.0, Version # 1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US (B) SUBMISSION DATE: (C) CLASSIFICATION: (viii) EMPLOYEE / AGENT INFORMATION: (A) NAME: Walsh, Andrea C. (B) ) REGISTRATION NUMBER: 34,988 (C) REFERENCE NUMBER / FILE: AHP-97147 (ix) INFORMATION FOR TELECOMMUNICATIONS (A) TELEPHONE: (973) 683-2169 (B) TELEFAX. (973) 683-4117 (2) INFORMATION FOR SEQ ID NO: l: (i) Characteristics OF THE SEQUENCE: (A) LENGTH: 606 base pairs (B) TYPE: nucleic acid (C) CHAIN: single (D) ) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 1.606 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l: ATG GAC CCG TTC CTG GTG CTG CTG CAC TCG GTG TCG TCC AGC CTG TCG 48 Met Asp Pro Phe Leu Val Leu Leu His Ser Val Ser Ser Ser Leu Ser 1 5 10 15 AGC AGC GAG CTG ACC GAG CTC AAG TTC CTA TGC CTC GGG CGC GTG GGC 96 Ser Ser Glu Leu Thr Glu Leu Lys Phe Leu Cys Leu Gly Arg Val Gly 20 25 30 AAG CGC AAG CTG GAG CGC GTG CAG AGC GGC CTA GAC CTC TTC TCC ATG 144 Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Leu Asp Leu Phe Ser Met 35 40 45 CTG CTG GAG CAG AAC GAC CTG GAG CCC GAG CTG CTC GCC TCC CTG CGG 192 Leu Leu Glu Gln Asn Asp Leu Glu Pro Glu Leu Leu Wing Ser Leu Arg 50 55 60 CGC CAC GAC CTG CTG CGG CGC GTC GAC GTC TTC GAG GCG GGG GCG GCG 240 Arg His Asp Leu Leu Arg Arg Val Asp Asp Phe Glu Wing Gly Wing 65 70 75 80 GCC GGG GCC GCG CCT GGG GAA GAA GAC CTG TGT GCA GCA TTT AAC GTC 288 Wing Gly Wing Wing Pro Gly Glu Glu Asp Leu Cys Wing Wing Phe Asn Val 85 90 95 ATA TGT GAT AAT GTG GGG AAA GAT TGG AGA AGG CTG GCT CGT CAG CTC 336 lie Cys Asp Asn Val Gly Lys Asp Trp Arg Arg Leu Ala Arg Gln Leu 100 105 110 AAA GTC TCA GAC ACC AAG ATC GAC AGC ATC GAG GAC AGA TAC CCC CGC 384 Lys Val Ser Asp Thr Lys lie Asp Ser lie Glu Asp Arg Tyr Pro Arg 115 120 125 AAC CTG AC GAG CGT GTG CGG GAG TCA CTG AGA ATC TGG AAG AAC ACÁ 432 Asn Leu Thr Glu Arg Val Arg Glu Ser Leu Arg He Trp Lys Asn Thr 130 135 140 GAG AAG GAG AAC GCA ACA GTG GCC CAC CTG GTG GGG GCT CTC AGG TCC 480 Glu Lys Glu Asn Wing Thr Val Wing His Leu Val Gly Wing Leu Arg Ser 145 150 155 160 TGC CAG ATG AAC CTG GTG GCT GAC CTG GTA CA GGG GTT CAG CAG GCC 528 Cys Gln Met Asn Leu Val Wing Asp Leu Val Gln Gly Val Gln Gln Wing 165 170 175 CGT GAC CTC CAG AAC AGG AGT GGG GCC ATG TCC CCG ATG TCA TGG AAC 576 Arg Asp Leu Gln Asn Arg Ser Gly Wing Met Ser Pro Met Ser Trp Asn 180 185 190 TCA GAC GCA TCT ACC TCC GAA GCG TCC TGA 606 Ser Asp Wing Ser Thr Ser Glu Wing Ser 195 200 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 202 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein. { xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Met Asp Pro Phe Leu Val Leu Leu His Ser Val Ser Ser Ser Leu Ser 1 5 10 15 Be Ser Glu Leu Thr Glu Leu Lys Phe Leu Cys Leu Gly Arg Val Gly 20 25 30 Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Leu Asp Leu Phe Ser Met 35 40 45 Leu Leu Glu Gln Asn Asp Leu Glu Pro Glu Leu Leu Ala Ser Leu Arg 50 55 60 Arg His Asp Leu Leu Arg Arg Val Asp Asp Phe Glu Ala Gly Ala Wing 65 70 75 80 Ala Gly Ala Ala Pro Gly Glu Glu Asp Leu Cys Ala Ala Phe Asn Val 85 90 95 He Cys Asp Asn Val Gly Lys Asp Trp Arg Arg Leu Wing Arg Gln Leu 100 105 110 Lys Val Ser Asp Thr Lys He Asp Ser He Glu Asp