AU765741B2

AU765741B2 - Sag: sensitive to apoptosis gene

Info

Publication number: AU765741B2
Application number: AU19180/99A
Authority: AU
Inventors: Yi Sun
Original assignee: Warner Lambert Co LLC
Current assignee: Warner Lambert Co LLC
Priority date: 1997-12-19
Filing date: 1998-12-15
Publication date: 2003-09-25
Anticipated expiration: 2018-12-15
Also published as: CA2303483A1; WO1999032514A3; KR20010033299A; EP1044217A2; WO1999032514A2; AU1918099A; NZ503417A; JP2001526063A; BR9813757A

Description

-1- SAG: SENSITIVE TO APOPTOSIS GENE Background of the Invention IThe p 11reeL iIVenILUn relates t LU Inov gIee a polypepUtdes derived therefrom encoding a redox-sensitive protein that protects cells from apoptosis and promotes cell growth, as well as antibodies directed against the polypeptide. The invention also describes methods for using the novel gene, polypeptides, and antibodies in the detection of genetic deletions of the gene, subcellular localization of the polypeptide, isolation of discrete classes of RNA, inhibition of apoptosis, scavenging of oxygen radicals, reversion of tumor phenotype, and therapeutic applications by gene therapy.

Summary of the Related Art The following description of the prior art is provided so that the present invention may be more fully understood and appreciated in its technical context and its 15 significance more fully appreciated. Unless clearly indicated to the contrary, however, this discussion is not, and should not be interpreted as, an express or implied admission that any of the prior art referred to is widely known or forms part of the common general knowledge in the field.

Apoptosis, also referred to as programmed cell death, is a genetically programmed process for maintaining homeostasis under physiological conditions and for responding to various stimuli (Thompson (1995) Science 267, 1456-1462). This form of cell death is characterized by cell membrane blebbing, cytoplasmic shrinkage, nuclear chromatin condensation, and DNA fragmentation (Wyllie (1980) Int. Rev. Cytol. 68, 251-306). The process of apoptosis can be divided into three distinct phases: initiation, 25 effector molecule stimulation and DNA degradation (Kroemer et al. (1995) FASEB J. 9, 1277-1287; Vaux and Strasser (1996) Proc. Natl. Acad. Sci. USA 93, 2239-2244).

Apoptosis can be initiated in various cell types by a wide variety of physical, chemical, and biological stimuli (both internal and external), including diverse cancer therapeutic drugs, oxidative DNA damage reagents, and cytokines (Kroemer (1997) Nature Med. 3, 416AUPOO.DOC -2ainduction will allow improved design of therapeutic drugs that either induce (anticancer) or inhibit (anti-aging) apoptosis.

Summary of the Invention The present invention provides novel genes and polypeptides derived therefrom encoding a redox-sensitive protein that protects cells from apoptosis, scavenges oxygen radicals, and can be used for the reversion of a tumor phenotype.

According to a first aspect, the present invention provides an isolated and purified DNA sequence having more than 70% sequence identity to SEQ ID NO: 1.

According to a second aspect, the present invention provides an isolated and purified DNA sequence that hybridizes to the DNA sequence shown in SEQ ID 1 under high stringency hybridization conditions.

According to a third aspect, the present invention provides an isolated and purified DNA sequence encoding a protein that protects cells from apoptosis and wherein said sequence is substantially similar to the DNA sequence shown in SEQ ID NO:1.

According to a fourth aspect, the present invention provides a recombinant DNA molecule comprising the isolated and purified DNA sequence according to any one of the first to third aspects subcloned into an extra-chromosomal vector.

20 According to a fifth aspect, the present invention provides a recombinant host cell comprising a host cell transfected with the recombinant DNA molecule according to the fourth aspect.

According to a sixth aspect, the present invention provides a recombinant host cell deposited with the ATCC under accession number 98402.

25 According to a seventh aspect, the present invention provides an isolated and purified DNA sequence having more than more than 70% sequence identity to SEQ ID •oooeo NO:3.

According to an eighth aspect, the present invention provides an isolated and "0.i purified DNA sequence that hybridizes to the DNA sequence shown in SEQ ID NO:3 under high stringency hybridization conditions.

2b According to a ninth aspect, the present invention provides an isolated and purified DNA sequence encoding a protein that protects cells from apoptosis and wherein said sequence is substantially similar to the DNA sequence shown in SEQ ID NO:3.

According to a tenth aspect, the present invention provides a recombinant DNA molecule comprising the isolated and purified DNA sequence according to any one of the seventh to ninth aspects subcloned into an extra-chromosomal vector.

According to an eleventh aspect, the present invention provides a recombinant host cell comprising a host cell transfected with the recombinant DNA molecule according to the tenth aspect.

According to a twelfth aspect, the present invention provides a recombinant host cell deposited with the ATCC under accession number 98403.

According to a thirteenth aspect, the present invention provides a recombinant host cell deposited with the ATCC under accession number 98404.

According to a fourteenth aspect, the present invention provides a recombinant host cell deposited with the ATCC under accession number 98405.

According to a fifteenth aspect, the present invention provides an isolated and purified DNA sequence selected from the group consisting of 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 20 NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID S•NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, and SEQ ID NO:49.

According to a sixteenth aspect, the present invention provides a recombinant DNA molecule comprising an isolated and purified DNA sequence according to the 25 fifteenth aspect, subcloned into an extra-chromosomal vector.

According to a seventeenth aspect, the present invention provides a recombinant host cell comprising a host cell transfected with a recombinant DNA molecule according to the sixteenth aspect.

According to an eighteenth aspect, the present invention provides a substantially purified recombinant polypeptide, encoding a protein that protects cells from apoptosis, and wherein the amino acid sequence of the substantially purified recombinant polypeptide is substantially similar to the amino acid sequence shown in SEQ ID NO:2.

2c According to a nineteenth aspect, the present invention provides a substantially purified recombinant polypeptide, wherein the amino acid sequence of the substantially purified recombinant polypeptide has more than 70% sequence identity to SEQ ID NO:2.

According to a twentieth aspect, the present invention provides a substantially purified recombinant polypeptide, encoding a protein that protects cells from apoptosis, and wherein the amino acid sequence of the substantially purified recombinant polypeptide is substantially similar to the amino acid sequence shown in SEQ ID NO:4.

According to a twenty-first aspect, the present invention provides a substantially purified recombinant polypeptide, wherein the amino acid sequence of the substantially purified recombinant polypeptide has more than 70% sequence identity to SEQ ID NO:4.

According to a twenty-second aspect, the present invention provides a substantially purified recombinant polypeptide, wherein the amino acid sequence of the polypeptide is selected from the group consisting of 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 According to a twenty-third aspect, the present invention provides an antibody that selectively binds polypeptides with an amino acid sequence substantially similar to the amino acid sequence according to any one of the eighteenth to twenty-second 20 aspects.

According to a twenty-fourth aspect, the present invention provides a method of detecting SAG protein in cells, comprising contacting cells with the antibody according to the twenty-third aspect and incubating the cells in a manner that allows for detection of the SAG protein-antibody complex.

25 According to a twenty-fifth aspect, the present invention provides a diagnostic assay for detecting cells containing SAG mutations, comprising isolating total genomic DNA from the cell and subjecting the genomic DNA to PCR amplification using primers o derived from the isolated and purified DNA sequence according to any one of the first, second, third, seventh, eighth, ninth or fifteenth aspects, and determining whether the resulting PCR product contains a mutation.

2d- According to a twenty-sixth aspect, the present invention provides a diagnostic assay for detecting cells containing SAG mutations, comprising isolating total cell RNA, subjecting the RNA to reverse transcription-PCR amplification using primers derived from the isolated and purified DNA sequence according to any one of the first, second, third, seventh, eighth, ninth or fifteenth aspects, and determining whether the resulting PCR product contains a mutation.

According to a twenty-seventh aspect, the present invention provides a method of isolating RNA containing stretches of polyA or polyC residues, comprising: contacting an RNA sample with SAG protein in RNA binding buffer in the presence of a reducing agent; incubating the RNA-SAG protein mixture with the antibody according to the twenty-third aspect; isolating the antibody-SAG protein-RNA complexes; and purifying the RNA away from the antibody-SAG protein complex.

According to a twenty-eighth aspect, the present invention provides a method of isolating RNA containing stretches of polyU residues, comprising contacting an RNA sample with SAG protein in RNA binding buffer in oo ~the absence of reducing agents; incubating the RNA-SAG protein mixture with the antibody according to S 20 the twenty-third aspect; isolating the antibody-SAG protein-RNA complexes; and purifying the RNA away from the antibody-SAG protein complex.

According to a twenty-ninth aspect, the present invention provides a method for isolating genes induced during cell apoptosis, comprising: 25 treating one set of cells with OP and not treating a control set of cells; isolating RNA from each set of cells; subjecting the RNA from each set of cells to the differential display procedure, wherein the RNA is reverse transcribed into cDNA and the S"cDNA is subjected to the polymerase chain reaction; o e identifying cDNAs that are expressed in the OP-treated set of cells and not in the control set of cells; and cloning the OP-induced cDNAs.

2e According to a thirtieth aspect, the present invention provides a method for protecting cells from apoptosis induced by redox reagents, comprising introducing into the cells an expression vector comprising the isolated and purified DNA sequence according to any one of the first, second, third, seventh, eighth, ninth or fifteenth aspects, which is operatively linked to a DNA sequence that promotes the high level expression of the isolated and purified DNA sequence in the cells.

According to a thirty-first aspect, the present invention provides a method for inhibiting the growth of tumor cells, comprising introducing into the tumor cells an expression vector comprising the isolated and purified DNA sequence according to any one of the first, second, third, seventh, eighth, ninth or fifteenth aspects, which isoperatively linked to a DNA sequence that promotes the high level expression of the antisense strand of the isolated and purified DNA sequence in the cells.

According to a thirty-second aspect, the present invention provides a method for purifying SAG protein from bacterial cells comprising: a) transfecting a bacterial host cell with a vector comprising the isolated and purified DNA sequence according to any one of the first, second, third, seventh, eighth, ninth or fifteenth aspects operatively linked to a promoter S. capable of directing gene expression in a bacterial host cell; b) inducing expression of the isolated and purified DNA sequence in the 20 bacterial cells; c) lysing the bacterial cells; d) isolating bacterial inclusion bodies; e) purifying SAG protein from the isolated inclusion bodies.

According to a thirty-third aspect, the present invention provides a 25 pharmaceutical composition comprising the substantially purified recombinant polypeptide according to any one of the eighteenth to twenty-second aspects and a pharmaceutically acceptable carrier.

:o°.oAccording to a thirty-fourth aspect, the present invention provides a method of S' oxygen radical scavenging in an organism comprising administering an oxygen radicalreducing amount of the pharmaceutical composition according to the thirty-third aspect to the organism.

2f- According to a thirty-fifth aspect, the present invention provides a method of promoting the healing of a wound comprising administering the DNA sequence according to the first aspect to cells associated with the wound.

According to a thirty-sixth aspect, the present invention provides a method of promoting or inhibiting the growth of plant cells comprising administering the DNA sequence according to the first aspect or a DNA sequence which is complementary to the DNA sequence according to the first aspect to plant cells.

According to a thirty-seventh aspect, the present invention provides use of an expression vector comprising the isolated and purified DNA sequence according to any one of the first, second, third, seventh, eighth, ninth or fifteenth aspects in the manufacture of a medicament for protecting cells from apoptosis induced by redoxreagents, wherein the isolated and purified DNA sequence according to any one of the first, second, third, seventh, eighth, ninth or fifteenth aspects is operatively linked to a DNA sequence that promotes the high level expression of the isolated and purified DNA sequence in the cells.

According to a thirty-eighth aspect, the present invention provides use of an expression vector comprising the isolated and purified DNA sequence according to any o ne of the first, second, third, seventh, eighth, ninth or fifteenth aspects in the manufacture of a medicament for inhibiting the growth of tumor cells, wherein the 20 isolated and purified DNA sequence according to any one of the first, second, third, seventh, eighth, ninth or fifteenth aspects is operatively linked to a DNA sequence that promotes the high level expression of the antisense strand of the isolated and purified DNA sequence in the cells.

According to a thirty-ninth aspect, the present invention provides use of the DNA 25 sequence according to the first aspect in the manufacture of a medicament for promoting the healing of a wound, wherein the DNA sequence is administered to cells associated with the wound.

According to a fortieth aspect, the present invention provides SAG protein when detected in cells by a method according to the twenty-fourth aspect.

According to a forty-first aspect, the present invention provides cells containing SAG mutations when detected using the diagnostic assay according to the twenty-fifth or twenty-sixth aspect.

2g- According to a forty-second aspect, the present invention provides RNA containing stretches of polyA or polyC residues identified by a method according to the twenty-seventh aspect when used to bind to a SAG protein wherein the ability of the RNA to bind to SAG protein was not previously known, wherein the stretch of polyA or polyC residues was not previously known.

According to a forty-third aspect, the present invention provides RNA containing stretches of polyU residues when isolated by a method according to the twenty-eighth aspect.

According to a forty-fourth aspect, the present invention provides a gene induced during cell apoptosis when isolated by a method according to the twenty-ninth aspect, wherein said gene was not previously known.

According to a forty-fifth aspect, the present invention provides SAG protein when purified from bacterial cells by a method according to the thirty-second aspect.

In a further aspect, the present invention provides novel isolated and purified DNA sequences (referred to herein as "mouse SAG" and "human SAG") as shown in SEQ ID NO:1 and SEQ ID NO:3, and their gene products (referred to herein as "mouse SAG protein" and "human SAG protein") as shown in SEQ ID NO:2 and SEQ ID NO:4, that are induced during 1,10-phenanthroline ("OP")-induced apoptosis. (Mouse cDNA Sencoding SAG (mSAG) and human cDNA encoding SAG (hSAG) were deposited with 20 the American Type Culture Collection on 10 April 1997 under Accession Numbers 98402 and 98405, respectively.) In another embodiment, the present invention comprises a nucleotide sequence that hybridizes to the nucleotide sequence shown in SEQ ID NO:1 and SEQ ID NO:3 under high stringency hybridization conditions. In a preferred embodiment, the isolated and purified DNA sequence consists essentially of 25 the DNA sequence of SEQ ID NO:1 or SEQ ID NO:3.

q WO 99/32514 PCT/US98/6705 In another aspect, the invention provides novel recombinant DNA molecules, comprising SAG subcloned into an extra-chromosomal vector. In a further aspect, the present invention provides recombinant host cells that are stably transfected with a recombinant DNA molecule comprising SAG subcloned into an extra-chromosomal vector.

In a different aspect, the present invention provides a substantially purified recombinant protein comprising a polypeptide substantially similar to the SAG protein shown in SEQ ID 2 and SEQ ID 4. In a further aspect, the present invention provides a polyclonal antibody that selectively binds to proteins with an amino acid sequence substantially similar to the amino acid sequence shown in SEQ ID 2 and SEQ ID 4.

Additional aspects of the present invention provide a method of detecting the SAG protein in cells, comprising contacting cells with a polyclonal antibody that recognizes the SAG protein; a method of detecting cells containing SAG deletions, comprising isolating total genomic DNA from the cell and subjecting the genomic DNA to PCR amplification using primers derived from the DNA sequence of SEQ ID 1 and SEQ ID 3; and a method of detecting cells containing SAG deletions, comprising isolating total cell RNA and subjecting the RNA to reverse transcription-PCR amplification using primers derived from the DNA sequence of SEQ ID 1 and SEQ ID 3.

In another aspect, the present invention further provides methods of isolating RNA containing stretches of polyA, polyC, or polyU residues from cells, contacting the total cell RNA with the SAG protein, and incubating the RNA-SAG protein mixture with an antibody that recognizes the SAG protein.

In another aspect of the present invention, a method for isolating genes induced during cell apoptosis is provided, comprising treating cells with OP, subjecting the OPinduced RNA to the differential display procedure, and cloning the OP-induced genes.

A further aspect of the invention provides a method for protecting mammalian and/or non-mammalian cells from apoptosis induced by redox reagents, comprising introducing into mammalian and/or non-mammalian cells an expression vector comprising a DNA sequence substantially similar to the DNA sequence shown in SEQ ID 1 and SEQ ID 3, which is operatively linked to a DNA sequence that promotes the expression of the DNA sequence, wherein the isolated and purified DNA sequence of SEQ ID 1 and SEQ ID 3 will be expressed at high levels in the mammalian and/or non-mammalian cells.

An additional aspect of the present invention provides a method for treatment of mammalian and/or non-mammalian tumor cells, comprising introducing into mammalian and/or non-mammalian tumor cells an expression vector comprising a DNA sequence substantially similar to the DNA sequence shown in SEQ ID 1 and SEQ ID 3, which is operatively linked to a DNA sequence that promotes the expression of the antisense strand of the DNA sequence, wherein the antisense strand of the DNA sequence of SEQ D i and SEQ D 3 will be expressed at high levels in the mammalian andior nonmammalian cells.

Another aspect of the present invention provides a method for oxygen radical scavenging in an organism, comprising administering an oxygen radical-reducing amount of a pharmaceutical composition comprising SAG protein and a pharmaceutically acceptable carrier.

A further aspect of the present invention provides for gene therapy applications of SAG, including but not limited to methods of promoting the closure healing) of a wound in a patient.

The foregoing is not intended and should not be construed as limiting the invention in any way. All patents and publications cited herein are incorporated by reference in their entirety.

Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

Brief Description of the Drawings 9o,: Figure 1A. Predicted structural features of the deduced protein sequence of the mouse and human SAG cDNA.

:Figure lB. Description of human SAG protein mutants.

i 25 Figure 2. Bar graph depiction of soft agar colony growth of various SAG-transfected stable cell lines.

Figure 3. Graphical representation of tumor mass in SCID mice per days post implant with SAG transfectants.

Detailed Description of the Invention The present invention provides novel genes and polypeptides derived therefrom encoding a redox-sensitive protein that protects cells from apoptosis, scavenges oxygen 416AUPOO.DOC 4a radicals, and can be used for the reversion of a tumor phenotype. The present invention also comprises genes and their gene products involved in OP-induced apoptosis. The isolation of such genes and their gene products permits a detailed analysis of the OPinduced apoptotic pathway, thus providing laboratory tools useful to identify the mechanisms of OP-induced apoptosis and enabling improved design of therapeutic drugs to regulate apoptosis.

Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual

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416AUPOO.DOC WO 99/32514 PCT/US98/26705 (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology Deutshcer, ed., (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, 2 n d Ed. Freshney. 1987. Liss, Inc. New York, NY), and Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, In one aspect, the present invention provides novel isolated and purified DNA sequences, hereinafter referred to as Sensitive to Apoptosis Genes encoding SAG proteins. In one embodiment, the invention comprises DNA sequences substantially similar to those shown in SEQ ID 1 (mouse SAG) or SEQ ID 2 (human SAG), respectively. As defined herein, "substantially similar" includes identical sequences, as well as deletions, substitutions or additions to a DNA, RNA or protein sequence that maintain the function of the protein product and possess similar zinc-binding motifs. Preferably, the DNA sequences according to the invention consist essentially of the DNA sequence of SEQ ID 1 or SEQ ID 3, or are selected from the group consisting of SEQ ID 11, SEQ ID 13, SEQ ID 21, SEQ ID 23, SEQ ID 25, SEQ ID 27, SEQ ID 29, SEQ ID 31, SEQ ID 33, SEQ ID 35, SEQ ID 37, SEQ ID 39, SEQ ID 41, SEQ ID 43, SEQ ID 45, SEQ ID 47 and SEQ ID 49. These novel purified and isolated DNA sequences can be used to direct expression of the SAG protein and for mutational analysis of SAG protein function.

Mutated sequences according to the invention can be identified in a routine manner by those skilled in the art using the teachings provided herein, as described in Example 8, infra, and techniques well known in the art.

In another embodiment, the invention comprises a nucleotide sequence that hybridizes to SEQ ID 1 and/or SEQ ID 3 under high stringency hybridization conditions. As used herein, the term "high stringency hybridization conditions" refers to hybridization at 65 0 C in a low salt hybridization buffer to the probe of interest at 2 x 108 cpm/g for between about 8 hours to 24 hours, followed by washing in 1% SDS, 20 mM phosphate buffer and 1 mM EDTA at 65 0 C, for between about 30 minutes to 4 hours. In a preferred embodiment, the low salt hybridization buffer comprises between, 0.5-10% SDS, and 0.05M and 0.5 M sodium phosphate. In a most preferred embodiment, the low salt hybridization buffer comprises, 7% SDS, and 0.125M sodium phosphate. These DNA sequences can be used to direct expression WO 99/32514 PCT/US98/26705 of the SAG protein and for mutational analysis of SAG protein function, and are isolated via hybridization as described.

In another aspect, the invention provides novel recombinant DNA molecules, comprising SAG or a sequence substantially similar to it subcloned into an extrachromosomal vector. This aspect of the invention allows for in vitro expression of the SAG gene, thus permitting an analysis of SAG gene regulation and SAG protein structure and function. As used herein, the term "extra-chromosomal vector" includes, but is not limited to, plasmids, bacteriophages, cosmids, retroviruses and artificial chromosomes. In a preferred embodiment, the extra-chromosomal vector comprises an expression vector that allows for SAG protein production when the recombinant DNA molecule is inserted into a host cell. Such vectors are well known in the art and include, but are not limited to, those with the T3 or T7 polymerase promoters, the SV40 promoter, the CMV promoter, or any promoter that either can direct gene expression, or that one wishes to test for the ability to direct gene expression. These recombinant vectors are produced via standard recombinant DNA protocols as described in the references cited above. This aspect of the invention allows for high level expression of the SAG protein.

In a further aspect, the present invention provides recombinant host cells that are stably transfected with a recombinant DNA molecule comprising SAG subcloned into an extra-chromosomal vector. The host cells of the present invention may be of any type, including, but not limited to, non-eukaryotic bacterial), and eukaryotic such as fungal yeast), plant, non-human animal, non-human mammalian rabbit, porcine, mouse, horse) and human cells. Transfection of host cells with recombinant DNA molecules is well known in the art (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2 nd ed., Cold Spring Harbor Press, 1989) and, as used herein, includes, but is not limited to calcium phosphate transfection, dextran sulfate transfection, electroporation, lipofection and viral infection. This aspect of the invention allows for in vitro and in vivo expression of SAG and its gene product, thus enabling high-level expression of SAG protein, as described in Example 6, infra.

