KR20010033299A - SAG: Sensitive to Apoptosis Gene - Google Patents

Abstract

The present invention provides novel genes and polypeptides derived from redox-sensitive proteins that can be used to promote cell growth, protect cells from apoptosis, remove oxygen radicals, and restore tumor traits. To identify key gene (s) of apoptosis induced by 1,10-phenanthroline (OP) in tumor cells, genes induced by OP (SAG) using differential expression techniques (SAG) , Sensitive to Apoptosis Gene) was cloned. SAG encodes a novel redox-sensitive, heme-binding protein with zinc ring finger domains. SAG protein has a calculated molecular weight of 12.7 kDa and consists of 113 amino acids. Sequence homology analysis revealed that SAG is highly conserved between species, suggesting its functional significance. This proved that yeast with SAG collapsed would die. Two mutants have been found in human cancer cell lines of colon and testicular origin, indicating that they may play a role in human carcinogenesis. Overexpression of the SAG protein in human colon carcinoma cell line DLD1 and human neuroblastoma cell line SY5Y protected cells from apoptosis induced by OP, zinc and copper ions. In addition, transfection of antisense SAG inhibited certain tumor cells in human DLD1 cell lines, and microinjection of SAG RNA stimulated cell growth. Thus, we propose that SAG protein is a cell protective protein that stimulates the growth of cells as a growth factor as well as the function of buffering damage caused by oxidation-stress as a redox detector. SAG protein will be an ideal molecular target for the development of drugs for the promotion of degenerative neuropathy, cancer, muscle malnutrition and wound healing.

Description

SAO: Apoptosis sensitive gene {SAG: Sensitive to Apoptosis Gene}

Apoptosis, also known as planned cell death, is a genetically planned process for maintaining homeostasis in physiological conditions and responding to various stimuli (Thompson (1995) Science 267, 1456-1462). This type of cell death is characterized by cell membrane saturation, cytoplasmic contraction, condensation of chromatin in the nucleus and fragmentation of DNA (Wyllie et al. (1995) Int. Rev. Cytol. 68, 251-306). . Apoptosis processes include initiation, stimulation of effector molecules and DNA degradation (Kroemer et al. (1995) FASEB J. 9, 1277-1287) and [Vaux and Strasser (1996). Proc. Acad. Sci. USA 93, 2239-2244). Apoptosis is initiated by various physical, chemical, and biological stimuli (internal and external) in various types of cells, which include various cancer therapeutics, oxidative DNA damaging agents, and cytokines (Kroemer (1997)). Nature Med. 3, 614-620, White (1996) Genes Dev. 10, 1-15, Dive and Hickman (1991) Br. J. Cancer 64, 192 -196 and Yuan et al. (1993) Cell 75, 641-652). Such initiators promote the induction of apoptosis signal transduction and amplification of effector molecules in cells, resulting in irreversible DNA degradation and cell death.

Many genes are involved in the apoptosis process. Typically, the products of these genes are classified as inducers or inhibitors of apoptosis. Whether a cell passes through the apoptosis pathway or not is determined by the activity balance between the inducer and suppressor of apoptosis in that cell. Among the genes that regulate apoptosis, the best known are p53 tumor suppressor gene, Bcl-2 gene family (consisting of both apoptosis inducer and suppressor), interleukin 1β converting enzyme (ICE) gene family and FAS / Fas ligands (Kroemer (1997), White (1996), Yuan et al. (1993) and Nagata and Golstein (1995) Science 267, 1449- 1456]). During the process of apoptosis, there is substantial interaction between products of the apoptosis regulatory genes, which includes the formation of hetero-dimer between Bcl-2 gene family products, p53 activation by expression of Bax (Olt Olvai et al. (1993) Cell 74, 609-619 and Miyashita and Reed (1995) Cell 80, 293-299).

The inventors of the present invention recently discovered that 1,10 phenanthroline ("OP"), a metal chelating agent, can activate p53 and induce apoptosis in tumor cells of two mice with wild type p53 therein. (Sun et al. (1997) Oncogene 14, 385-393). OP is a common chelating agent that chelates Fe (II) to interfere with the formation of Fe (II) -mediated hydroxyl radicals via the Fenton reaction (Halliwell et al. (1989), Free Radicals in Biology and Medicine, 2nd Edition, Clarendon Press, Oxford, and Auld (1988), Methods in Enzymology, Vol. 158 (Editor: JF Riordan and B. L. Bell. (BL Valle) PP. 110-114, Academic Press, New York]). OP is known to interfere with DNA damage by hydroxyl radicals in a myriad of cellular systems (Sun, Y. Free Radic. Biol. Med. 8: 583-599 (1990)), Martins ( Martins) and Menegini, Biochem J. 299: 137-140 (1994) and Morgan et al., Biochem. Pharmacol. 44: 215-221 (1992). Although not a condition required for subsequent apoptosis apoptosis, activation of p53 by OP is known to contribute significantly to apoptosis apoptosis (Sun et al. (1997) Oncogene 14: 385-). 393 and Sun (1997) FEBS Lett. 408, 16-20). Thus, the critical genes associated with apoptosis induced by OP and its gene products remain a challenge to be characterized. A more detailed understanding of the molecular mechanisms for the induction of apoptosis requires the design of improved therapeutics to induce (anticancer) or inhibit (anti-aging) apoptosis.

<Summary of invention>

The present invention provides novel genes and polypeptides derived therefrom that encode redox-sensitive proteins that can be used to protect cells from apoptosis, remove oxygen radicals, and restore tumor traits.

In one aspect, the invention provides an isolated and purified novel DNA sequence described in SEQ ID NO: 1 and SEQ ID NO: 3 referred to herein as "rat SAG" and "human SAG" and 1,10-phenanthroline (" OP ") to provide their gene products (referred to herein as" murine SAG proteins "and" human SAG proteins ") as described in SEQ ID NO: 2 and SEQ ID NO: 4. In other embodiments, the nucleotide sequences hybridize with the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 3 even under stringent hybridization conditions. In a more preferred embodiment, the isolated and purified DNA sequence consists essentially of the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

In another aspect, the invention provides a recombinant DNA molecule comprising SAG subcloned into an extrachromosomal vector. In a further aspect, the present invention provides host cells stably transformed with recombinant DNA molecules subcloned into an extrachromosomal vector.

In another aspect, the present invention provides a substantially purified recombinant protein comprising a polypeptide substantially similar to the SAG protein of SEQ ID NO: 2 and SEQ ID NO: 4. In a further aspect, the present invention provides a polyclonal antibody that selectively binds to a protein having an amino acid sequence substantially similar to the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4.

In a further aspect, the present invention provides a method for detecting SAG protein in a cell, including contacting the cell with a polyclonal antibody that recognizes the SAG protein, isolating whole genomic DNA and using primers derived from SEQ ID NOS: 1 and 3 A method of detecting a cell comprising a deletion of SAG, including amplifying the genomic DNA by PCR, and separating the entire RNA of the cell to reverse transcription-PCR of the RNA using primers derived from SEQ ID NO: 1 and SEQ ID NO: 3 Provided are methods for detecting cells comprising a deletion of SAG, including amplifying.

In another aspect, the present invention provides RNA comprising an extension of a polyA, polyC or polyU residue in a cell by contacting the entire RNA of the cell with a SAG protein and reacting the RNA-SAG protein mixture with an antibody that recognizes the SAG protein. Provides a further isolation method.

In another aspect of the invention, apoptosis comprising treating cells with OP, performing differential expression of RNA induced by the OP, and then cloning the gene induced by the OP It provides a method for isolation of genes induced in the liver.

In a further aspect of the invention, a DNA sequence substantially similar to the DNA sequences of SEQ ID NO: 1 and SEQ ID NO: 3 (Isolated and purified DNA of SEQ ID NO: 1 and SEQ ID NO: 3 is operably linked to the DNA sequence which proceeds with the expression of the DNA sequence). Mammalian from apoptosis induced by a redox agent, comprising introducing into an mammalian and / or non-mammalian cell an expression vector comprising a sequence of which is expressed at high levels in mammalian and non-mammalian cells). Provided are methods for protecting animal and / or non-mammalian cells.

In an additional aspect of the invention, a DNA sequence substantially similar to the DNA sequences of SEQ ID NO: 1 and SEQ ID NO: 3 (Isolated and purified sequence 1 and operably linked to a DNA sequence that proceeds the expression of the antisense chain of the DNA sequence) Mammalian comprising introducing into a tumor cell of a mammal and / or a non-mammalian an expression vector comprising the DNA sequence of the antisense chain for SEQ ID NO: 3 is expressed at high levels in mammalian and non-mammalian cells). Methods of treating tumor cells in animals and non-mammals are provided.

In another aspect of the present invention, there is provided a method of removing oxygen radicals in an organism, wherein the pharmaceutical composition comprising the SAG protein and a pharmaceutically acceptable carrier comprises controlling the amount of reduction of oxygen radicals.

In a further aspect of the present invention, the application of gene therapy of SAG is provided, not limited to methods that promote wound closure (ie, treatment) in a patient.

The foregoing is not intended to limit the invention in any way and should not be so interpreted. All patents and documents cited herein are hereby incorporated by reference in their entirety.

The present invention relates to novel genes and polypeptides thereof that encode redox-sensitive proteins that protect cells from apoptosis and promote cell growth, as well as antibodies to such polypeptides. The invention also uses the novel genes, polypeptides and antibodies to determine the deletion of the gene, to locate the polypeptide in the cytoplasm, to isolate discrete RNA species, to inhibit apoptosis, to remove oxygen radicals, to restore tumor traits, and Therapeutic methods by gene therapy are described.

1A shows the expected structural properties of the deduced protein sequences of murine and human SAG cDNAs.

1B is a description of human SAG protein mutations.

2 is a bar graph showing the soft agar colony growth of various cell lines stably transfected with SAG.

3 is a graph showing the mass of tumors on a daily basis after transplanting SAG transfectants into mice with SCID.

The present invention provides novel genes and polypeptides derived from the genes that protect the cells from apoptosis, remove oxygen radicals, and encode redox-sensitive proteins that can be used to restore tumor traits. The present invention also encompasses genes involved in OP-induced apoptosis and gene products thereof. Isolation of these genes and their gene products allows for detailed analysis of the OP-induced apoptosis pathway, thus providing a useful experimental tool to confirm the mechanism of OP-induced apoptosis and an improved control of apoptosis. Therapies can be devised.

Unless otherwise stated in such applications, the techniques used are Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, D. Goeddel, 1991. Academic Press, San Diego, Calif.], "Guide to Protein Purification" by Methods in Enzymology (published by MPDeutshcer, (1990) Academic Press, Inc.), [PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990, Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2nd Edition (RI Freshney, 1987, Siss, Inc. New York State). New York City) and Gene Transfer and Expression Protocols, pp. 109-128, E.J. Several well-known references, such as Murray Publishing, The Humana Press Inc., and Clifton, NJ.

In one aspect, the present invention provides isolated and purified novel DNA sequences encoding SAG proteins, hereinafter referred to as apoptosis sensitive genes ("SAG"). In one embodiment, the present invention comprises DNA sequences that are substantially similar to the sequences set forth in SEQ ID NO: 1 (rat SAG) or SEQ ID NO: 2 (human SAG), respectively. As used herein, “substantially similar” includes not only identical sequences, but also sequences deleted, substituted or added to DNA, RNA or protein sequences that maintain the function of the protein and have similar zinc-binding motifs. Preferably, the DNA sequence according to the present invention consists of the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 49. The purified and isolated novel DNA sequences can be used directly for expression of SAG proteins and mutation analysis of SAG protein function.

Mutant sequences according to the present invention can be identified in a customary manner by those skilled in the art using the methods described in Example 8 below and techniques known in the art.

In other embodiments, the invention includes nucleotide sequences that hybridize to SEQ ID NO: 1 and / or SEQ ID NO: 3 under highly stringent hybridization conditions. The term “highly stringent hybridization conditions” as used herein refers to 1% SDS, 20 mM after hybridization at 65 ° C. for 8 to 24 hours in a low salt concentration hybridization buffer using a 2 × 10 ⁸ cpm / μg probe. Washing at 65 ° C. for 30 minutes to 4 hours with phosphate buffer and 1 mM EDTA. In a preferred embodiment, the low salt concentration hybridization buffer comprises 0.5-10% SDS and 0.05M-0.5M sodium phosphate. In the most preferred embodiment, the low salt concentration hybridization buffer comprises 7% SDS and 0.125M sodium phosphate. The DNA can be used for direct expression of SAG protein and mutation analysis of SAG protein function and is isolated by hybridization described herein.

In another aspect, the present invention provides a novel recombinant DNA molecule comprising a SAG or substantially similar sequence subcloned into an extrachromosomal vector. This aspect of the present invention enables the ex vivo expression of the SAG gene, and thus allows the regulation of the SAG gene and the structural and functional analysis of the SAG protein. The term "extrachromosomal vector" as used herein includes, but is not limited to, plasmids, bacteriophages, cosmids, retroviruses, and artificial chromosomes. In a preferred embodiment, the extrachromosomal vector comprises an expression vector that allows the recombinant DNA molecule to produce SAG protein when introduced into a host cell. Such vectors are well known in the art and include, but are not limited to, T3 or T7 polymerase promoters, SV40 promoters, CMV promoters, or any promoter that intends to test the ability to express a gene directly, or to directly express a gene. It doesn't work. Such recombinant vectors can be made via standard recombinant DNA methods described in the references cited above. This aspect of the invention allows for the expression of high levels of SAG protein.

In a further aspect, the present invention provides a recombinant host cell stably transfected with a recombinant DNA molecule comprising SAG subcloned into an extrachromosomal vector. Host cells of the invention include non-eukaryotic cells (eg bacteria) and bacteria (eg yeast), plant cells, non-human animal cells, non-human mammalian cells (eg rabbits, pigs, Can be of any type, including, but not limited to, eukaryotic cells such as rats, horses) and human cells. Methods for transfecting host cells with recombinant DNA molecules are well known in the art (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, 1989), Calcium phosphate transfection, dextran sulfate transfection, electroporation, lipofectin and viral infections as used herein, include, but are not limited to. This aspect of the invention allows for the expression of SAG in vivo and ex vivo, and as such the SAG protein is expressed at high levels as described in Example 6 below.