Arg Tyr Pro Arg 115 120 125 Asn Leu Thr Glu Arg Val Arg Glu Ser Leu Arg He Trp Lys Asn Thr 130 135 140 Glu Lys Glu Asn Wing Thr Val Wing His Leu Val Gly Wing Leu Arg Ser 145 150 155 160 Cys Gln Met Asn Leu Val Wing Asp Leu Val Gln Gly Val Gln Gln Wing 165 170 175 Arg Asp Leu Gln Asn Arg Ser Gly Wing Met Ser Pro Met Ser Trp Asn 180 185 190 Ser Asp Wing Ser Thr Ser Glu Wing Ser 195 200 (2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE : (A) LENGTH: 606 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) Linear TOPOLOGY (Ü) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY: CDS ( B) LOCATION: 1.606 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: ATG GAC CCG TTC CTG GTG CTG CTG CAC TCG GTG TCG TCC AGC CTG TCG 48 Met Asp Pro Phe Leu Val Leu Leu His Ser Val Ser Ser Ser Leu Ser 1 5 10 15 AGC AGC GAG CTG ACC GAG CTC AAG TTC CTA TGC CTC GGG CGC GTG GGC 96 Ser Ser Glu Leu Thr Glu Leu Lys Phe Leu Cys Leu Gly Arg Val Gly 20 25 30 AAG CGC AAG CTG GAG CGC GTG CAG AGC GGC CTA GAC CTC TTC TCC ATG 144 Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Leu Asp Leu Phe Ser Met 35 40 45 CTG CTG GAG CAG AAC GAC CTG GAG CCC GAG CTG CTC GCC TCC CTG CGG 192 Leu Leu Glu Gln Asn Asp Leu Glu Pro Glu Leu Leu Wing Ser Leu Arg 50 55 60 CGC CAC GAC CTG CTG CGG CGC GTC GAC GAC TTC GAG GCG GGG GCG GCA 240 Arg His Asp Leu Leu Arg Arg Val Asp Asp Phe Glu Ala Gly Ala Wing 65 70 75 80 GCC GGG GCC GCG CCT GGG GAA GAA GAC CTG TGT GCA GVT TTT AAC GTC 288 Wing Gly Wing Wing Pro Gly Glu Glu Asp Leu Cys Wing Wing Phe Asn Val 85 90 95 ATA TGT GAT AAT GTG GGG AAA GAT TGG AGA AGG CTG GCT CGT CAG CTC 336 He Cys Asp Asn Val Gly Lys Asp Trp Arg Arg Leu Wing Arg Gln Leu 100 105 110 AAA GTC TCA GAC ACC AAG ATC GAC AGC ATC GAG GAC AGA TAC CCC CGC 384 Lys Val Ser Asp Thr Lys He Asp Ser He Glu Asp Arg Tyr Pro Arg 115 120 125 AAC CTG ACÁ GAG CGT GTG CGG GAG TCA CTG AGA ATC TGG AAG AAC ACÁ 432 Asn Leu Thr Glu Arg Val Arg Glu Ser Leu Arg He Trp Lys Asn Thr 130 135 140 GAG AAG GAG AAC GCA ACÁ GTG GCC CAC CTG GTG GGG GCT CTC AGG TCC 480 Glu Lys Glu Asn Wing Thr Val Wing His Leu Val Gly Wing Leu Arg Ser 145 150 155 160 TGC CAG ATG AAC CTG GCG GCT GAC CTG GTA CA GAG GTT CAG CAG GCC 528 Cys Gln Met Asn Leu Ala Ala Asp Leu Val Gln Glu Val Gln Gln Wing 165 170 175 CGT GAC CTC CAG AAC AGG AGT GGG GCC ATG TCC CCG ATG TCA TGG AAC 576 Arg Asp Leu Gln Asn Arg Ser Gly Wing Met Ser Pro Met Ser Trp Asn 180 185 190 TCA GAC GCA TCT ACC TCC GAA GCG TCC TGA 606 Ser Asp Ala Ser Thr Ser Glu Ala Ser 195 200 (2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 4; (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 202 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (i) DESCRIPTION OF SEQUENCE: SEQ ID NO: 4: Met Asp Pro Phe Leu Val Leu Leu His Ser Val Ser Ser Ser Leu Ser 1 5 10 15 Be Ser Glu Leu Thr Glu Leu Lys Phe Leu Cys Leu Gly Arg Val Gly 20 25 30 Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Leu Asp Leu Phe Ser Met 35 40 45 Leu Leu Glu Gln Asn Asp Leu Glu Pro Glu Leu Leu Ala Ser Leu Arg 50 55 60 Arg His Asp Leu Leu Arg Arg Val Asp Asp Phe Glu Ala Gly Ala Wing 65 70 75 80 Ala Gly Ala Ala Pro Gly Glu Glu Asp Leu Cys Ala Ala Phe Asn Val 85 90 95 He Cys Asp Asn