In another aspect, the present invention provides a substantially purified recombinant 30 protein comprising a polypeptide substantially similar to the SAG polypeptides shown in SEQ ID 2 and SEQ ID 4. Furthermore, this aspect of the invention enables the use of SAG protein in several in vitro assays described below. As used herein, the term "substantially similar" includes deletions, substitutions and additions to the sequences of SEQ IDs 1-4 (as WO 99/32514 PCT/US98/26705 appropriate) introduced by any in vitro means. As used herein, the term "substantially purified" means that the protein should be free from detectable contaminating protein, but the SAG protein may be co-purified with an interacting protein, or as an oligomer. Preferably, the protein sequences according to the invention comprise an amino acid sequence selected from the group consisting of SEQ ID 2, SEQ ID 4, SEQ ID 12, SEQ ID 14, SEQ ID 22, SEQ ID 24, SEQ ID 26, SEQ ID 28, SEQ ID 30, SEQ ID 32, SEQ ID 34, SEQ ID 36, SEQ ID 38, SEQ ID 40, SEQ ID 42, SEQ ID 44, SEQ ID 46, SEQ ID 48, and SEQ ID 50. In a most preferred embodiment, the protein sequences according to the invention comprise an amino acid sequence selected from the group consisting of SEQ ID 2 and SEQ ID 4. Mutated sequences according to the invention can be identified in a routine manner by those skilled in the art using the teachings provided herein and techniques well known in the art. This aspect of the invention provides a novel purified protein that can be used for in vitro assays, as described in Examples 12, infra, and as a component of a pharmaceutical composition for oxygen radical scavenging, described infra.

In a further aspect, the present invention provides antibodies and methods for detecting antibodies that selectively bind polypeptides with an amino acid sequence substantially similar to the amino acid sequence of SEQ ID 2 and SEQ ID 4. The antibody of the present invention can be a polyclonal or a monoclonal antibody, prepared by using all or part of the sequence of SEQ ID 2 or SEQ ID 4, or modified portions thereof, to elicit an immune response in a host animal according to standard techniques (Harlow and Lane (1988), eds. Antibody: A Laboratory Manual, Cold Spring Harbor Press). In a preferred embodiment, the entire polypeptide sequence of SEQ ID 2 or SEQ ID 4 is used to elicit the production of polyclonal antibodies in a host animal.

The method of detecting SAG antibodies comprises contacting cells with an antibody that recognizes SAG protein and incubating the cells in a manner that allows for detection of the SAG protein-antibody complex. Standard conditions for antibody detection of antigen can be used to accomplish this aspect of the invention (Harlow and Lane, 1988). This aspect of the invention permits the detection of SAG protein both in vitro and in vivo, as described in Examples 12 and 14, infra.

In a further aspect, the present invention provides a diagnostic assay for detecting cells containing SAG deletions, comprising isolating total genomic DNA from the cell and subjecting the genomic DNA to PCR amplification using primers derived from the DNA sequence of SEQ ID 1 SEQ ID 3, SEQ ID 11, SEQ ID 13, SEQ ID 21, SEQ ID 23, SEQ WO 99/32514 PCT/US98/26705 ID 25, SEQ ID 27, SEQ ID 29, SEQ ID 31, SEQ ID 33, SEQ ID 35, SEQ ID 37, SEQ ID 39, SEQ ID 41, SEQ ID 43, SEQ ID 45, SEQ ID 47 and SEQ ID 49.

This aspect of the invention enables the detection of SAG deletions in any type of cell, and can be used in genetic testing or as a laboratory tool. The PCR primers can be chosen in any manner that allows the amplification of a SAG gene fragment large enough to be detected by gel electrophoresis. Detection can be by any method, including, but not limited to ethidium bromide staining of agarose or polyacrylamide gels, autoradiographic detection of radio-labeled SAG gene fragments, Southern blot hybridization, and DNA sequence analysis. In a preferred embodiment, detection is accomplished by polyacrylamide gel electrophoresis, followed by DNA sequence analysis to verify the identity of the deletions. PCR conditions are routinely determined based on the length and base-content of the primers selected according to techniques well known in the art (Sambrook et al., 1989).

An additional aspect of the present invention provides a diagnostic assay for detecting cells containing SAG deletions, comprising isolating total cell RNA and subjecting the RNA to reverse transcription-PCR amplification using primers derived from the DNA sequence of SEQ ID 1 SEQ ID 3, SEQ ID 11, SEQ ID 13, SEQ ID 21, SEQ ID 23, SEQ ID 25, SEQ ID 27, SEQ ID 29, SEQ ID 31, SEQ ID 33, SEQ ID 35, SEQ ID 37, SEQ ID 39, SEQ ID 41, SEQ ID 43, SEQ ID 45, SEQ ID 47 and SEQ ID 49. This aspect of the invention enables the detection of SAG deletions in any type of cell, and can be used in genetic testing or as a laboratory tool.

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

In another aspect, the present invention provides methods of isolating RNA containing stretches of polyA (adenine), polyC (cytosine) or polyU (uridine) residues, comprising contacting an RNA sample with SAG protein, incubating the RNA-SAG protein mixture with an antibody that recognizes the SAG polypeptide, isolating the antibody-SAG protein-RNA complexes, and purifying the RNA away from the antibody-SAG protein complex. This aspect of the invention provides a novel in vitro method for isolating a discrete class of RNA. In a preferred embodiment, the RNA sample is contacted with SAG protein in the presence (for preferential isolation of polyA and polyC-containing RNAs), or absence (for preferential isolation of polyU-containing RNAs), of a reducing agent. Preferred reducing agents for use in this aspect of the invention include, but are not limited to DTT and WO 99/32514 PCT/US98/26705 P-mercaptoethanol. The reducing agents are preferably used at a concentration of between about 50 mM and 1 M. Isolation of antibody-SAG protein-RNA complexes can be accomplished via standard techniques in the art, including, but not limited to the use of Protein-A conjugated to agarose or cellulose beads.

In a further aspect of the present invention, a method for isolating genes induced during cell apoptosis is provided, comprising treating one set of cells with OP and not treating a control set of cells, isolating RNA from each set of cells, subjecting the RNA from each set of cells to reverse transcription and PCR ("differential display"), identifying cDNAs that are expressed in the OP-treated set of cells and not in the control set of cells, and cloning the OP-induced cDNAs. This aspect of the invention provides a tool for isolating other genes that control the OP-induced apoptotic pathway and is useful both as a way to enable the design of therapeutic drugs that regulate apoptosis and as a laboratory tool to identify the mechanisms of OP-induced apoptosis. Details of the differential display technique, including selection of primers, are well known in the art (Liang and Pardee, Science 257:967-971, 1992). Reverse transcription and PCR conditions are routinely determined based on the length and base-content of the primers selected according to techniques well known in the art (Sambrook et al., 1989). In a preferred embodiment, OP is used at a concentration of between 50 pM and 300 gM. In a most preferred embodiment, OP is used at a concentration of between 100 gM and 150 gM.

A further aspect of the invention provides a method for protecting mammalian and/or non-mammalian cells from apoptosis induced by redox reagents, comprising introducing into mammalian and/or non-mammalian cells an expression vector comprising a DNA sequence substantially similar to the DNA sequence shown in SEQ ID 1 or SEQ ID 3, that is operatively linked to a DNA sequence that promotes the expression of the DNA sequence and incubating the cells under conditions wherein the DNA sequence of SEQ ID 1 or SEQ ID 3 will be expressed at high levels in the mammalian and/or non-mammalian cells. In a preferred embodiment, the DNA sequence consist essentially of SEQ ID 1 or SEQ ID 3.

Suitable expression vectors are as described above. In a preferred embodiment, the coding region of the human SAG gene is subcloned into an expression vector under the transcriptional control of the cytomegalovirus (CMV) promoter to allow for constitutive SAG gene expression.

An additional aspect of the present invention provides a method for inhibiting the growth of mammalian and/or non-mammalian tumor cells, comprising introducing into WO 99/32514 PCT/US98/26705 mammalian and/or non-mammalian tumor cells an expression vector comprising a DNA that is antisense to a sequence substantially similar to the DNA sequence shown in SEQ ID 1 or SEQ ID 3 that is operatively linked to a DNA sequence that promotes the expression of the antisense DNA sequence. The cells are then grown under conditions wherein the antisense DNA sequence of SEQ ID 1 or SEQ ID 3 will be expressed at high levels in the mammalian and/or non-mammalian cells. In a preferred embodiment, the DNA sequence consists essentially of SEQ ID 1, SEQ ID 3, SEQ ID 11, SEQ ID 13, SEQ ID 21, SEQ ID 23, SEQ ID SEQ ID 27, SEQ ID 29, SEQ ID 31, SEQ ID 33, SEQ ID 35, SEQ ID 37, SEQ ID 39, SEQ ID 41, SEQ ID 43, SEQ ID 45, SEQ ID 47 and SEQ ID 49.

In a most preferred embodiment, the DNA sequence consists essentially of SEQ ID 1 or SEQ ID 3. In a further preferred embodiment, the expression vector comprises an adenoviral vector wherein SAG cDNA is operatively linked in an antisense orientation to a cytomegalovirus (CMV) promoter to allow for constitutive expression of the SAG antisense cDNA in a host cell. In a preferred embodiment, the SAG adenoviral expression vector is introduced into mammalian tumor cells by injection into a mammalian tumor cell mass.

An additional aspect of the present invention provides a method for oxygen radical scavenging in an organism, comprising introducing into mammalian and/or non-mammalian cells an expression vector comprising a DNA sequence substantially similar to the DNA sequence shown in SEQ ID 1 or SEQ ID 3 which is operatively linked to a DNA sequence that promotes the expression of the DNA sequence, and the cells are grown under conditions wherein the DNA sequence of SEQ ID 1 or SEQ ID 3 will be expressed at high levels in the mammalian and/or non-mammalian cells. In a preferred embodiment, the DNA sequence consists essentially of SEQ ID 1 or SEQ ID 3. In a preferred embodiment, the SAG cDNA is operatively linked to a cytomegalovirus (CMV) promoter, to allow for constitutive expression of the SAG cDNA in a host cell.

Another aspect of the present invention provides pharmaceutical compositions and methods for oxygen radical scavenging in an organism, comprising administering an oxygenreducing amount of a pharmaceutical composition comprising the SAG protein of SEQ ID 2 or SEQ ID 4 and a pharmaceutically acceptable carrier.

Chimeric gene constructs of the present invention expression vectors) containing SAG polynucleotide sequences may be used in gene therapy applications to achieve expression of SAG or anti-sense SAG polynucleotide sequences in selected target cells, including non-eukaryotic cells plant) and eukaryotic cells. Gene therapy applications typically involve identifying target host cells or tissues in need of the therapy, WO 99/32514 PCT/US98/26705 designing vector constructs capable of expressing a desired gene product in the identified cells, and delivering the constructs to the cells in a manner that results in efficient transduction of the target cells.

SThe cells or tissues targeted by gene therapy are typically those that are affected by the disease that the vector construct is designed to treat. For example, in the case of cancer, the targeted tissues are malignant tumors.

In one embodiment, the present invention provides a method of promoting the closure healing) of a wound in a patient. This method involves transferring exogenous SAG to the region of the wound whereby a product of SAG is produced in the region of the wound to promote the closure healing) of the wound.

The present inventive method promotes closure healing) of both external surface) and internal wounds. Wounds to which the present inventive method is useful in promoting closure healing) include, but are not limited to, abrasions, avulsions, blowing wounds, burn wounds, contusions, gunshot wounds, incised wounds, open wounds, penetrating wounds, perforating wounds, puncture wounds, seton wounds, stab wounds, surgical wounds, subcutaneous wounds, tangential wounds, or traumatopneic wounds.

Preferably, the present inventive methods are employed to close chronic open wounds, such as non-healing external ulcers and the like.

Exogenous SAG can be introduced into the region of the wound by any appropriate means, such as, for example, those means described herein. For example, where the wound is a surface wound, SAG can be supplied exogenously by topical administration of SAG protein to the region of the wound.

Preferably, exogenous SAG is provided to the wound by transferring a vector comprising an SAG expression cassette to cells associated with the wound. Upon expression of SAG within the cells in the region of the wound, a product of SAG is produced to promote wound closure healing). Transferring a vector comprising an SAG expression cassette to cells associated with the wound is preferred as such procedure is minimally invasive, supplies SAG products locally within the region of the wound, and requires no reapplication of salves, solutions, or other extrinsic media. Furthermore, SAG activity remains expressed during wound closure and will inactivate following healing.

The vector comprising the SAG expression cassette can be transferred to the cells associated with the wound in any manner appropriate to transfer the specific vector type to the cells, such as those methods discussed herein.

WO 99/32514 PCT/US98/26705 As discussed above, the cells associated with the wound to which the vector is transferred are any cells sufficiently connected with the wound such that expression of SAG within those cells promotes wound closure healing), such as cells within the wound or cells from other sources. In one embodiment, the cells are cells of the wound, and the present inventive method comprises transfer of the vector to the cells in situ.

In other embodiments, the cells are not the cells of the wound, but can be cells in an exogenous tissue, such as a graft, or can be cells in vitro. For example, to promote the healing of certain types of wounds, the cells associated with the wound can be cells within a graft, such as a skin graft. Transfer of the vector to the cells associated with the wound, thus involves transferring the vector to the cells within the graft ex vivo. For other wounds, the cells associated with the wound are cells in vitro, and the cells are transferred to the region of the wound following transfer to them of a vector containing the SAG expression cassette.

The present inventive method applies to any patient having a wound. For example, the patient can be any animal, such as a mammal. Preferably, the patient is human.

In another embodiment, the present invention provides a method of inhibiting or promoting plant cell growth. The method involves the use of chimeric gene constructs to achieve expression of SAG, in the case of promoting growth of plants, or anti-sense SAG, in the case of inhibiting plants weeds), polynucleotide sequences in selected target plant cells.

The dosage regimen for in vivo oxygen radical scavenging by the administration of SAG protein is based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary 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 a most preferred embodiment, the pharmaceutical composition comprises between 1 and 10 mg of SAG protein.

The SAG protein may be made up in a solid form (including granules, powders or suppositories) or in a liquid form solutions, suspensions, or emulsions). The SAG protein may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc.

While the SAG protein can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other agents. When administered as a WO 99/32514 PCT/US98/26705 combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.

For administration, the SAG protein is ordinarily combined with one or more adjuvants appropriate for the indicated route of administration. The SAG protein may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, the SAG protein may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.

In a preferred embodiment of the present invention, the SAG protein pharmaceutical composition is administered intramuscularly (IM) or intravenously A suitable IM or IV dose of active ingredient of SAG protein is 5 mg/mL administered daily. For IM or IV administration, the active ingredient may comprise from 0.001% to 10% w/w, from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from 0.1% to 1% of the formulation.

The present invention may be better understood with reference to the accompanying examples that are intended for purposes of illustration only and should not be construed to limit the scope of the invention, as defined by the claims appended hereto.

Examples Example 1. Identification of an OP-inducible gene The differential display (DD) technique was employed to isolate genes responsible for or associated with OP-induced apoptosis in two murine tumor lines. Since OP inducedapoptosis can be visually detected at 12 hours post exposure (Sun, (1997) FEBS Lett. 408:16- 20), it was reasoned that gene(s) responsible for apoptosis induction should be up- or downregulated prior to the appearance of apoptosis. Six hours of OP treatment was conducted, therefore, in one of these tumor lines followed by the DD analysis.

WO 99/32514 PCT/US98/26705 Mouse JB6 tumor line L-RT101 (an epidermal originated tumor cell line) was cultured in Minimal Essential Medium with Earle's salts (BRL) containing 5% fetal calf serum (Sigma). H-Tx cells, a spontaneously transformed mouse liver line, were cultured in Dulbecco's Modified Eagle Medium containing 10% fetal calf serum and 1 mM sodium pyruvate. Human colon carcinoma line DLD-1 was grown in 10% DMEM.

L-RTI01 cells were treated with 150 uM OP for 6 hours and subjected to differential display analysis using DMSO-treated cells as a control. Briefly, total RNA was isolated from both OP-treated and control cells using RNAzol solution (Tel-Test) according to the manufacturer's instructions, and subjected to reverse transcription performed as previously described (Sun et al. (1993) Mol. Carcinogenesis 8, 49-57), followed by the polymerase chain reaction (PCR). The primer used for reverse transcription (P1) consisted of the sequence 5 AAGCTTTTTTTTTTTTTR (SEQ ID wherein R consists of either adenine, guanine or cytosine. P1 was used as the downstream primer in the subsequent PCR while the upstream primer consisted of the sequence AAGCTTNNNNNNN (SEQ ID 6), wherein N consists of adenine, cytosine, guanine, or thymine.

Primers PI and P2 reproducibly detected differential expression between the control and OP-treated cells. The fragments reproducibly showing differential expression were PCR amplified using the same primers and used as probes for Northern analysis (Sun et al. (1992) Cancer Res. 52:1907-1915) of both L-RT101 and H-Tx cells treated with OP (Sun (1997) FEBS Letters 408:16-20). Those fragments that were induced by OP (as determined by Northern analysis) were then subcloned into TA cloning vectors (In Vitrogen) according to the manufacturer's instructions, and sequenced by DNA Sequenase Version 2.0, according to the manufacturer's instructions (Amersham). The resulting clones comprise OP-inducible cDNA fragments.

Example 2. cDNA library screening and One of the OP-inducible clones was used as a probe to screen a mouse lung cDNA library to clone the full length mouse SAG cDNA. Briefly, 1 x 106 recombinant plaques were plated onto 1% NZY in 150 mm plates (a total of 20). The recombinant phage DNA was transferred to nitrocellulose membrane and hybridized with mouse SAG probe (2X108 cpm/gg) in a hybridization solution containing 5X SSC, 5X Denhardt solution, 50 mM sodium phosphate, and 100 gg/mL denatured DNA at 60 0 C for 16-18 hours. The filter was WO 99/32514 PCT/US98/26705 then washed once for 5 min in a solution of 2XSSC/0.1% SDS, once for 5 min in 0.5XSSC/0.1% SDS, and twice 0.1XSSC/0.1% SDS for 15 min.

The longest clone isolated was a 1.0 kilobase fragment consisting of a partial open reading frame and the entire 3'-end untranslated region. A mouse brain Marathon- Ready cDNA (ClonTech) was screened via PCR amplification using a primer derived from the 1 kb fragment and another primer derived from the vector sequence, according to the protocol supplied with the cDNA library. This yielded a further 100 bp fragment consisting of 5'-end untranslated sequence and some of the coding sequence. The derived cDNA clone consists of 1140 base pairs (SEQ ID 1) that encode a novel deduced protein of 113 amino acids, containing 12 cysteine residues (SEQ ID The open reading frame was preceded by 17 bp upstream sequence. The start codon was located in a context that conformed 100% to the Kozak consensus sequence (Kozak,M. (1991) J. Biol. Chem. 266, 19867-19870). An in-frame stop codon was identified 72 bp upstream of the start codon in the 5' untranslated region in one genomic clone (not shown). The 3'-end untranslated region consists of 792 bp sequence with two polyadenylation signals (AATAAA). These data indicate that a near full length cDNA was isolated.

The mouse cDNA was used as a probe to screen a human HeLa cell cDNA library (Strategene) as described above. One positive clone was isolated and purified through two more cycle of screening. In this manner, a 754 bp clone containing a polyadenylation signal at the 3' end was isolated (SEQ ID The human cDNA also contains an open reading frame encoding a novel predicted 113 amino acid polypeptide containing 12 cysteine residues (SEQ ID The sequence identity between the isolated mouse and human cDNAs is 82% in overall sequence and 94% in the coding region. At the protein level, they shared 96.5% identity, with all 12 cysteine residues being conserved. Computer analysis of protein databases using the GCG program (Genetics Computing Group, Madison, WI) revealed that the encoded proteins share 70% identity with hypothetical proteins from yeast (accession #Z74876) and C-elegans (accession #80449).

Motif searching of the deduced protein sequences using the GCG program did not reveal any known functional domains. However, they each contain two imperfect heme binding sites (CXXCH, at codons 47-51 and 50-54) (Matthews, Prog. Biophys. Mol. Biol.

45:1-56, 1985) and one imperfect C 3

HC

4 zinc ring finger domain (Freemont et al., Cell 64:483-484, 1991) at the C-terminal of the molecule (Fig. 1A) among other consensus motifs.

The second potential heme binding domain (Fig. 1A) contains a substitution of arginine to WO 99/32514 PCT/US98/26705 histidine (amino acid 54). Since these two amino acids are structurally similar, this may constitute an authentic heme binding site. The zinc ring finger domain mismatch involves substitution of cysteine by histidine at amino acid 85. The ring finger domain in this protein is a C 3

H

2

C

3 structure, rather than the consensus C 3

HC

4 structure. Since cysteine and histidine residues are interchangeable in zinc hinding (Berg and Shi, Science 271:1081, 1996; Inouye et al., Science 278:103-106, 1997), the C 3

H

2

C

3 domain in these proteins may comprise authentic zinc-binding sites. Significantly, these heme and zinc ring finger domains are 100% conserved among C. elegans, mouse and human. In yeast, only the last cysteine residue in C 3

H

2

C

3 motif was not conserved. This evolutionary conservation of the heme and zinc-binding domains suggest their functional importance.

Other motifs identified in the deduced sequence of the SAG protein, when allowing for a single mismatch, include an aminoacyl-transfer RNA synthetase class II motif (codons 54-63), a Kazal serine protease inhibitor family motif (codons 85-107), a Ly-6/U-par domain (codons 65-107), a prokaryotic membrane lipoprotein lipid attachment site (codons 16-27), and somatotropin, prolactin and related hormone motifs (codons 49-66).

These experiments thus resulted in the cloning of novel mouse and human genes that encode nearly identical, evolutionarily conserved protein that contain distinct heme and zinc binding motifs.

Example 3. SAG is inducible by OP in both mouse and human tumor cells To confirm that the cloned cDNAs are subject to OP induction, a Northern analysis was performed with RNAs isolated from mouse tumor lines L-RT101 and H-Tx, and human colon carcinoma line DLD-1. Subconfluent cells were treated with 150 pM OP for various times up to 24 hours and subjected to total RNA isolation. Fifteen gg of total RNA was subjected to Northern analysis using mouse SAG or human SAG cDNA as probes.

Both cloned mouse and human cDNAs detected an OP inducible transcript with a size of 1.2 kb and 0.9 kb, respectively. Since these genes were induced in the OP-induced apoptosis pathway, the genes were named Sensitive to Apoptosis Genes (hereinafter referred to as which encode SAG proteins.

Example 4. Tissue distribution and embryonic expression ofSAG SAG expression was next examined in multiple human tissues. The assays were performed as detailed previously (Sun et al. (1993) Mol. Carcinogenesis 8, 49-57; Sun et al., Proc. Natl. Acad. Sci. USA 90:2827-2831, 1993). Briefly, total RNA was isolated from WO 99/32514 PCTIUS98/26705 multiple human tissues (ClonTech) and then subjected to Northern blot analysis using the mouse or human SAG cDNA as probes. SAG RNA was detected in all tissue examined including heart, brain, pancreas, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocytes. A very high expression level was detected in heart, skeleton muscle and testis, which consume high levels of oxygen. Its tissue distribution and high level expression in oxygen-consuming tissues, and its induction by a redox sensitive compound implies that SAG encodes a redox sensitive protein.

Since SAG protein is evolutionarily conserved, the possible developmental role of SAG was tested by measuring SAG expression in mouse embryonic tissue (provided by Dr.