In another aspect, the present invention provides a substantially purified recombinant protein comprising a polypeptide substantially similar to the SAG polypeptide shown in SEQ ID NO: 2 and SEQ ID NO: 4. In addition, this aspect of the invention allows the use of SAG proteins in several in vitro assays described below. The term "substantially similar" as used herein includes the deletions, substitutions, and additions of SEQ ID NO: 1 to SEQ ID NO: 4 (suitable sequences) introduced by ex vivo means. As used herein, the term "substantially purified" means a protein in a state in which no contaminated protein is detected, but the SAG protein may be purified with the interacting protein or as an oligomer. Preferably, the protein sequence according to the present invention is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48 and SEQ ID NO: 50 comprising an amino acid sequence selected from the group consisting of. In the most preferred embodiment, the protein sequence according to the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4. Mutated sequences according to the present invention can be identified in a customary manner by one skilled in the art using the methods provided herein and techniques well known in the art. This aspect of the invention provides a purified novel protein that can be used for in vitro assays as described in Example 12 below, and can be used as a component of the pharmaceutical composition for oxygen radical removal described below.

In a further aspect, the present invention provides antibodies and methods for detecting the antibodies that selectively bind to polypeptides substantially similar to the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4. Antibodies of the invention can be prepared in accordance with standard techniques (Harlow and Lane (1988), eds. Antibody: A Laboratory Manual, Cold Spring Harbor Press) to elicit an immune response in a host animal. It may be a polyclonal antibody or monoclonal antibody prepared using the full or partial sequence of SEQ ID NO: 4 or using a modified portion thereof. In a preferred embodiment, the entire polypeptide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 is used to produce polyclonal antibodies in the host animal.

Methods of detecting SAG antibodies include contacting the cells with an antibody that recognizes the SAG protein and culturing the cells in a manner capable of detecting the SAG protein-antibody complex. To carry out this aspect of the invention, standard conditions of antibody detection against antigens (Harlow and Lane, 1988) can be used. This aspect of the invention allows for the detection of SAG proteins in vitro and in vivo as described in Examples 12 and 14 below.

In a further aspect, the present invention isolates genomic DNA from cells and amplifies the genomic DNA by PCR (SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, Sequence). 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and the use of primers derived from SEQ ID NO: 49). Provide the method.

This aspect of the invention allows detection of SAG deletion in any type of cell, which can be used as a genetic test or laboratory tool. PCR primers can be selected in any manner that allows amplification of SAG gene fragments that are large enough to be detectable by gel electrophoresis. To any method, including but not limited to brominated ethidium staining of agarose or polyacrylamide gels, autoradiographic detection of radiolabeled SAG gene fragments, Southern blot hybridization and DNA sequencing Detection is possible. In a preferred embodiment, polyacrylamide gel electrophoresis followed by DNA sequence analysis demonstrates the identity of the deletion. PCR conditions are routinely determined based on the length and base content of the selected primers according to techniques well known in the art (Sambrook et al., Supra).

An additional aspect of the present invention is to isolate total RNA of a cell and amplify the RNA by reverse transcription-PCR (SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 49). Provide diagnostic assays. This aspect of the invention allows detection of SAG deletion in any type of cell, which can be used as a genetic test or laboratory tool.

Reverse transcription is routinely performed via standard techniques (published by Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., 1994), and PCR is performed as described above.

In another aspect, the invention provides an antibody-SAG protein-RNA complex isolated from an antibody-SAG protein complex after contacting the RNA sample with the SAG protein and incubating the RNA-SAG protein mixture with the SAG polypeptide-cognitive antibody. Provided is a method for isolating RNA comprising a polyA (adenine), polyC (cytosine) or polyU (freedine) extension, comprising the step of purifying. This aspect of the invention provides a novel method for isolation of discrete RNA species in vitro. In a preferred embodiment, the RNA sample is contacted with the SAG protein in the presence (selective isolation of RNA comprising polyA and polyC) or absence (selective isolation of RNA comprising polyU) of a reducing agent. Preferred reducing agents for use in this aspect of the invention include, but are not limited to, DTT and β-mercaptoethanol. The reducing agent is preferably used at a concentration between about 50 mM and 1M. Isolation of the antibody-SAG protein-RNA complex can be accomplished through standard techniques, including but not limited to methods using agarose or cellulose beads conjugated with protein-A.

In a further aspect of the present invention, after treating experimental cells with OP and not control cells with OP and isolating RNA from each cell group, the RNA is reverse transcribed and PCR (“differential display”). By identifying cDNA expressed in the cell group treated with OP but not expressed in the control group, cloning the cDNA induced by OP, it provides a method for isolating genes derived from cells undergoing apoptosis. . This aspect of the invention provides a tool for isolating other genes that control the OP-induced apoptosis pathway, and is useful for both designing therapeutics to control apoptosis and laboratory tools for identifying the OP-induced apoptosis pathway. Do. Details of differential expression assays, including the selection of primers, are well known in the art (Liang and Pardee, Science 257: 967-971, 1992). Reverse transcription and PCR conditions are customarily determined based on the length and base content of the chosen primer according to well known techniques (Sambrook et al., Supra). In a preferred embodiment, the OP is used at a concentration between 50 μM and 300 μM. In the most preferred embodiment, the OP is used at a concentration between 100 μM and 150 μM.

A further aspect of the present invention provides a method for a mammalian and / or non-mammalian cell comprising a DNA sequence substantially similar to the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3 (operably linked to a DNA sequence that facilitates expression of the DNA sequence). Apoptosis induced by redox agents, comprising introducing an expression vector and culturing the cells under conditions where the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3 is expressed at high levels in said mammalian and / or non-mammalian cells A method of protecting said mammalian and / or non-mammalian cells is provided. In a preferred embodiment, the DNA sequence consists of SEQ ID NO: 1 or SEQ ID NO: 3. Suitable expression vectors are described above. In a preferred embodiment, the coding region of the human SAG gene is subcloned into an expression vector in which the SAG gene is constitutively expressed under the transcriptional control of the cytomegalovirus (CMV) promoter.

An additional aspect of the invention involves introducing into a mammalian and / or non-mammalian tumor cell an expression vector comprising antisense DNA for a sequence substantially similar to the DNA sequence set forth in SEQ ID NO: 1 or SEQ ID NO: Operably linked to a DNA sequence that promotes expression of the sequence), thereby providing a method of inhibiting growth of said mammalian and / or non-mammalian tumor cells. The cells are then cultured under conditions in which the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3 is expressed at high levels in mammalian and / or non-mammalian cells. In a preferred embodiment, the DNA sequence is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39 , SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 49.

In the most preferred embodiment, the DNA sequence consists of SEQ ID NO: 1 or SEQ ID NO: 3. In a further preferred embodiment, the expression vector comprises an adenovirus vector in which the SAG cDNA is operably linked to the cytomegalovirus (CMV) promoter in the antisense direction, thereby constitutively expressing the antisense cDNA of SAG in the host cell. do. In a preferred embodiment, the vector is introduced into mammalian tumor cells by injecting adenovirus expression vectors of SAG into the mammalian tumor cell mass.

A further aspect of the invention includes a DNA sequence substantially similar to the DNA sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 in mammalian and / or non-mammalian cells (operably linked to a DNA sequence that facilitates expression of said DNA sequence). A method of removing oxygen radicals in an organism comprising introducing an expression vector to the cell and culturing the cell under conditions where the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3 is expressed at high levels in said mammalian and / or non-mammalian cells. To provide. In a preferred embodiment, the DNA sequence consists of SEQ ID NO: 1 or SEQ ID NO: 3. In a preferred embodiment, the SAG cDNA is operably linked with a cytomegalovirus (CMV) promoter such that the SAG cDNA is constitutively expressed in a host cell.

Another aspect of the invention provides a pharmaceutical composition, comprising administering a pharmaceutical composition comprising a SAG protein of SEQ ID NO: 2 or SEQ ID NO: 4 and a pharmaceutically acceptable carrier in an amount of reduced oxygen Also provides.

Chimeric gene constructs (e.g., expression vectors) of the invention comprising SAG polypeptides express expression of SAG or antisense SAG polynucleotide sequences in selected target cells, including non-eukaryotic cells (ie, plant cells) and eukaryotic cells. Can be used for gene therapy. Typically, application to gene therapy involves finding a target host cell or tissue in need of treatment, designing a vector construct suitable for expression of the desired gene product in said cell, and inducing efficient transduction in the target cell. In connection with providing such a construct.

Typically, the target cells or tissues of gene therapy are affected by the disease to which the designed vector construct is to be treated. For example, in the case of cancer the target tissue is a malignant tumor.

In one embodiment, the present invention provides a method of promoting closure (ie, treatment) of a patient wound. The method provides an exogenous SAG to the wound site such that the product of the SAG promotes closure (ie, treatment) of the wound at the wound site.

The present invention promotes suture (ie, treatment) of both trauma (eg, surface) and internal wounds. Wounds useful in the methods of the present invention to promote suture (ie, treatment) include abrasions, avulsion fractures, open pneumothorax, burns, bruises, wounds, incisions, open windows, penetrating windows, perforated windows, magnetic windows, charity windows, cuts, Surgical wounds, subcutaneous windows, tangential windows, or traumatic dyspnea. Preferably, the method of the present invention is used for the treatment of chronic open windows, such as poorly treated external ulcers.

Exogenous SAG can be introduced to the wound site by any suitable method, such as the methods described herein. For example, in the case of a surface wound, SAG can be supplied externally by topical administration of the SAG protein to the wound site.

Preferably, the exogenous SAG delivers the vector comprising the SAG expression cassette to a cell associated with the wound and provides it to the wound site. In the case of SAG expression in wound site cells, the product of SAG is made to promote closure (ie treatment) of the wound. Delivering a vector containing a SAG expression cassette to cells associated with the wound minimally invades the cells, locally supplies the SAG product only to the wound site, and requires reapplication of slaves, solutions or other external media. It is preferable because it does not. In addition, the activity of SAG will remain during the treatment of the wound and will be inactivated after treatment.

A vector comprising a SAG expression cassette can be delivered to a cell associated with a wound in any suitable way to deliver a particular vector type to a cell, as described herein.

As discussed above, the cell associated with the wound to which the vector is delivered is any cell that is sufficiently associated with the wound, and the expression of SAG in the cell (cell inside the wound or cell derived elsewhere) may result in the closure of the wound ( That is, to promote treatment). In one embodiment, said cell is a cell at a wound site and the method of the invention comprises delivering a vector to a cell in situ.

In other embodiments, the cells may not be cells at the wound site, but cells of exogenous tissue, such as transplanted tissue, or cells ex vivo. For example, to facilitate the treatment of certain types of wounds, the cells associated with the wound may be transplanted tissues such as skin graft tissue. Thus, delivering the vector to cells associated with the wound involves delivering the vector to exogenous ex vivo tissue cells. For other wounds, the cells associated with the wound may be ex vivo cells, and the cells are transplanted to the wound site after delivering a vector comprising a SAG expression cassette to the cells.

The method of the present invention is used in any patient with a wound. Preferably, the patient is a human.

In another embodiment, the present invention provides a method for inhibiting or promoting the growth of plant cells. The method uses a chimeric gene configured to express SAG when promoting plant growth, and a chimeric gene configured to express SAG of antisense when inhibiting the growth of plants (ie weeds) to selected target plant cells. do.

The method of administering SAG proteins for the removal of oxygen radicals in vivo is based on a variety of factors including the type of injury, age, weight, sex, medical condition of the individual, the severity of symptoms, route of administration and the compound to be used. Thus, the method of administration varies widely, but can be determined routinely using standard methods. In a preferred embodiment, the pharmaceutical composition comprises between 0.1 and 100 mg of SAG protein. In the most preferred embodiment, the pharmaceutical composition comprises between 1 and 10 mg SAG protein.

SAG proteins may be prepared in solid form, including particulates, powders or suppositories, or in liquid form such as solutions, suspensions or emulsions. SAG proteins may be subjected to pharmaceutical manipulations such as sterilization and / or may include conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers.

SAG proteins may be administered alone or in combination with one or more other agents. When used in combination, the therapeutic agent may be a therapeutic agent that is formulated in separate compositions that are administered simultaneously or at different times, or that is administered in a single composition.

SAG proteins are usually administered in combination with one or more adjuvants suitable for the designated route of administration. SAG proteins include lactose, starch powder, cellulose esters of alkanoic acid, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfuric acid, gum arabic, gelatin, sodium alginate, polyvinylpyrrolidone and ( Or) mixed with polyvinyl alcohol, tableted or encapsulated and administered by conventional methods. Alternatively, the SAG protein may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solution, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, transcancan rubber and / or various buffers. Can be. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include a time delay material of glyceryl monostearate or glyceryl distearate alone, or a time delay material in the form of a mixture with wax or other materials known in the art.

In a preferred embodiment of the invention, the pharmaceutical composition of the SAG protein may be administered intramuscularly (IM) or intravenously (IV). A suitable IM or IV dose of the active ingredient of the SAG protein is 5 mg / ml daily. In IM or IV administration, the active ingredient may comprise from 0.001% to 10% (w / w), for example from 1% to 2% by weight, up to 10% (w / w). However, it preferably comprises less than 5% (w / w), more preferably 0.1% to 1% of the formulation.

The following examples are merely intended to illustrate the invention and should not be construed as limiting the invention as defined in the appended claims, which will be better understood by reference to the examples.

Example 1 Identification of Genes Induced by OP

Differential display (DD) techniques in two mouse tumor cell lines were used to isolate genes responsible for or associated with OP-induced apoptosis. Since OP-induced apoptosis is visibly detected only 12 hours after exposure (Sun, (1997) FEBS Lett. 408: 16-20), the genes responsible for apoptosis induction are After apoptosis, expression must increase or decrease. Therefore, the tumor cell lines were treated with OP for 6 hours, followed by DD analysis.

Murine JB6 tumor cell line L-RT101 (tumor cell line derived from epithelium) was cultured in Minimal Essential Media with Earle's salt, including 5% calf fetal serum (Sigma). H-Tx cells, spontaneously transformed rat liver cell lines, were cultured in Dulbecco's Modified Eagle Media containing 10% fetal calf serum and 1 mM sodium pyruvate. Human colon carcinoma cell line DLD-1 was cultured in 10% DMEM.

