Note: Descriptions are shown in the official language in which they were submitted.
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/Z6705
SAG: SENSITIVE TO APOPTOSIS GENE
Background of the Invention
The present invention relates to a novel gene and polypeptides 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
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
I S condensation, and DNA fragmentation (Wyllie ( 1980) Int. Rev. Cytol. 68,
251-306). The
process of apoptosis can be divided into three distinct phases: initiation,
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, 614-620, White (1996)
Genes Dev.
10, 1-15; Sen and D'Incalci (1992) FEBS Lett. 307, 122-127; Dive and Hickman
(1991) Br.
J. Cancer 64, 192-196; Yuan et al. (1993) Cell 75, 641-652). These initiators
trigger the
effector molecules in cells leading to apoptotic signal transduction and
amplification, which
ultimately results in irreversible DNA degradation and cell death.
Many genes are involved in the apoptotic process. In general, the products of
these
genes are classified as either inducers or inhibitors of apoptosis. The
balance between the
activities of apoptosis inducers and inhibitors in a given cell determines
whether that cell
undergoes apoptosis. Among the growing list of apoptotic regulatory genes, the
most well
characterized are the p53 tumor suppressor gene, the Bcl-2 gene family
(consisting of both
inducers and inhibitors of apoptosis), the interleukin 1 (3 converting enzyme
(ICE) gene
family, and FAS/Fas ligand (Kroemer ( 1997), White ( 1996); Yuan et al. ( 1993
); Nagata and
1
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
Golstein (1995) Science 267, 1449-I456). During apoptosis, there are
substantial interactions
involving the products of apoptotic regulatory genes, including heterodimer
formation among
the gene products of the Bcl-2 gene family, and p53 activation of Bax
expression (Oltvai
et al. (I993) Cell 74, 609-619; Miyashita and Reed (1995) Cell 80, 293-299).
The inventor has recently found that 1,10 phenanthroline ("OP"), a metal
cheIating
agent, can activate p53 activity and induce apoptosis in two marine tumor cell
lines that
harbor endogenous wild-type p53 (Sun et al. (1997) Oncogene 14, 385-393). OP
is a typical
metal chelating reagent in that it chelates Fe(II) and prevents Fe(II)-
mediated hydroxyl
radical formation through the Fenton reaction (Halliwell et al. (1989) in:
Free Radicals in
Biology and Medicine, 2nd ed., Clarendon Press, Oxford; Auld (1988) in Methods
in
Enzymology, Vol. 158 (J. F. Riordan and B. L. Valle, Eds.) PP. 110-114,
Academic Press,
New York). OP has been shown to prevent hydroxyl radical-induced DNA damage in
a
number of cellular systems (Sun, Y. Free Radic. Biol. Med. 8:583-599 (1990);
Martins and
Meneghini, Biochem J. 299:137-140 (1994}; Morgan et al., Biochem. Pharmacol.
44:215-221
(1992)). Activation of p53 by OP was found to significantly contribute to, but
was not
required for subsequent apoptotic cell death (Sun et al., (1997) Oncogene 14:
385-393; Sun
(1997) FEBS Lett. 408, 16-20). Thus, the critical genes and gene products
responsible for
OP-induced apoptosis remain to be characterized. A better understanding of the
molecular
mechanisms of apoptotic induction will allow improved design of therapeutic
drugs that
either induce (anti-cancer) 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.
In one 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 1
and SEQ ID 3, and their gene products (referred to herein as "mouse SAG
protein" and
"human SAG protein") as shown in SEQ ID 2 and SEQ ID 4, that are induced
during
I,IO-phenanthroline ("OP")-induced apoptosis. In another embodiment, the
present
invention comprises a nucleotide sequence that hybridizes to the nucleotide
sequence shown
in SEQ ID I and SEQ ID 3 under high stringency hybridization conditions. In a
preferred
embodiment, the isolated and purified DNA sequence consists essentially of the
DNA
sequence of SEQ ID 1 or SEQ ID 3.