Tom Glaser, University of Michigan), using reverse transcription of total RNA followed by PCR with the following primers: SAGTA.01 3' (SEQ ID 7) and SAGT.02 5'-CGGGATCCTCATTTGCCGATTCTTTG-3' (SEQ ID 8), which flank the entire SAG coding region. The PCR reaction mixture for 11 samples contained 55 gL of 10X buffer, 22 lgL of 1.25 mM dNTP, 1.1 glL of SAGTA.01 and SAGT.02, respectively, 5.5 gL of Taq DNA polymerase, 5.5 gL of 3 2 P-dCTP and sterile water up to 495 pl. Into each tube which contains 5 gL of first strand cDNA reversetranscribed for total RNA isolated from mouse embryonic tissues (Sun et al. (1997), Mol.

Carcinogenesis 8:49-57), 45 gL of reaction mixture was added and PCR was performed for 25 cycles (95°C for 45 sec, 60 0 C for 1 min and 72 0 C for 2 min). A 5 gpL aliquot of the PCR product was denatured and separated on a sequencing gel, which was dried and exposed to X-ray film.

SAG RNA was expressed in 9.5 day old to 19.5 day old whole mouse embryos, with a higher level of expression detected between days 9.5 and 11.5. These results suggest that SAG plays a role in embryonic development.

Example 5. Cellular localization by immunofluorescence NIH3T3 cells (ATCC CRL 1658) were plated on coverslips in 24-well culture dishes and transfected by the calcium phosphate method according to standard techniques (Sambrook et al, 1989) with the following constructs: pcDNA3.1 (Invitrogen vector pcDNA 3 with a myc-his-tag); pcDNA3.1-SAG (human SAG cDNA subcloned into the BamHI site of pcDNA3.1, downstream from the CMV promoter and upstream and in-frame with the myc-his-tag, such that upon expression, the resulting fusion protein consists of the SAG WO 99/32514 PCT/US98/26705 protein followed by the myc-his tag at the carboxy-end of SAG); or pcDNA3.1-LacZ (Invitrogen). Two days post-transfection, cells were washed once with cold PBS and then fixed with 3% formaldehyde in PBS for 10 minutes followed by 5 minutes in 1:1 methanol:acetone. The fixed cells were washed 4 times in PBS and incubated with antibody directed against the Myc-tag (Invitrogen 1:200 dilution) in PBS containing 1% BSA, 0.1% saponin, 2 gg/mL DAPI for 1 hour in the dark with shaking. Cells were then washed 4 times with 0.1% saponin in PBS and incubated with FITC-conjugated goat anti-mouse antibody (Jackson Laboratory, 1:100 dilution) for 1 hour in the same conditions as the first antibody.

After incubation cells were washed 4 times with 0.1% saponin in PBS and twice with PBS.

The coverslips were then mounted to glass slides with non-fade mounting medium and analyzed using a Leita Dialux 20 microscope.

SAG fusion protein was detected in both the cytoplasm and nucleus, while the Pgalactosidase control was expressed predominately in the cytoplasm. No immunofluorescence staining was detected with the vector-only control. The cytoplasmic/ nuclear localization of SAG was confirmed also in a SAG stable transfectant using both SAG and myc-tag antibodies. These data demonstrate that exogenously expressed SAG fusion proteins can be detected within transfected cells by using antibodies directed against an epitope fused to SAG protein.

Example 6. Expression and purification ofSAG protein in bacteria The entire open reading frame of the human SAG cDNA was PCR amplified as described above and subcloned into the pET11 expression vector (Novogen) under control of the T7 promoter, yielding construct pETlla-hSAG. The sequence and orientation of the SAG DNA insert were confirmed by DNA sequencing. pETlla-hSAG was used to transform E. coli strain BL21 (Novagen, Inc.). Transformed cells were grown in LB media containing ampicillin (50 gg/mL). SAG expression was induced by 0.5 mM IPTG and SAG protein was found in inclusion bodies, which were subsequently isolated as follows.

Following IPTG induction, four liters of cells were grown for 4.5 hours at 37 0 C at a shaker setting of 150 rpm. Cell pellets were obtained by centrifugation at 5000 rpm for minutes, and were resuspended in 100 mL TN buffer (20 mM Tris-HC1, pH 7.5, 50 mM NaCl) containing 100 gM PMSF. The resuspended cell pellet was subsequently sonicated sec/round for 5 rounds at a setting of 15 on Model 50 sonic dismembrator, Fisher Scientific) and subjected to pressure of 2500 pounds/square inch on a French cell press, WO 99/32514 PCT/US98/26705 followed by addition of 1 mM MgC12 and 10 mg of DNase I. The cell lysate was placed on ice for 30-60 minutes and then centrifuged at 18,000 rpm and the supernatant was disposed.

The pellet was seen to have 2 layers. The white layer on the top was carefully blown loose with TN buffer and removed. The remaining dark brown layer on the bottom was resuspended thoroughly in 15 mL of urea buffer (7 M urea, 20 mM Tris-HC1, pH 200 mM NaC1) and allowed to sit overnight at room temperature. The resuspended cell pellet was vigorously homogneized with a serological pipette and then centrifuged at 40,000 rpm for 40 minutes using an SW50 ultracentrifuge rotor. The supernatant was collected and concentrated using a Centricon-10 concentrator to a volume of 5 mL and loaded onto a Sephacryl-100 column (100 cm long with a diameter of 2.5 cm) that had been equilibrated with urea buffer. The column was run at a rate of 0.25 mL/min and fractions were collected.

The early fractions containing a brownish color consisted of mostly the large molecular weight protein, as expected. They also contained a protein with the same size of SAG protein (approximately 13 kDa). Since SAG protein contains 12 cysteine residues, it follows that SAG protein may form oligomers when expressed in bacteria and thus may elute as a SAG protein oligomer. Since SAG is a redox-sensitive protein, the DTT present in SDS sample buffer reduces SAG protein oligomers to monomer, leading to the detection of a fast migrating band. When early fractions were run in SDS-PAGE without DTT, the 13 kDa SAG protein band disappeared, and a 260 kDa band was detected, representing a SAG protein 20-mer. This unique feature helped us to purify SAG protein. Early fractions were pooled and loaded on the same Sephacryl-100 column pre-equilibrated with 7M urea and

DTT.

SAG protein oligomer was reduced to monomer by using DTT in the loading buffer and was eluted in the later fractions, thus separating it from high molecular weight contaminant proteins (eluted earlier). The brownish fractions were pooled and concentrated using a Centricon-10 to a volume of 5 mL. DTT was added to a concentration of 5 mM. The combined fractions were loaded onto an S-100 column (100 cm long with a diameter of cm), that had been equilibrated with urea buffer plus 5 mM DTT. The column was run at a rate of 0.25 mL/min and fractions were collected. The fractions containing SAG protein are brownish in color, highly suggesting that SAG is a heme-containing protein. The SAG protein containing fractions and their sensitivity to DTT were confirmed by Western blot using SAG antibody. The brownish fractions were pooled and concentrated using a concentrator to a volume of 2 mL. The resulting sample was dialyzed against 4 liters of dialysis buffer (150 mM KC1, 20 mM Tris-HCl, pH 7.5) at 4 0 C overnight to WO 99/32514 PCT/US98/26705 remove urea and DTT to yield refolded SAG protein. The dialyzed sample was loaded onto an S-100 column (100 cm long with a diameter of 2.5 cm), that had been equilibrated with dialysis buffer. The brownish fractions were pooled and concentrated using a Centricon-10 to a volume of 1 mL. The resulting sample was stored at 4 0 C. The protein concentration was determined by a BioRad protein assay. The purity of the samples was demonstrated in 10-20% SDS-PAGE. These data demonstrate the purification of recombinant SAG protein.

Example 7. Redox Sensitivity ofSAG Protein To confirm that purified recombinant SAG protein possesses the same redox sensitivity as it shows during protein purification, the sensitivity of refolded SAG to redox reagents was examined next. SAG protein (1 ig) was exposed to various concentrations of DTT (1 M, 300 mM, 100 mM, or 30 mM) or H 2 0 2 (15 mM, 50 mM, 150 mM or 450 mM) for 10 min before being separated by polyacrylamide gel electrophoresis (PAGE), followed by Western blot analysis. Alternatively, 10 gLg of SAG protein was incubated with 50 mM

H

2 0 2 for 10, 30, 60 or 120 minutes followed by PAGE separation and Coomassie Blue staining.

Dimers of SAG protein are rather resistant to reducing reagent DTT since no significant dimer was reduced to monomer after DTT treatment. However, as little as 15 mM

H

2 0 2 induces oligomerization of SAG protein, possibly through the formation of intermolecular disulfide bonds. The oligomerization is incubation-time dependent, as higher order SAG protein oligomers were detected upon increased incubation time. Interestingly, a band migrating faster than the monomer form is observed upon H 2 0 2 treatment, and the monomer form of SAG protein becomes a doublet, possibly due to the formation of intramolecular disulfide bonds.

In order to determine whether H 2 0 2 -induced SAG protein oligomerization can be reversed by DTT treatment, 1 gig of purified SAG protein was incubated with 50 mM H 2 0 2 for 10 minutes, followed by a 10 minute incubation with either H 2 0 2 50 mM DTT, 100 mM, 500 mM, or 1 M DTT. The samples were separated via PAGE followed by Western analysis.

The results demonstrated that H 2 0 2 -induced SAG protein oligomerization can be reversed by subsequent incubation with DTT in a dose dependent manner, indicating that SAG protein oligomerization is subject to redox regulation.

WO 99/32514 PCT/US98/26705 To confirm that SAG protein oligomerization and doublet formation is due to interand intra-molecular disulfide bond formation, respectively, SAG protein was treated, prior to

H

2 02 exposure, with 50 mM N-ethylmaleimide (NEM), an alkylating reagent that will alkylate the free SH-groups in SAG protein. Purified SAG protein (1 gg) was pre-incubated with 50 mM NEM or DMSO, or buffer only, for 10 minutes prior to ,202 treatment. The samples were separated via PAGE, followed by Western blot analysis. Pre-incubation of SAG protein with DMSO did not affect H 2 0 2 -induced oligomerization and doublet formation, whereas NEM pre-treatment abolished H 2 0 2 activity. Neither inter- (oligomerization) nor intra- (doublet monomer) disulfide bonds were formed, demonstrating that alkylation of the free SAG protein SH groups abolishes H 2 0 2 sensitivity. These data demonstrate that SAG protein is redox sensitive. It is subjected to both intra- and intermolecular disulfide bond formation upon exposure to H 2 0 2 as evidenced by both doublet and oligomer formation. These H 2 0 2 -induced changes can be reversed by subsequent treatment with reducing reagents, including DTT, or can be prevented by NEM pretreatment.

It has also been observed that zinc can promote H 2 0 2 -induced oligomerization, although zinc itself did not induce oligomerization.

Example 8: Production ofSAG mutants In order to understand the role of each particular cysteine residues in heme binding and SAG oligomerization, a series of single and double SAG mutants were made in heme binding sites as well as the zinc ring finger motif (see Figure 1B). To generate single point mutations in SAG cDNA, 15 pairs of sense and antisense primers were designed, which are partially complimentary and contain a desired point mutation. The wildtype SAG cDNA cloned into the pET1 la vector at the Nhe I/Bam HI sites was used as the template for PCR amplification. Two separate PCR reactions were conducted using a) primer SAG P.01 (5'-TATGGCTAGC ATGGCCGACGTGGAGG-3) (SEQ ID 9) and each of antisense primers and b) each of sense primers and SAG T.02 (SEQ ID respectively. The resultant PCR products that overlap with each other and contain a desired point mutation were mixed and served as templates for a third PCR. The primers used were SAG P.01 and SAG T.02, which flank the entire encoding region of SAG cDNA. The PCR was performed as previously described (Sun et al. (1992) BioTechniques 12:639-640). The PCR products were digested with restriction enzymes Nhe I and Bam HI and subcloned into the pETl la vector, which was digested with the same restriction enzymes. To generate SAG double mutants WO 99/32514 PCT/US98/26705 MM13, MM14, see Figure 1B), a QuickChange site-directed mutagenesis kit was purchased from Strategene (La Jolla, CA) and used as instructed. All SAG mutants generated were verified by DNA sequencing (SEQ ID 21, SEQ ID 23, SEQ ID 25, SEQ ID 27, SEQ ID 29, SEQ ID 31, SEQ ID 33, SEQ ID 35, SEQ ID 37, SEQ ID 39, SEQ ID 41, SEQ ID 43, SEQ ID 45, SEQ ID 47 and SEQ ID 49). The predicted mutant SAG proteins encoded by these mutant SAGs are shown in SEQ ID 22, SEQ ID 24, SEQ ID 26, SEQ ID 28, SEQ ID 30, SEQ ID 32, SEQ ID 34, SEQ ID 36, SEQ ID 38, SEQ ID 40, SEQ ID 42, SEQ ID 44, SEQ ID 46, SEQ ID 48, and SEQ ID Individual SAG mutant-expressing vectors were used to transform E.coli strain BL21 (Novagen, Inc.). Mutant SAG protein was expressed and purified as detailed in Example 6.

The fractions after a Sephacryl-100 column were collected and analyzed on 8-25% Phast gels followed by Coomassie blue protein staining. The pure fraction containing mutant SAG protein was dialyzed in 4 liters of 20 mM Tris-HC1, pH 7.5 and used for SAG protein oligomerization studies.

Purified wildtype SAG protein is a heme-containing brownish protein (See Example Some of the purified SAG protein mutants were found to have either lost the brownish color (MM3 and MM13) or had decreased brownish color (MM1) compared to wildtype SAG protein. This color change indicates the loss or decrease of heme binding (Table 1).

WO 99/32514 PCT/US98/6705 TABLE 1. SUMMARY OF SAG MUTANTS NAME MUTATION SITE(S) HEME BINDING OLIGOMERIZA TION WT None Yes MM1 CA/heme Yes MM2 Cw/heme +t Yes MM3 CA+B/heme Yes MM4 C 1 /Zn-ring finger 1 Yes

C

3 /Zn-ring finger 1 Yes MM6 H 4 /Zn-ring finger 1 Yes MM7 H5/Zn-ring finger 2 Yes MM8 C 6 /Zn-ring finger 2 Yes MM9 C 7 /Zn-ring finger 2 Yes

H

4 5 /Zn-ring fingers 1&2 Yes MM11 C 2 /Zn-ring finger 1 Yes MM12 Cc/protease inhibitor Yes MM13 C 1 2 /Zn-ring finger 1 Yes MM14 C 7 8 /Zn-ring finger 2 No GADPH binding site Yes To examine mutant SAG protein oligomerization, each mutant SAG protein as well as wildtype SAG was'treated with 50 mM H 2 0 2 for 10 min. All of the SAG mutants, except MM14, can be oligomerized upon exposure to H 2 0 2 The mutant 14, which is a double mutants in positions of C7 and C8 in the zinc ring finger domain, becomes insensitive to oligomerization (Table indicating that these two positions are important for intermolecular disulfide bond formation.

Example 9. Heme measurement ofSAG protein Heme content in SAG protein was measured as previously described (Rieske (1967) Methods in Enzymol. 76, 488-493). Briefly, 1 mg of purified SAG protein, along with cytochrome C, catalase, and BSA as controls, was extracted with cold acetone (0.5 mLs) WO 99/32514 PCT/US98/26705 After centrifugation the pellet was extracted sequentially with 0.5 mL of chloroform:methanol 0.5 mL of cold acetone, and finally 0.5 mL of cold acetone containing 5 jgL of 2.4 N HC1. The acetone extracts were dried under speed-vac and dissolved in 0.5 mL of pyridine. After addition of 0.5 mL of 0.2 N NaOH, the solution was centrifuged briefly and clear supernatant was recovered. One drop of diluted potassium ferricyanide (0.05 M) was added to the supernatant and the absorbance was read at 556 nm in mL quartz cuvettes using water as a blank. The solution was then reduced by adding uL of 2 M DTT and absorbance was read at 556 nm, 587 nm and 550 nm, respectively.

Heme absorbance at 556, 587, and 550 nm was observed in SAG protein, as well as in cytochrome C and catalase, but not in BSA. This result demonstrated that SAG protein contains heme, but did not reveal the molar ratio between SAG protein and heme molecule.

Example 10. SAG protein antibody production Two polyclonal antibodies against SAG protein were generated using standard methods [by Zymed Laboratories, Inc. (San Francisco) under a service agreement with Warner-Lambert]. Briefly, the peptide antibody was generated as following. A 16-aminoacid peptide (SAG-Pepl: QNNRCPLCQQDWVVQR) (SEQ ID 10) located in the C terminus of SAG protein (codons 95-110) was synthesized and purified via standard techniques. The purified peptide was conjugated to keyhole limpet hemocyanin (KLH) via cysteine residues. The conjugated peptide (0.5 mg) was emulsified with equal volume of Complete Freund Adjuvant (CFA) and subcutaneously injected into rabbit, followed by 4 boosts with 0.5 mg each in Incomplete Freund Adjuvant (IFA) at 3 week intervals. Rabbits were bled 10 days after the final boost and antiserum was collected. The same protocol was used for protein antibody production using purified human SAG protein as the antigen, prepared as described above.

Example 11. Analysis ofSAG protein transcriptional regulatory activity SAG protein belongs to the zinc ring finger protein families by virtue of its C 3

H

2

C

3 motif (Saurin et al. (1996) TIBS 21, 208-214). Some zinc ring finger proteins have been shown to bind to DNA and function as transcriptional repressors (for example, RING1) (Satijn et al. (1997) Mol. Cell. Biol. 17, 4105-4113), whereas others function as transcriptional activators (Chapman and Verma (1996) Nature 382, 678-679; Monteiro et al.

(1996) Proc. Natl. Acad. Sci. USA 93, 13595-13599). To examine the transcriptional regulatory activity of SAG protein, the cDNA encoding the entire open reading frame of WO 99/32514 PCT/US98/26705 human SAG was PCR amplified and fused both in frame and as an antisense fusion, downstream of the Gal-4 DNA binding domain (encoding amino acids 1-147) in the pG4 vector (Sadowski et al., Nature 335:563-564, 1988). The resulting construct was sequenced to confirm in frame fusion and freedom from PCR-generated mutation. The construct was co-transfected along with a chloramphenicol acetyltransferase (CAT)-reporter-expressing vector (Sadowski et al., Nature 335:563-564, 1988) as well as a (3-galactosidase reporter whose expression is driven by a CMV promoter for normalization of transfection efficiency into human kidney 293 cells (ATCC accession number CRL1573) by the calcium phosphate method. CAT activity was measured 36 hours post-transfection using a CAT assay kit (Quan-T-CAT; Amersham) according to the manufacturer's instructions. PG4-VP16, a known transcription factor (Triezenberg et al., Genes and Develop. 2:718-729, 1988), fused downstream of the Gal4 DNA binding domain was used as a positive control. Activation was calculated by arbitrarily choosing CAT activity from the vector control as 1 and comparing the other constructs to it. Three independent transfections and assays were performed.

SAG protein showed no transactivation activity. The positive control, VP16 showed 300-fold activation of CAT activity. To test for transrepression activity, SAG constructs (both sense and antisense) were co-transfected with pG4-VP16. Again, neither orientation of SAG induced significant expression of VP16-induced transactivation. These results demonstrated that SAG protein lacks transcriptional regulatory activity when fused downstream Gal-4 DNA binding domain.

Example 12. SAG is an RNA binding protein The zinc-ring finger domain of the MDM2 protein has been shown to bind to RNA (Elenbaas et al. (1996) Mol. Med. 2, 439-445). Since SAG protein showed no transcriptional regulatory activity, it was tested whether SAG protein could bind to RNA or DNA. Binding of purified SAG protein to different nucleic acid cellulose conjugates was performed as described (Elenbaas et al. (1996)). Briefly, 0.5 gg of SAG protein was incubated in 300 gL RNA binding buffer for 1 hour at 4°C with double-stranded calf thymus DNA, denatured calf thymus DNA (ssDNA), or one of 4 RNA homopolymer columns (Sigma) conjugated to agarose or cellulose beads (Sigma), and used according to the manufacturer's instructions.

RNA binding buffer consisted of 20 mM Tris, pH 7.5, 150 mM NaCI, 5 mM MgCl 2 0.1% nonidet P-40, 50 gM ZnCl 2 2% glycerol, and 1 mM DTT. The columns were washed with 3 mL RNA binding buffer to remove non-specifically bound protein from the beads, which -26were then boiled in SDS sample buffer. The protein so eluted from the beads was separated by SDS-PAGE, transferred to nitrocellulose for Western blot analysis using the polyclonal antibody directed against SAG protein described previously detected by ECL chemiluminescence (Amersham) according to the manufacturer's instructions.

Purified SAG bound to polyU, polyA, and polyC RNA, respectively. No binding was seen with polyG RNA or ssDNA. A band showing dsDNA binding did not agree with SAG molecular weight. Oligomeric SAG protein bound to polyU RNA, whereas the monomeric form of SAG binds to polyA and polyC RNA. Purified SAG protein was run as a marker. These results suggest that SAG is an RNA binding protein and that binding specificity is determined by the oligomeric form of SAG protein.

Example 13. Identification of two deletion mutants of SAG in cancer cell lines Total RNA was isolated from DLD-1 colon carcinoma cells (ATCC Accession Number CCL221) and subjected to RT-PCR using primers SAG TA.01 and SAG T.02.

15 The resulting PCR fragments were subcloned into the TA cloning vector (Invitrogen).

During sequence verification of the resulting clones, it was found that several clones S° contained either a 7 bp or a 48 bp deletion at nucleotide 170 or 177, respectively, assigning the first A at the start codon as nucleotide Both SAG deletions encode the potential heme-binding sites. The 7 base pair deletion (SAG mutant 1) (SEQ ID 11) is a frame shift deletion that abolishes the downstream encoded zinc-ring finger motif in the resulting protein (SEQ ID 12), whereas the 48 base pair deletion (SAG mutant 2) (SEQ ID 13) is an in-frame deletion that eliminates 16 amino acids in the encoded protein (SEQ ID 14), but retains the zinc-ring finger motif. (Human cDNA encoding mutated SAG, (hSAG-mutant 1) and human cDNA encoding mutated SAG, (hSAG-mutant 2) 25 were deposited with the America Type Culture Collection on 10 April 1997 under Accession Numbers 98403 and 98404, respectively.) Total RNA was isolated from a total of 20 human tumor lines and transformed lines originating from lung, brain, kidney, prostate, testis, nasopharynx, bone, cervix and foreskin and subjected to RT-PCR analysis as described previously (Sun et al. (1993) Mol. Carcinogenesis 8, 49-57). Genomic DNA was also isolated from these cell lines and subjected to PCR amplification as described (Sun et al. (1992) BioTechniques 416AUPOO.DOC 26a 12:639-640). The primers used for PCR were hSAG.M1, GCCATCTGCAGGGTCCAG-3- (SEQ ID 15), starting at nt 151 of hSAG cDNA, and SAGT.02-1 5'-GGATCCTCATFE[GCCGATTrCT1TGGAC-3' (SEQ ID 16), including stop codon (underlined). The resulting fragment is 200 bp for wildtype SAG.

The PCR was conducted in the presence of 35 S-dATP (Amersham) and PCR products were resolved in 6% denaturink sequencing gels, as described previously (Sun et al.