L-RT101 cells were treated with 150 μM of OP for 6 hours, and DMSO treated cells were subjected to differential expression analysis as controls. Briefly, total RNA was isolated from OP-treated cells and control cells using RNAzol solution (Tel-Test) according to the manufacturer's instructions, see Sun et al., (1993) Mol. Carcinogenesis 8, 49-57] followed by reverse transcription (RT) followed by polymerase chain reaction (PCR). The primer (P1) used for reverse transcription was AAGCTTTTTTTTTTTTTR (SEQ ID NO: 5), where R is one of adenine, guanine or cytosine. P1 was used as a downstream primer in subsequent PCR, and the upstream primer was AAGCTTNNNNNNN (SEQ ID NO: 6), where N is adenine, cytosine, guanine or thymine.

P1 and P2 primers reproducibly detected differential expression between control and OP-treated cells. Fragments that exhibit reproducibly differential expression were amplified using these primers and were analyzed by Northern analysis in both L-RT101 and H-Tx cells treated with OP (Sun et al. (1992) Cancer Res. 52: 1907- 1915]) as a probe (Sun (1997) FEBS Letters 408: 16-20). The fragments derived by OP (measured by Northern analysis) were subcloned into TA cloning vectors (Invitrogen) according to the manufacturer's instructions and sequenced by DNA Sequencer Version 2.0 (Amersham) according to the manufacturer's instructions. . The resulting clones contain OP-derived cDNA fragments.

Example 2. cDNA Library Screening and 5 'Race (RACE)

To clone the full-length murine SAG cDNA, one of the OP-induced clones was used as a probe to screen the murine lung cDNA library. Briefly, 1 × 10 ⁶ recombinant plaques were inoculated into 150% 1% NZY plates (20 in total). Recombinant plaques were transferred to nitrocellulose membranes and mouse SAG probes (2 × 10 ⁸ cpm / μg) in hybridization solution containing 5 × SSC, 5 × Denhardt solution, 50 mM sodium phosphate and 100 μg / ml denatured DNA Hybridized at 60 ° C. for 16-18 h. The filter is washed once for 5 minutes with a solution of 2X SSC and 0.1% SDS, once for 5 minutes with a solution of 0.5X SSC and 0.1% SDS, then for 15 minutes with a solution of 0.1X SSC and 0.1% SDS. Wash twice.

The longest clone isolated was a fragment of 1.0 kilobase ("kb") consisting of a partial open reading frame and a full 3'-terminal untranslated portion. Murine brain Marathon ready cDNA (ClonTech) was screened according to the protocol provided in the cDNA library via PCR amplification using primers derived from the 1 kb fragment and other primers derived from the vector sequence. The result was an additional 100 bp fragment consisting of the 5'-terminal untranslated sequence and some coding sequences. The derived cDNA clone consisted of 1140 base pairs (“bps”) (SEQ ID NO: 1), consisting of 113 amino acids presumed to be novel proteins and encoding proteins comprising 12 cysteine residues (SEQ ID NO: 2). The open reading frame preceded the 17 bp upstream sequence. The start codon was located in the Kozak consensus sequence (Kozak, M. (1991) J. Bilo. Chem. 266, 19867-19870), which is 100% certain in context. there was. A stop codon of the same frame was identified at 72 bp upstream of the start codon, which is the 5 'untranslated portion in some genomic clones (not shown). The 3'-terminal untranslated portion consisted of a sequence of 792 bp and contained two polyadenylation signals (AATAAA). The data means that almost full length cDNA was isolated.

Human HeLa cell cDNA libraries (Strategene) were screened by the method described above using mouse cDNA as a probe. One clone of the positive reaction was isolated and purified through an additional two step screen. In this manner, a 754 bp clone (SEQ ID NO: 3) containing a polyadenylation signal at the 3 'end was isolated. Human cDNA also included an open reading frame encoding a protein consisting of 113 amino acids that were assumed to be novel proteins and comprising 12 cysteine residues (SEQ ID NO: 4). The identity of the isolated murine and human cDNA sequences was 82% in total sequence and 94% in coding region. At the protein level, the sequence showed 96.5% identity and 12 cysteine residues were conservative. Computer analysis of the protein database using the GCG program (Genetics Computing Group, Madison, Wisconsin) shows that the coding protein is the theoretical protein of the yeast (approval number Z74876) and the theoretical protein of C-elegans (approval number) 70804) and 70% identity.

Motif search of the putative protein sequence using the GCG program revealed no known functional domains. However, the sequence contains two incomplete heme binding sites (CXXCH, codons 47-51 and 50-54, (Matthews, Prog. Biophys, Mol. Biol. 45: 1-56, 1985)). Canine and incomplete C ₃ HC ₄ zinc ring finger domains (Freemont et al., Cell 64: 483-484, 1991) intersect other consensus motifs at the C-terminus of the molecule (FIG. 1A). Included in. The second potential heme binding domain (FIG. 1A) had arginine substituted with histidine (amino acid 54). Since the two amino acids are structurally similar, it is possible to form a heme binding site. The zinc ring finger domain had cysteine substituted for histidine at the 85th amino acid position. The ring finger domains in the protein were C ₃ H ₂ C ₃ structures, not the C ₃ HC ₄ structures that are common. For zinc bonds, cysteine and histidine are interchangeable residues (Berg and Shi, Science 271: 1081, 1996 and Inouye et al., Science 278: 103-106, 1997). As such, there is evidence that the C ₃ H ₂ C ₃ domain in the protein may comprise a zinc-binding site. Importantly, the heme and zinc ring finger domains are seeded. 100% conservative among C. elegans, mice and humans. For yeast, only the last cysteine residue in the C ₃ H ₂ C ₃ motif is not conservative. The evolutionary conservation of heme and zinc-binding domains suggests their functional importance.

Disregarding one amino acid mismatch, other motifs identified in the deduced sequence of the SAG protein include aminoacyl-tRNA synthetase clease II motifs (codons 54-63) and Kazal serine protease inhibitor family motifs ( Codon 85-107), Ly-6 / U-par domain (codon 65-107), prokaryotic membrane lipoprotein lipid binding site (codon 16-27) and somatotropin, prolactin and its associated hormone motifs (codon 49 -66).

Thus, as a result of these experiments, cloned mouse and human novel genes encoding almost identical and evolutionarily conserved proteins, including unique heme and zinc binding motifs.

Example 3 SAG in Rat and Human Tumor Cells is Induced by OP

To confirm that the cloned cDNA was due to the induction of OP, Northern analysis was performed with RNA isolated from mouse tumor cell lines L-RT101 and H-Tx and human colon carcinoma cell line DLD-1. Cells grown with subconfluency were treated with 150 μM OP for various times (up to 24 hours) and total RNA was isolated. 15 μg of total RNA was used for Northern analysis using murine SAG or human SAG cDNA as probe.

OP cloned transcripts were detected in both cloned rat and human cDNAs, which were 1.2 kb and 0.9 kb in size, respectively. Since the gene is derived from the apoptotic pathway induced by OP, it was named Apoptosis Sensitive Genes (SAG), which encodes the SAG protein.

Example 4 Tissue Distribution and Embryo Expression of SAG

The expression of SAG was then examined in various human tissues. Analytical methods were performed as detailed above (Sun et al., (1993) Mol. Carcinogenesis 8, 49-57) and Sun et al., Proc. Natl. Acad. Sci. USA 90 : 2827-2831, 1993]. Briefly, total RNA was isolated from various human tissues (ClonTech) and then Northern analysis was performed using either mouse or human SAG cDNA as a probe. SAG RNA was detected in all tissues examined, including heart, brain, pancreas, lung, liver, skeletal muscle, kidney, spleen, thymus, prostate, small intestine, colon and peripheral vascular leukocytes. It has been found to be expressed at very high levels in the oxygen consuming heart, skeletal muscle and testes. Tissue distribution and high levels of expression in oxygen-consuming tissues and induction by redox-sensitive compounds (OP) suggest that SAG encodes redox-sensitive proteins.

Since SAG proteins are evolutionarily conserved, the possible developmental function of SAG was tested by measuring the expression of SAG in mouse embryonic tissue using reverse transcription of total RNA followed by PCR. The primers used in the PCR were SAGTA.01 5'-CGGGATCCCCATGGCCGACGTGAGG-3 '(SEQ ID NO: 7) and SAGT.02 5'-CGGGATCCTCATTTGCCGATTCTTTG-3' (SEQ ID NO: 8), which could synthesize the entire SAG coding region. The PCR reaction mixture of 11 samples consisted of 55 μl 10 × buffer, 22 μl 1.25 mM dNTP, 1.1 μl SAGTA.01, 1.1 μl SAGTA.02, 5.5 μl Taq DNA polymerase, 5.5 μl ³² P-dCTP And sterile water (final 495 μl). 45 μL in each tube (Sun et al., (1997), Mol. Carcinogenesis 8: 49-57) containing 5 μL of primary strand cDNA reverse transcribed from total RNA isolated from rat embryo tissue. Reaction mixture was added and PCR was performed for 25 cycles (45 seconds at 95 ° C., 1 minute at 60 ° C. and 2 minutes at 72 ° C.). 5 μl of a portion of the PCR product was denatured and separated on sequencing gels, dried and exposed to X-ray film.

SAG RNA was expressed in the embryonic embryos of mice grown for 9.5-19.5 days and detected higher levels between 9.5-11.5 days. The results suggest that SAG plays a role in embryonic development.

Example 5 Intracellular Positioning by Immunofluorescence

NIH3T3 cells (ATCC CRL 1658) were inoculated in a cover glass in a 24-well culture dish, pcDNA3.1 (pcDNA 3 vector of Invitrogen with myc-his-tag), pcDNA3.1-SAG (BamHI of pcDNA3.1) Human SAG cDNA sub-cloned at the site, downstream of the CMV promoter and upstream of the myc-his-tag of the same frame, resulting in a protein in the form of a fusion of the SAG protein followed by the myc-his-tag at the C-terminus) Or pcDNA3.1-LacZ (Invitrogen) was transfected with calcium phosphate according to standard techniques (Sambrook et al., 1989). After 2 days of transfection, cells were washed once with cold PBS and fixed for 3 minutes with 3% formaldehyde in PBS and then fixed for 5 minutes in a mixture of methanol and ecetone in a one-to-one relationship. Fixed cells were washed 4 times with PBS and 1 hour in the dark with antibody to Myc-tag in PBS containing 1% BSA, 0.1% saponin, 2 μg / ml DAPI (Invitrogen, diluted 1: 200). Incubation while stirring. Cells were then washed 4 times with 0.1% saponin in PBS and incubated for 1 hour in FITC conjugated goat's anti-mouse antibody (Jackson Labroatory, diluted 1: 100) under the same conditions as the primary antibody. After incubation, cells were washed four times with 0.1% saponin in PBS and twice with PBS. The cover glass was then placed on slide glass with a non-fade mounting medium and analyzed with a Leita Dialux 2000 microscope.

The control β-galactosidase was mainly expressed in the cytoplasm, while the SAG fusion protein was detected in both the cytoplasm and the nucleus. No immunofluorescence staining was detected in cells transfected with the vector only. Positioning of the SAG protein into the cytoplasm and nucleus was confirmed in cells stably transfected with SAG using SAG and myc-tag antibodies. The data means that externally expressed SAG fusion proteins can be detected using antibodies against epitopes fused to SAG in transfected cells.

Example 6 Expression and Purification of SAG Protein in Bacteria

The entire open reading frame of human SAG cDNA was amplified by PCR by the method described above and subcloned into PET11 expression vector (Novagen) under the control of the T7 promoter to generate pET11a-hSAG. The sequence and orientation of the SAG DNA insert was confirmed by DNA sequencing. E. coli strain BL21 (Novagen, Inc.) was transformed with pET11a-hSAG. Transduced cells were cultured in LB medium containing ampicillin (50 μg / ml). When SAG expression was induced by 0.5 mM IPTG, SAG protein was found as an inclusion body, which was isolated by the following method.

Following IPTG induction, 4 liters of cells were incubated at 37 ° C. for 4.5 hours on a shaker set at 150 rpm. Cell pellets were obtained by centrifugation at 5000 rpm for 10 minutes and resuspended in TN buffer (20 mM Tris-HCl, pH 7.5 and 50 mM NaCl) containing 100 μM PMSF. The resuspended cell pellet was sonicated (15 seconds / circuit 5 times using Fisher Scientific's Model 50 sonic dismembrator set to 15) and at a pressure of 2500 pounds / inch ² in a French cell press. After crushing the cells, 1 mM MgCl ₂ and 10 mg of DNase I were added. The cell lysate was placed on ice for 30 to 60 minutes, then centrifuged at 18,000 rpm to discard the supernatant.

The pellet appeared in two layers. The upper white layer was blunted with TN buffer and carefully removed. The dark brown layer remaining at the bottom was resuspended vigorously in 15 ml of urea buffer (7M urea, 20 mM Tris-HCl, pH 7.5 and 200 mM NaCl) and left overnight at room temperature. Resuspended cell pellet was homogenized vigorously with a serum pipette and then centrifuged for 40 minutes at 40,000 rpm using a SW50 ultracentrifuge rotor. Supernatant was collected, concentrated to 5 ml volume using Centricon-10 concentrator, and then Sephacryl-100 column (100 cm diameter 2.5 cm) in equilibrium with urea buffer. Loaded on. The column was run at a flow rate of 0.25 ml / min and fractions collected. As expected, the initial fraction consisted of large brown molecular weight proteins. In addition, the fraction contained SAG protein of constant size (about 13 kDa). Since the SAG protein contains 12 cysteine residues, the SAG protein can form oligomers upon expression in bacteria and thus elute as SAG protein oligomers. Since the SAG protein is a redox-sensitive protein, the DTT of the SDS sample buffer can reduce the SAG protein oligomer to the monomers so that a rapidly moving band can be detected. When the initial fraction was electrophoresed on SDS-PAGE without DTT, the SAG protein of 13 kDa disappeared and the band of 260 kDa was detected, indicating that the SAG protein is 20-mer. Initial fractions were collected and loaded onto a Sephacryl-100 column previously equilibrated with 7M urea and 5 mM DTT.