2
CA 02303483 2000-03-08
WO 99/32514 PCTNS98/26705
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 l 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 OP-
induced 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
3
CA 02303483 2000-03-08
WO 99132514 PCT/US98/26705
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 ID 1
and SEQ
ID 3 will be expressed at high levels in the mammalian and/or non-mammalian
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 (i.e.,
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.
Brief Description of the Drawings
Figure lA. Predicted structural features of the deduced protein sequence of
the mouse and
human SAG cDNA.
Figure 1B. Description of human SAG protein mutants.
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
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 OP-
induced 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
4
CA 02303483 2000-03-08
WO 99/32514 PCTNS98/26705
(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 (M.P.
Deutshcer,
ed., (I990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and
Applications
S (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal
Cells: A Manual of
Basic Technigue, 2nd Ed. (R.I. Freshney. 198?. Liss, Inc. New York, NY), and
Gene
Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humans
Press Inc.,
Clifton, N.J.).
In one aspect, the present invention provides novel isolated and purified DNA
sequences, hereinafter referred to as Sensitive to Apoptosis Genes ("SAG"),
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°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°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 O.OSM 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
5
CA 02303483 2000-03-08
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
extra-
chromosomal 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 pmtein 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 polymerise 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 (e.g., bacterial), and
eukaryotic such as fungal
(e.g., yeast), plant, non-human animal, non-human mammalian (e.g., 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, 2nd
ed., Cold
Spring Harbor Press, 1989) and, as used herein, includes, but is not limited
to calcium
phosphate transfection, dextrin 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
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
6
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
appropriate) introduced by any in vitro means. As used herein, the tenor
"substantially
purified" means that the protein should be free finm detectable contaminating
protein, but the
SAG protein may be co-purif ed 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 LD 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 fiu~ther 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
7
CA 02303483 2000-03-08
WO 99/32514 PC"T/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 (Sarnbrook
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
8
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
(3-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 ~M and 300 ~,M. In a most preferred embodiment, OP is used at a
concentration
of between 100 ~t,M and 150 N.M.
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
9
CA 02303483 2000-03-08
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
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. -
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 oxygen-
reducing 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 (e.g., 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 (i.e., plant) and eukaryotic cells. Gene
therapy
applications typically involve identifying target host cells or tissues in
need of the therapy,
CA 02303483 2000-03-08
WO 99/32514 PC"T/ttS98l26705
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.
The 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
closwe
(i.e., 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 closwe (i.e., healing) of the wound.
The present inventive method promotes closwe (i.e., healing) of both external
(e.g.,
surface) and internal wounds. Wounds to which the present inventive method is
useful in
promoting closwe (e.g., 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 (i.e., healing). Transferring a vector comprising an SAG
expression cassette
to cells associated with the wound is prefen~ed as such procedwe 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 closwe 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.
11
CA 02303483 2000-03-08
WO 99/32514 PCTNS98I26705
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 (i.e., 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 ceps 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 (i.e., 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 (e.g., 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
12
CA 02303483 2000-03-08
WO 99132514 PCTNS98/26705
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 (IV). 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,
e.g., 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 I % 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 marine tumor lines. Since
OP induced-
apoptosis can be visually detected at 12 hours post exposure (Sun, (1997) FEBS
Lett. 408:16-
20), it was reasoned that genes) responsible for apoptosis induction should be
up- or down-
regulated 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.
13
CA 02303483 2000-03-08
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-RT101 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 RNAzoI solution (Tel-Test) according
to the
manufacturer's instructions, and subjected to reverse transcription (RT),
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 (P
1 ) consisted of
the sequence 5 AAGCTTTTTTITTI'TTTR (SEQ ID 5), 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 AAGCTTrII~INNNNN (SEQ ID
6),
1 S wherein N consists of adenine, cytosine, guanine, or thymine.
Primers P 1 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 Vitmgen)
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 S'RACE
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/P,g) in a hybridization solution containing SX SSC, SX Denhardt solution,
50 mM
sodium phosphate, and 100 ~.g/mL denatured DNA at 60°C for 16-18 hours.