(1995) Cancer Epidemiology, Biomarkers Prevention, 4, 261-267). The bands corresponding to wildtype as well as the 416AUPOO.DOC WO 99/32514 PCT/US98/26705 two deletion mutants were cut out from the gel, PCR amplified using the same set of primers, and sequenced to verify the DNA sequence of the resulting PCR fragments.

Both the 7 base pair and the 48 base pair deletions were detected in RNA from only the CATES-1B cell line, a testicular carcinoma line obtained from ATCC (accession number HTB104). This tumor line also contains the wildtype SAG DNA sequence. The identity of these three bands was confirmed by DNA sequencing after PCR amplification and TA cloning. HONE-1, a nasopharyngeal carcinoma line which only contains wildtype SAG was included for comparison.

It was next examined whether these SAG deletions were detectable at the DNA level.

Genomic DNA was isolated from CATES-1B cells and subjected to PCR analysis, as described previously (Sun et al. (1992) BioTechniques 12:639-640). The primers used were hSAG.MI and SAG T.02 (see above for sequences). Genomic DNA from CATES-1B cells possesses only wildtype SAG and no SAG deletion mutants were detected. These results indicate that the SAG deletion mutations occur very rarely in human cancer lines. Detection of the mutations in SAG RNA, but not genomic DNA, may reflect an RNA editing modification of SAG messenger RNA.

Example 14. Production ofstable SAG transfected mammalian cells The potential biological function of human SAG protein was examined next by its overexpression in cells. DLD-1 cells were transfected with the following plasmids: the neo control pcDNA-3 (Invitrogen) (identical to pcDNA3.1 described above, except that it lacks the myc-his tag), pcDNA-SAG, pcDNA-SAG-mutant-1, and pcDNA-SAG-mutant-2 (pcDNA3 with SAG, SAG 1 or SAG 2 subcloned into the BamHI site, respectively, using methods well known in the art). The SAG mutant constructs were generated by RT-PCR as follows. Total RNA was isolated from DLD-1 cells, and subjected to reverse transcription, followed by PCR amplification. The primers used were SAG.TA01 (SEQ ID 7) and SAGT.02 (SEQ ID which flank the entire coding region of SAG gene. The PCR products were digested with restriction enzyme Bam HI, and subcloned into pcDNA3 (In Vitrogen, San Diego), a mammalian expression vector under the transcriptional control of the CMV promoter, which drives gene expression constitutively. The resultant clones were sequenced to confirm both sense and antisense orientation and freedom of PCR-generated mutations.

DNA sequencing revealed wildtype SAG clone as well as two deletion mutants: SAGmutant-1 (7 bp deletion, SEQ ID 11) and SAG-mutant-2 (48 bp deletion, SEQ ID 13) in DLD-1 tumor cells.

WO 99/32514 PCT/US98/26705 DLD-1 cells were transfected by lipofectamine (BRL) with plasmids expressing wildtype (both sense and antisense orientation), SAG mutant-1, and SAG mutant-2, along with the neo control vector. Neomycin resistant colonies were identified by G418 selection (600 gg/mL) for 18 days. Stable clones were ring-isolated by well known methods (Sun et al. (1993) Proc. Natl. Acad. Sci. USA. 90: 2827-2831) and SAG expression was monitored by Northern analysis. Selected clones were examined for SAG protein expression by immunoprecipitation, as described below.

Total RNA was isolated from the cloned cell lines and subjected to Northern analysis.

Cell lines transfected with the following constructs were analyzed: vector controls Dl-3 and D1-6; SAG-wildtype D12-1 and D12-8; SAG-mutant-1 D3-3 and D3-4; and SAG-mutant-2 D4-2 and Northern blot analysis of RNA from selected stable SAG-expressing clones probed with the human SAG cDNA demonstrated that all SAG transfectants express SAG mRNA, while very low levels of endogenous SAG message were detected in the neo control cells.

The vector control lines and SAG wildtype and SAG deletion mutant transfectants were subsequently subjected to immunoprecipitation using standard techniques (Sun et al.

(1993) Proc. Natl. Acad. Sci. USA. 90: 2827-2831, Sun et al. (1993) Mol. Carcinogenesis 8, 49-57). Subconfluent SAG transfectants were subjected to methionine starvation for 1 hour and then metabolically labeled with 3 5 S-translabel (0.2 mCi/mL) for 3 hours. Cells were then lysed on ice for 30 minutes in a lysis buffer comprising 2% Nonidet P40, 0.2% SDS, sodium deoxycholate, 1 mM sodium orthovanadate, 5 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, and 1 gl/mL leupeptin, and centrifuged at 12,000 x g. The TCA precipitable radioactivity in the supernatant (1 x 108 cpm) was immunoprecipitated using rabbit anti-human SAG antibody (generated as described above). The immunoprecipitates were collected, washed, and analyzed on a 10-20% SDSpolyacrylamide gel, followed by autoradiography. High SAG protein expression was detected only in the wildtype transfectants. The antibody used did not recognize the two SAG protein mutants. These data demonstrate the production of stably transfected cells expressing either wildtype or mutant SAG protein Example 15. Morphological appearance of SAG transfectants after exposure to redox reagents WO 99/32514 PCT/US98/26705 Two neo controls (D1-3 and D1-6) and two SAG-producing lines (D12-1 and D12-8) were chosen to examine their sensitivity to redox compounds by morphological observation.

After exposure to 150 pM OP, 200 JM H 2 0 2 or 125 iM zinc for 24 hours, the neo-control cells were shrunken and detached, a sign of apoptosis, while SAG-expressing cells appeared morphologically normal. These results indicate that SAG production protects cells from apoptosis induced by redox compounds. Expression of SAG, however, did not offer the protection against copper. No difference in morphological signs of apoptosis was observed with CuSO 4 treatment (up to 750 gM) between the vector controls and SAG transfectants.

Higher doses induced apoptosis in all lines.

Example 16. SAG expression protects cells from DNA fragmentation The sensitivity of these SAG-transfected cells to OP-induced apoptosis was examined next by monitoring DNA fragmentation, a hallmark of apoptosis. Subconfluent (80-90%) SAG transfected cells expressing wildtype SAG, SAG mutant-1, SAG mutant-2, or vector control cells, were seeded at 3.5 x 106 per 100 mm dish and exposed after 16-24 hours to 150 JgM OP, 125 JIM zinc sulfate, or 200 JM H 2 0 2 for 24 hours. Both detached and attached cells in 2 x 100 mm dishes were harvested and subjected to DNA fragmentation analysis as follows. Cells were collected by centrifugation and lysed with lysis buffer (5 mM Tris-HCL, pH 8; 20 mM EDTA; 0.5% Triton-X100) on ice for 45 minutes. Fragmented DNA in the supernatant of a 14,000 rpm centrifugation (45 minutes at 4 0 C) was extracted twice with phenol/chloroform and once with chloroform and precipitated by ethanol and salt. The DNA pellet was washed once with 70% ethanol and resuspended in TE buffer with 100 gg/mL RNase at 37°C for 2 hours. The fragmented DNA was separated in 1.8% agarose gel electrophoresis, stained with ethidium bromide, and visualized under ultraviolet light.

OP induced apoptosis in the vector control cells. Less DNA fragmentation was observed in wild type SAG transfected cells compared to control cells. SAG mutant 1, which does not encode the zinc ring-finger motif, did not show any protection against OP-induced DNA fragmentation, whereas SAG mutant 2, which retains the zinc ring finger domain, still showed protection. These results suggest that overexpression of SAG protein protects cells against OP-induced apoptosis, and the zinc ring finger domain is required for this protective activity.

Since SAG protein contains a zinc ring finger motif, the sensitivity of SAG transfectants to zinc treatment was examined next. Zinc induced apoptosis in DLD-1 cells WO 99/32514 PCT/US98/26705 transfected with the vector only. Induction of apoptosis was limited by SAG overexpression, which showed much less DNA fragmentation than the control lines. This data suggests that the SAG protein binds to and chelates zinc through the zinc ring finger domain and thus provides increased resistance to zinc toxicity compared to non-transfected cells.

Another feature of SAG is the formation of oligomers after exposure to H202. Cells may be protected from H 2 0 2 induced toxicity by SAG oligomerization. SAG-transfected cells were, therefore, treated with H 2 0 2 followed by assays for DNA fragmentation. H 2 0 2 induced apoptosis in DLD-1 cells. SAG protein overexpression partially protected cells from

H

2 0 2 -induced apoptosis, as evidenced by a reduction in DNA fragmentation. Taken together, these results demonstrate that SAG affords at least some protection against apoptosis induced by redox compounds such as OP and H 2 0 2 and also against apoptosis caused by zinc.

Example 17. Antisense SAG expression inhibits tumor cell growth To test the growth effects induced by SAG expression, DLD-1 cells were transfected with the neo control vector, or vectors expressing SAG, SAG mutants 1 or 2, or antisense SAG, as described above. Neomycin resistant colonies were selected with G418 (600 pg/mL) for 18 days and stained with 50% methanol/10% acetic acid/0.25% Coomassie Blue.

A stable DLD-1 transfectant expressing antisense SAG mRNA (D15-1) was cloned after G418 selection in order to examine potential changes in tumor cell phenotype caused by decreased SAG expression. Subconfluent D15-1 cells, along with the vector control cell (D and SAG (sense) overexpressing cells (D12-1 and D12-8) were metabolically labeled and subjected to immunoprecipitation using SAG protein antibody as described above.

Densitometric quantitation of SAG protein expression using a computing densitometer, (Molecular Dynamics) was performed according to the manufacturer's instructions. The number was calculated by arbitrarily choosing the value from the vector control cell D1-6 as 1. Antisense SAG transfected cells (D15-1) exhibited a 60% reduction in endogenous SAG protein. Monolayer growth of DLD-1 cells was significantly inhibited by antisense SAG transfection. None of the other transfectants were growth-inhibited, as compared to the neo control.

It was next examined whether antisense SAG-transfected cells would exhibit growth inhibition in soft agar. D15-1 cells, along with transfectants expressing wildtype SAG (D12-8), SAG mutant-1 SAG mutant-2 as well as the neo control (D1-3) WO 99/32514 PCT/US98/26705 were grown in 0.25% agar medium for 14 days. Colonies containing greater than 16 cells were counted. Three independent experiments, each run in duplicate, were performed.

Shown is the mean standard error of the mean. As shown in Figure 2, down-regulation of SAG in D15-1 cells did cause significant growth inhibition of DLD-1 cells as reflected by 75% reduction of soft agar colony number when compared to the neo control SAG (sense) expressing line, D12-8, and SAG mutants (D3-3, D4-2).

In a further study, 4 x 106 confluent D15-1 cells along with parental DLD-1 cells, the vector control Dl-6, and SAG wildtype transfectant D12-1 cells were inoculated subcutaneously into SCID mice (Taconic Farms, Germantown, New York), 10 mice per group. Tumor growth was observed twice a week. The average tumor size/mass for 10 mice was plotted against time post injection up to 24 days. When implanted into SCID mice, antisense expressing line D15-1 failed to form tumors up to 24 days after inoculation, whereas substantial tumor growth was observed in parental DLD-1 cells, the neo control Dl- 6 cells, and SAG (sense) expressing D12-1 cells (Figure All these experiments demonstrate that downregulation of SAG expression leads to growth inhibition of tumor cells, and further indicates that SAG is a cellular protective molecule.

Example 18. Cancer gene therapy using adenovirus expressing antisense SAG Since antisense SAG expression has been shown to inhibit tumor growth both in vitro and in vivo (example 17), SAG can be used as a target for cancer gene therapy. Methods for conducting cancer gene therapy are well known in the art (see Zhang and Fang, Exp. Opin, Invest. Drugs 4: 487-514, 1995 and Zhang et al., Adv. Pharmacol. 32: 289-341, 1995).

Tumor cell lines with endogenous SAG expression, including, but not limited to DLD-1 (colon), Du145 (prostate), G401 (kidney), H2009 (lung) and HONET-1 (nasopharynx), are used to establish the tumor models,. Tumor cells from tissue culture are suspended in PBS at a concentration of 5 x 10 7 /mL and stored on ice. 0.2 mL of the cell suspension (containing approximately ten million cells) is subcutaneously injected into the flank of 6- to 8-week-old athymic nude mice and tumors are allowed to grow for 30-40 days or until the average tumor size reaches 5 mm.

Recombinant adenoviral vectors expressing antisense human SAG, driven by the CMV promoter (Ad.CMV-SAG) were produced by co-transfecting a shuttle plasmid (pJM17, circularized Ad5 genome) and a recombinant plasmid (pEC-SAG; a CMV driven plasmid containing left arm of Ad5 genome) into 293 cells.

WO 99/32514 PCT/US98/26705 Tumors are injected with either 0.1 mL of recombinant adenoviral solution (1-5 x 1010 pfu/mL) or 0.1 mL of PBS alone as a control. Daily treatment is performed for 2 days and after 1 week without treatment, daily treatment is resumed for 3 days. The tumor size is measured daily for 2 weeks. To test combinatorial therapy with oxygen radical-generating reagents or irradiation, the treated group is subdivided into three sub-groups (10 mice per subgroup): group A receives adenovirus alone (see above); group B receives adenovirus and at the same time receives an intraperitoneal injection of adriamycin (3 mg/kg) an oxygen radical-generating reagent, and group C receives adenovirus plus irradiation at 350cGy of cesium-137. Some tumor-bearing mice will only receive the same dose of adriamycin or irradiation as drug or irradiation controls,.

Expression of antisense SAG blocks endogenous SAG synthesis, which renders tumor cells supersensitive to oxygen radicals. Significant tumor shrinkage in treated tumors with or without drugs or radiation, as compared with the vehicle control, indicates the efficacy of this therapy. The tumors in both control and treated groups can be further examined histologically. Samples can be immediately embedded in optimal cutting temperature compound (Miles, Inc. Elkhart, Indiana) and snap-frozen in liquid nitrogen for frozen section preparation (3-5 gm) for enzymatic staining terminal deoxynucleotidyl transferase (Boehringer Manheim, Indianapolis, Indiana) staining for apoptosis) or immunohistochemical staining for expression of the antisense SAG. Alternatively, the samples may be fixed in formalin for histologic sectioning and analyze with hematoxylin-eosin (Sigma, St. Louis, Missouri) staining.

Example 19. SAG functions as a oxygen radical scavenger to prevent oxygen radical induced damages SAG protein contains 12 cysteine residues and forms disulfide bonds both intermolecularly and intramolecularly after exposure to hydrogen peroxide. SAG protein also binds to heme, which can modulate oxidants by oxidation/reduction of This oxidative buffering activity may qualify SAG as an oxygen radical scavenger.

Yeast cells having deletions in antioxidant enzyme genes [superoxide dismutase (SOD) and catalase (CAT)] are supersensitive to superoxide anion and hydrogen peroxide (Longo et al. (1997), J. Cell Biol. 137:1581-1588). Yeast cells that lack Cu, Zn-SOD, Mn-SOD, both Cu, Zn-SOD and Mn-SOD, and CAT have been transfected with human SAG expression plasmids. Sensitivity of these transfected cells to oxygen radical producing compounds such as paraquat (a superoxide anion generating compound) and WO 99/32514 PCT/US98/26705 hydrogen peroxide are tested in yeast growth assays and compared to the growth of the same host cells transfected with vector controls. Rescue of these yeast cells from oxygen radicalinduced cell killing indicates that SAG is an effective oxygen radical scavenger.

Example 20. Prevention of IL-J/3 induced brain injury during ischemia by SAG administration It has been previously shown that middle cerebral artery occlusion in rats causes overexpression of interleukin-l which induces brain injury by the release of free radicals (Yang et al., Brain Research 751:181-188, (1997)). Two experiments are conducted to test whether SAG, by scavenging free radicals released, will prevent brain damage.

In the first experiment, human SAG is subcloned into an adenovirus vector driven by RSV promoter (AdRSV-SAG). The adenoviral suspension is injected stereotactically into the lateral ventricle to ensure SAG expression in brain. Five days after administration of adenovirus, middle cerebral artery is occluded in animals for 24 hours as described (Yang etal., Brain Research 751:181-188, (1997)). Brain edema (as measured by brain water content) and cerebral infarct size, measured by histological techniques (Yang et al., Stroke 23:1331-1336, (1992)) is determined. As compared to the vector control, any reduction of brain edema and infarction size indicates SAG protection against free radical induced damage.

In the second experiment, middle cerebral artery occlusion is performed with the rat suture model, allowing either permanent (6 hours) or temporary occlusion (3 hours of occlusion and 3 hours of reperfusion) (Yang and Betz, Stroke, 25:1658-1665, (1994)). Rats then receive an injection of purified SAG protein at the size of occlusion. Brain water, ion contents, and infarct volume are measured to determine brain infarction and blood-brain barrier disruption. As compared to injection of the vehicle control, reduction in brain infarction size and blood-brain barrier disruption indicates a SAG protective effect.

Example 21. Human cancer diagnosis using SAG as a marker: Two SAG deletion mutants in human cancer cell lines originating from colon and testis have been identitifed. Twelve pairs of colon carcinomas and adjacent normal tissues were collected from 12 patients. Genomic DNA and total RNA are isolated from these samples and subjected to PCR amplification. The resulting amplification products are analyzed for detection of SAG deletion mutations by methods well known in the art, including but not limited to RNA protection assays, DNA sequencing, hybridization, and gel WO 99/32514 PCTIUS98/6705 electrophoresis for deletion mutants. Mutations detected in tumor tissues but not in normal adjacent tissues indicate that they are tumor specific mutations and can be used as a diagnostic tool in the clinic for colon as well as testicular carcinomas.

Example 22: The yeast homolog of human SAG gene is essential for yeast growth To further understand the function of SAG, yeast SAG knock-out mutants were constructed by homologous recombination. The construct used to knockout yeast SAG was made by PCR of a kanamycin cassette from kanMX4 plasmid (Wach et al., Yeast 10:1793- 1808, 1994). The primers used for PCR were CGTACGCTGCAGGTCGAC-3' (SEQ ID 17), and SAGKanMX4-3:

TGATTTAAATGTTTACGGGCAATTCATTTTT

ATCGATGAATTCGAGCTCG-3' (SEQ ID 18). The primer SAGKanMX4-5 consists of yeast SAG DNA sequence (ATCC Accession number Z74876) immediately upstream of the initiation codon ATG (underlined) and the upstream kanamycin cassette sequence at its 3'end. Primer SAGKanMX4-3 consists of yeast SAG DNA sequence immediately downstream of the stop codon TGA (underlined) and the downstream kanamycin cassette sequence at its 3'-end.

PCR was conducted for 5 cycles at 94°C 1 min, 50°C, 1.5 min, 72 0 C 2 min, followed by 25 cycles at 94C, 1 min, 56 0 C, 1.5 min, 72 0 C 2 min, followed by a 10 min extension at 72°C. The resulting PCR product (1.5 kb) was gel-purified using Qiaex II gel-purification kit (Qiagen) according to the manufacturer's instruction, and was used to transfect the diploid yeast strain Y21 using the YEASTMAKER yeast Transformation System (ClonTech Laboratory, Inc.) according to the manufacturer's instruction. Following transfection, yeast cells were grown in YPD media (Difco) containing G418 (200 gg/mL, BRL) to select transfectants containing the kanamycin cassette, which have had the yeast SAG deleted by homologous recombination.

Several G418-resistant clones were selected and assayed to determine whether heterozygous or homozygous deletions had been produced. The primers used are SAGPCR- 5'-TTCTCCAGTGGCAGAGAAC-3' (SEQ ID 19) and SAGPCR-3: ATGATTTAAATGTTTACGGGC-3' (SEQ ID 20). These primers constitute fragments of and SAGKanMX4-3, respectively, and flank the entire yeast SAG coding region. PCR of wildtype yeast SAG produces a 0.35 kb band, whereas PCR of SAG deletion WO 99/32514 PCT/US98/26705 mutants give rise to 1.5kb band, consisting of the kanamycin cassette. Both the 0.35 kb and kb fragments were generated in all of the clones tested, indicating that heterozygous mutants were produced. Identical knock-out experiments were conducted with haploid yeast cells (InvSC1 from In Vitrogen) and no G418-resistant clone was isolated.

The failure to isolate homozygous yeast SAG deletion mutants suggests that yeast SAG is essential for growth. To confirm this, 12 individual heterozygous yeast strains (y21ySAG/ySAG::Kan) were sporulated to determine if yeast SAG-kan haploids were viable. The strains were inoculated into minimal potassium acetate sporulation media, supplemented with uracil, lysine, adenine and tryptophan (Kassir, and Simchen, G. Method Enzymol. 194, 94- 110, 1991) and grown at 30 0 C for 7 days. Tetrads was dissected into 4 haploid offspring from each strain. For dissection, a clamp of cells from the sporulation plate was suspended in 100 pgL of 1 M glycerol containing 0.5 mg/mL zymolase T20. After 30 min at 37 0 C, the suspension was diluted with 800 gL sterile water and put on ice. A loop of suspension was struck across a YPD plate and examined under a Zeiss Tetrad microscope for tetrads. The glass microneedle of the scope was used to dissect 4 tetrads from each strain. Two of these four haploid cell should contain wildtype SAG, while the other two should contain a yeast SAG deletion. In all 12 clones, only two out of four dissected cell grew, and none were viable in YPD medium supplemented with G418, indicating that viable cells did not contain the kanamycin cassette or the SAG deletion. The experiment clearly demonstrate that SAG is essential for yeast growth, further demonstrating its evolutionary importance.

To determine ifySAG is required for normal growth or simply for germination, hSAG was cloned into a yeast expression vector with URA3 selectable marker. The hSAG-URA plasmid was then transformed into heterozygous ySAG knockout cells, and transformants were selected on URA-minus plates. Clones expressing hSAG (measured by Western blot analysis) were sporulated and tetrads were dissected. Viable colonies were then screened on either YPD alone, or YPD+G418, or YPD+5-fluoroorotic acid (5-FOA; used to select against the URA3-containing centromere plasmid (Boeke et al., Mol. Gen. Genet, 1984;197:345).

Again the hSAG-URA3 plasmid complemented the ySAG::kan allele, as all four haploids from four individual tetrads grew. When grown on YPD+G418 plates, two haploids from each tetrad die, indicating that they contain the wildtype ySAG gene. Other two haploids from each tetrads survived, indicating they contained ySAG::kan allele. When these latter colonies were grown on YPD+5-FOA plates, which selects against URA3 plasmid, all failed WO 99/32514 PCT/US98/26705 to grow, indicating that ySAG is essential for normal vegetative growth and not simply for sporulation.

Example 23: Human SAG rescue ofyeast SAG knockout phenotype To examine whether human SAG can rescue death phenotype of yeast SAG knockout, wildtype human SAG, along with the SAG mutants (MM3, sequence ID 25; MMIO, sequence ID 39; and MM14, sequence ID 47, Figure 1A) were constructed into a plasmid with Trp selection marker and transfected into heterozygous yeast strain (y21-SAG/ySAG::Kan) as described above. The clones grown in Trp-minus/G418-plus plates were examined by Western blot analysis for SAG expression. The clones expressing human SAG were sporulated and dissected. In 10 wildtype human SAG clones, 3 or 4 haploids are viable. Some of them contain yeast SAG, whereas the others contain ySAG K/O plus human SAG, indicating human wildtype SAG can complement yeast SAG knockout.