Val Gly Lys Asp Trp Arg Arg Leu Wing Arg Gln Leu 100 105 110 Lys Val Ser Asp Thr Lys He Asp Ser He Glu Asp Arg Tyr Pro Arg 115 120 125 Asn Leu Thr Glu Arg Val Arg Glu Ser Leu Arg He Trp Lys Asn Thr 130 135 140 Glu Lys Glu Asn Wing Thr Val Wing His Leu Val Gly Wing Leu Arg Ser 145 150 155 160 Cys Gln Met Asn Leu Wing Wing Asp Leu Val Gln Glu Val Gln Gln Wing 165 170 175 Arg Asp Leu Gln Asn Arg Ser Gly Wing Met Ser Pro Met Ser Trp Asn 180 185 190 Ser Asp Wing Ser Thr Ser Glu Wing Ser 195 200 (2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 627 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MMOLECULATION: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 1.667 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: ATG GAC CCG TTC CTG GTG CTG CTG CAC TCG GTG TCG TCC AGC CTG TCG 48 Met Asp Pro Phe Leu Val Leu Leu His Ser Val Ser Ser Ser Leu Ser 1 5 10 15 AGC AGC GAG CTG ACC GAG CTC AAG TTC CTA TGC CTC GGG CGC GTG GGC 96 Ser Ser Glu Leu Thr Glu Leu Lys Phe Leu Cys Leu Gly Arg Val Gly 20 25 30 AAG CGC AAG CTG GAG CGC GTG CAG AGC GGC CTA GAC CTC TTC TCC ATG 144 Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Leu Asp Leu Phe Ser Met 35 40 45 CTG CTG GAG CAG AAC GAC CTG GAG CCC GAG ACC ACC GAG CTC CTG CGC 192 Leu Leu Glu Gln Asn Asp Leu Glu Pro Glu His Thr Glu Leu Leu Arg 50 55 60 GAG CTG CTC GCC TCC CTG CGG CGC CAC GAC CTG CTG CGG CGC GTC GAC 240 Glu Leu Leu A the Ser Leu Arg Arg His Asp Leu Leu Arg Arg Val Asp 65 70 75 80 GAC TTC GAG GCG GGG GCG GCA GCC GGG GCC GCG CCT GGG GAA GAA GAC 288 Asp Phe Glu Wing Gly Wing Wing Wing Gly Wing Wing Pro Gly Glu Glu Asp 85 90 95 CTG TGT GCA GCA TTT AAC GTC ATA TGT GAT AAT GTG GGG AAA GAT TGG 336 Leu Cys Ala Ala Phe Asn val He Cys Asp Asn Val Gly Lys Asp Trp 100 105 110 AGA AGG CTG GCT CGT CAG CTC AAA GTC TCA GAC ACC AAG ATC GAC AGC 384 Arg Arg Leu Wing Arg Gln Leu Lys Val Ser Asp Thr Lys He Asp Ser 115 120 125 ATC GAG GAC AGA TAC CCC CGC AAC CTG ACA GAG CGT GTG CGG GAG TCA 432 He Glu Asp Arg Tyr Pro Arg Asn Leu Thr Glu Arg Val Arg Glu Ser 130 135 140 CTG AGA ATC TGG AAG AAC ACA GAG AAG GAG AAC GCA AC GTG GCC CAC 480 Leu Arg He Trp Lys Asn Thr Glu Lys Glu Asn Wing Thr Val Wing His 145 150 155 160 CTG GTG GGG GCT CTC AGG TCC TGC CAG ATG AAC CTG GCG GCT GAC CTG 528 Leu Val Gly Wing Leu Arg Ser Cys Gln Met Asn Leu Wing Wing Asp Leu 165 170 175 GTA CA GAG GTT CAG CAC GCC CGT GAC CTC CAG AAC AGG AGT GGG G CC 576 Val Gln Glu Val Gln Gln Wing Arg Asp Leu Gln Asn Arg Ser Gly Wing 180 185 190 ATG TCC CCG ATG TCA TGG AAC TCA GAC GCA TCT ACC TCC GAA GCG TCC 624 Met Ser Pro Met Ser Trp Asn Ser Asp Wing Ser Thr Ser Glu Ala Ser 195 200 205 TGA 627 (2) INFORMATION FOR THE SEQUENCE SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 209 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: Met Asp Pro Phe Leu Val Leu Leu His Ser Val Ser Ser Ser Leu Ser 1 5 10 15 Ser Ser Glu Leu Thr Glu Leu Lys Phe Leu Cys Leu Gly Arg Val Gly 20 25 30 Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Leu Asp Leu Phe Ser Met 35 40 45 Leu Leu Glu Gln Asn Asp Leu Glu Pro Glu His Thr Glu Leu Leu Arg 50 55 60 Glu Leu Leu Wing Ser Leu Arg Arg His Asp Leu Leu Arg Arg Val Asp 65 70 75 80 Asp Phe Glu Wing Gly