The filter was
14
CA 0230348312000-03-08
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
O.SXSSC/0.1% SDS, and twice O.1XSSC/0.1% SDS for 15 min.
The longest clone isolated was a 1.0 kilohase ("kb") 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 I kb fragment and another primer derived from the vector sequence,
according to the
protocol supplied with the cDNA library. This yielded a further 100 by
fragment consisting
of 5'-end untranslated sequence and some of the coding sequence. The derived
cDNA clone
consists of 1140 base pairs ("bp") (SEQ ID 1 ) that encode a novel deduced
protein of
113 amino acids, containing 12 cysteine residues (SEQ ID 2). The open reading
frame was
preceded by 17 by 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 by upstream of the
start codon in
the 5' untranslated region in one genomic clone (not shown). The 3'-end
untranslated region
consists of 792 by 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 by clone containing a
polyadenylation signal
at the 3' end was isolated (SEQ ID 3). The human cDNA also contains an open
reading
frame encoding a novel predicted 113 amino acid polypeptide containing 12
cysteine residues
(SEQ ID 4). 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 C3HC4 zinc ring finger domain (Freemont et
al., Cell
64:483-484, 1991) at the C-terminal of the molecule (Fig. lA) among other
consensus motifs.
The second potential heme binding domain (Fig. 1 A) contains a substitution of
arginine to
CA 02303483 2000-03-08
WO 99/32514 PCTNS98/26705
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 C3H2C3 structure, rather than the consensus C3HC4 structure. Since
cysteine and
histidine residues are interchangeable in zinc binding (Berg and Shi, Science
271:1081, 1996;
Inouye et al., Science 278:103-106, 1997), the C3H2C3 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 C3H2C3 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 N,M OP
for various
times up to 24 hours and subjected to total RNA isolation. Fifteen ~,g 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 "SAG"), which encode SAG proteins.
Example 4. Tissue distribution and embryonic expression of SAG
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
16
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
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 (OP), 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.O1 5'-CGGGATCCCCATGGCCGACGTGAGG-
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 p,L of lOX buffer, 22 ~,L of 1.25 mM dNTP, 1.1 ~,L of SAGTA.O1
and
SAGT.02, respectively, 5.5 ~,L of Taq DNA polymerase, 5.5 P,I, of 32P-dCTP and
sterile
water up to 495 Er.l. Into each tube which contains 5 ~.I, of first strand
cDNA reverse-
transcribed for total RNA isolated from mouse embryonic tissues (Sun et al.
(1997), Mol.
Carcinogenesis 8:49-57), 45 ~,L of reaction mixture was added and PCR was
performed for
25 cycles (95°C for 45 sec, 60°C for 1 min and 72°C for 2
min). A 5 ~,L 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 S. 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
17
CA 02303483 2000-03-08
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 ~,g/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
~i-
galactosidase 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 of SAG protein in bacteria
The entire open reading frame of the human SAG cDNA was PCR amplified as
described above and subcloned into the pETl l 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 pg/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°C at a
shaker setting of 150 rpm. Cell pellets were obtained by centrifugation at
5000 rpm for
10 minutes, and were resuspended in 100 mL TN buffer (20 mM Tris-HCI, pH 7.5,
50 mM
NaCI) containing 100 ~.M PMSF. The resuspended cell pellet was subsequently
sonicated
(15 sec/round for S rounds at a setting of 15 on Model 50 sonic disrnembrator,
Fisher
Scientific) and subjected to pressure of 2500 pounds/square inch on a French
cell press,
18
CA 02303483 2000-03-08
WO 99/32514 PCTNS98lZ6705
followed by addition of 1 mM MgCl2 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-HCI, pH
7.5,
200 mM NaCI) 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
SmM 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
2.5 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
Centricon-10 concentrator to a volume of 2 mL. The resulting sample was
dialyzed against
4liters of dialysis buffer (150 mM KCI, 20 mM Tris-HCI, pH 7.5) at 4°C
overnight to
19
CA 02303483 2000-03-08
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°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 of SAG 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 ~,g) was exposed to various
concentrations of
DTT (1 M, 300 mM, 100 mM, or 30 mM) or H202 (15 mM, SO mM, 150 mM or 450 mM)
for 10 min before being separated by polyacrylamide gel electrophoresis
(PAGE), followed
by Western blot analysis. Alternatively, 10 ~g of SAG protein was incubated
with 50 mM
H202 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
H202 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 H202 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 H202-induced SAG protein oligomerization can be
reversed by DTT treatment, 1 ~.g of purified SAG protein was incubated with SO
mM H202
for 10 minutes, followed by a 10 minute incubation with either H202, 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 H202-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.