All three mutant clones (total of 41 tested) gave rise to 1 or 2 haploids and all survival haploids contains yeast SAG, indicating that human SAG mutants cannot complement yeast SAG knockout.

Example 24: SAG binds to metals Since SAG contains a zinc-ring finger domain, it has the potential to bind with metals.

To measure potential metal binding of SAG, electrospray ionization mass spectrometry (ESI-MS) (Fenn et al., 1989) was used to compare the molecular mass of SAG under denaturing and non-denaturing solution conditions (Loo, 1997; Witkowska et al., 1995).

ESI-MS was performed with a double focusing hybrid mass spectrometer (Finnigan MAT 900Q, Bremen, Germany) with a mass-to-charge range of 10,000 at 5 kV full acceleration potential. A position-and-time-resolved-ion-counting (PATRIC) scanning array detector was used. An ESI interface based on a heated metal capillary inlet and a low flow micro-EsI source (150 nL/min analyte flowrate) were used (Sannes-Lowery et al., 1997).

The metal capillary temperature was maintained around 150-200°C for metal-protein complex studies. Recombinant protein under 7 M urea-denaturing solution was refolded by dialyzing in 50 gtM ZnCl 2 for 3 days with three changes of buffer. Prior to ESI-MS measurement, the SAG solution was washed with a solution of 10 mM ammonium bicarbonate (pH 7) and 1 mM DTT, and excess zinc was removed by centrifugal ultrafiltration by passing through a 10 kDa molecular weight cut-off centrifugal filtration cartridge (Microcon-10 microconcentrator, Amicon, Beverly, MA). For the ESI-MS WO 99/32514 PC TIUS98/6705 analysis, a small portion of the filtered SAG protein solution was diluted into either a denaturing solvent (80:15:5 acetonitrile:water:acetic acid v/v/v, pH 2.5) or a non-denaturing solution (10 mM ammonium bicarbonate and 1 mM DTT, pH 7).

Zinc binding of SAG was first measured. Under a denaturing acidic solution (pH and high organic concentration) where the protein is not expected to retain metal-binding characteristics even in the presence of zinc, the molecular mass of SAG was measured to be 12550, in close agreement with the expected mass for the apo-protein (12552 Da). The ESI-MS analysis of the SAG protein in a non-denaturing aqueous solution (pH 7) resulted in an increase in mass to 12733 and 12800 Da. These masses are consistent for the holo-protein binding 3 and 4 zinc metal ions, respectively.

Copper binding to SAG was also measured. As little as 1 jRM CuSO 4 in the dialysis solution causes SAG precipitation with a blue (copper) color, suggesting a copper binding.

Next, using ESI-MS, the potential copper binding of SAG was measured in a non-denaturing solution described above. Addition of copper acetate to a final concentration of 10 pM resulted in a further inccrease in mass to approximately 12929 Da. However, a precise mass could not be obtained, as a wide distribution of copper adducts appears to bind to SAG protein. Adding copper to higher concentrations resulted in precipitation of the protein.

Example 25: SAG minimizes or prevents LDL oxidation induced by copper ion or a free radical generator Due to its H 2 0 2 buffering and metal binding, it was reasoned that SAG may prevent oxidation of macromolecules induced by metal or free radical generator. An LDL (low density lipoprotein) oxidation induced by copper ion or a free radical generator, AAPH (2,2azobis-2-amidinopropane hydrochloride), was used as a model to test potential protection activity of SAG against lipid peroxidation.

Lipoproteins (100 gig of protein/mL, Intraocel) were incubated with 10 pM CuSO4 or with 5 mM AAPH for 4 hours at 37 0 C in the presence of various concentrations of purified SAG protein. AAPH is a water-soluble azo compound that thermally decomposes and generates water soluble peroxyl radicals at a constant rate (Frei et al., 1988). Oxidation was terminated by the addition of 10 gpM butylated hydrozytoluence (BHT) and refrigeration at 4 0 C. The extent of lipoprotein oxidation was measured by the TBARS assay, using malondialdehyde (MDA) for the standard curve, as described (Buege Aust, 1978).

WO 99/32514 PCTIUS98/26705 Copper-induced LDL oxidation, as measured by the formation of thio barbituric acid reactive substances (TBARS), was slightly enhanced by SAG at low concentrations. At higher SAG concentrations, however, a dose-dependent inhibition (up to 90%) of LDL oxidation was observed. Inhibition was heat-resistant since heat-treated (60 0 C for 15 min) SAG still retains the activity, suggesting that enzymatic activity is not involved. Inhibitory activity was, however, completely or partially abolished by pretreatment of SAG with alkylating reagents NEM and p-hydroxy mercury benzoate (PHMB), respectively. The results indicated that free SH groups in SAG are the major contributors to this activity.

Furthermore, metallothionein, a small metal binding protein consisting of 20 cysteine residues out of 61 amino acids (Nordberg Kojima, 1979) showed a similar inhibitory curve as SAG. Glutathion (GSH), an additional cysteine containing peptide showed a inhibition at a concentration of 100 gM. Inhibition of copper-induced LDL oxidation was, however, not observed in other known antioxidant enzymes such as superoxide dismutase, catalase or other proteins such as BSA, and cytochrome C. These results clearly showed that by binding and chelating copper ion through its free SH groups, SAG prevents copperinitiated free radical reactions leading to LDL oxidation and superoxide or hydrogen peroxide appear not to be involved in the process. To test whether SAG protection against LDL oxidation was solely mediated through copper binding, we initiated LDL oxidation by AAPH, a free radical generator. In this metal-ion free system, SAG also protects LDL oxidation (up to 85%) at a concentration of 59 l.M (750 gjg/mL). Thus, by metal binding and free radical scavenging, SAG acts as a protector against lipid peroxidation.

Example 26. SAG protects cytochrome C release and caspase activation induced by metal ions Since cytochrome C release from mitochondria and caspase activation are the key events in apoptosis (Liu et al., 1996; Yang et al., 1997; Li et al., 1997; Hengartner, 1998, for review, see Mignotte Vayssiere, 1998), the levels of cytochrome C released into cytoplasm and potential activation of caspase upon metal treatments were measured. Treatment of cells with ZnSO 4 induces a time-dependent release of cytochrome C in cytoplasm. Compared to the vector control cell the SAG overexpressing cell (D12-1) has much less cytoplasmic release of cytochrome C. Likewise, activation of caspase 7, shown as disappearance of pro-enzyme form, was seen in a time-dependent manner post zinc treatment.

More activation was seen in vector control cell (D1-6) than that in the SAG overexpressing WO 99/32514 PCT/US98/26705 cell (D12-1). A similar result was obtained with CPP32 (caspase 3) activation. A significant difference, however, was not seen in cytochrome C release or caspase activation between D1- 6 and D12-1 cells upon copper treatment. This is consistent with the lack of difference in morphological changes between the two lines upon copper treatment, although DNA fragmentation was obvious only in the vector control cells. To further examine potential protection of SAG against metal-induced cytochrome C release and CPP32 activation, cytochrome C release and CPP32 activation was measured in 293 cells transiently transfected with SAG expressing plasmid followed by exposure to copper. A significant amount of cytochrome C started to release 6 hours post CuSO 4 (2.0 mM) treatment and lasted up to 12 hours. Expression of SAG delayed cytochrome C release for up to 16 hours. Activation of caspase 7 was seen in the vector control cells 12 hours and 16 hours post copper treatment.

No significant activation was seen in SAG transfectants. The similar result was seen with CPP32 antibody. For zinc treatment, no difference was detected in cytochrome C release and caspase activation between control cells and SAG transfectants, consistent with the lack of difference in morphological signs of apoptosis. These results indicate that metal treatment induces cytochrome C release and caspase activation during apoptosis which can be largely prevented or delayed by SAG and there is a good correlation between morphological signs of apoptosis and cytochrome C release/caspase activation.

Example 27: SAG protects against neuronal apoptosis SAG was transfected into HY5Y human neuroblastoma cells and a few stable lines were selected which expressed exogenous SAG as determined by Western blot. One SAGtransfectant (SYW-20) and a vector control (SYV-3) were used to determine their sensitivity to metal ions, zinc and copper. Treatment with 1.25 mM CuSO 4 or 200 gM ZnSO 4 for 16 hours induced cell shrinkage and detachment in the neo control cells, but to a less extent in SAG-expressing cells. The morphological difference was more obviously seen with the zinc treatment. To determine the nature of cell death, we performed TUNEL assay, a fluorescein labelling assay of free 3'-OH termini generated from cleavage of genomic DNA during apoptosis.

In Situ cell death assay (TUNEL assay) was performed according to the manufacturer's instructions (Boehringer Mannheim). Briefly, 5 x 104 cells were plated into the 8-well glass slides. After treatment with 1.25 mM copper (CuSO 4 or 200 gM zinc (ZnSO 4 for 16 hours, cells were fixed with 0.5% glutaraldehyde for 10 min, then washed WO 99/32514 PCT/US98/26705 with PBS twice. The fixed cells were incubated in permeabilization solution Triton X-100, 0.1% sodium citrate) for 2 min on ice. The TUNEL reaction mixture (50 gL) was added to samples and incubated for 1 hour at 37 0 C followed by 3 times wash with PBS.

Samples were embedded with antifade prior to analysis under a fluorescence microscope.

Substantially more fluorescein staining was seen in the vector control cells after 16 hours treatment with 1.25 mM CuSO 4 or 200 gM ZnSO 4 The results indicate that expression of SAG protects neuronal cells from apoptosis.

Example 28. SAG stimulates proliferation To test potential growth stimulation activity, SAG RNA (8 pg/mL or 25 gg/mL), along with the control P-galactosidase (25 gg/mL), was injected into serum-starved NIH 3T3 fibroblast monolayer. Approximately 50 cells attached to the glass coverslip within an etched circle were injected. A 3-hour pulse of 3 H]thymidine (5 gCi/mL, Amersham) was performed to 24 hours after injection. Cultures were washed with isotonic phosphate-buffered saline and fixed in 3.7% (vol/vol) formaldehyde. Induction of 3 H]thymidine incorporation (an indicator of DNA synthesis) into the nuclei of serum-starved fibroblast cells was obviously observed in SAG-injected cells. In contrast, injection of P-galactosidase does not induce DNA synthesis and no 3 H]thymidine incorporation was observed. The results clearly indicate that human SAG has proliferative activity to stimulate cell growth.

Growth promotion activity of SAG was also examined in human neuroblastoma cells (SY5Y), overexpressing hSAG protein by hSAG cDNA transfection. Both the vectorexpressing control cells and SAG overexpressing cells were first serum-starved for 48 hours, followed by 3H-thymidine labelling for 16 hours in either serum-starved or 1% serum conditions. Cells were washed, lysed and counted in a liquid scintillation counter for 3H, an assay for the measurement of 3H-thymidine incorporation into DNA (S-phase entry).

Compared to the vector control cells, SAG-expressing cells have 10-fold more 3H-thymidine incorporation in both conditions (serum-free or 1% serum), indicating that SAG stimulates cell proliferation/growth.

Growth promotion activity of SAG was also examined in yeast. As described in Example 22, the yeast homolog of human SAG gene is essential for yeast growth. To correlate yeast growth rate with SAG expression, hSAG expressing plasmid was constructed under control of Gal promoter. The plasmid was transformed into heterozygous ySAG knockout and transformants were sporulated and dissected. Haploid ySAG knockout clone WO 99/32514 PCT/US98/26705 that contained hSAG plasmid was identified and analyzed. In the uninduced condition, little SAG expression due to the leakness of the promoter led to formation a tiny clone compared to the full size wildtype clone. Under induced condition, SAG expression level increased and clone size also increased. This experiment clearly demonstrated that SAG promotes cell growth in a dose-dependent manner.

It is to be understood that the invention is not to be limited to the exact details of operation, or to the exact compounds, compositions, methods, procedures or embodiments shown and described, as obvious modifications and equivalents will be apparent to one skilled in the art, and the invention is therefore to be limited only by the full scope of the appended claims.

EDITORIAL NOTE A TUWT Ifl A 'W'3I'%ILT ILTWTI1 fU3-"I I""I1Irr kAjrjr1JALA I I"IN rI uiII The following Sequence Listing pages 1 to 37 are part of the description. The claims pages follow on pages 42 to 54.

WO 99/32514 PCT/US98/26705 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Yi Sun STREET: 4841 Hillway Court CITY: Ann Arbor STATE: Michigan COUNTRY: USA POSTAL CODE (ZIP): 48105 TELEPHONE: (313) 996-1959 TELEFAX: (313) 996-7158 (ii) TITLE OF INVENTION: Sensitive to Apoptosis Gene (SAG) (iii) NUMBER OF SEQUENCES: (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 1140 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:17..355 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION:17..355 (ix) FEATURE: NAME/KEY: misc_feature LOCATION:1..1140 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 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 20 WO 99/32514 WO 9932514PCTIUS98/26705

AAG

Lys

GAC

Asp

GCC

Ala.

GTC

Val

ATG

Met

GTT

Val

TGC

Cys

TCT

Ser

TGC

Cys

AAG

Lys

ACC

Thr

CAA

Gin 65

AAC

Asn

AAC

Asn

ATC

Ile

GCG

Ala

TGC

Cys

GCC

Ala

GTC

Val

GAG

,l Iu L

ATG

Met

CAG

Gin

AGC

Ser

ATG

Met

TGG

Trp,

GAT

Asp

GTG

Va~l

CTG

Leu CTT CGA TGT rLeu Arg Cys GAA AAC AAG GAC TGT GTT AAsp Cys Val TGG GGA GAG Trp Gly Glu CAT TCC TTC His Ser Phe

CAC

His TGC TGC ATG Cys Cys Met

TCC

Ser TGG GTA rrp Val TGG GTG AAA Trp Val Lys GTC CAA AGA Val Gin Arg 110 CAG AAC AAT Gin Asn Asn ATC GGC AAA Ile Gly Lys CGC TGC CCT CTG TGC CAG CAG GAC Arg Cys Pro Leu Cys Gin Gin Asp 100 105 TGAGAGGTGG CCCAGGCGCT CCTGGTGTGG 385

TTGCTGACCC

GCTGTGCGCC

GAAATTCTCT

GTGTGATACG

CCACGACTGG

AAAATAGATA

AATCCTTTTT

AATGAGGATT

GCGTAACTGT

AGCCTGGATT

TATTGTATGT

TTCATTATGC

TCTTAAAATC

TGGACAAAGA

TTTGAGACTC

ACAATTAAGA

AATGCATAGA

AACATTGTGT

AACGAATGTT

GTGTTGGGAG

TTAACCTGCA

CGGGTAAACG

GTTCAACCAC

TTTAGTCAAA

AATGTTTTAA

ATTAAACTAA

CTAAACACTG

ACCAAAGGCT

TAATTTGTTA

AGAGCGAGAA

TCACAGAAGA

ACAGTAACAA

AGAGGCAAGC

CTCAGTGAAG

GCTTTGTCTC

TTAGTTCTAA

AATATTAGTA

TAAAATATTG

TTCATCAATT

CAGGGGATTC

TGCTTTATTA

AAAATGGCCT

CACCAGAAAA

ACATTGTTTG

ATAAAATGCA

GAGGCCACCC

AGGCGTAACT

CTGACTTCTC

AGAACTGTTT

GGAAAATGGC

TGCTTTGAGT

AAATG

ATCCTTGAGA

ATTTGTCTGT

TTCCTACCTC

TGATCTTTGT

TGTTTATGCT

TTGAAAAGCC

TGCTGTCTTC

GTCGGGTAAA

CATCTTTGAC

TCTGTTTTTG

TTACTAGTAT

TATTAAAGTT

GAGAGAGGAT

TTAGTTTTGG

TGGTGTGTGT

TTATCTGTAC

TGAGGGTTAA

GACTCCTCCT

ATTTGCTGTG

CTGTAATATG

TTGGCCAGGA

CCGAAGGTTG

AACACTGAAG

TGATATATAC

445 505 565 625 685 745 805 865 925 985 1045 1105 1140 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 113 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: WO 99/32514 Met Ala Asp 1 Ser Gly Ser Lys Lys Trp 1 Thr Cys Ala Gin Ala Glu 1 Asn His Ser I Asn Arg Cys Lys /al Ala Asn Ile Asn ?he Pro

LOO

Glu 5 Gly Ala Cys Lys His Leu Asp Ser Val Arg Gin 70 Asn Cys Gly Lys Ala Val Glu Cys Gin Glu Ser Met 40 Gin Asp Cys Gin Glu Gly Trp Val Cys Met Asp 105 Pro 10 Gly Ser Met Val Ser 90 Trp Cys Asp Trp Asp Val 75 Leu Val Leu Met Val Cys Trp Val Gin Ser Ser Cys Arg Glu Gin Ile PCT/US98/26705 His Leu Asp Cys Cys Asn Gly (2) INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 754 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:1..339 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION:1..339 (ix) FEATURE: NAME/KEY: misc_feature LOCATION:1..754 OTHER INFORMATION:/note= "Human SAG" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His 5 10 GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu 25 AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT

ATG

Met 1

TCC

Ser

AAG

WO 99/32514 Lys Lys Trp Asn ACG TGC GCC ATC Thr Cys Ala Ile CAA GCT GAA AAC Gin Ala Giu Asn Ala Val Ala TGC AGG GTC Cys Arg Val 55 AAA CAA GAG Lys Gin Giu Trp Ser Trp Asp Val

TGT

Cys PCT/US98/26705 Giu Cys Asp CTT AGA TGT 192 Leu Arg Cys GTG ATG GAT Val Met Asp

GCC

Ala

GTC

V, ~I 240 GAC TGT GTT Asn 11;41~

GTG

75 TGG GGA GAA

AAT

Asn

TGT

AAC

Asn CAT TCC TTC His Ser Phe AAC TGC TGC ATG Asn Cys Cys Met CTG TGG GTG AAA Leu Trp Val Lys 288 AAT CGC TGC CCT Asn Arg Cys Pro TGC CAG CAG Cys Gin Gin

GAC

Asp 105 TGG GTG GTC Trp Vai Val CAA AGA ATC GGC Gin Arg Ile Giy 110

CCCTGGTGGA.

336

AAA

Lys TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG 389

TCTTGTAATC

GAGCCGATGG

ATCTTCAGAA

TGTGGTGTGT

GAATCACCTT

TTTCGATGCT

TTAAA

CAGTGCCCTA

ATCTGTGGTC

ATTCTCTGTG

GTCGCGCACA

ATAATTTACC

TATGGTTGAT

CAAAGGCTAG

TTTGGACTCA

ATTAAGAAGA

CAGCTTAGAA

CATTTCTATA

CAGTTAAAAA

AACACTACAG

TCAAAGCCTT

TAATTTATTA

GTGCTATAAA

CAACAGGCAG

AGAATGTTAC

GGGATGAATT

GGTTAGCATT

AAGGTGGTCC

AAAGGAAAGA

TGGAAGCAGT

AGTAACAAAT

CTTCAAATAG

TGTCAGTTTT

TTCCTACCTC

GCTCCAAATT

TTCGAGACTT

AAAGTGCAGT

449 509 569 629 689 749 754 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 113 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala i 5 10 Leu Ala Ser His Ser Giy Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys 25 Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp 40 Thr Cys Ala Ile Cys Arg Val Gin Val Met Asp Ala 55 Met Val Cys Phe Ser Leu Glu Cys Asp Leu Arg Cys WO 99/32514 PCT/US98/26705 Gin Ala Glu Asn Lys Gin Glu Asp Cys Val Val Val Trp Gly Glu Cys 70 75 Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gin Asn 90 Asn Arg Cys Pro Leu Cys Gin Gin Asp Trp Val Val Gin Arg Ile Gly 100 105 110 Lys INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide P1 downstream primer" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AAGCTTTTTT TTTTTTTR 18 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Oligonucleotide: P2 upstream primer" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: AAGCTTNNNN NNN 13 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Oligonucleotide SAG TA.01" WO 99/32514 PCT/US98/26705 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CGGGATCCCC ATGGCCGACG TGAGG INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide SAG T.02" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CGGGATCCTC ATTTGCCGAT TCTTTG 26 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide P.01" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: TATGGCTAGC ATGGCCGACG TGGAGG 26 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Gin Asn Asn Arg Cys Pro Leu Cys Gin Gin Asp Trp Val Val Gin Arg 1 5 10 WO 99/32514 PCT/US98/26705 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 747 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:1..270 (ix) FEATURE: NAME/KEY: matpeptide LOCATION:1..270 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC Met 1

TCC

Ser

AAG

Lys Ala Asp Val Giu Asp Gly Giu Glu TGC GCC CTG GCC TCT CAC Cys Ala Leu Ala Ser His GAC AAG ATG TTC TCC CTC Asp Lys Met Phe Ser Leu GGG AGC TCA Gly Ser Ser AAG TGG AAC Lys Trp Asn GGC TCC AAG TCG Gly Ser Lys Ser

OGA

Gly 25

TGG

Trp GCG GTG GCC Ala Val Ala ACG TGC GCC Thr Cvs Ala

ATG

Met

CAG

Gin AGC TGG GAC Ser Trp Asp GAG TGC GAT Glu Cys Asp GTC AAG CTG Val Lys Leu ATC TGC AGG Ile Cys Arg ATG CCT GTC Met Pro Val AAA ACA Lys Thr

TTA

Leu

GAG

Glu AAC AAG AGG Asn Lys Arg

ACT

Thr 70 GTG TTG TGG TCT Val Leu Trp Ser

GGG

Gly AAT GTA ATC Asn Val Ile

CCT

Pro TCC ACA ACT Ser Thr Thr GCA TGT CCC TGT Ala Cys Pro Cys TGAAACAGAA CAATCGCTGC

CCTCTCTGCC

TTCTTAGCGC

AGAACACTAC

CATCAAAGCC

GATAATTTAT

AAGTGCTATA

TACAACAGGC

AGCAGGACTG

AGTTGTTCAG

AGGGGATGAA

TTGGTTAGCA

TAAAGGTGGT

AAAAAGGAAA

AGTGGAAGCA

GGTGGTCCAA

AGCCCTGGTG

TTCTTCAAAT

TTTGTCAGTT

CCTTCCTACC

GAGCTCCAAA

GTTTCGAGAC

AGAATCGGCA

GATCTTGTAA

AGGAGCCGAT

TTATCTTCAG

TCTGTGGTGT

TTGAATCACC

TTTTTCGATG

AATGAGAGTG

TCCAGTGCCC

GGATCTGTGG

AAATTCTCTG

GTGTCGCGCA

TTATAATTTA

CTTATGGTTG

GTTAGAAGGC

TACAAAGGCT

TCTTTGGACT

TGATTAAGAA

CACAGCTTAG

CCCATTTCTA

ATCAGTTAAA

WO 99/32514 AAAGAATGTT ACAGTAACAA ATAAAGTGCA GTTTAAA INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 90 amino acids TYPE: amino acid 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 1 5 Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys 25 Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp 40 Thr Cys Ala Ile Cys Arg Val Gin Met Pro Val Leu 55 Lys Thr Asn Lys Arg Thr Val Leu Trp Ser Gly Glu 70 75 Pro Ser Thr Thr Ala Ala Cys Pro Cys Gly INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 706 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:1..291 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION:1..291 PCT/US98/26705 747 Leu Met Val Asp Asn Ala Phe Glu Val Val His Leu Asp Leu Ile (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His 1 5 10 TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu 25 WO 99/32514 PCT/US98/26705 AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp 40 ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GTG GTC Thr Cys Ala Ile Cys Arg Val Gin Vai Met Vai Val 55 AAT CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp 70 AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC Asn Arg Cys Pro Leu Cys Gin Gin Asp Trp Val Val 90 AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG Lys GTG GAG TGC GAT Vai Giu Cys Asp TGG GGA GAA TGT Trp Giy Giu Cys 144 192 GTG AAA CAG Vai Lys Gin CAA AGA ATC Gin Arg Ile