Wing Wing Wing Gly Wing Wing Pro Giv Glu Glu Asp 85 90 95 Leu Cys Wing Wing Phe Asn Val He Cys Asp Asn Val Gly Lys Asp Trp 100 105 110 Arg Arg Leu Wing Arg Gln Leu Lys Val Ser Asp Thr Lys He Asp Ser 115 120 125 He Glu Asp Arg Tyr Pro Arg Asn Leu Thr Glu Arg Val Arg Glu Ser 130 135 140 Leu Arg He Trp Lys Asn Thr Glu Lys Glu Asn Wing Thr Val Wing His 145 150 155 160 Leu Val Gly Ala Leu Arg Ser Cys Gln Met Asn Leu Wing Wing Asp Leu 165 170 175 Val Gln Glu Val Gln Gln Wing Arg Asp Leu Gln Asn Arg Ser Gly Wing 180 185 190 Met Ser Pro Met Ser Trp Asn Ser Asp Wing Ser Thr Ser Glu Wing Ser 195 200 205

Claims (7)

1. Novelty of the Invention 1. A composition comprising an isolated polynucleotide from NTERA2 cells selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of MORT1? 21 set forth in SEQ. ID NO. 1, filed under accession number ATCC 209013; (b) a polynucleotide comprising the nucleotide sequence encoding a protein that interacts with the death domain of Fas / AP01 or fragments thereof; (c) a polynucleotide that codes for a protein that interacts with
2. MACHal or other members of the ICE / Ced3 (Caspasa) family of proteins or fragments thereof; (d) a polynucleotide that is an allelic variant of the polynucleotide of (a) - (c) above; and (e) a polynucleotide capable of hybridizing under astringent conditions to any one of the polynucleotides specified in (a) - (d). 2. A composition comprising an isolated polynucleotide from a human brain selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence set forth in SEQ. ID NO. 3, filed under accession number ATCC 209018; (b) a polynucleotide that encodes a protein that interacts with the death domain of Fas / AP01 or fragments thereof; (c) a polynucleotide that encodes a protein that interacts with MACHal or other members of the 1CE / Ced3 (Caspase) family of proteins or fragments thereof; (d) a polynucleotide that is an allelic variant of the polynucleotide of (a) - (c) above; and (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a) - (d).
3. 3. A composition comprising an isolated polynucleotide from a human brain selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence set forth in SEQ. ID NO. 5, filed under accession number ATCC 209019; (b) a polynucleotide encoding a prolein that interacts with the death domain of Fas / AP01 or fragments thereof; (c) a polynucleotide that encodes a protein that interacts with MACHal or other members of the ICE / Ced3 (Caspasa) family of proteins or fragments thereof; (d) a polynucleotide that is an allelic variant of the polynucleotide of (a) - (c) above; and (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a) - (d).
4. 4. An isolated MORT1 nucleic acid isoform, wherein the isoform is a human NTERA2 neuronal cell isoform or human brain coding for a protein comprising the amino acid sequences selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO.
5. 5. An isolated MORT1 nucleic acid isoform comprising the amino acid sequence of SEQ ID NO. 2.
6. 6. An isolated MORT1 nucleic acid isoform comprising the amino acid sequence of SEQ ID NO. 4. An isolated MORT1 nucleic acid isoform comprising the amino acid sequence of SEQ ID NO. 6