SAG protein oligomers were reduced to monomers using DTT in loading buffer and eluted in later fractions to separate from high molecular weight contaminating proteins (eluted initially). The brown fractions were collected and concentrated to 5 ml volume using a Centricon-10 concentrator. DTT was added at a concentration of 5 mM. The mixed fractions were loaded onto an S-100 column (100 cm long with a diameter of 2.5 cm) previously equilibrated with urea buffer with 5 mM DTT. The run was run at a flow rate of 0.25 ml / min and fractions collected. Fractions containing SAG protein were brown, indicating that SAG is a protein containing heme. SAG proteins including fractions and susceptibility to DTT were identified by Western blot using SAG antibodies. The brown fractions were collected and concentrated to 2 ml volume using a Centricon-10 concentrator. The resulting sample was dialyzed overnight at 4 ° C. against 4 liters of dialysis buffer (150 mM KCl and 20 mM Tris-HCl, pH 7.5) to remove urea and DTT and yield a refolded SAG protein. The dialyzed sample was loaded onto an S-100 column (100 cm long, 2.5 cm in diameter) in equilibrium with the dialysis buffer. The brown fractions were collected and concentrated to 1 ml volume using a Centricon-10 concentrator. The resulting sample was stored at 4 ° C. Protein concentrations were determined by BioRad protein analysis. The purity of the sample was confirmed by SDS-PAGE of 10-20%. The data shows that the recombinant SAG protein was purified.

Example 7 Redox Sensitivity of SAG Proteins

To confirm that the purified recombinant SAG protein had the same redox sensitivity during the purification process, the sensitivity of the refolded SAG to redox agent was investigated. Prior to isolation by polyacrylamide gel electrophoresis (PAGE), SAG protein (1 μg) was immersed in various concentrations of DTT (1M, 300 mM, 100 mM or 30 mM) or hydrogen peroxide (15 mM, 50 mM, 150 mM or 450 mM) for 10 minutes. Exposed and western blot analysis was performed. Alternatively, 10 μg of SAG protein was incubated in 50 mM hydrogen peroxide for 10, 30, 60 or 120 minutes, followed by PAGE isolation and staining with Coomassie Blue.

Since no dimer was converted to monomer after DTT treatment, the dimer of SAG protein is more resistant to reducing agent DTT. However, the low concentration of 15 mM hydrogen peroxide also induces the oligomerization of SAG protein, which can be expected to form disulfide bonds between molecules. Oligomerization was a phenomenon dependent on incubation time, where more SAG protein oligomers were detected as the incubation time increased. Interestingly, bands traveling faster than the monomeric form were observed in the samples treated with hydrogen peroxide, and the monomeric form of the SAG protein appeared to be paired by disulfide bonds inside the molecule.

To confirm that oligomerization of SAG protein induced by hydrogen peroxide reacted inversely by DTT treatment, 1 μg of purified SAG protein was incubated for 10 minutes in 50 mM hydrogen peroxide and then in 50 mM DTT, 100 mM DTT, 500 mM DTT or 1M DTT. Each was incubated. Samples were separated by PAGE and western analysis performed. As a result of the experiment, the hydrogen peroxide-induced SAG protein oligomerization was reacted upside down in proportion to the amount by subsequent incubation with DTT, so SAG protein oligomerization was a phenomenon due to redox control.

In order to confirm that oligomerization and dimer formation of SAG proteins are due to molecular interactions and intramolecular disulfide bonds, 50 mM concentration of N-ethylmaleimide (NEM), which alkylates the free SH group of SAG protein, prior to exposure to hydrogen peroxide Treated with SAG protein. Purified SAG protein (1 μg) was preincubated for 10 minutes in 50 mM NEM or DMSO, or just buffer before treatment with hydrogen peroxide. Samples were separated by PAGE and western blot analysis performed. The samples incubated with SAG protein in DMSO had no effect on oligomerization and pair formation induced by hydrogen peroxide, but the NEM preincubated samples showed the effect of deactivating hydrogen peroxide. That is, it was found that neither the intermolecule (oligomerization) nor the disulfide bond inside the molecule (monomer pair) was formed, and thus alkylation of the free SH group of the SAG protein eliminated its sensitivity to hydrogen peroxide. The data indicate that SAG protein is sensitive to redox. As evidenced by pair and oligomer formation, exposure to hydrogen peroxide forms disulfide bonds within and between molecules. The change induced by hydrogen peroxide may be reacted upside down by subsequent reducing agent treatment, including DTT, or may be previously treated with NEM to hinder the reaction. Zinc does not in itself induce oligomerization, but it has been known that zinc can promote induction of oligomerization by hydrogen peroxide.

Example 8 Preparation of SAG Mutations

To understand the role of each cysteine residue in heme binding and oligomerization of SAG, a series of single and double SAG mutants were made at the heme binding site as well as the zinc finger motif (see FIG. 1 B). To generate single point mutations in SAG cDNA, 15 pairs of sense and antisense primers were designed that were partially complementary and contained the desired point mutations. Wild type SAG cDNA cloned into pET11a vector at NheI and BamHI sites was used as template for PCR amplification. a) two PCR reactions isolated using primers SAG P.01 (5'-TATGGCTAGC ATGGCCGACGTGGAGG-3) (SEQ ID NO: 9) and each antisense primer, and b) each sense primer and SAG T.02 (SEQ ID NO: 8), respectively. Was performed. The resulting PCR products, overlapping each other and containing the desired point mutations, were mixed and used as template for the third PCR. Primers used were SAG P.01 and SAG T.02, which were able to synthesize the entire region of SAG cDNA. PCR reactions were performed as described above (Sun et al. (1992) BioTechniques 12: 639-640). PCR products were cut with restriction enzymes NheI and BamHI and subcloned into pET11a vector cut with the same restriction enzyme. To generate dual SAG mutants (MM10, MM13 and MM14, see FIG. 1B), the QuickChange position-directed mutagenesis kit was produced by Strategene, La Jolla, CA. It was purchased from and used as directed. All generated SAG mutants were confirmed by DNA sequence analysis (SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 49). The sequence of the mutant SAG protein encoded by the SAG mutant sequence is SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 , SEQ ID NO: 46, SEQ ID NO: 48, and SEQ ID NO: 50.

E. coli strain BL21 (Novagen, Inc.) was transformed using vectors expressing individual SAG mutations. Mutant SAG protein was expressed and purified by the method described in detail in Example 6. Fractions separated on Sephacryl-100 columns were collected and analyzed on 8-25% Phast gel and then stained with Coomassie Blue. Pure fractions containing mutant SAG protein were dialyzed at 4 liters of 20 mM Tris-HCl, pH7.5, which was used for SAG protein oligomerization studies.

The purified wild-type SAG protein was a brown protein containing heme (see Example 9). Compared to wild type SAG protein, certain purified SAG protein mutants lost brown (MM3 and MM13), or reduced brown tint (MM1). This color change means loss or decrease of heme binding capacity (Table 1).

Summary of SAG Mutants name Mutation site (s) Heme bonding Oligomerization Wild type none +++ being MM1 C _A / hem ++ being MM2 C _B / hem +++ being MM3 C _{A + B} / hem +/- being MM4 C ₁ / Zinc-ring finger 1 +++ being MM5 C ₃ / Zinc-ring finger 1 +++ being MM6 H ₄ / zinc-ring finger 1 +++ being MM7 H ₅ / Zinc-ring finger 2 +++ being MM8 C ₆ / Zinc-ring finger 2 +++ being MM9 C ₇ / Zinc-ring finger 2 +++ being MM10 H _{4 + 5} / Zinc-ring fingers 1 and 2 +++ being MM11 C ₂ / Zinc-ring finger 1 +++ being MM12 C _C / Protease Inhibitors +++ being MM13 C _{1 + 2} / Zinc-ring finger 1 +/- being MM14 C _{7 + 8} / Zinc-ring finger 2 +++ no MM15 GAPDH binding site +++ being

To investigate oligomerization of the mutant SAG protein, each mutant SAG protein and wild type SAG protein were treated with 50 mM hydrogen peroxide for 10 minutes. All SAG mutants except MM14 oligomerized upon exposure to hydrogen peroxide. Mutant 14 mutated double at the C7 and C8 positions of the zinc ring finger domains lost sensitivity to oligomerization (Table 1), meaning that these two positions are important for the formation of disulfide bonds between molecules. .

Example 9 Determination of Heme Content of SAG Proteins

Rieske (1967) Methods in Enzymol. 76, 488-493, measured the content of heme in the SAG protein. Briefly, 1 mg of purified SAG protein with cytochrome C, hydrogen peroxide degrading enzyme and BSA as a control was extracted with cold acetone (0.5 ml). The pellet was centrifuged and extracted into a mixture of chloroform and methanol in a 2: 1 mixture, extracted with 0.5 ml of cold acetone, and finally with 0.5 ml of cold acetone containing 2.4N HCl. The acetone extracts were dried under speed-vac and dissolved in 0.5 ml of pyridine. After addition of 0.5 ml 0.2N NaOH, the solution was briefly centrifuged and a clear supernatant was recovered. One drop of diluted potassium ferricyanide (0.05M) was added to the supernatant, and the absorbance at 556 nm was measured with zero water in a 1.0 ml quartz cuvette. Subsequently, 10 μl of 2M DTT was added to the solution, and the absorbances of 556 nm, 587 nm, and 550 nm were measured, respectively.

Absorbances were measured for sheet chrome C and hydrogen peroxide degrading enzymes except BSA, as well as for 556 nm, 587 nm, and 550 nm heme absorbances in SAG proteins. As a result, the SAG protein was found to contain heme, but the molar ratio between the SAG protein and the heme molecule was not found.

Example 10 Antibody Preparation Against SAG Protein

Two polyclonal antibodies against SAG proteins were prepared using standard methods (Zymed Labroatories, Inc., San Francisco, in agreement with Warner-Lambert). Briefly, peptide antibodies were generated as follows. A peptide of 16 amino acids (codon 95-110) located at the C-terminus of the SAG protein (SAG-Pepl: QNNRCPLCQQDWVVQR) (SEQ ID NO: 10) was synthesized and purified by standard techniques. Purified peptides were conjugated with hemocyanin (KLH) of a keyhole-shaped hatchfish through cysteine residues. Conjugated peptide (0.5 mg) was emulsified with Eastern Freund's Complete Freund Adjuvant (CFA), subcutaneous injection in rabbit, and then emulsified with Incomplete Freund Adjuvant (IFA) for 3 weeks. The immune response was boosted by four injections with 0.5 mg of peptide. Antiserum was collected by bleeding rabbits 10 days after the last immune booster. Protein antibodies were prepared as described above using the same protocol as above using purified human SAG protein as antigen.

Example 11 Analysis of Transcription Regulatory Activity of SAG Proteins

Thanks to the C ₃ H ₂ C ₃ motif, the SAG protein belongs to the zinc ring finger protein family (Saurin et al. (1996) TIBS 21, 208-214). Some of the zinc ring finger proteins bind to DNA and act as transcription inhibitors (eg, RING1) (Satijn et al. (1997) Mol. Cell. Biol. 17, 4105-4113), while others Act as transcriptional activators (Chapman and Verma (1996) Nature 382, 678-679) and Monteiro et al. (1996) Proc. Natl. Acad. Sci. USA 93, 13595-13599). To investigate the transcriptional regulatory activity of SAG proteins, PCR amplified cDNA encoding the entire open reading frame of human SAG, followed by pG4 vectors (Sadowski et al., Nature 335: 563-564, 1988). Downstream of the Gal-4 DNA binding domain (coding amino acids 1-147), both in the same frame fusion and antisense fusion. The resulting vector constructs were sequenced to confirm that they were in the same frame and that no mutations occurred during the PCR process. The vector composition is a vector expressing the chloramphenicol acetyltransferase (CAT) reporter (Sadowski et al., Nature 335: 563-564), as well as a β-galactosidase reporter whose expression is induced by the CMV promoter. Human kidney 293 cell line (ATCC CRL 1573) was transfected with calcium phosphate method (to standardize transfection efficiency). 36 hours after transfection the CAT activity was measured using the CAT Assay Kit (Quan-T-CAT; Amersham) as instructed by the manufacturer. PG4-VP16 (Trizenberg et al., Genes and Develop. 2: 718-729, 1988), a known transcription factor fused downstream of the Gal4 DNA binding domain, was used as a positive control. Optionally, the CAT activity of the vector control was calculated to be 1, based on which the value of the other vector composition was determined. Three independent transfections and assays were performed.

SAG protein did not activate transcription at all. VP16, a positive control, increased CAT activity 300-fold. To test transcription inhibitory activity, SAG vector constructs (sense and antisense) were transfected with pG4-VP16. Likewise, neither direction of SAG affected the transcriptional activity induced by VP16. The results indicate that SAG proteins fused downstream of the Gal-4 DNA binding domain lack transcriptional regulatory activity.

Example 12. SAG is a protein that binds to RNA.

Zinc-ring finger domains of the MDM2 protein have been known to bind RNA (Elenbaas et al. (1996) Mol. Med. 2, 439-445). Since the SAG protein lacked transcriptional regulatory activity, it was tested if the protein could bind to DNA or RNA. Experiments in which the purified SAG protein binds to different nucleic acid cellulose conjugates were performed as described in Elenbaas et al. (1996). Briefly, 0.5 μg of SAG protein is a double-stranded calf thymus DNA column conjugated to agarose or cellulose beads (Sigma), denatured calf thymus DNA (ssDNA) column or 300 μl of RNA binding buffer at 4 ° C. Incubate for one hour according to the manufacturer's instructions on one column of four RNA isoforms. RNA binding buffer consists of 20 mM Tris, pH 7.5, 150 mM NaCl, 5 mM MgCl ² , 0.1% NP-40, 50 μM ZnCl ₂ , 2% glycerol and 1 mM DTT. The column was washed with 3 ml of RNA binding buffer to remove nonspecifically bound protein from the beads and then boiled in SDS sample buffer. The proteins eluted as above in the beads were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot analysis using the polyclonal antibody of the SAG protein described above, followed by ECL chemiluminescence (Amersham). Detection was in accordance with the instructions.