CCCTGGTGGA

240 288

TCTTGTAATC

GAGCCGATGG

ATCTTCAGAA

TGTGGTGTGT

GAATCACCTT

TTTCGATGCT

TTAAA

CAGTGCCCTA

ATCTGTGGTC

ATTCTCTGTG

GTCGCGCACA

ATAATTTACC

TATGGTTGAT

CAAAGGCTAG

TTTGGACTCA

ATTAAGAAGA

CAGCTTAGAA

CATTTCTATA

CAGTTAAAAA

AACACTACAG

TCAAAGCCTT

TAATTTATTA

GTGCTATAAA

CAACAGGCAG

AGAATGTTAC

GGGATGAATT

GGTTAGCATT

AAGGTGGTCC

AAAGGAAAGA

TGGAAGCAGT

AGTAACAAAT

CTTCAAATAG

TGTCAGTTTT

TTCCTACCTC

GCTCCAAATT

TTCGAGACTT

AAAGTGCAGT

INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 97 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Met Ala Asp Val Glu Asp Gly Giu Giu Thr Cys Ala 1 5 10 Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Leu Ala Ser His 25 Lys Lys Trp Asn Ala Val Ala Met Trp 40 Thr Cys Ala Ile Cys Arg Val Gin Val 55 Asn His Ser Phe His Asn Cys Cys Met 70 Ser Trp Asp Met Val Val Ser Leu Trp Met Val Trp Phe Ser Leu Glu Cys Asp Gly Giu Cys Val Lys Gin WO 99/32514 PCT/US98/26705 Asn Arg Cys Pro Leu Cys Gin Gin Asp Trp Val Val Gin Arg Ile Gly 90 Lys INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide hSAG. Ml" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GCCATCTGCA GGGTCCAG 18 INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide SAG T.02L" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GGATCCTCAT TTGCCGATTC TTTGGAC 27 INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 58 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: TTCTCCAGTG GCAGAGAACT TTAAAGAGAA ATAGTTCAAC CGTACGCTGC AGGTCGAC 58 WO 99/32514 PCT/US98/26705 INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide SAGKan MX 4-3" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: ACCTCGGTAT GATTTAAATG TTTACGGGCA ATTCATTTTT ATCGATGAAT TCGAGCTCG 59 INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide SAG per (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: TTCTCCAGTG GCAGAGAAC 19 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "oligonucleotide SAG per 3" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATGATTTAAA TGTTTACGGG C 21 INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 754 base pairs TYPE: nucleic acid STRANDEDNESS: double WO 99/32514 CD) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA PCT[US98/26705 (ix) FEATURE: NAME/KEY: CDS LOCATION:l. .339 (ix) FEATURE: NAME/KEY: mat-peptide LOCATION:l. .339 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: ATG GCC GAC GTG GAA Met 1

TCC

Ser

AAG

Lys Ala Asp Val GGG AGC TCA Gly Ser Ser AAG TGG AAC Lys Trp Asn GAO GGA GAG GAA ACC Asp Gly Glu Giu Thr 10 TCC AAG TOG GGA GGC Ser Lys Ser Gly Gly TGC GCC CTG GCO Cys Ala Leu Ala TOT CAC Ser His GAC AAG ATG Asp Lys Met 25

TGG

Trp GCG GTG GCC Ala Val Ala AGO TGG GAO Ser Trp Asp ACG AGO GCC Thr Ser Ala

GTG

Val

TGT

Cys TTC TCC OTO Phe Ser Leu GAG TGC GAT Glu Cys Asp OTT AGA TGT Leu Arg Cys ATO TGC AGG Ile Cys Arg CAA GCT Gin Ala

GTC

Val 55

GAG

Giu GTG ATG GAT Val Met Asp

GCC

Ala

GTC

Val1 GAA AAO AAA Giu Asn Lys

CAA

Gin GAC TGT GTT Asp Cys Val TGG GGA GAA Trp Gly Glu 240

AAT

Asn CAT TCC TTO CAC AAC TGO TGC ATG His Ser Phe His Asn Cys Cys Met

TCC

Ser

TGG

Trp CTG TGG Leu Trp GTG GTO Val Val AAT OGO TGC CCT OTO TGC CAG CAG Asn Arg Cys Pro Leu Cys Gln Gin

GAC

Asp 105 GTG AAA CAG AAO Val Lys Gin Asn CAA AGA ATO GGC Gin Arg Ile Gly 110

CCCTGGTGGA

288 336 389

AAA

Lys TGAGAGTGGT TAGAAGGCTT CTTAGOGOAG TTGTTCAGAG

TCTTGTAATC

GAGOOGATGG

ATCTTCAGAA

TGTGGTGTGT

GAATCAC.CTT

TTTOGATGOT

TTAAA

OAGTGOOCTA

ATOTGTGGTO

ATTOTOTGTG,

GTOGCGOAOA

ATAATTTAOC

TATGGTTGAT

CAAAGGCTAG

TTTGGAOTCA

ATTAAGAAGA

CAGCTTAGAA

CATTTOTATA

CAGTTAAAAA

AACACTACAG

TCAAAGCCTT

TAATTTATTA

GTGCTATAAA

CAACAGGCAG

AGAATGTTAC

GGGATGAATT

GGTTAGOATT

AAGGTGGTCC

AAAGGAAAGA

TGGAAGCAGT

AGTAACAAAT

CTTCAAATAG

TGTOAGTTTT

TTOOTAOOTO

GOTCCAAATT

TTOGAGAOTT

AAAGTGCAGT

449 509 569 629 689 749 754 WO 99/32514 PCT/US98/26705 INFORMATION FOR SEQ ID NO': 22: SEQUENCE CHARACTERISTICS: LENGTH: 113 amino acids TYPE: amino acid 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 Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu 25 Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp 40 Thr Ser Ala Ile Cys Arg Val Gin Val Met Asp Ala Cys Leu Arg Cys 55 Gin Ala Glu Asn Lys Gin Glu Asp Cys Val Val Val Trp Gly Glu Cys 70 75 Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gin Asn 90 Asn Arg Cys Pro Leu Cys Gin Gin Asp Trp Val Val Gin Arg Ile Gly 100 105 110 Lys INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 754 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:1..339 (ix) FEATURE: NAME/KEY: mat_peptide 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 WO 99/32514 WO 9932514PCTIUS98/26705 TCC GGG AGC Ser Gly Ser AAG AAG TGG Lys Lys Trp ACG TGC GCC Thr Cys Ala

TCA

Ser

AAC

Asn GGC TCC AAG TCG Gly Ser Lys Ser

GGA

Gly 25

TGG

Trp GGC GAG AAG ATG Gly Asp Lys Met TTC TCC CTC Phe Ser Leu GAG TGC GAT Glu Cys Asp GCG GTG GCC Ala Val Ala AGC TGG GAC Ser Trp Asp

GTG

Val ATC AGC AGG Ile Ser Arg CAA GCT Gin Ala

GTC

Val

GAG

Glu GTG ATG GAT GCC TGT CTT AGA TGT Val Met Asp Ala Cys Leu Arg Cys GAA AAC AAA Glu Asn Lys GAG TGT GTT Asp Cys Val

GTG

Val GTC TGG GGA GAA Val Trp Gly Glu

AAT

Asn CAT TCC TTC His Ser Phe

CAC

His

CTC

Leu AAC TGC TGC ATG Asn Cys Cys Met TGC GAG GAG GAG Cys Gin Gin Asp 105

TCC

Ser 90

TGG

Trp CTG TGG GTG AAA Leu Trp Val Lys GAG AAC Gin Asn AAT CGC TGC CCT Asn Arg Cys Pro GTG GTC Val Val CAA AGA AT Gin Arg Il 110

CCCTGGTGGA

CGGC

e Gly

AAA

Lys TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG

TCTTGTAATC

GAGCCGATGG

ATCTTCAGAA

TGTGGTGTGT

GAATCACCTT

TTTCGATGCT

TTAAA

CAGTGCCCTA

ATCTGTGGTC

ATTCTCTGTG

GTCGCGCACA

ATAATTTACC

TATGGTTGAT

CAAAGGCTAG

TTTGGACTCA

ATTAAGAAGA

CAGCTTAGAA

CATTTCTATA

CAGTTAAAAA

AACACTACAG

TCAAAGCCTT

TAATTTATTA

GTGCTATAAA

CAACAGGCAG

AGAATGTTAC

GGGATGAATT

GGTTAGCATT

AAGGTGGTCC

AAAGGAAAGA

TGGAAGCAGT

AGTAACAAAT

CTTCAAATAG

TGTCAGTTTT

TTCCTACCTC

GCTCCAAATT

TTCGAGACTT

AAAGTGCAGT

INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 113 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: Met Ala Asp Val Glu Asp Gly Glu Giu Thr Cys Ala Leu Ala Ser His 1 5 10 Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu 25 WO 99/32514 Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp 40 Thr Cys Ala Ile Ser Arg Val Gin Val Met Asp Ala 55 Gin Ala Glu Asn Lys Gin Glu Asp Cys Val Val Val 70 75 Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp 90 Asn Arg Cys Pro Leu Cys Gin Gin Asp Trp Val Val 100 105 Lys INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 754 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:1..339 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION:1..339 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala 1 5 10 TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys 25 AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp 40 ACG AGC GCC ATC AGC AGG GTC CAG GTG ATG GAT GCC Thr Ser Ala Ile Ser Arg Val Gin Val Met Asp Ala 55 CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC Gin Ala Glu Asn Lys Gin Glu Asp Cys Val Val Val 70 75 AAT CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG Val Cys Trp Val Gin

CTG

Leu

ATG

Met

GTG

Val

TGT

Cys

TGG

Trp

GTG

Glu Leu Gly Lys Arg 110

GCC

Ala

TTC

Phe

GAG

Glu

CTT

Leu

GGA

Gly

AAA

Cys Arg Glu Gin Ile

TCT

Ser

TCC

Ser

TGC

Cys

AGA

Arg

GAA

Glu

CAG

PCT/US98/26705 Asp Cys Cys Asn Gly

CAC

His

CTC

Leu

GAT

Asp

TGT

Cys

TGT

Cys

AAC

WO 99/32514 PCT/US98/26705 Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gin Asn 90 AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336 Asn Arg Cys Pro Leu Cys Gin Gin Asp Trp Val Val Gin Arg Ile Giy i00 105 AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389 Lvs

TCTTGTAATC

GAGCCGATGG

ATCTTCAGAA

TGTGGTGTGT

GAATCACCTT

TTTCGATGCT

TTAAA

CAGTGCCCTA

ATCTGTGGTC

ATTCTCTGTG,

GTCGCGCACA

ATAATTTACC

TATGGTTGAT

CAAAGGCTAG

TTTGGACTCA

ATTAAGAAGA

CAGCTTAGAA

CATTTCTATA

CAGTTAAAAA

AACACTACAG

TCAAAGCCTT

TAATTTATTA

GTGCTATAAA

CAACAGGCAG

AGAATGTTAC

GGGATGAATT

GGTTAGCATT

AAGGTGGTCC

AAAGGAAAGA

TGGAAGCAGT

AGTAACAAAT

CTTCAAATAG

TGTCAGTTTT

TTCCTACCTC

GCTCCAAATT

TTCGAGACTT

AAAGTGCAGT

449 509 569 629 689 749 754 INFORMATION FOR SEQ ID NO: 26: SEQUENCE CHARACTERISTICS: LENGTH: 1i3 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: Met Aia Asp Val Giu Asp Gly Giu Giu Thr Cys Aia Leu Ala 1 Ser Ser His Gly Ser Ser Giy Ser Lys Ser Giy 25 Trp Gly Asp Lys Met Lys Lys Trp Asn Ala Val Ala Met 40 Gin Ser Trp Asp Val Cys Phe Ser Leu Giu Cys Asp Leu Arg Cys Thr Ser Gin Ala Ala Ile Ser Arg Val 55 Giu Val Met Asp Ala Val Giu Asn Lys Gin 70 Asp Cys Val Vai Leu Trp Gly Giu Asn His Ser Phe His Leu Asn Cys Cys Met Trp Val Lys Gin Asn Ile Gly Asn Arg Cys Pro 100 Cys Gin Gin Asp 105 Val Vai Gin INFORMATION FOR SEQ ID NO: 27: WO 99/32514 PCT/US98/26705 SEQUENCE CHARACTERISTICS: LENGTH: 754 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:l..339 (ix) FEATURE: NAME/KEY: matpeptide LOCATION:l..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 1

TCC

Ser

AAG

Lys

ACG

Thr

CAA

Gln

AAT

Asn

AAT

AGC

Ser

TGG

Trp

GCC

Ala

GAA

Glu

TCC

Ser

TGC

TCA

Ser

AAC

Asn

ATC

Ile

AAC

Asn

TTC

Phe

CCT

TCC

Ser

GTG

Val

AGG

Arg

CAA

Gln 70

AAC

Asn

TGC

Gly

AAG

Lys

GCC

Ala

GTC

Val

GAG

Glu

TGC

Cys

CAG

Glu Glu Thr

TCG

Ser

ATG

Met 40

CAG

Gln

GAC

Asp

TGC

Cys

CAG

GGA

Gly 25

TGG

Trp

GTG

Val

TGT

Cys

ATG

Met

GAC

10

GGC

Gly

AGC

Ser

ATG

Met

GTT

Val

TCC

Ser

TGG

Cys

GAC

Asp

TGG

Trp

GAT

Asp

GTG

Val 75

CTG

Leu

GTG

Ala

AAG

Lys

GAC

Asp

GCC

Ala

GTC

Val

TGG

Trp

GTC

Leu Ala Ser ATG TTC TCC Met Phe Ser GTG GAG TGC Val Glu Cys AGT CTT AGA Ser Leu Arg TGG GGA GAA Trp Gly Glu GTG AAA CAG Val Lys Gln CAA AGA ATC Gin Arg Ile 110

CCCTGGTGGA

His

CTC

Leu

GAT

Asp

TGT

Cys

TGT

Cys

AAC

Asn

GGC

Gly 96 144 192 240 288 336 Asn Arg Cys Pro Leu Cys Gin Gin Asp Trp Val Val 100 105 AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG 389 TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA GGGATGAATT CTTCAAATAG GGTTAGCATT TGTCAGTTTT AAGGTGGTCC TTCCTACCTC AAAGGAAAGA GCTCCAAATT WO 99/32514 PCT/US98/26705 GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689 TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749 TTAAA 754 INFORMATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: 113 amino acids TYPE: amino acid 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 Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu 25 Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp 40 Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Ser Leu Arg Cys 55 Gin Ala Glu Asn Lys Gin Glu Asp Cys Val Val Val Trp Gly Glu Cys 70 75 Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn 90 Asn Arg Cys Pro Leu Cys Gin Gin Asp Trp Val Val Gin Arg Ile Gly 100 105 110 Lys INFORMATION FOR SEQ ID NO: 29: SEQUENCE CHARACTERISTICS: LENGTH: 754 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:1..339 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION:1..339 WO 99/32514 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: PCTIUS98/26705 ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC Met 1

TCC

Ser Ala Asp Val Glu Asp Gly Giu Glu Thr 10

GGC

Gly TGC GCC CTG GCC TCT CAC Cys Ala Leu Ala Ser His GAC AAG ATG TTC TCC CTC Asp Lys Met Phe Ser Leu GGG AGC TCA Gly Ser Ser GGC TCC AAG TCG Gly Ser Lys Ser AAG AAG TGG Lys Lys Trp ACG TGC GCC Thr Cvs Ala

AAC

Asn GCG GTG GCC ATG TGG AGC TGG GAC Ala Val Ala Met Trp Ser Trp Asp

GTG

Val GAG TGC GAT Glu Cys Asp ATO TGC AGG Ile Cys Arg CAG GTG ATG GAT GCC TGT CTT AGA TGT Gin Val Met Asp Ala Cys Leu Arg Cys

CAA

Gin

AAT

Asn GAA AAC AAA Glu Asn Lys

CAA

Gin GAG GAC TGT GTT Glu Asp Cys Val GTC TGG GGA GAA Val Trp Gly Glu CAT TCC TTC His Ser Phe

CAC

His AAC TGC TGC ATG Asn Cys Cys Met

TCC

Ser 90 CTG TGG GTG AAA Leu Trp Val Lys CAG AAC Gin Asn 288 AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA Asn Arg Cys Pro Leu Cys Gin Gin Asp Trp Val Val Gin 100

CCC

AGA AT Arg Ii 110

[GGTGGA

C GGC e Gly

AAA

Lys TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG

TCTTGTAATC

GAGCCGATGG

ATCTTCAGAA

TGTGGTGTGT

GAATCACCTT

TTTCGATGCT

TTAAA

CAGTGCCCTA

ATCTGTGGTC

ATTCTCTGTG

GTCGCGCACA

ATAATTTACC

TATGGTTGAT

CAAAGGCTAG

TTTGGACTCA

ATTAAGAAGA

CAGCTTAGAA

CATTTCTATA

CAGTTAAAAA

AACACTACAG

TCAAAGCCTT

TAATTTATTA

GTGCTATAAA

CAACAGGCAG

AGAATGTTAC

GGGATGAATT

GGTTAGCATT

AAGGTGGTCC

AAAGGAAAGA

TGGAAGCAGT

AGTAACAAAT

CTTCAAATAG

TGTCAGTTTT

TTCCTACCTC

GCTCCAAATT

TTCGAGACTT

AAAGTGCAGT

INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 113 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Ala Asp Val Glu Asp Giy Giu Giu Thr Cys Ala Leu Ala Ser His 1 5 10 WO 99/32514 Ser Gly Ser Lys Lys Trp A Thr Cys Ala I Gin Ala Glu I Asn His Ser F Asn Arg Cys I 1 Lys Ser sn :le %sn Phe >ro .00 Gly Ala Cys Lys His Leu Ser Val Arg Gin 70 Asn Cys Lys Ala Val 55 Glu Cys Gin Ser Met 40 Gin Asp Cys Gin Gly 25 Trp Val Cys Met Asp 105 Gly Ser Met Val Ser 90 Trp Asp Trp Asp Val 75 Leu Val Lys Asp Ala Val Trp Val (2) INFORMATION FOR SEQ ID NO: 31: SEQUENCE CHARACTERISTICS: LENGTH: 754 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:1..339 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION:1..339 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala 5 10 GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys 25 AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp 40 TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC Cys Ala Ile Cys Arg Val Gin Val Met Asp Ala 55 GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC Met Val Cys Trp Val Gln

CTG

Leu

ATG

Met

GTG

Val

TGT

Cys

TGG

Phe Glu Leu Gly Lys Arg 110

GCC

Ala

TTC

Phe

GAG

Glu

CTT

Leu

GGA

Ser Cys Arg Glu Gln Ile

TCT

Ser

TCC

Ser

TGC

Cys

AGA

Arg

GAA

PCT/US98/26705 Leu Asp Cys Ser Asn Gly CAC 48 His CTC 96 Leu GAT 144 Asp TGT 192 Cys TGT 240

ATG

Met 1

TCC

Ser

AAG

Lys

ACG

Thr

CAA

WO 99/32514 PCT/US98/26705 Gin Ala Glu Asn Lys Gin Giu Asp Cys Val Val Val Trp Gly Glu Cys 70 75 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 Gin Asn 90 AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336 Asn Arg Cys Pro Leu Cys G1 n G In Asp Trp Val Val Gin Arg Ile Gly 100 105 110 AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389 Lys

TCTTGTAATC

GAGCCGATGG

ATCTTCAGAA

TGTGGTGTGT

GAATCACCTT

TTTCGATGCT

TTAAA

CAGTGCCCTA

ATCTGTGGTC

ATTCTCTGTG

GTCGCGCACA

ATAATTTACC

TATGGTTGAT

CAAAGGCTAG

TTTGGACTCA

ATTAAGAAGA

CAGCTTAGAA

CATTTCTATA

CAGTTAAAAA

AACACTACAG

TCAAAGCCTT

TAATTTATTA

GTGCTATAAA

CAACAGGCAG

AGAATGTTAC

GGGATGAATT

GGTTAGCATT

AAGGTGGTCC

AAAGGAAAGA

TGGAAGCAGT

AGTAACAAAT

CTTCAAATAG

TGTCAGTTTT

TTCCTACCTC

GCTCCAAATT

TTCGAGACTT

AAAGTGCAGT

INFORMATION FOR SEQ ID NO: 32: SEQUENCE CHARACTERISTICS: LENGTH: 113 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: Met Ala Asp Val Glu Asp Gly Giu Glu Thr Cys Ala Leu Ala 1 Ser 5 Gly 10 Gly Ser His Gly Ser Ser Ser Lys Ser Asp Lys Met Phe Ser Leu Glu Cys Asp Lys Lys Trp Thr Cys Ala Asn Ala Val Ala Trp Ser Trp Asp Val Ile Cys Arg Gin Ala Val Glu Val Met Asp Cys Leu Arg Cys Glu Asn Lys Gin 70 Asn Asp Cys Val Val1 Trp Gly Glu Asn Lys Ser Phe His Cys Cys Met Ser 90 Leu Trp Val Lys Gin Asn Arg Cys Pro Leu Cys Gin Gin Asp Trp, Val Val Gin Arg Ile Gly WO 99/32514 WO 9932514PCT[US98/26705 Lys INFORMATION FOR SEQ ID NO: 33: SEQUENCE CHARACTERISTICS: LENGTH: 754 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:l. .339 (ix) FEATURE: NAME/KEY: mat-peptide LOCATION:l. .339 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala 1