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
To confirm that SAG protein oligomerization and doublet formation is due to
inter-
and infra-molecular disulfide bond formation, respectively, SAG protein was
treated, prior to
H202 exposure, with 50 mM N-ethylmaleimide (NEM), an alkylating reagent that
will
alkylate the free SH-groups in SAG protein. Purified SAG protein ( 1 ~,g) was
pre-incubated
with 50 mM NEM or DMSO, or buffer only, for 10 minutes prior to H202
treatment. The
samples were separated via PAGE, followed by Western blot analysis. Pre-
incubation of
SAG protein with DMSO did not affect H202-induced oligomerization and doublet
formation, whereas NEM pre-treatment abolished H202 activity. Neither inter-
(oligomerization) nor infra- (doublet monomer) disulfide bonds were formed,
demonstrating
that alkylation of the free SAG protein SH groups abolishes H202 sensitivity.
These data
demonstrate that SAG protein is redox sensitive. It is subjected to both infra-
and inter-
molecular disulfide bond formation upon exposure to H202, as evidenced by both
doublet
and oligomer formation. These H202-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 H202-induced oligomerization,
although
zinc itself did not induce oligomerization.
Example 8: Production of SAG 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 pETI 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.OI
(S'-TATGGCTAGC ATGGCCGACGTGGAGG-3) (SEQ ID 9) and each of antisense
primers and b) each of sense primers and SAG T.02 (SEQ ID 8), 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.O1 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
21
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/Z6705
(MM 10, MM 13, MM 14, see Figure 1 B), 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 50.
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-I00 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-HCI, pH 7.5 and used for SAG
protein
oligomerization studies.
Purified wildtype SAG protein is a heme-containing brownish protein (See
Example 9}. Some of the purified SAG protein mutants were found to have either
lost the
brownish color (MM3 and MM13) or had decreased brownish color (MMI) compared
to
wildtype SAG protein. This color change indicates the loss or decrease of heme
binding
(Table I ).
22
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
TABLE 1. SUMMARY OF SAG MUTANTS
NAME MUTATION SITES) HEMS BINDING OLIGOMERIZATION
WT None +++ Yes
MMl CA/heme ++ Yes
MM2 CB/heme +++ Yes
MM3 CA+g/heme +/- Yes
MM4 C I /Zn-ring finger +++ Yes
1
MMS C3/Zn-ring finger +++ Yes
1
MM6 H4/Zn-ring finger +++ Yes
1
MM7 HS/Zn-ring finger +++ Yes
2
MM8 C6/Zn-ring finger +++ Yes
2
MM9 C7/Zn-ring finger +++ Yes
2
MM I 0 H4+5/Zn-ring fingers+++ Yes
1 &2
I i C2~Zn-ring finger +++ Yes
1
MM12 Cc/protease inhibitor+++ Yes
MM 13 C I +2/Zn-nng forger+/- Yes
1
MM14 C7+g/Zn-ring finger +++ No
2
MM15 I GADPH binding site +++ Yes
To examine mutant SAG protein oligomerization, each mutant SAG protein as well
as
wildtype SAG was treated with 50 mM H202 for 10 min. All of the SAG mutants,
except
MM14, can be oligomerized upon exposure to H202. 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 I), indicating that these two positions are important
for intermolecular
disulfide bond formation.