Purified SAG protein bound to polyU, polyA and polyC RNA, respectively. No binding to polyG or ssDNA. The band of protein bound to dsDNA did not match the molecular weight of SAG. Oligomeric SAG protein bound to polyU RNA, while SAG in monomeric form bound to polyA and polyC RNA. Purified SAG protein was electrophoresed as a marker. The results show that SAG is an RNA binding protein and the binding specificity is determined by the oligomeric form of the SAG protein.

Example 13. Identification of Two SAG Deletion Mutations in Cancer Cell Lines

Total RNA was isolated from DLD-1 colon carcinoma cell line (ATCC CCl221), and RT-PCR was performed with primers SAG TA.01 and SAG T.02. The resulting PCR fragment was subcloned into a TA cloning vector (Invitrogen). In the course of identifying the clones, 7 or 48 bp were deleted, respectively, at the 170 or 177 nucleotide positions of several clones (if the first A of the start codon was designated as nucleotide 1). Mutants lacking the 7 base pair (SAG mutant 1, SEQ ID NO: 11) are deletion mutations that shift the frame, resulting in no zinc-ring finger motif encoded downstream of the resulting protein (SEQ ID NO: 12). On the other hand, a mutant lacking 48 base pairs (SAG mutant 2, SEQ ID NO: 13) is a deletion mutation of the same frame, which removes 16 amino acids of the resulting protein (SEQ ID NO: 14) but contains a zinc-ring finger motif .

Total RNA was isolated from 20 human tumor cell lines and transformed cell lines of lung, brain, kidney, prostate, testes, nasopharynx, bone, cervical and foreskin origin, described above (Sun et al. (1993) Mol. Carcinogenesis 8, 49-57]) was performed RT-PCR analysis. Genomic DNA was isolated from this cell line and PCR amplified as described in Sun et al. (1992) BioTechniques 12: 639-640. Primers used for PCR were SAGT.02-1 5'-GGATCCTCATTTGCCGATTCTTTGGAC-3, including hSAG.M1 starting at 151 nucleotides of hSAG cDNA, 5'-GCCATCTGCAGGGTCCAG-3 '(SEQ ID NO: 15), and a stop codon (underscore). (SEQ ID NO: 16). For wild-type SAG the resulting fragment was 200 bp. PCR was performed in the presence of ³⁵ S-dATP (Amersham), and the PCR product was subjected to 6% denaturation sequence analysis by the method described in Sun et al. (1995) Cancer Epidermiology, Biomarkers & Prevention, 4, 261-267. Separated on gel. The bands corresponding to wild type and two mutants were cut out of the gel, PCR amplified using the same primer set, and the DNA of the resulting PCR fragment was confirmed by sequencing.

Both 7 base pair and 48 base pair deletion mutants were detected only in the RNA of the CATES-1B cell line (ATCC HTB104), a testicular carcinoma cell line. The tumor cell line also contained wild type SAG DNA sequences. The identity of the three bands was confirmed by DNA sequencing after cloning into TA vectors by PCR. HONE-1, a nasal pharyngeal tumor cell line containing only wild-type SAG, was included for comparison.

It was then examined if SAG deletion mutations were detected at the DNA level. Genomic DNA was isolated from CATES-1B cells and subjected to PCR analysis as described in Sun et al. (1992) BioTechniques 12: 639-640. Primers used were hSAG.M1 and SAG T.02 (see sequence above). Genomic DNA isolated from CATES-1B cells contained only wild type SAG and no SAG deletion mutations were detected. The results showed that SAG deletion mutations are very rare in human carcinoma cell lines. The detection of mutations only in SAG RNA, not in genomic DNA, may reflect changes by RNA editing of SAG mRNA.

Example 14. Preparation of mammalian cells stably transfected with SAG

Overexpression in cells examined the potential biological function of human SAG protein. DLD-1 cells were subjected to plasmids (pcDNA-3 under neomycin regulation (consistent with pcDNA3.1 above except for the absence of the myc-his tag), pcDNA-SAG, pcDNA-SAG-mutant-1 and pcDNA-SAG transfected with -mutant-2 (pcDNA3 with SAG, SAG 1 or SAG 2. SAG mutant constructs were generated by RT-PCR as follows: After reverse transcription by isolation of total RNA from DLD-1 cells, PCR amplification The primers used were SAG.TA01 (SEQ ID NO: 7) and SAGT.02 (SEQ ID NO: 8), which are capable of synthesizing the entire coding region of the SAG gene. Subcloning was carried out in pcDNA3 (Invitrogen, San Diego), a mammalian expression vector under transcriptional control of the CMV promoter that expresses the resulting clones were sequenced to confirm both the sense and antisense directions, and no mutation occurred during PCR. Confirmed DNA sequencing revealed not only wild-type SAG clones but also two deletion mutants (SAG mutant-1 (7 bp deletion, SEQ ID NO: 11) and SAG mutant-2 (48 bp deletion, SEQ ID NO: 13)) in the DLD-1 tumor cell line. I knew it existed.

DLS-1 cells were transfected with lipofectamine (BRL) wildtype (sense and antisense directions), SAG mutant-1 and SAG mutant-2 along with neomycin control vector. Colonies resistant to neomycin were identified by G418 (600 μg / ml) selection for 18 days. Stable clones were ring-isolated by known methods (Sun et al. (1993) Proc. Natl. Acad. Sci. USA 90: 2827-2831) to monitor expression of SAG by Northern analysis. Selected clones were examined for expression of SAG protein by immunoprecipitation as described below.

Total RNA was isolated from cloned cell lines for Northern analysis. Vector constructs of control vectors D1-3 and D1-6, SAG wild type D12-1 and D12-8, SAG mutant-1 D3-3 and D3-4, and SAG mutant-2 D4-2 and D4-5 Transfected cell lines were analyzed.

Northern blot analysis of RNA isolated from selected stable SAG-expressing clones using human SAG cDNA as a probe showed that SAG mRNA was found in all SAG transfectants, but little in neomycin control cells.

Subsequently, the vector control cell line and the SAG wild type and SAG deletion mutant transformants were standardized (Sun et al. (1993) Proc. Natl. Acad. Sci. USA. 90: 2827-2831) and Sun et al. (1993) Mol. Carcinogenesis 8, 49-57]. SAG transfectants grown with subconfluency were incubated for 1 hour with methionine deficient, then given ³⁵ S-labeled methionine (0.2 mCi / ml) and incubated for 3 hours. 2% NP-40, 0.2% SDS, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 5 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride over 30 minutes and Cells were lysed in lysis buffer containing 1 μl / ml leupeptin and centrifuged at 12,000 × g. Radioactivity precipitated in TCA in the supernatant (1 × 10 ⁸ cpm) was immunoprecipitated using rabbit anti-human SAG antibody (prepared by the method described above). Immunoprecipitated samples were collected, washed and analyzed on 10-20% SDS-polyacrylamide gels, followed by autoradiography. Only high levels of SAG protein expression were detected in wild-type transfectants. The antibody used did not recognize both SAG protein mutants. The data means that stably transfected cells were prepared expressing wild type or mutant SAG proteins.

Example 15 Morphological Aspects of SAG Transfectants After Exposure to Redox Agents

Sensitivity to redox compounds was examined by morphological observation by selecting two neomycin controls (D1-3 and D1-6) and two SAG-producing cell lines (D12-1 and D12-8). After 24 hours of exposure to 150 μM OP, 200 μM hydrogen peroxide or 123 μM zinc, neomycin control cells fell in corrugated form (signal of apoptosis), while SAG-expressing cells were morphologically normal. The results indicate that production of SAG protects cells from apoptosis induced by redox compounds. However, expression of SAG did not protect cells against copper. There was no difference in the morphological signal of apoptosis between vector control treated with CuSO ₄ (up to 750 μM concentration) and SAG transfectants. Increasing the dose induced apoptosis in all cell lines.

Example 16 Expression of SAG Protects Cells from DNA Degradation

The sensitivity of SAG-transfected cells to OP-induced apoptosis was investigated by monitoring DNA degradation, a phenomenon of apoptosis. SAG transfected cells or vector control cells expressing wild type SAG, SAG mutant-1, SAG mutant-2, or vector control cells grown to subconfluency (in a range of 80 to 90%) inoculated in 3.5 × 10 ⁶ cells in a 100 mm culture dish. And 16 to 24 hours later exposed to 150 μM OP, 125 μM zinc sulfate or 200 μM hydrogen peroxide for 24 hours. Both the detached cells and the attached cells in two 100 mm culture dishes were harvested, and the following DNA digestion assays were performed. Cells were harvested by centrifugation and lysed for 45 minutes on ice with lysis buffer (5 mM Tris-HCl, pH 8, 20 mM EDTA and 0.5% Triton-X100). As a result of centrifugation at 14,000 rpm (45 minutes at 4 ° C), the digested DNA present in the supernatant was extracted twice with phenol and chloroform and once with chloroform and precipitated with ethanol and salt. The DNA pellet was washed with 70% ethanol and resuspended at 37 ° C. for 2 hours with TE buffer containing 100 μg / ml RNase. The digested DNA was isolated by 1.8% agarose gel electrophoresis, stained with ethidium bromide and observed under ultraviolet light.

OP in vector control cells induced apoptosis. Compared to control cells, DNA was less degraded in wild-type SAG transfected cells. SAG mutant 1, which does not encode a zinc ring-finger motif, did not protect the cells from DNA degradation induced by OP at all, whereas SAG mutant 2, including a zinc ring finger domain, from DNA degradation induced by OP Cells were protected. The results indicate that overexpression of SAG protein protects cells from DNA degradation induced by OP, and that zinc ring finger domains are required for this protective activity.

Since the SAG protein contains a zinc ring finger motif, the sensitivity of the SAG transformant to zinc treatment was investigated. Zinc induced apoptosis in DLD-1 cells transfected with vectors only. Induction of apoptosis by overexpression of SAG was inhibited, resulting in much less DNA degradation than control cell lines. The data indicate that the SAG protein chelated zinc through the zinc ring finger domain, resulting in increased resistance to zinc toxicity compared to untransfected cells.

Another feature of SAG is the formation of oligomers after exposure to hydrogen peroxide. By oligomerization of SAG cells can be protected from toxicity induced in hydrogen peroxide. Therefore, SAG-transfected cells were treated with hydrogen peroxide, followed by DNA digestion analysis. Hydrogen peroxide induced apoptosis in DLD-1 cells. As evidenced by the reduction in DNA degradation, overexpression of the SAG protein partially protected the cells from apoptosis induced by hydrogen peroxide. Considering the above results, it can be seen that SAG protects the cells to some extent from apoptosis induced by redox compounds such as OP and hydrogen peroxide, and to some extent from apoptosis due to zinc. have.

Example 17 Expression of Antisense SAG Inhibits Growth of Tumor Cells

To test the growth effects induced by SAG expression, the methods described above were used to transduce neomycin control vectors or vectors expressing SAG, SAG mutant-1, SAG mutant-2 or antisense SAG in DLD-1 cells. Infected. Neomycin resistant colonies were selected with G418 (600 μg / ml) and stained with 50% methanol / 10% acetic acid / 0.25% Coomassie Blue.

To observe potential changes in tumor cell traits due to decreased SAG expression, stable DLD-1 transformants (D15-1) expressing antisense SAG mRNA were cloned after G418 selection. Together with vector control cells (D1-6) and SAG (sense) overexpressing cells (D12-1 and D12-8) grown with subconfluency, D15-1 cells were labeled with metabolic material and described above. Immunoprecipitated using SAG protein antibody as described. Concentration quantification of SAG protein expression using computer dynamics (Molecular Dynamics) was performed according to the manufacturer's instructions. A randomly selected value from the vector control cells D1-6 was designated as 1. Antisense SAG-transfected cells (D15-1) had a 60% reduction in internal SAG protein. Transfection of antisense SAG significantly inhibited monolayer growth of DLD-1 cells. None of the other transformants inhibited growth compared to the neomycin vector control.

Antisense SAG-transfected cells were examined for growth inhibition in soft agar medium. 0.25% agar medium for D15-1 cells with wild type SAG (D12-8), SAG mutant-1 (D3-3), SAG mutant-2 (D4-2) as well as neomycin control (D1-3) Incubated for 14 days. More than 16 cells were counted in the colonies of the plate. Three independent experiments were conducted, each of which was doubled. The values stated are the standard deviation of the mean +/- mean. As shown in FIG. 2, down-regulation of SAG expression in D15-1 cells results in significant growth inhibition of DLD-1 cells, such as neomycin control (D1-3), SAG (sense) expressing cell lines D12-8 and Compared to SAG mutants (D3-3 and D4-2), the number of colonies in soft agar medium was reduced to 70%.

In a further study, D15-1 cells grown in 4 × 10 ⁶ confluence with parental DLD-1 cells, vector control D1-6 and wild-type SAG transformant D12-1 were obtained from SCID mice (Taconic Farms, Germantown, Subcutaneously (10 rats in each group). Tumor proliferation was observed twice a week. Tumor size per mass (average for 10 rats) is plotted against time up to 24 days post-infusion. When transplanted into SCID mice, antisense expressing cell line D15-1 did not form tumors until 24 days post inoculation, whereas in parental DLD-1 cells, vector control D1-6 and SAG (sense) expressing D12-1 cells Substantial tumor proliferation was observed (FIG. 3). All the above experiments indicate that down-regulation of SAG expression induces growth inhibition of tumor cells, and further that SAG proteins are cell protective molecules.

Example 18 Carcinoma Gene Therapy With Adenovirus Expressing Antisense SAG

Since expression of antisense SAG in vivo and ex vivo inhibited tumor proliferation, SAG can be used as a target for carcinoma gene therapy. Methods of performing carcinoma gene therapy are well known in the art (Zhang and Fang, Exp. Opin, Invest.Drugs 4: 487-514, 1995) and Zhang et al., Adv. Pharmacol. 32: 289-341, 1995).