TCC

Ser

AAG

Lys

ACG

Thr

CAA

Gln

AAT

Asn

AAT

GGG

Gly

AAG

Lys

TGC

Cys

GCT

Ala

CAT

His

CGC

TCA

Ser

AAC

Asn

ATC

Ile

AAC

Asn

TTC

Phe

CCT

5

GGC

Gly

GCG

Ala

TGC

Cys

AAA

Lys

AAG

Lys

CTC

TCC

Ser

GTG

Val

AGG

Arg

CAA

Gln 70

AAC

Asn

TGC

AAG

Lys

GCC

Ala

GTC

Val 55

GAG

Glu

TGC

Cys

CAG

TCG

Ser

ATG

Met

CAG

Gln

GAC

Asp

TGC

Cys

CAG

GGA

Gly 25

TGG

Trp

GTG

Val

TGT

Cys

ATG

Met

GAC

10

GGC

Gly

AGC

Ser

ATG

Met

GTT

Val

TCC

Ser 90

TGG

GAC

Asp

TGG

Trp,

GAT

Asp

GTG

Val

CTG

Leu

GTG

AAG

Lys

GAC

Asp

GCC

Ala

GTC

Val

TGG

Trp

GTC

Leu Ala Ser ATG TTC TCC Met Phe Ser GTG GAG TGC Val Glu Cys TGT CTT AGA Cys Leu Arg TGG GGA GAA Trp Gly Glu GTG AAA CAG Val Lys Gln CAA AGA ATC Gln Arg Ile 110

CCCTGGTGGA

96 144 192 240 288 336 Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val

AAA

Lys TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG 389 TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 449 509 WO 99/32514 PCT/US98/26705 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 INFORMATION FOR SEQ ID NO: 34: SEQUENCE CHARACTERISTICS: LENGTH: 113 amino acids TYPE: amino acid 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 Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu 25 Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp 40 Thr Cys Ala Ile Cys Arg Val Gin Val Met Asp Ala Cys Leu Arg Cys 55 Gin Ala Glu Asn Lys Gin Glu Asp Cys Val Val Val Trp Gly Glu Cys 70 75 Asn His Ser Phe Lys Asn Cys Cys Met Ser Leu Trp Val Lys Gin Asn 90 Asn Arg Cys Pro Leu Cys Gln Gin Asp Trp Val Val Gin Arg Ile Gly 100 105 110 Lys INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 754 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:1..339 (ix) FEATURE: WO 99/32514 NAME/KEY: matpeptide LOCATION:l..339 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: PCTIUS98/26705 ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC Met 1

TCC

Ser Ala Asp Val Glu Asp Gly Glu Glu GGG AGC TCA GGC Gly Ser Ser Gly TCC AAG TCG Ser Lys Ser

GGA

Gly 25

TGG

Trp Thr Cys GGC GAC Gly Asp AGC TGG Ser Trp GCC CTG GCC TCT CAC Ala Leu Ala Ser His AAG ATG TTC TCC CTC Lys Met Phe Ser Leu GAC GTG GAG TGC GAT Asp Val Giu Cys Asp 48 96 144 192 AAG AAG TGG Lys Lys Trp ACG TGC GCC Thr Cys Ala AAC GCG GTG GCC Asn Ala Val Ala

ATG

Met 40

CAG

Gln

TGT

Cys ATC TGC AGG Ile Cys Arg GTG ATG GAT Val Met Asp CTT AGA TGT Leu Arg Cys CAA GCT Gln Ala GAA AAC AAA Glu Asn Lys GAC TGT GTT Asp Cys Val

GTG

Val TGG GGA GAA Trp Gly Glu 240

AAT

Asn CAT TCC TTC His Ser Phe

CAC

His

CTC

Leu AAC TGC AGC ATG Asn Cys Ser Met CTG TGG GTG AAA Leu Trp Val Lys CAG AAC Gin Asn AAT CGC TGC CCT Asn Arg Cys Pro TGC CAG CAG GAC TGG GTG GTC CAA Cys Gln Gin Asp Trp Val Val Gin

CCC

AGA AT( Arg I1 110

PGGTGGA

GGC

Gly

AAA

Lys TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG 389

TCTTGTAATC

GAGCCGATGG

ATCTTCAGAA

TGTGGTGTGT

GAATCACCTT

TTTCGATGCT

TTAAA

CAGTGCCCTA

ATCTGTGGTC

ATTCTCTGTG

GTCGCGCACA

ATAATTTACC

TATGGTTGAT

CAAAGGCTAG

TTTGGACTCA

ATTAAGAAGA

CAGCTTAGAA

CATTTCTATA

CAGTTAAAAA

AACACTACAG

TCAAAGCCTT

TAATTTATTA

GTGCTATAAA

CAACAGGCAG

AGAATGTTAC

GGGATGAATT

GGTTAGCATT

AAGGTGGTCC

AAAGGAAAGA

TGGAAGCAGT

AGTAACAAAT

CTTCAAATAG

TGTCAGTTTT

TTCCTACCTC

GCTCCAAATT

TTCGAGACTT

AAAGTGCAGT

449 509 569 629 689 749 754 INFORMATION FOR SEQ ID NO: 36: SEQUENCE CHARACTERISTICS: LENGTH: 113 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Me Se Ly Th Gl1 6 As As: Ly (2

ATI

Me

TC

Se

AA(

Ly

AC

WO 99/32514 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: t Ala Asp Val Glu Asp Gly Glu Glu Thr Cys A 1 5 r Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp L 25 s Lys Trp Asn Ala Val Ala Met Trp Ser Trp A 40 r Cys Ala Ile Cys Arg Val Gin Val Met Asp A 55 n Ala Glu Asn Lys Gin Glu Asp Cys Val Val V 5 70 75 n His Ser Phe His Asn Cys Ser Met Ser Leu T: n Arg Cys Pro Leu Cys Gin Gin Asp Trp Val V 100 105 la ys sp la al rp al s

G

t 1

C

r

G

s

G

INFORMATION FOR SEQ ID NO: 37: SEQUENCE CHARACTERISTICS: LENGTH: 754 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:I..339 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION:I..339 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala 5 GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys 25 AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp 40 TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC PCT/US98/26705 Leu Met Val Cys Trp Val Gln

CTG

Leu

ATG

Met

GTG

Val

TGT

Ala Phe Glu Leu Gly Lys Arg 110

GCC

Ala

TTC

Phe

GAG

Glu

CTT

His Leu Asp Cys Cys Asn Gly

CAC

His

CTC

Leu

GAT

Asp

TGT

WO 99/32514 Thr Cys Ala Ile Cys Arg Vai Gin Val Met Asp Aia 55 CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC Gin Ala Giu Asn Lys Gin Giu Asp Cys Val Val Val 70 AAT CAT TCC TTC CAC AAC TGC TGC ATG TCC CTG TGG Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp 90 AAT CGC AGC CCT CTC TGC CAG CAG GAC TGG GTG GTC Asn Arg Ser Pro Leu Cys Gin Gin Asp Trp Vai Val i00 105 AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG Lys PCT/IJ598/26705 Cys Leu Arg Cys TGG GGA GAA Trp Giy Giu 240 GTG AAA CAG AAC Val Lys Gin Asn CAA AGA ATC GGC Gin Arg Ile Gly 110 CCC TGGTGGA 288 336 389

TCTTGTAATC

GAGCCGATGG

ATCTTCAGAA

TGTGGTGTGT

GAATCACCTT

TTTCGATGCT

TTAAA

CAGTGCCCTA

ATCTGTGGTC

ATTCTCTGTG

GTCGCGCACA

ATAATTTACC

TATGGTTGAT

CAAAGGCTAG

TTTGGACTCA

ATTAAGAAGA

CAGCTTAGAA

CATTTCTATA

CAGTTAAAAA

AACACTACAG

TCAAAGCCTT

TAATTTATTA

GTGCTATAAA

CAACAGGCAG

AGAATGTTAC

GGGATGAATT

GGTTAGCATT

AAGGTGGTCC

AAAGGAAAGA

TGGAAGCAGT

AGTAACAAAT

CTTCAAATAG

TGTCAGTTTT

TTCCTACCTC

GCTCCAAATT

TTCGAGACTT

AAAGTGCAGT

449 509 569 629 689 749 754 INFORMATION FOR SEQ ID NO: 38: SEQUENCE CHARACTERISTICS: LENGTH: i13 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: Met Ala Asp Vai Giu Asp Giy Giu Giu Thr Cys Ala i 5 10 Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Leu Ala Ser His Met 25 Trp Lys Lys Trp Asn Ala Val Ala Met 40 Thr Cys Aia Ile Cys Arg Val Gin 55 Gin Ala Giu Asn Lys Gin Glu Asp Ser Trp Asp Val Cys Phe Ser Leu Giu Cys Asp Leu Arg Cys Val Met Asp Ala Cys Val Val Trp Gly Glu 70 Cys Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gin Asn 90 WO 99/32514 PCT/US98/26705 Asn Arg Ser Pro Leu Cys Gin Gin Asp Trp Val Val Gin Arg Ile Gly 100 105 110 Lys INFORMATION FOR SEQ ID NO: 39: SEQUENCE CHARACTERISTICS: LENGTH: 754 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:1..339 (ix) FEATURE: NAME/KEY: mat_peptide 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 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 25 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 40 ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192 Thr Cys Ala Ile Cys Arg Val Gin Val Met Asp Ala Cys Leu Arg Cys 55 CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240 Gin Ala Glu Asn Lys Gin Glu Asp Cys Val Val Val Trp Gly Glu Cys 70 75 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 Gin Asn 90 AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336 Asn Arg Cys Pro Leu Cys Gin Gin Asp Trp Val Val Gin Arg Ile Gly 100 105 110 AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389 Lys WO 99/32514 TCTTGTAATC CAGTGCCCTA GAGCCGATGG ATCTGTGGTC ATCTTCAGAA ATTCTCTGTG TGTGGTGTGT GTCGCGCACA ,a GAATCACCTT ATAATTTACC TTTCGATGCT TATGGTTGAT

TTAAA

CAAAGGCTAG

TTTGGACTCA

ATTAAGAAGA

CAGCTTAGAA

CAGTTAAAAA

AACACTACAG

TCAAAGCCTT

TAATTTATTA

GTGCTATAAA

AGAATGTTAC

GGGATGAATT

GGTTAGCATT

AAGGTGGTCC

AAAGGAAAGA

AGTGGAACAGT

AGTAACAAAT

PCT/US98/26705 CTTCAAATAG 449 TGTCAGTTTT 509 TTCCTACCTC 569 GCTCCAAATT 629 TTCGAGACTT 689 AAAGTGCAGT 749 754 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 113 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala 1 Ser 10 Gly Ser His Gly Ser Ser Gly Ser Lys Ser Gly 25 Trp Asp Lys Met Lys Lys Trp Thr Cys Ala Asn Ala Val Ala Ser Trp Asp Val Cys Phe Ser Leu Glu Cys Asp Leu Arg Cys Ile Cys Arg Val Glu Val Met Asp Ala Gin Ala Glu Asn Lys Asp Cys Val Val 75 Leu Val Trp Gly Glu Cys Asn Lys Ser Phe Asn Cys Cys Met Ser Trp Trp Val Lys Gin Asn Asn Arg Cys Pro 100 Leu Cys Gin Gin Asp 105 Val Val Gin Arg Ile Gly 110 Lys INFORMATION FOR SEQ ID NO: 41: SEQUENCE CHARACTERISTICS: LENGTH: 754 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: WO 99/32514 WO 9932514PCT/US98/26705 NAME/KEY: CDS LOCATION:l. .339 (ix) FEATURE: NAME/KEY: mat-peptide LOCATION:l. .339 SEQUENCE DESCRIPTION: SEQ 1D NO: 41: ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC Met Ala Asp Val Glu Asp Gly Glu Glu Thr

GGC

Gly TGC GCC CTG GCC TCT CAC Cys Ala Leu Ala Ser His GAC AAG ATG TTC TCC CTC Asp Lys Met Phe Ser Leu TCC GGG AGC Ser Gly Ser AAG AAG TGG Lys Lys Trp ACG TGC GCC Thr Cys Ala

TCA

Ser

AAC

Asn GGC TCC AAG TCG Gly Ser Lys Ser

GGA

Gly

TGG

Trp GCG GTG GCC Ala Val Ala

ATG

Met 40

CAG

Gln AGC TGG GAC Ser Trp Asp

GTG

Val

TGT

Cys GAG TGC GAT Glu Cys Asp CTT AGA AGT Leu Arg Ser ATC TGC AGG Ile Cys Arg GTG ATG GAT Val Met Asp CAA GCT Gln Ala

GCC

Ala

GTC

Val GAA AAC AAA Glu Asn Lys

CAA

Gln GAC TGT GTT Asp Cys Val TGG GGA GAA Trp Gly Glu AAT CAT TCC TTC Asn His Ser Phe AAT CGC TGC CCT Asn Arg Cys Pro AAC TGC TGC ATG Asn Cys Cys Met

TCC

Ser CTG TGG GTG AAA Leu Trp Val Lys CAG AAC Gln Asn TGC CAG CAG GAC TGG GTG GTC CAA Cys Gln Gln Asp Trp Val Val Gln 105 ccc' AGA AT Arg Il 110

EGGTGGA

CGGC

e Gly

AAA

Lys TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG

TCTTGTAATC

GAGCCGATGG

ATCTTCAGAA

TGTGGTGTGT

GAATCACCTT

TTTCGATGCT

TTAAA

CAGTGCCCTA

ATC!TGTGGTC

ATTCTCTGTG

GTCGCGCACA

ATAATTTACC

TATGGTTGAT

CAAAGGCTAG

TTTGGACTCA

ATTAAGAAGA

CAGCTTAGAA

CATTTCTATA

CAGTTAAAAA

AACACTACAG

TCAAAGCCTT

TAATTTATTA

GTGCTATAAA

CAACAGGCAG

AGAATGTTAC

GGGATGAATT

GGTTAGCATT

AAGGTGGTCC

AAAGGAAAGA

TGGAAGCAGT

AGTAACAAAT

CTTCAAATAG

TGTCAGTTTT

TTCCTACCTC

GCTCCAAATT

TTCGAGACTT

AA.AGTGCAGT

INFORMATION FOR SEQ ID NO: 42: SEQUENCE CHARACTERISTICS: LENGTH: 113 amino acids Me Se Ly Th G1 6 As As WO 99/32514 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: t Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala 1 5 10 r Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys 25 s Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp 40 r Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala 55 n Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val 5 70 75 n His Ser Phe His Asn Cys Cys Met Ser Leu Trp 90 n Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val 100 105 PCTIUS98/26705 Leu Met Val Cys Trp Val Gin Ala Phe Glu Leu Gly Lys Arg 110 Ser Ser Cys Arg Glu Gin Ile His Leu Asp Ser Cys Asn Gly Lys (2)

ATG

Met 1

TCC

Ser

AAG

INFORMATION FOR SEQ ID NO: 43: SEQUENCE CHARACTERISTICS: LENGTH: 754 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:1..339 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION:1..339 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His 5 10 GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu 25 AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144 WO 99/32514 Lys Lys Trp Asn Ala Val Ala ACG TGC GCC ATC TGC AGG GTC Thr Cys Ala Ile Cys Arg Val 55 CAA GCT GAA AAC AAA CAA GAG Gin Al a Giu Asrn Lys Gln Giu Trp Ser Trp Asp Val

TGT

GCC

Ala GAC AGT GTT Asp Ser Val GTC TGG GGA GAA Val Trp Gly Giu AAT CAT TCC TTC Asn His Ser Phe AAT CGC TGC CCT Asn Arg Cys Pro 100

CAC

His

CTC

Leu AAC TGC TGC ATG Asn Cys Cys Met

TCC

Ser 90 CTG TGG Leu Trp TGC CAG CAG GAC TGG GTG GTC Cys Gin Gin Asp Trp Val Vai GTG AAA CAG AAC Val Lys Gin Asn CAA AGA ATC GGC Gin Arg Ile Gly 110

CCCTGGTGGA

240 288 336

AAA

Lys TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG

TCTTGTAATC

GAGCCGATGG

ATCTTCAGAA

TGTGGTGTGT

GAATCACCTT

TTTCGATGCT

TTAAA

CAGTGCCCTA

ATCTGTGGTC

ATTCTCTGTG

GTCGCGCACA

ATAATTTACC

TATGGTTGAT

CAAAGGCTAG

TTTGGACTCA

ATTAAGAAGA

CAGCTTAGAA

CATTTCTATA

CAGTTAAAAA

AACACTACAG

TCAAAGCCTT

TAATTTATTA

GTGCTATAAA

CAACAGGCAG

AGAATGTTAC

GGGATGAATT

GGTTAGCATT

AAGGTGGTCC

AAAGGAAAGA

TGGAAGCAGT

AGTAACAAAT

CTTCAAATAG

TGTCAGTTTT

TTCCTACCTC

GCTCCAAATT

TTCGAGACTT

AAAGTGCAGT

449 509 569 629 689 749 754 INFORMATION FOR SEQ ID NO: 44: SEQUENCE CHARACTERISTICS: LENGTH: 113 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ, ID NO: 44: Met Ala Asp Val Glu Asp Gly Giu Glu Thr Cys Ala 1 5 10 Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys 25 Leu Ala Ser His Met Phe Ser Leu Val Giu Cys Asp Asn Lys Lys Trp Thr Cys Ala Ala Val Ala Met 40 Trp Ser Trp Asp Cys Ile Cys Arg Val 55 Gin Val Met Asp Ala Leu Arg Cys WO 99/32514 PCT/US98/26705 Gin Ala Glu Asn Lys Gin Glu Asp Ser Val Val Val Trp Gly Glu Cys 70 75 Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gin Asn 90 Asn Arg Cys Pro Leu Cys Gin Gin Asp Trp Val Val Gin Arg Ile Gly 100 105 110 Lys INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 754 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:1..339 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION:1..339 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 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 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 25 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 40 ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC AGT CTT AGA AGT 192 Thr Cys Ala Ile Cys Arg Val Gin Val Met Asp Ala Ser Leu Arg Ser 55 CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240 Gin Ala Glu Asn Lys Gin Glu Asp Cys Val Val Val Trp Gly Glu Cys 70 75 f 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 Gin Asn 90 AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336 Asn Arg Cys Pro Leu Cys Gin Gin Asp Trp Val Val Gin Arg Ile Gly 100 105 110 WO 99/32514 AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA Lys PCT/US98/26705

TCTTGTAATC

GAGCCGATGG

ATCTTCAGAA

TGTGGTGTGT

GAATCACCTT

TTTCGATGCT

TTAAA

CAGTGCCCTA

ATCTGTGGTC

ATTCTCTGTG

GTCGCGCACA

ATAATTTACC

TATGGTTGAT

CAAAGGCTAG

TTTGGACTCA

ATTAAGAAGA

CAGCTTAGAA

CATTTCTATA

CAGTTAAAAA

AACACTACAG

TCAAAGCCTT

TAATTTATTA

GTGCTATAA.A

CAACAGGCAG

AGAATGTTAC

GGGATGAATT

GGTTAGCATT

AAGGTGGTCC

AAAGGAAAGA

TGGAAGCAGT

AGTAACAAAT

CTTCAAATAG

TGTCAGTTTT

TTCCTACCTC

GCTCCAAATT

TTCGAGACTT

AAAGTGCAGT

449 509 569 629 689 749.

754 INFORMATION FOR SEQ ID NO: 46: SEQUENCE CHARACTERISTICS: LENGTH: 113 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: Met 1 Ser Lys Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala 10 Gly Ser His Gly Ser Ser Lys Trp Asn Gly Ser Lys Ser Asp Lys Met Ala Val Ala Thr Cys Ala Met 40 Gln Ser Trp Asp Val Ser Phe Ser Leu Glu Cys Asp Leu Arg Ser Ile Cys Arg Gln Ala Val1 Glu Val Met Asp Glu Asn Lys Asp Cys Val Asn Val 75 Trp Gly Glu Cys His Ser Phe Asn Cys Cys Met Ser Trp Leu Trp Val Lys Gln Asn Ile Gly Asn Arg Cys Pro 100 Cys Gln Gln Val Val Gln Arg 110 INFORMATION FOR SEQ ID NO: 47: SEQUENCE CHARACTERISTICS: LENGTH: 754 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear WO 99/32514 WO 9932514PCT/US98/26705 (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:l. .339 NAME/KEY: matpeptide LOCATION:1. .339 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC Cys Ala Leu Ala Met 1

TCC

Ser

AAG

Lys Ala Asp Val GGG AGC TCA Gly Ser Ser AAG TGG AAC Lys Trp Asn Giu 5

GGC

Giy Asp Gly Giu Giu Thr 1.0

GGC

Gly TCT CAC Ser His TCC AAG TCG Ser Lys Ser GAC AAG ATG Asp Lys Met GCG GTG GCC Ala Val Ala ACG TGC GCC Thr Cys Ala

ATG

Met 40

CAG

Gin AGC TGG GAC Ser Trp Asp

GTG

Val

TOT

Cys TTC TCC CTC Phe Ser Leu GAG TGC GAT Giu Cys Asp CTT AGA TOT Leu Arg Cys ATO TGC AGO Ile Cys Arg CAA OCT Gin Ala

GTC

Val

GAG

Glu OTO ATG OAT Val Met Asp 192 GAA AAC AAA Olu Asn Lys GAC TGT GTT Asp Cys Val TGG GGA GAA Trp Gly Glu 240

AAT

Asn CAT TCC TTC His Ser Phe

CAC

His

CTC

Leu AAC TOC TGC ATG Asn Cys Cys Met

TCC

Ser 90 TGG GTO AAA Trp Val Lys

CA

01: 9 G AAC n Asn

CGC

e Gly 288 AAT CGC AGC CCT Asn Arg Ser Pro AGC CAG CAG Ser Gin Gin

GAC

Asp 1.05 TOO GTG GTC Trp Val Val CAA AGA ATi Gin Arg Ii 110

CCCTGGTGGA

336

AAA

Lys TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG 389

TCTTGTAATC

GAGCCGATGG

ATCTTCAGAA

TGTGGTGTGT

GAATCACCTT

TTTCGATGCT

TTAAA

CAGTGCCCTA

ATCTGTGGTC

ATTCTCTGTG

OTCGCGCACA

ATAATTTACC

TATGGTTGAT

CAAAGGCTAG

TTTGGACTCA

ATTAAGAAGA

CAGCTTAGAA

CATTTCTATA

CAGTTAAAAA

AACACTACAG

TCAAAGCCTT

TAATTTATTA

GTGCTATAAA

CAACAGGCAG

AGAATGTTAC

GGGATGAATT

GGTTAOCATT

AAGGTGGTCC

AAAGGAAAGA

TGGAAGCAGT

AGTAACAAAT

CTTCAAATAG

TGTCAGTTTT

TTCCTACCTC

GCTCCAAATT

TTCGAGACTT

AAAGTGCAGT

449 509 569 629 689 749 754 WO 99/32514 INFORMATION FOR SEQ ID NO: 48: SEQUENCE CHARACTERISTICS: LENGTH: 113 amino acids TYPE: amino acid 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 1 5 10 Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys 25 Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp 40 Thr Cys Ala Ile Cys Arg Val Gin Val Met Asp Ala 55 Gin Ala Glu Asn Lys Gin Glu Asp Cys Val Val Val 70 75 Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp 90 Asn Arg Ser Pro Leu Ser Gln Gin Asp Trp Val Val 100 105 Lys INFORMATION FOR SEQ ID NO: 49: PCT[US98/26705 Leu Met Val Cys Trp Val Gin Ala Phe Glu Leu Gly Lys Arg 110 Ser Ser Cys Arg Glu Gln Ile His Leu Asp Cys Cys Asn Gly SEQUENCE CHARACTERISTICS: LENGTH: 754 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION:1..339 (ix) FEATURE: NAME/KEY: mat_peptide 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 Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His 1 5 10 WO 99/32514 WO 9932514PCT/US98/26705 TCC GGG AGC Ser Gly Ser AAG AAG TGG Lys Lys Trp Thr Cys Ala CAA GCT GAA Gin Ala Glu