Models using tumor cell lines expressing SAG internally, including but not limited to DLD-1 (colon), Du145 (prostate), G401 (kidney), H2009 (lung) and HONET-1 (nasal pharynx) Was established. Tumor cells obtained by tissue culture were suspended in PBS at 5 × 10 ⁷ / ml and stored on ice. 0.2 ml of cell suspension (containing about 10 ⁷ cells) was injected subcutaneously into the flanks of athymic nude mice over 6 to 8 weeks old, so that the tumors could last 30 to 40 days or the average size of the tumors. Proliferation was performed until 5 mm was obtained.

Shuttle plasmids (pJM17, a circular Ad5 genome) and recombinant plasmids (pEC-SAG, including the left arm of the Ad5 genome, affected by the CMV promoter) were transformed together in 293 cells to express expression by the CMV promoter. A recombinant adenovirus vector (Ad.CMV-SAG) expressing an antisense human SAG was prepared.

0.1 ml of recombinant adenovirus solution (1-5 × 10 ¹⁰ pfu / ml) or 0.1 ml of PBS (control) were injected into the tumor. The treatment was carried out daily for 2 days and again for 3 days after 1 week of no treatment. Tumor size was measured daily for two weeks. To test the combination therapy of oxygen radical-generating agent or radiation, the treatment group was divided into three groups (10 rats per group). Group A infected only adenovirus (see above), group B was injected intraperitoneally with adenovirus infection, adriamycin (3 mg / kg), and group C adenovirus infection and cesium-137 350cGy was investigated. Some mice with tumors were treated with the same amount of adriamycin alone or irradiated as drug treatment or radiation control.

Expression of antisense SAG interfered with internal SAG synthesis, which makes tumor cells extremely sensitive to oxygen radicals. The contraction of tumor cells treated with or without drugs or radiation relative to the vehicle control indicates the efficacy of the treatment. In addition, tumors from control and treatment groups were examined histologically. Samples were immediately buried in optimal cleavage temperature compounds (Miles, Inc. Elkhart, Indiana) and stained with enzymes (e.g., terminal deoxynucleodydyl transferase (Boehringer Manheim, Indianapolis, Indiana) for apoptosis confirmation). ) Or cooling flakes to confirm expression of antisense SAG by immunohistochemical staining were prepared by snap-cooling in liquid nitrogen. Alternatively, samples were fixed with 10% formalin and histological sections were made and analyzed by hematoxylin-eosin (Sigma, St. Louis, Missouri) staining.

Example 19 Function of SAG as an Oxygen Radical Scavenger to Prevent Damage Induced by Oxygen Radicals

SAG proteins contain 12 cysteine residues and can form disulfide bonds within and between molecules when exposed to hydrogen peroxide. In addition, SAG protein can bind to heme and change the oxidant by oxidation and reduction of Fe ⁺² . The above oxidation buffering activity can qualify the SAG as an oxygen radical scavenger.

Yeast cells defective in antioxidant enzyme genes (peroxide dismutase (SOD) and hydrogen peroxide degrading enzyme (CAT)) are highly sensitive to superoxide anions and hydrogen peroxide (Longo et al. (1997), J. Cell Biol. 137: 1581-1588]. Yeast cells lacking (a) Cu, Zn-SOD, (b) Mn-SOD, (c) Cu, Zn-SOD and Mn-SOD, and (d) CAT were transfected with human SAG expressing plasmids. The sensitivity of the transfected cells to oxygen radical producing compounds (e.g., paraquat producing hydrogen peroxide and superoxide anions) was tested by yeast growth assay and compared to the same host cells transfected with the vector control. . The rescue of the yeast cells from oxygen radical-induced cell death means that SAG is an effective oxygen radical scavenger.

Example 20 Prevention of Brain Injury Induced by IL-1β During Ischemia by SAG Administration

Central cerebral artery occlusion in rats leads to overexpression of interleukin-1, resulting in brain damage by the release of free radicals (Yang et al., Brain Research 751: 181-188, (1997)). This is known. Two experiments were conducted to test whether the SAG prevented brain damage by eliminating the free radicals released.

In the first experiment, human SAG was subcloned into an adenovirus vector (AdRSV-SAG) regulated by the RSV promoter. To confirm the expression of SAG in the brain, adenovirus suspensions were injected stereoscopically into the lateral cavities. Five days after adenovirus administration, the central cerebral artery of the animal was occluded for 24 hours as described in Yang et al., Brain Research 751: 181-188, (1997). Brain edema (measured by brain water content) and cerebral infarct size (measured by histological techniques (Yang et al., Stroke 23: 1331-1336 (1992))) were measured. Hydrocephalus and infarct size were somewhat reduced compared to the vector control, which means that SAG functions opposite to the damage induced by free radicals.

Second, the rat structural model (Yang and Betz, Stroke, 25: 1658-1665, (1994), either permanent occlusion (6 hours) or transient occlusion (3 hours reperfusion after 3 hours infarction). ] Was performed in the middle cerebral artery occlusion experiment. The purified SAG protein was then injected into the occlusion site of the rat. Brain water, ion content and infarct volume were measured to determine cerebral infarction and blood-brain barrier disruption. Reduction in cerebral infarct size and blood-brain barrier disruption compared to vehicle-injected control means that SAG exhibits a protective effect.

Example 21.Diagnosis of Human Cancers Using SAG as a Marker

Two SAG deletion mutants were identified in human cancer cell lines from colon and testes. Twelve pairs of colon carcinoma and adjacent normal tissue were collected from 12 patients. Genomic DNA and total RNA were isolated from the samples and PCR amplified. The resulting amplified product was analyzed by methods known in the art for detecting SAG deletion mutations. Such methods include, but are not limited to, RNA protection assays, DNA sequencing, hybridization and gel electrophoresis of deletion mutants. Mutations that are detected only in tumor tissue but not in normal tissue adjacent thereto are mutations specific for the tumor, which can be used as diagnostic tools in testicular carcinoma as well as colon carcinoma.

Example 22. Yeast homologues of the human SAG gene are essential for the growth of yeast.

To understand the additional function of SAG, yeast SAG knockout mutants were prepared by homologous recombination. The vector construct used to knock out yeast SAG was prepared by PCR of the kanamycin cassette of the kanMX4 plasmid (Wach et al., Yeast 10: 1793-1808, 1994). Primers used for PCR were SAGKanMX4-5: 5'-TTCTCCAGTGGCAGAGAACTTTAAAGAGAAATAGTTCAACCGTACGCTGCAGGTCGAC-3 '(SEQ ID NO: 17) and SAGKanMX4-3: 5'-ACCTCGGTATGATTTAAATGTTTACGGGCAATTCATTTTTATCGATGAATTCGA (SEQ ID NO: 18). The SAGKanMX4-5 primer consists of the yeast SAG DNA sequence immediately upstream of the start codon ATG (ATCC Z74876, underlined) and the 3′-end upstream kanamycin cassette sequence. The SAGKanMX4-3 primer consists of the yeast SAG DNA sequence immediately underlined by the stop codon TGA (underlined) and the downstream kanamycin cassette sequence 3'-end.

PCR was performed for 5 cycles of 94 ° C 1 minute, 50 ° C 1.5 minutes, 72 ° C 2 minutes, followed by 25 cycles of 94 ° C 1 minute, 56 ° C 1.5 minutes, 72 ° C 2 minutes, followed by 10 at 72 ° C The synthesis step was performed for minutes. The PCR product (1.5 kb) was gel-purified using the Qiaex II gel-purification kit (Qiagen) and used for transformation of diploid yeast strain Y21. The transformation was performed according to the manufacturer's instructions using the YEASTMAKER yeast transformation system (ClonTech Laboratory, Inc.). After transformation, cultured in YPD medium (Difco) containing G418 (200 μg / ml, BRL) to select a transformant from which the SAG of yeast was removed by homologous recombination, including a kanamycin cassette.

Several G418-resistant clones were selected and analyzed to determine whether they are heterozygous deletion mutations or homozygous deletion mutations. Primers used were SAGPCR5: 5'-TTCTCCAGTGGCAGAGAAC-3 '(SEQ ID NO: 19) and SAGPCR-3: 5'-ATGATTTAAATGTTTACGGGC-3' (SEQ ID NO: 20). The primers constitute fragments of SAGKanMX4-5 and SAGKanMX4-3, respectively, and can synthesize the entire SAG coding sequence of yeast. Wild-type yeast SAG produced a band of 0.35 kb as a result of PCR, while the SAG deletion mutant produced a band of 1.5 kb consisting of a kanamycin cassette. 0.35 kb and 1.5 kb fragments were generated in all clones tested, which means preparation of heterozygous mutations. The same knockout experiments were performed with haploid yeast cells (InvSC1 obtained from Invitrogen) and could not isolate G418-resistant clones.

Failure to isolate SAG deletion mutants by homozygous means that yeast SAG is essential for growth. To confirm this, spores were formed with 12 heterozygous yeast strains (y21-ySAG / ySAG :: Kan) to investigate whether the SAG-kan fraction of yeast survived. The strains were inoculated in uracil, lysine, adenine and tryptophan minimal potassium acetate sporulation media (Kassir and Simchen, G. Method Enzymol. 194, 94-110, 1991 ]) And incubated at 30 ° C. for 7 days. A four minute chromosome from each strain was excised into four haploid offspring. To dissect, the cell mass of the spore forming plate was suspended in 100 [mu] l of 1M glycerol containing 0.5 mg / ml of geolase T20. After 30 minutes of incubation at 37 ° C., the suspension was diluted with 800 μl of sterile water and placed on ice. The suspension was streaked onto YPD plates using an inoculation loop and the 4 minute chromosome was observed under a Zeiss Tetrad microscope. Microscopic glass microneedles were used to excise the 4 minute chromosome of each strain. Two of the four haploid cells should contain wild-type SAG and the other two should contain SAG deletion mutations. In all 12 clones, only two of the four incised cells grew and no cells were observed in GPD containing YPD medium. Thus, it was found that the surviving cells did not contain the kanamycin cassette or the SAG deletion. The experiment clearly explains that SAG is essential for yeast growth and further explains its evolutionary importance.

To investigate whether ySAG is required for normal growth or only for germination, hSAG was cloned into yeast expression vectors with URA3 selection markers. hSAG-URA plasmids were transformed into heterozygous ySAG knockout cells and transformants were selected in URA-deficient plates. Clones expressing hSAG (measured by Western blot analysis) were allowed to form spores and a 4 minute chromosome was excised. Survived colonies were used for the selection of isotopic plasmids containing YPD, YPD + G418 or YPD + 5-fluoroorroic acid (5-FOA, URA3, Boeke et al., Mol. Gen. Genet. 1984, 197-345). From each of the four 4-minute chromosomes, all four haploids, the hSAG-URA3 plasmid supplemented with the ySAG :: kan allele, were grown. Two haploids of each four-minute chromosome died when fed in YPD + G418 medium, indicating the inclusion of the wild type ySAG :: kan gene. The other two haploids of each 4 minute chromosome survived, indicating the inclusion of the ySAG :: kan allele. When the latter colonies were cultured in YPD + 5-FOA plates, not all cells grew, which means that ySAG is not simply necessary for sporulation but for normal growth.

Example 23 Rescue for Yeast SAG Knockout Traits in Human SAG

To investigate whether human SAG rescues the killing traits of yeast SAG knockout, wild-type human SAGs were converted to SAG mutants (MM3 of SEQ ID NO: 25, MM10 of SEQ ID NO: 39 and MM14 of SEQ ID NO: 47, FIG. Consisting of the plasmid, which was transformed into a heterozygous yeast strain (y21-SAG / ySAG :: Kan) in the same manner as above. Srp expression of clones growing on Trp-deficient and G418 plates was examined by Western blot analysis. Sponsored and dissected with clones expressing human SAG. Of the 10 wild-type human SAG clones, only 3 or 4 haploids were grown. Some of them contain yeast SAG, while the remaining clones have added human SAG to ySAG knockout, which means that human wild-type SAG can complement yeast SAG knockout. Clones (all 41 tested) of all three mutants survived one or two haploids, and all surviving haploids contained yeast SAG. The results indicate that human SAG mutants cannot compensate for SAG knockout of yeast.

Example 24. A SAG binds to a metal.

Since SAG contains zinc-ring finger domains, it has the potential to bind metals. In order to determine the metal binding potential of SAG, electrospray ionization mass spectrometry (ESI-MS) was used to denature and denature the solution conditions (Loo's 1997 literature and Witcove). The molecular weight of SAG was compared under the literature of Witkowska et al. (1995).

ESI-MS was performed using a double focusing hybrid mass spectrometer (Finnigan MAT 900Q, Bremen, Germany) with a mass-to-charge (m / z) range of 10,000 and a maximum acceleration potential of 5 kV. . A position to time-decomposition-ion-coefficient (PATRIC) scan array detector was used. An ESI interface based on a heated metal capillary inlet and a low flow rate micro-EsI source (150 nl / min analyte flow rate) was used (Sannes-Lowery et al., 1997). The temperature of the metal capillary was maintained at about 150-200 ° C. for the study of metal-protein complexes. Recombinant protein under denaturation solution containing 7M urea was dialyzed and refolded with 50 μM ZnCl ₂ , with three exchanges of buffer. Prior to ESI-MS measurement, the SAG solution was submerged with 10 mM ammonium bicarbonate (pH 7) and 1 mM DTT and centrifuged through a centrifugal filter cartridge (Microcon-10 microconcentrator, Amicon, Beverly, MA) that 10 kDa would not pass. Excess zinc was removed by separable ultrafiltration. For ESI-MS analysis, a portion of the filtered SAG protein solution was either denatured solvent (acetonitrile: water: acetic acid = 80: 15: 5, volume / volume / volume, pH 2.5) or non-modified solvent (10 mM bicarbonate) Diluted with ammonium and 1 mM DTT, pH 7).

First, zinc binding of SAG was measured. In denaturing acidic solutions (pH 2.5 and high concentrations of organic solvents) that are believed to have no metal-bonding properties even if zinc is present, the molecular weight of SAG is 12550, which closely matches the expected mass of the apo-protein (12552 Da). Was measured. ESI-MS analysis of SAG protein in a non-denaturing aqueous solution (pH 7) resulted in an increase in mass to 12733 and 12800 Da. The mass was consistent with the molecular weight of the holo-protein to which three and four metal ions bound, respectively.

In addition, the copper bonds of SAG were measured. Even if only 1 μM of CuSO ₄ was present in the dialysis solution, SAG precipitated blue (copper), which means bonding with copper. Subsequently, the potential copper binding of SAG in the non-modified solution was measured using ESI-MS as above. The addition of copper acetate to a final concentration of 10 μM further increased the mass to about 12929 Da. However, the exact mass could not be obtained because the copper adduct appeared to be widely distributed and bound. Higher concentrations of copper precipitated the protein.

Example 25. SAG minimizes or hinders LDL oxidation induced by copper ions or free radical generators.

Due to the hydrogen peroxide buffering action and metal binding properties, it is natural to think that SAG will prevent the oxidation of macromolecules induced by metals or free radical generators. LDL (low density lipoprotein) oxidation induced by copper or free radical generator (AAPH, 2,2-azobis-2-amidinopropane hydrochloride) tests potential protective activity against lipid peroxidation of SAG It was used as a model of.

Lipoproteins (100 μg protein per ml, Intraocel) were incubated at 37 ° C. for 4 hours with 10 μM CuSO ₄ or 5 mM AAPH in the presence of various concentrations of purified SAG protein. AAPH is a water-soluble azo compound that is decomposed by heat to generate water-soluble peroxyl radicals at a constant rate (Frei et al. 1988). 10 μM of butylated hydrochitoluene (BHT) was added to terminate the oxidation and refrigerated at 4 ° C. As described in Buege and Aust, 1978, the degree of oxidation of lipoproteins was determined by TBARS analysis using malondialdehyde (MDA) as a standard curve.

Copper-induced LDL oxidation, as measured by the formation of thiobarbituric acid reactive material (TBARS), was slightly increased by low concentrations of SAG. However, at higher SAG concentrations, dose-dependent inhibition (up to 90%) of LDL oxidation was observed. Since the heat treatment (for 15 minutes at 60 ° C.) SAG was still active, the inhibition phenomenon was heat resistant, meaning that the enzyme activity was not related. However, treatment of SAG with alkylating agents NEM and p-hydroxy mercury benzoic acid (PHMB), respectively, in advance completely or partially eliminated the inhibitory activity. The above results indicate that the free SH groups of SAG mainly contribute to this activity. In addition, metallothionein (Nordberg and Kojima, 1979), a small metal binding protein consisting of 61 amino acids containing 20 cysteine residues, also showed a similar inhibition curve to SAG. Glutathione (GSH), a peptide containing additional cysteine, showed 25% inhibition at 100 μM concentration. However, inhibition of copper-induced LDL oxidation was not observed with other known antioxidant enzymes such as superoxide dismutase, hydrogen peroxide degrading enzymes or other proteins (BSA and cytochrome C). The results indicate that by binding and chelating copper ions through free SH groups, SAG initiates a free radical reaction to induce LDL oxidation and superoxide or hydrogen peroxide may not be involved in the process. To test whether LDL oxidation prevention of SAG is completely mediated through the binding of copper, oxidation of LDL was initiated by free radical generator AAPH. The SAG also prevented LDL oxidation (up to 85%) at concentrations of 59 μM (750 μg / ml) even in the metal-ion free system. Thus, by the removal of metal bonds and free radicals, SAG prevents peroxidation of lipids.

Example 26. SAG prevents the release of cytochrome C and caspase activation by metal ions.

The release of cytochrome C and the activation of caspase in mitochondria were the major events of apoptosis (Liu et al., 1996, Yang et al., 1997, Li et al., 1997). ], [Hengartner, 1998] and for review (Mignotte and Bayssiere, 1998), caspase by release of cytochrome C into the cytoplasm and by metal treatment The potential level of activation of was determined. Treatment of the cells with zinc sulfate released cytochrome C into the cytoplasm in proportion to time. Compared to the vector control cells (D1-6), cells overexpressing SAG (D12-1) released much less cytochrome C. Similarly, the activation of caspase 7, which results in the disappearance of pro-enzyme, increased with time after zinc treatment. Compared to the vector control cells (D1-6), they were more activated in cells overexpressing SAG (D12-1). Similar results were obtained with the activation of CPP32 (caspase 3). However, there was a significant difference in cytochrome C release or caspase activation between D1-6 cells and D12-1 cells after treatment with copper. This is consistent with the fact that when the two cell lines were treated with copper, DNA degradation was obvious but no difference in morphological changes compared to the vector control cells. To further investigate metal-induced cytochrome C release and prevention of CPP32 activation of potential SAG, plasmids expressing SAG were transiently transfected to 293 cells and exposed to copper, followed by cytochrome C release and CPP32 activation. . After 6 hours of treatment with CuSO ₄ (2.0 mM) a significant amount of cytochrome C began to be released and lasted up to 12 hours. Expression of SAG delayed the release of cytochrome C by up to 16 hours. Activation of caspase 7 was observed in vector control cells 12 and 16 hours after treatment with copper. No special activation was observed in SAG transformants. Similar results were obtained with assays with CPP32 antibodies. When treated with zinc, no difference was detected in cytochrome C and caspase activation between control and transformant cells, consistent with the fact that there was no difference in the morphological signals of apoptosis. The results indicate that during apoptosis, metal treatment induces the release of cytochrome C and caspase activation, the phenomenon may be prevented or delayed by SAG, and the phenomenon (release of cytochrome C and caspase activation) is apoptotic. It is closely related to the morphological signal of the sheath.

Example 27. SAG protects neurons from apoptosis.

Human neuroblastoma cell HY5Y was transfected with SAG and a stable cell line (determined by Western blot) expressing exogenous SAG was selected. Sensitivity to metal ions (zinc and copper) was measured using one SAG-transformer (SYW-20) and vector control (SYV-3). Treatment with 1.25 mM CuSO ₄ or 200 μM ZnSO ₄ for 16 hours resulted in shrinking and falling of cells as compared to neomycin control cells, but slightly reduced in SAG-expressing cells. Morphological differences were more apparent when treated with zinc. To determine the nature of cell death, TUNEL analysis was performed to label the free 3′-OH terminus generated by cleavage of genomic DNA during apoptosis with fluorescein.

Cell death assay (TUNEL assay) in situ was performed according to manufacturer's instructions (Boehringer Mannheim). Briefly, 5 × 10 ⁴ cells were plated on 8-well glass slides. After treatment with 1.25 mM copper (CuSO ₄ ) or 200 μM zinc (ZnSO ₄ ), the cells were fixed for 0.5 min glutaraldehyde for 10 minutes and then washed twice with PBS. Fixed cells were incubated for 2 min in permeabilization solution (0.1% Triton X-100 and 0.1% sodium citrate) on ice. TUNEL reaction mixture (50 μl) was added to the sample, incubated at 37 ° C. for 1 hour, and then washed three times with PBS. Samples were embedded with antifade prior to analysis under fluorescence microscopy. After 16 hours of treatment with 1.25 mM CuSO ₄ or 200 μM ZnSO ₄ , substantially more fluorescein staining was observed in the vector control. The results indicate that expression of SAG protects neurons from apoptosis.

Example 28. SAG stimulates proliferation of cells.

To test for potential growth stimulating activity, SAG RNA (8 μg / ml or 25 μg / ml) was injected into serum-deficient NIH3T3 fibroblasts with β-galactosidase (25 μg / ml) control. Injected into approximately 50 cells attached within a clear circle of glass cover glass. A 3-hour pulse experiment with [ ³ H] thymidine (5 μCi / ml, Amersham) was performed 10-24 hours after injection. Cultures were washed in isotonic phosphate-buffered saline and fixed with 3.7% (volume / volume) of formaldehyde. The phenomenon of inducing [ ³ H] thymidine incorporation (which is an indicator of DNA synthesis) into the nucleus of serum-deficient fibroblasts was clearly observed in SAG-injected cells. In contrast, injection of β-galactosidase did not induce DNA synthesis and no incorporation of [ ³ H] thymidine was observed. The results clearly indicate that human SAG has proliferative activity that stimulates the growth of cells.

In addition, the growth promoting activity of SAG was investigated in human neuroblastoma cells overexpressing hSAG protein by transfection of hSAG cDNA. Both vector-expressing control cells and SAG overexpressing cells were first deficient in blood serum for 48 hours and then labeled with [ ³ H] thymidine for 16 hours in serum-deficient or 1% serum-supplied state. The cells were washed and lysed and the [ ³ H] count was counted with a liquid scintillation counter (analysis to determine the incorporation of [ ³ H] thymidine into DNA, indicating the beginning of the S-group). Compared to the vector control cells, SAG-expressing cells had a 10-fold increase in the incorporation of [ ³ H] thymidine under both conditions (serum-deficient or 1% serum), which SAG stimulated the proliferation and growth of cells. Means.

In addition, the growth promoting activity of SAG in yeast was investigated. As described in Example 22, yeast homologues of the human SAG gene are essential for the growth of yeast. To correlate the growth rate of yeast with the expression of SAG, hSAG expression plasmids were cloned under the control of the Gal promoter. The plasmid was transformed with a heterozygous ySAG knockout and excised to form a spore with a transformant. Haploid ySAG knockout clones containing hSAG plasmids were identified and analyzed. In the absence of inducing expression, small amounts of SAG expression due to promoter leakage induced the formation of small clones compared to full size wild type clones. In the expression-induced state, the expression level of SAG increased and clone size increased. The experiment clearly demonstrated that SAG promotes cell growth in proportion to dose.

Because obvious variations and equivalents will be apparent to those skilled in the art, it is to be understood that the invention is not limited to the details of the operation itself, or to the precise compounds, compositions, methods, processes or embodiments described, and therefore the invention is directed to the appended claims. Note that you are limited to the full scope of.

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Yi Sun

(B) STREET: 4841 Hillway Court

(C) CITY: Ann Arbor

(D) STATE: Michigan

(E) COUNTRY: USA

(F) POSTAL CODE (ZIP): 48105

(G) TELEPHONE: (313) 996-1959

(H) TELEFAX: (313) 996-7158

(ii) TITLE OF A: Sensitive to Apoptosis Gene (SAG)

(iii) NUMBER OF SEQUENCES: 50

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patent In Release # 1.0, Version # 1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1140 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 17..355

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 17..355

(ix) FEATURE:

(A) NAME / KEY: misc_feature

(B) LOCATION: 1..1140

(D) OTHER INFORMATION: / note = "Mouse SAG"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GTTCTGCGCC GCCGCC ATG GCC GAC GTG GAG GAC GGC GAG GAA CCC TGC 49

Met Ala Asp Val Glu Asp Gly Glu Glu Pro Cys

1 5 10

GTC CTT TCT TCG CAC TCC GGG AGC GCA GGC TCC AAG TCG GGA GGC GAC 97

Val Leu Ser Ser His Ser Gly Ser Ala Gly Ser Lys Ser Gly Gly Asp

15 20 25

AAG ATG TTC TCT CTC AAG AAG TGG AAC GCG GTA GCC ATG TGG AGC TGG 145

Lys Met Phe Ser Leu Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp

30 35 40

GAC GTT GAG TGC GAT ACC TGT GCC ATC TGC AGG GTC CAG GTG ATG GAT 193

Asp Val Glu Cys Asp Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp

45 50 55

GCC TGC CTT CGA TGT CAA GCT GAA AAC AAG CAA GAG GAC TGT GTT GTG 241

Ala Cys Leu Arg Cys Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val

60 65 70 75

GTC TGG GGA GAG TGT AAC CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG 289

Val Trp Gly Glu Cys Asn His Ser Phe His Asn Cys Cys Met Ser Leu

80 85 90

TGG GTG AAA CAG AAC AAT CGC TGC CCT CTG TGC CAG CAG GAC TGG GTA 337

Trp Val Lys Gln Asn Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val

95 100 105

GTC CAA AGA ATC GGC AAA TGAGAGGTGG CCCAGGCGCT CCTGGTGTGG 385

Val Gln Arg Ile Gly Lys

110

TTGCTGACCC TGGACAAAGA CTAAACACTG CAGGGGATTC ATCCTTGAGA GAGAGAGGAT 445

GCTGTGCGCC TTTGAGACTC ACCAAAGGCT TGCTTTATTA ATTTGTCTGT TTAGTTTTGG 505

GAAATTCTCT ACAATTAAGA TAATTTGTTA AAAATGGCCT TTCCTACCTC TGGTGTGTGT 565

GTGTGATACG AATGCATAGA AGAGCGAGAA CACCAGAAAA TGATCTTTGT TTATCTGTAC 625

CCACGACTGG AACATTGTGT TCACAGAAGA ACATTGTTTG TGTTTATGCT TGAGGGTTAA 685

AAAATAGATA AACGAATGTT ACAGTAACAA ATAAAATGCA TTGAAAAGCC GACTCCTCCT 745

AATCCTTTTT GTGTTGGGAG AGAGGCAAGC GAGGCCACCC TGCTGTCTTC ATTTGCTGTG 805

AATGAGGATT TTAACCTGCA CTCAGTGAAG AGGCGTAACT GTCGGGTAAA CTGTAATATG 865

GCGTAACTGT CGGGTAAACG GCTTTGTCTC CTGACTTCTC CATCTTTGAC TTGGCCAGGA 925

AGCCTGGATT GTTCAACCAC TTAGTTCTAA AGAACTGTTT TCTGTTTTTG CCGAAGGTTG 985

TATTGTATGT TTTAGTCAAA AATATTAGTA GGAAAATGGC TTACTAGTAT AACACTGAAG 1045

TTCATTATGC AATGTTTTAA TAAAATATTG TGCTTTGAGT TATTAAAGTT TGATATATAC 1105

TCTTAAAATC ATTAAACTAA TTCATCAATT AAATG 1140

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Ala Asp Val Glu Asp Gly Glu Glu Pro Cys Val Leu Ser Ser His

1 5 10 15

Ser Gly Ser Ala Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: misc_feature

(B) LOCATION: 1..754

(D) OTHER INFORMATION: / note = "Human SAG"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

AAT CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG GTG AAA CAG AAC 288

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749

TTAAA 754

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide P1

downstream primer "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

AAGCTTTTTT TTTTTTTR 18

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "Oligonucleotide: P2

upstream primer "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

AAGCTTNNNN NNN 13

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "Oligonucleotide SAG TA.01"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