TCA

Ser 2 b

AAC

Asn GGC TCC AAG TCG Gly Ser Lys Ser

GGA

Gly 25

TGG

Trp GGC GAC AAG ATG Gly Asp Lys Met GCG GTG GCC Ala Val Ala AGC TGG GAC Ser Trp Asp TTC TOG CTC Phe Ser Leu GAG AGC GAT Glu Ser Asp CTT AGA rul' Leu Arg Cys Ile Cys Arg AAG AAA CAA Asn Lys Gin 70 '3TLL Val 55

GAG

Glu Val Met Asp

GCC

Ala

GTC

Val GAC TGT GTT Asp Cys Val TGG GGA GAA Trp Gly Glu

AAT

Asn

TGT

Gys CAT TGG TTC His Ser Phe

GAC

His AAC TGC TGC ATG Asn Cys Gys Met

TCC

Ser 90

TGG

Trp CTG TGG GTG AAA Leu Trp Val Lys GAG AAC Gin Asn AAT CGC TGG CGT GTG TGC GAG GAG GAG Asn Arg Cys Pro Leu Gys Gin Gin Asp GTG GTG Val Val CAA AGA AT( Gin Arg Il 110

CCCTGGTGGA

CGGC

e Gly

AAA

Lys TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG

TCTTGTAATC

GAGCGGATGG

ATCTTCAGAA

TGTGGTGTGT

GAATCACCTT

TTTCGATGGT

TTAAA

CAGTGCGCTA

ATGTGTGGTC

ATTCTCTGTG

GTCGGGACA

ATAATTTACC

TATGGTTGAT

GAAAGGCTAG

TTTGGAGTGA

ATTAAGAAGA

GAGCTTAGAA

CATTTGTATA

CAGTTAAAAA

AACACTACAG

TCAAAGCCTT

TAATTTATTA

GTGCTATAAA

CAACAGGCAG

AGAATGTTAC

GGGATGAATT

GGTTAGCATT

AAGGTGGTCC

AAAGGAAAGA

TGGAAGCAGT

AGTAACAAAT

CTTCAAATAG

TGTCAGTTTT

TTCGTAGCTC

GCTCCAAATT

TTGGAGACTT

AAAGTGCAGT

449 509 569 629 689 749 754 INFORMATION FOR SEQ ID NO: SEQUENGE GHARACTERISTICS: LENGTH: 113 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Ala Asp Val Glu Asp Gly Giu Glu Thr Gys Ala Leu Ala Ser His 1 5 10 Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu 25 Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Ser Asp 40 WO 99/32514 PCTIUS98/26705 Thr Cys Ala Ile Cys Arg Val Gin Val. Met Asp Ala Cys Leu Arg Cys 55 Gin Ala Giu Asn Lys Gin Giu Asp Cys Val. Val. Val. Trp Gly Giu Cys 70 75 Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val. Lys Gin Asn Asn Arg Cys Pro Leu Cys Gin Gin Asp Trp Val. Val. Gin Arg Ile Giy 100 105 110 Lys

Claims

1. An isolated and purified DNA sequence having more than 70% sequence identity to SEQ ID NO:1.

2. An isolated and purified DNA sequence that hybridizes to the DNA sequence shown in SEQ ID NO:1 under high stringency hybridization conditions.

3. An isolated and purified DNA sequence encoding a protein that protects cells from apoptosis and wherein said sequence is substantially similar to the DNA sequence shown in SEQ ID NO:1.

4. A recombinant DNA molecule comprising the isolated and purified DNA sequence of Claim 1, 2 or 3 subcloned into an extra-chromosomal vector.

A recombinant host cell comprising a host cell transfected with the recombinant DNA molecule of Claim 4.

6. A recombinant host cell deposited with the ATCC under accession number 15 98402.

7. An isolated and purified DNA sequence having more than more than sequence identity to SEQ ID NO:3.

8. An isolated and purified DNA sequence that hybridizes to the DNA sequence shown in SEQ ID NO:3 under high stringency hybridization conditions. 20

9. An isolated and purified DNA sequence encoding a protein that protects cells from apoptosis and wherein said sequence is substantially similar to the DNA sequence shown in SEQ ID NO:3.

10. A recombinant DNA molecule comprising the isolated and purified DNA sequence of Claim 7, 8 or 9 subcloned into an extra-chromosomal vector.

11. A recombinant host cell comprising a host cell transfected with the recombinant DNA molecule of Claim -43-

12. A recombinant host cell deposited with the ATCC under accession number

98403.

13. A recombinant host cell deposited with the ATCC under accession number

98404.

14. A recombinant host cell deposited with the ATCC under accession number

98405. An isolated and purified DNA sequence selected from the group consisting of 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. 16. A recombinant DNA molecule comprising an isolated and purified DNA sequence of Claim 15, subcloned into an extra-chromosomal vector. 17. A recombinant host cell comprising a host cell transfected with a recombinant DNA molecule of Claim 16. 18. A substantially purified recombinant polypeptide, encoding a protein that protects Scells from apoptosis, and wherein the amino acid sequence of the substantially purified recombinant polypeptide is substantially similar to the amino acid sequence shown in SEQ ID NO:2. 19. A substantially purified recombinant polypeptide, wherein the amino acid sequence of the substantially purified recombinant polypeptide has more than sequence identity to SEQ ID NO:2. 20. A substantially purified recombinant polypeptide, encoding a protein that protects cells from apoptosis, and wherein the amino acid sequence of the substantially purified recombinant polypeptide is substantially similar to the amino acid sequence shown in SEQ ID NO:4. -43a- 21. A substantially purified recombinant polypeptide, wherein the amino acid sequence of the substantially purified recombinant polypeptide has more than sequence identity to SEQ ID NO:4. 22. A substantially purified recombinant polypeptide, wherein the amino acid sequence of the polypeptide is selected from the group consisting of 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 SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, and SEQ ID 23. An antibody that selectively binds polypeptides with an amino acid sequence substantially similar to the amino acid sequence of Claim 18, 19, 20, 21 or 22. 24. A method of detecting SAG protein in cells, comprising contacting cells with the antibody of Claim 23 and incubating the cells in a manner that allows for detection of the SAG protein-antibody complex. S 15 25. A diagnostic assay for detecting cells containing SAG mutations, comprising isolating total genomic DNA from the cell and subjecting the genomic DNA to PCR amplification using primers derived from the isolated and purified DNA sequence of Claim 1, 2, 3, 7, 8, 9 or 15, and determining whether the resulting PCR product contains a mutation. 26. A diagnostic assay for detecting cells containing SAG mutations, comprising isolating total cell RNA, subjecting the RNA to reverse transcription-PCR amplification using primers derived from the isolated and purified DNA sequence of Claim 1, 2, 3, 7, 8, 9 or 15, and determining whether the resulting PCR product contains a mutation. 27. A method of isolating RNA containing stretches of polyA or polyC residues, 25 comprising: contacting an RNA sample with SAG protein in RNA binding buffer in the presence of a reducing agent; incubating the RNA-SAG protein mixture with the antibody of Claim 23; isolating the antibody-SAG protein-RNA complexes; and WO 99/32514 PCT/US98/26705 purifying the RNA away from the antibody-SAG protein complex. 28. A method of isolating RNA containing stretches of polyU residues, comprising contacting an RNA sample with SAG protein in RNA binding buffer in the absence of reducing agents; incubating the RNA-SAG protein mixture with the antibody of Claim 23; isolating the antibody-SAG protein-RNA complexes; and purifying the RNA away from the antibody-SAG protein complex. 29. A method for isolating genes induced during cell apoptosis, comprising: treating one set of cells with OP and not treating a control set of cells; isolating RNA from each set of cells; subjecting the RNA from each set of cells to the differential display procedure, wherein the RNA is reverse transcribed into cDNA and the cDNA is subjected to the polymerase chain reaction; identifying cDNAs that are expressed in the OP-treated set of cells and not in the control set of cells; and cloning the OP-induced cDNAs. A method for protecting cells from apoptosis induced by redox reagents, comprising introducing into the cells an expression vector comprising the isolated and purified DNA sequence of Claim 1, 2, 3, 7, 8, 9, or 15, which is operatively linked to a DNA sequence that promotes the high level expression of the isolated and purified DNA sequence in'the cells. 31. A method for inhibiting the growth of tumor cells, comprising introducing into the tumor cells an expression vector comprising the isolated and purified DNA sequence of Claim 1, 2, 3, 7, 8, 9, or 15, which is operatively linked to a DNA sequence that promotes the high level expression of the antisense strand of the isolated and purified DNA sequence in the cells. 32. A method for purifying SAG protein from bacterial cells comprising: a) transfecting a bacterial host cell with a vector comprising the isolated and purified DNA sequence of Claim 1, 2, 3, 7, 8, 9, or 15 operatively linked to a promoter capable of directing gene expression in a bacterial host cell; b) inducing expression of the isolated and purified DNA sequence in the bacterial cells; c) lysing the bacterial cells; d) isolating bacterial inclusion bodies; e) purifying SAG protein from the isolated inclusion bodies. 33. A pharmaceutical composition comprising the substantially purified recombinant polypeptide of any one of Claims 18, 19, 20, 21, or 22 and a pharmaceutically acceptable carrier. 34. The pharmaceutical composition of Claim 33 wherein the substantially purified recombinant polypeptide comprises an oligomer. A method of oxygen radical scavenging in an organism comprising administering an oxygen radical-reducing amount of the pharmaceutical composition of Claim 33 or Claim 34 to the organism. 36. A method of promoting the healing of a wound comprising administering the DNA sequence of Claim 1 to cells associated with the wound. 37. A method of promoting or inhibiting the growth of plant cells comprising administering the DNA sequence of Claim 1 or a DNA sequence which is complementary to the DNA sequence of Claim 1 to plant cells. 38. Use of an expression vector comprising the isolated and purified DNA sequence go•• of any one of Claims 1, 2, 3, 7, 8, 9 or 15 in the manufacture of a medicament for protecting cells from apoptosis induced by redox reagents, wherein the isolated and purified DNA sequence of any one of Claims 1, 2, 3, 7, 8, 9 or 15 is operatively linked to ooooo a DNA sequence that promotes the high level expression of the isolated and purified DNA sequence in the cells. 39. Use of an expression vector comprising the isolated and purified DNA sequence of any one of Claims 1, 2, 3, 7, 8, 9 or 15 in the manufacture of a medicament for °inhibiting the growth of tumor cells, wherein the isolated and purified DNA sequence of *oo° ~any one of Claims 1, 2, 3, 7, 8, 9 or 15 is operatively linked to a DNA sequence that promotes the high level expression of the antisense strand of the isolated and purified 25 DNA sequence in the cells. Use of the DNA sequence of Claim 1 in the manufacture of a medicament for promoting the healing of a wound, wherein the DNA sequence is administered to cells associated with the wound. -46- 41. SAG protein when detected in cells by a method according to Claim 24. 42. Cells containing SAG mutations when detected using the diagnostic assay of Claim 25 or Claim 26. 43. RNA containing stretches of polyA or polyC residues identified by a method according to Claim 27 when used to bind to a SAG protein wherein the ability of the RNA to bind to SAG protein was not previously known, wherein the stretch of polyA or polyC residues was not previously known. 44. RNA containing stretches ofpolyU residues when isolated by a method according to Claim 28. 45. A gene induced during cell apoptosis when isolated by a method according to Claim 29, wherein said gene was not previously known. 46. SAG protein when purified from bacterial cells by a method according to Claim 32. 47. An isolated and purified DNA sequence substantially similar to the DNA -15 sequence shown in SEQ ID NO: 1, substantially as herein described with reference to any ""one of the examples but excluding comparative examples. S48. An isolated and purified DNA sequence that hybridizes to the DNA sequence shown in SEQ ID NO: 1 under high stringency hybridization conditions, substantially as herein described with reference to any one of the examples but excluding comparative examples. An isolated and purified DNA sequence that consists essentially of the DNA sequence shown in SEQ ID NO: 1, substantially as herein described with reference to any 6* one of the examples but excluding comparative examples. S' 50. A recombinant DNA molecule comprising an isolated and purified DNA sequence encoding a protein that protects cells from apoptosis and wherein said sequence is: substantially similar to the DNA sequence shown in SEQ ID NO: 1; (b) that hybridizes to the DNA sequence shown in SEQ ID NO:I under high stringency -47- hybridization conditions; or more than 70% in sequence identify to the DNA sequence shown in SEQ ID NO:; wherein said DNA sequence is subcloned into an extra-chromosomal vector, substantially as herein described with reference to any one of the examples but excluding comparative examples. 51. A recombinant host cell comprising a host cell transfected with a recombinant DNA molecule comprising an isolated and purified DNA sequence encoding a protein that protects cells from apoptosis and wherein said sequence is: substantially similar to the DNA sequence shown in SEQ ID NO: 1; that hybridizes to the DNA sequence shown in SEQ ID NO: 1 under high stringency hybridization conditions; or more than 70% in sequence identity to the DNA sequence shown in SEQ ID NO: wherein said DNA sequence is subcloned into an extra-chromosomal vector, substantially as herein described with reference to any one of the examples but excluding comparative examples. 52. An isolated and purified DNA sequence encoding a protein that protects cells from apoptosis and wherein said sequence is substantially similar to the DNA sequence shown in SEQ ID NO:3, substantially as herein described with reference to any one of the examples but excluding comparative examples. 53. An isolated and purified DNA sequence that hybridizes to the DNA sequence shown in SEQ ID NO:3 under high stringency hybridization conditions, substantially as herein described with reference to any one of the examples but excluding comparative examples. 54. An isolated and purified DNA sequence having more than 70% sequence identity to the DNA sequence shown in SEQ ID NO:3, substantially as herein described with o. reference to any one of the examples but excluding comparative examples. 55. A recombinant DNA molecule comprising an isolated and purified DNA L: sequence encoding a protein that protects cells from apoptosis and wherein said sequence is: substantially similar to the DNA sequence shown in SEQ ID NO:3; (b) that hybridizes to the DNA sequence shown in SEQ ID NO:3 under high stringency hybridization conditions; or more than 70% in sequence identity to the DNA -48- sequence shown in SEQ ID NO:3; wherein said DNA sequence is subcloned into an extra-chromosomal vector, substantially as herein described with reference to any one of the examples but excluding comparative examples. 56. A recombinant host cell comprising a host cell transfected with a recombinant DNA molecule comprising an isolated and purified DNA sequence encoding a protein that protects cells from apoptosis and wherein said sequence is: substantially similar to the DNA sequence shown in SEQ ID NO:3; that hybridizes to the DNA sequence shown in SEQ ID NO:3 under high stringency hybridization conditions; or more than in sequence identity to the DNA sequence shown in SEQ ID NO:3; subcloned into an extra-chromosomal vector, substantially as herein described with reference to any one of the examples but excluding comparative examples. 57. An isolated and purified DNA sequence selected from the group consisting of 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, substantially as herein described with reference to any one of the examples but excluding comparative examples. .58. A recombinant DNA molecule comprising an isolated and purified DNA sequence selected from the group consisting of 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, subcloned into an extra-chromosomal vector, substantially as herein described with reference to any one of the examples but excluding comparative examples. 25 59. A recombinant host cell comprising a host cell transfected with a recombinant DNA molecule comprising an isolated and purified DNA sequence selected from the group consisting of 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, subcloned into an extra-chromosomal -49- vector, substantially as herein described with reference to any one of the examples but excluding comparative examples. A substantially purified recombinant polypeptide encoding a protein that protects cells from apoptosis, and wherein the amino acid sequence of the substantially purified recombinant polypeptide is substantially similar to the amino acid sequence shown in SEQ ID 2, substantially as herein described with reference to any one of the examples but excluding comparative examples. 61. A substantially purified recombinant polypeptide, wherein the amino acid sequence of the substantially purified recombinant polypeptide has more than sequence identity to the amino acid sequence shown in SEQ ID NO:2, substantially as herein described with reference to any one of the examples but excluding comparative examples. 62. A substantially purified recombinant polypeptide encoding a protein that protects cells from apoptosis, and wherein the amino acid sequence of the substantially purified recombinant polypeptide is substantially similar to the amino acid sequence shown in SEQ ID NO:4, substantially as herein described with reference to any one of the examples but excluding comparative examples. 63. A substantially purified recombinant polypeptide, wherein the amino acid sequence of the substantially purified recombinant polypeptide has more than sequence identity to the amino acid sequence shown in SEQ ID NO:4, substantially as herein described with reference to any one of the examples but excluding comparative examples. *0 *0 0 0 0* 64. A substantially purified recombinant polypeptide, wherein the amino acid 06 sequence of the polypeptide is selected from the group consisting of SEQ ID NO:12, 0 00 25 SEQ ID NO:14, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ S: ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, and SEQ ID substantially as herein described with reference to any one of the examples but excluding comparative examples. An antibody that selectively binds polypeptides encoding a protein that protects cells from apoptosis and with an amino acid sequence substantially similar to the amino acid sequence of a polypeptide substantially similar to, or having more than sequence identity to the amino acid sequence shown in SEQ ID NO:2; substantially similar to, having more than 70% sequence identity to the amino acid sequence shown in SEQ ID NO:4; or selected from the group consisting of 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 SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, and SEQ ID NO:50, substantially as herein described with reference to any one of the examples but excluding comparative examples. 66. A method of detecting SAG protein in cells, substantially as herein described with reference to any one of the examples but excluding comparative examples. 67. A diagnostic assay for detecting cells containing SAG mutations, substantially as herein described with reference to any one of the examples but excluding comparative examples. 68. A method of isolating RNA containing stretches of polyA or polyC residues, substantially as herein described with reference to any one of the examples but excluding comparative examples. 69. A method of isolating RNA containing stretches of polyU residues, substantially as herein described with reference to any one of the examples but excluding comparative examples. 70. A method for isolating genes induced during cell apoptosis, substantially as herein described with reference to any one of the examples but excluding comparative 25 examples. 71. A method for protecting cells from apoptosis induced by redox reagents, substantially as herein described with reference to any one of the examples but excluding comparative examples. -51 72. A method for inhibiting the growth of tumor cells, substantially as herein described with reference to any one of the examples but excluding comparative examples. 73. A method for purifying SAG protein from bacterial cells, substantially as herein described with reference to any one of the examples but excluding comparative examples. 74. A pharmaceutical composition comprising a substantially purified recombinant polypeptide, wherein the amino acid sequence of the substantially purified recombinant polypeptide encoding a protein that protects cells from apoptosis and is substantially similar to, or having more than 70% sequence identity to the amino acid sequence shown in SEQ ID NO:2; substantially similar to, or having more than 70% sequence identity to the amino acid sequence shown in SEQ ID NO:4; or is selected from the group consisting of 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, substantially as herein described with reference to any one of the examples but excluding comparative examples. 75. A method of oxygen radical scavenging in an organism, substantially as herein described with reference to any one of the examples but excluding comparative examples. 76. A method of promoting the healing of a wound, substantially as herein described. 77. A method of promoting or inhibiting the growth of plant cells, substantially as herein described with reference to any one of the examples but excluding comparative examples. 25 78. Use of an expression vector comprising an isolated and purified DNA sequence encoding a protein that protects cells from apoptosis and that is substantially similar to the DNA sequence shown in SEQ ID NO:1; that hybridizes to the DNA sequence shown in SEQ ID NO:1 under high stringency hybridization conditions; that has more than 70% sequence identity to the DNA sequence shown in SEQ ID NO:1; that is -52- substantially similar to the DNA sequence shown in SEQ ID NO:3; that hybridizes to the DNA sequence shown in SEQ ID NO:3 under high stringency hybridization conditions; that has more than 70% sequence identity to the DNA sequence shown in SEQ ID NO:3; that is selected from the group consisting of 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 manufacture of a medicament for protecting cells from apoptosis induced by redox reagents, substantially as herein described. 79. Use of an expression vector comprising an isolated and purified DNA sequence encoding a protein that protects cells from apoptosis and that is substantially similar to the DNA sequence shown in SEQ ID NO:1; that hybridizes to the DNA sequence shown in SEQ ID NO:1 under high stringency hybridization conditions; that has more than 70% sequence identity to the DNA sequence shown in SEQ ID NO:1; that is substantially similar to the DNA sequence shown in SEQ ID NO:3; that hybridizes to the the DNA sequence shown in SEQ ID NO:3 under high stringency hybridization S. conditions; that has more than 70% sequence identity to the DNA sequence shown in SEQ ID NO:3; or that is selected from the group consisting of 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 manufacture of a medicament for inhibiting the growth of tumour cells, substantially as herein described. 80. Use of an isolated and purified DNA sequence encoding a protein that protects 25 cells from apoptosis and wherein said sequence; is substantially similar to the DNA .*o sequence shown in SEQ ID NO:1; has more than 70% sequence identity to SEQ ID NO:1 in the manufacture of a medicament for promoting the healing of a wound, substantially as herein described. 81. SAG protein when detected in cells by an antibody that selectively binds polypeptides encoding a protein that protects cells from apoptosis, with an amino acid -53- sequence substantially similar to the amino acid sequence of a polypeptide substantially similar to, or having more than 70% sequence identity to the amino acid sequence shown in SEQ ID NO:2; substantially similar to, or having more than sequence identity to the amino acid sequence shown in SEQ ID NO:4; or selected from the group consisting of 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, substantially similar to the amino acid sequence shown in SEQ ID 2; substantially as herein described with reference to any one of the examples but excluding comparative examples. 82. Cells containing SAG mutations when detected using a diagnostic assay, substantially as herein described with reference to any one of the examples but excluding comparative examples. 83. RNA containing stretches of polyA or polyC residues when isolated by a method comprising, in at least one step, contacting an RNA sample with SAG protein in RNA binding buffer in the presence of a reducing agent, substantially as herein described. 84. RNA containing stretches of polyU residues when isolated by a method •comprising, in at least one step, contacting an RNA sample with SAG protein in RNA binding buffer in the absence of reducing agents, substantially as herein described. 85. A gene induced during cell apoptosis when isolated by a method comprising: treating one set of cells with OP and not treating a control set of cells; isolating RNA from each set of cells; subjecting the RNA from each set of cells to the differential display procedure, wherein the RNA is reverse transcribed into cDNA and the cDNA is subject to the polymerase chain reaction; identifying cDNAs that are expressed in the OP-treated set of cells and not in the control set of cells; and cloning the OP-induced cDNAs, -54- substantially as herein described with reference to any one of the examples but excluding comparative examples. 86. SAG protein when purified from bacterial cells, substantially as herein described with reference to any one of the examples but excluding comparative examples. DATED this 25 th day of July 2003 BALDWIN SHELSTON WATERS Attorneys for: WARNER-LAMBERT COMPANY *o e