CGGGATCCCC ATGGCCGACG TGAGG 25

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide SAG T.02"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

CGGGATCCTC ATTTGCCGAT TCTTTG 26

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide P.01"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

TATGGCTAGC ATGGCCGACG TGGAGG 26

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Gln Asn Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 747 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..270

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..270

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG TGC GCC ATC TGC AGG GTC CAG ATG CCT GTC TTA GAT GTC AAG CTG 192

Thr Cys Ala Ile Cys Arg Val Gln Met Pro Val Leu Asp Val Lys Leu

50 55 60

AAA ACA AAC AAG AGG ACT GTG TTG TGG TCT GGG GAG AAT GTA ATC ATT 240

Lys Thr Asn Lys Arg Thr Val Leu Trp Ser Gly Glu Asn Val Ile Ile

65 70 75 80

CCT TCC ACA ACT GCT GCA TGT CCC TGT GGG TGAAACAGAA CAATCGCTGC 290

Pro Ser Thr Thr Ala Ala Cys Pro Cys Gly

85 90

CCTCTCTGCC AGCAGGACTG GGTGGTCCAA AGAATCGGCA AATGAGAGTG GTTAGAAGGC 350

TTCTTAGCGC AGTTGTTCAG AGCCCTGGTG GATCTTGTAA TCCAGTGCCC TACAAAGGCT 410

AGAACACTAC AGGGGATGAA TTCTTCAAAT AGGAGCCGAT GGATCTGTGG TCTTTGGACT 470

CATCAAAGCC TTGGTTAGCA TTTGTCAGTT TTATCTTCAG AAATTCTCTG TGATTAAGAA 530

GATAATTTAT TAAAGGTGGT CCTTCCTACC TCTGTGGTGT GTGTCGCGCA CACAGCTTAG 590

AAGTGCTATA AAAAAGGAAA GAGCTCCAAA TTGAATCACC TTATAATTTA CCCATTTCTA 650

TACAACAGGC AGTGGAAGCA GTTTCGAGAC TTTTTCGATG CTTATGGTTG ATCAGTTAAA 710

AAAGAATGTT ACAGTAACAA ATAAAGTGCA GTTTAAA 747

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 90 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Cys Ala Ile Cys Arg Val Gln Met Pro Val Leu Asp Val Lys Leu

50 55 60

Lys Thr Asn Lys Arg Thr Val Leu Trp Ser Gly Glu Asn Val Ile Ile

65 70 75 80

Pro Ser Thr Thr Ala Ala Cys Pro Cys Gly

85 90

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 706 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..291

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..291

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GTG GTC TGG GGA GAA TGT 192

Thr Cys Ala Ile Cys Arg Val Gln Val Met Val Val Trp Gly Glu Cys

50 55 60

AAT CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG GTG AAA CAG AAC 240

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

65 70 75 80

AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 288

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

85 90 95

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 341

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 401

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 461

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 521

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 581

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 641

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 701

TTAAA 706

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 97 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Cys Ala Ile Cys Arg Val Gln Val Met Val Val Trp Gly Glu Cys

50 55 60

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

65 70 75 80

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

85 90 95

Lys

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide hSAG. M1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

GCCATCTGCA GGGTCCAG 18

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide SAG T.02L"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

GGATCCTCAT TTGCCGATTC TTTGGAC 27

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 58 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide

SAGKanMX4-5 "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

TTCTCCAGTG GCAGAGAACT TTAAAGAGAA ATAGTTCAAC CGTACGCTGC AGGTCGAC 58

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 59 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide SAGKan MX

4-3 "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

ACCTCGGTAT GATTTAAATG TTTACGGGCA ATTCATTTTT ATCGATGAAT TCGAGCTCG 59

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide SAG pcr 5"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

TTCTCCAGTG GCAGAGAAC 19

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide SAG pcr 3"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

ATGATTTAAA TGTTTACGGG C 21

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG AGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192

Thr Ser Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

AAT CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG GTG AAA CAG AAC 288

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749

TTAAA 754

(2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Ser Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG TGC GCC ATC AGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192

Thr Cys Ala Ile Ser Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

AAT CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG GTG AAA CAG AAC 288

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749

TTAAA 754

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Cys Ala Ile Ser Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG AGC GCC ATC AGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192

Thr Ser Ala Ile Ser Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

AAT CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG GTG AAA CAG AAC 288

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749

TTAAA 754

(2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Ser Ala Ile Ser Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

Asn His SerPhe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC AGT CTT AGA TGT 192

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Ser Leu Arg Cys

50 55 60

CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

AAT CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG GTG AAA CAG AAC 288

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749

TTAAA 754

(2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Ser Leu Arg Cys

50 55 60

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA AGT 240

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Ser

65 70 75 80

AAT CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG GTG AAA CAG AAC 288

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749

TTAAA 754

(2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Ser

65 70 75 80

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

AAT AAA TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG GTG AAA CAG AAC 288

Asn Lys Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749

TTAAA 754

(2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

Asn Lys Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

AAT CAT TCC TTC AAG AAC TGC TGC ATG TCC CTG TGG GTG AAA CAG AAC 288

Asn His Ser Phe Lys Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749

TTAAA 754

(2) INFORMATION FOR SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

Asn His Ser Phe Lys Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

AAT CAT TCC TTC CAC AAC TGC AGC ATG TCC CTG TGG GTG AAA CAG AAC 288

Asn His Ser Phe His Asn Cys Ser Met Ser Leu Trp Val Lys Gln Asn

85 90 95

AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749

TTAAA 754

(2) INFORMATION FOR SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

Asn His Ser Phe His Asn Cys Ser Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

AAT CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG GTG AAA CAG AAC 288

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

AAT CGC AGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336

Asn Arg Ser Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749

TTAAA 754

(2) INFORMATION FOR SEQ ID NO: 38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Ser Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

AAT AAA TCC TTC AAG AAC TGC TGC ATG TCC CTG TGG GTG AAA CAG AAC 288

Asn Lys Ser Phe Lys Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749

TTAAA 754

(2) INFORMATION FOR SEQ ID NO: 40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

Asn Lys Ser Phe Lys Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA AGT 192

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Ser

50 55 60

CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

AAT CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG GTG AAA CAG AAC 288

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749

TTAAA 754

(2) INFORMATION FOR SEQ ID NO: 42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Ser

50 55 60

Gln Ala Glu AsnLys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

CAA GCT GAA AAC AAA CAA GAG GAC AGT GTT GTG GTC TGG GGA GAA TGT 240

Gln Ala Glu Asn Lys Gln Glu Asp Ser Val Val Val Trp Gly Glu Cys

65 70 75 80

AAT CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG GTG AAA CAG AAC 288

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749

TTAAA 754

(2) INFORMATION FOR SEQ ID NO: 44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

Gln Ala Glu Asn Lys Gln Glu Asp Ser Val Val Val Trp Gly Glu Cys

65 70 75 80

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC AGT CTT AGA AGT 192

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Ser Leu Arg Ser

50 55 60

CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

AAT CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG GTG AAA CAG AAC 288

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749

TTAAA 754

(2) INFORMATION FOR SEQ ID NO: 46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Ser Leu Arg Ser

50 55 60

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

AAT CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG GTG AAA CAG AAC 288

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

AAT CGC AGC CCT CTC AGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336

Asn Arg Ser Pro Leu Ser Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749

TTAAA 754

(2) INFORMATION FOR SEQ ID NO: 48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp

35 40 45

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Ser Pro Leu Ser Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

(2) INFORMATION FOR SEQ ID NO: 49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 754 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..339

(ix) FEATURE:

(A) NAME / KEY: mat_peptide

(B) LOCATION: 1..339

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:

ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG AGC GAT 144

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Ser Asp

35 40 45

ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

AAT CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG GTG AAA CAG AAC 288

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389

Lys

TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449

GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509

ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569

TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629

GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689

TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749

TTAAA 754

(2) INFORMATION FOR SEQ ID NO: 50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 113 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:

Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His

1 5 10 15

Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu

20 25 30

Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Ser Asp

35 40 45

Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys

50 55 60

Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys

65 70 75 80

Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn

85 90 95

Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly

100 105 110

Lys

Claims (37)

An isolated and purified DNA sequence substantially similar to the DNA sequence set forth in SEQ ID NO: 1. An isolated and purified DNA sequence that hybridizes to the DNA sequence set forth in SEQ ID NO: 1 under highly stringent hybridization conditions. An isolated and purified DNA sequence consisting essentially of the DNA sequence set forth in SEQ ID NO: 1. A recombinant DNA comprising the isolated and purified DNA sequence of any one of claims 1 to 3 subcloned into an extrachromosomal vector. A recombinant host cell comprising a host cell transfected with the recombinant DNA molecule of claim 4. Recombinant host cell deposited with ATCC under Accession No. 98402. An isolated and purified DNA sequence substantially similar to the DNA sequence set forth in SEQ ID NO: 3. An isolated and purified DNA sequence that hybridizes to the DNA sequence set forth in SEQ ID NO: 3 under highly stringent hybridization conditions. An isolated and purified DNA sequence consisting essentially of the DNA sequence set forth in SEQ ID NO: 3. A recombinant DNA comprising the isolated and purified DNA sequence of any one of claims 7 to 9 subcloned into an extrachromosomal vector. A recombinant host cell comprising a host cell transfected with the recombinant DNA molecule of claim 10. Recombinant host cell deposited with ATCC under Accession No. 98403. Recombinant host cell deposited with ATCC under Accession No. 98404. Recombinant host cell deposited with ATCC under Accession No. 98405. SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 49 Isolated and purified DNA sequence selected from the group consisting of. A recombinant DNA comprising the isolated and purified DNA sequence of claim 15 subcloned into an extrachromosomal vector. A recombinant host cell comprising a host cell transfected with the recombinant DNA molecule of claim 16. A substantially purified recombinant polypeptide, wherein the amino acid sequence of the substantially purified recombinant polypeptide is substantially similar to the amino acid sequence set forth in SEQ ID NO: 2. A substantially purified recombinant polypeptide, wherein the amino acid sequence of the substantially purified recombinant polypeptide consists essentially of the amino acid sequence set forth in SEQ ID NO: 2. A substantially purified recombinant polypeptide, wherein the amino acid sequence of the substantially purified recombinant polypeptide is substantially similar to the amino acid sequence set forth in SEQ ID NO: 4. A substantially purified recombinant polypeptide, wherein the amino acid sequence of the substantially purified recombinant polypeptide consists essentially of the amino acid sequence set forth in SEQ ID NO: 4. The amino acid sequence of the polypeptide is SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, A substantially purified recombinant polypeptide selected from the group consisting of SEQ ID NO: 48 and SEQ ID NO: 50. An antibody that selectively binds to a polypeptide having an amino acid sequence substantially similar to the amino acid sequence of claim 18. A method for detecting SAG protein in a cell, comprising culturing the cell in a manner capable of contacting the cell with the antibody of claim 23 and detecting the SAG protein-antibody complex. Isolating genomic DNA from a cell and using the primers derived from the isolated and purified DNA sequences of claims 1, 2, 3, 7, 8, 9 or 15. A diagnostic assay for detecting cells comprising a SAG mutation, comprising amplifying by PCR and examining whether the resulting PCR product contains a mutation. Isolating the entire RNA of the cell from the cell, the RNA with primers derived from the isolated and purified DNA sequences of claims 1, 2, 3, 7, 8, 9 or 15. A method of diagnostic analysis for detecting cells comprising a SAG mutation, comprising reverse transcription-PCR amplification to examine whether the resulting PCR product contains a mutation. (a) contacting the RNA sample with the SAG protein in RNA binding buffer in the presence of a reducing agent, (b) incubating the RNA-SAG protein mixture with the antibody of claim 23, (c) isolating the antibody-SAG protein-RNA complex, and (d) purifying RNA from the antibody-SAG protein complex A method for isolating RNA comprising an extension of a polyA or polyC residue comprising a. (a) contacting an RNA sample with a SAG protein in an RNA binding buffer free of reducing agents, (b) incubating the RNA-SAG protein mixture with the antibody of claim 23, (c) isolating the antibody-SAG protein-RNA complex, and (d) purifying RNA from the antibody-SAG protein complex A method of isolating RNA comprising an extension of a polyU residue, comprising. (a) treating a set of cells with OP and not a control set of cells, (b) isolating RNA from each set of cells, (c) using differential RNA isolated from each set of cells, undergoing a differential display process of reverse transcription of the RNA into cDNA and polymerase chain reaction thereof; (d) identifying cDNA not expressed in cells of the control set but expressed in cells of the set treated with OP, and (e) cloning the cDNA induced by OP Isolation method comprising the gene induced during cellular apoptosis, comprising. Claims 1, 2, 3, 7, 8, 9, or 15, operatively linked to DNA sequences that promote high levels of expression of isolated and purified DNA sequences in cells. A method of protecting a cell from apoptosis induced by a redox agent, comprising introducing into the cell an expression vector comprising the isolated and purified DNA sequence of the term. Claims 1, 2, 3, 7, 8, 9 operably linked to DNA sequences that promote high levels of expression of the antisense strands of isolated and purified DNA sequences in cells. Or introducing into the tumor cell an expression vector comprising the isolated and purified DNA sequence of claim 15. (a) Isolation of claims 1, 2, 3, 7, 8, 9 or 15, operatively linked to a promoter capable of inducing expression of a gene in a bacterial host cell. And transforming the bacterial host cell with a vector comprising the purified DNA sequence, (b) expressing said isolated and purified DNA sequence in a bacterial cell, (c) lysing the bacterial cells, (d) isolating an inclusion body of bacteria, and (e) purifying the SAG protein from the isolated inclusion body A method of purifying SAG protein from bacterial cells, comprising. A pharmaceutical composition comprising the substantially purified recombinant polypeptide of any one of claims 18-22 and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 33, wherein the substantially purified recombinant polypeptide comprises an oligomer. 35. A method for removing oxygen radicals from an organism comprising administering to the organism an amount of reducing the oxygen radicals of the pharmaceutical composition of claim 33 or 34. A method of promoting wound treatment comprising administering the DNA sequence of claim 1 to a cell associated with the wound. A method of promoting or inhibiting growth of a plant cell, comprising administering to the plant cell the DNA sequence of claim 1 or the DNA sequence complementary to claim 1.