Example 9. Heme measurement of SAG protein
Heme content in SAG pmtein 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)
23
CA 02303483 2000-03-08
WO 99/32514 PCTNS98/26705
After centrifugation the pellet was extracted sequentially with 0.5 mL of
chloroform:methanol (2:1); 0.5 mL of cold acetone, and finally 0.5 mL of cold
acetone
containing 5 p,L of 2.4 N HCI. 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
1.0 mL quartz cuvettes using water as a blank. The solution was then reduced
by adding 10
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 demonsh~ated 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-amino
acid peptide (SAG-Pepl: QNNRCPLCQQDWVVQR) (SEQ ID 10) located in the
C terminus of SAG protein (colons 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 I1. Analysis of SAG protein transcriptional regulatory activity
SAG protein belongs to the zinc ring finger protein families by virtue of its
C3H2C3
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,
RING 1 )
(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
24
CA 02303483 2000-03-08
WO 99/32514 PCTNS98/26705
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-VP
16, a
known transcription factor (Triezenberg et al., Genes and Develop. 2:718-729,
1988), fused
doam 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-VP 16. Again, neither
orientation of
SAG induced significant expression of VP 16-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 wg of SAG protein was
incubated in 300 p,L
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 MgCl2,
0.1%
nonidet P-40, 50 N,M ZnCl2, 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
CA 02303483 2000-03-08
WO 99/32514 PC"fNS98/26705
were 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 weigh. 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. Identif:cation 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.O1 and SAG T.02. 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
contained either a 7 by or
a 48 by deletion at nucleotide 170 or 177, respectively, assigning the first A
at the start codon
as nucleotide #1. 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.
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) BioTechruques
12:639-640).
The primers used for PCR were hSAG.Ml, 5' GCCATCTGCAGGGTCCAG-3'
(SEQ ID IS), starting at nt 151 of hSAG cDNA, and SAGT.02-1
S'-GGATCCTCATTTGCCGATTCTTTGGAC-3' (SEQ ID 16), including stop codon
(underlined). The resulting fragment is 200 by for wildtype SAG. The PCR was
conducted
in the presence of 35S-dATP (Amersham) and PCR products were resolved in 6%
denaturing 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
26
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/Z6705
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
HTB 104). 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-1 B cells and subjected to PCR analysis,
as
described previously (Sun et aI. (1992) BioTechniques 12:639-640). The primers
used were
hSAG.Ml 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 of stable 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 'n and
SAGT.02 (SEQ ID 8), 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 constitutiveiy. 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:
SAG-
mutant-1 (7 by deletion, SEQ ID 11 ) and SAG-mutant-2 (48 by deletion, SEQ ID
13) in
DLD-1 tumor cells.
27
CA 02303483 2000-03-08
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
6418 selection
(600 ~,g/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 D 1-3 and
D1-6; SAG-wildtype D12-l and D12-8; SAG-mutant-1 D3-3 and D3-4; and SAG-mutant-
2
D4-2 and D4-5.
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 35S-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,
0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 5 mM sodium fluoride, 5
mM
sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, and 1 ~,1/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% SDS-
polyacrylamide 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 1 S. Morphological appearance of SAG transfectants after exposure to
redox
reagents
28
CA 02303483 2000-03-08
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 p.M OP, 200 p,M H202, or 125 ~,M 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 CuS04 treatment (up to 750 ~ 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
lt.M OP, 125 p,M zinc sulfate, or 200 N.M H202 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°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
~,g/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
29
CA 02303483 2000-03-08
WO 99/32514 PCT/US98126~05
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 H202 induced toxicity by SAG oligomerization. SAG-
transfected
cells were, therefore, treated with H202 followed by assays for DNA
fragmentation. H202
induced apoptosis in DLD-1 cells. SAG protein overexpression partially
protected cells from
H202-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 H202 and also against
apoptosis
caused by zinc.
Example 17. Antisense SAG expression inhibits tumor cell growth
To test the growth elects 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 6418
(600
p,g/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 6418 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
(D1-6), 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 (D3-3), SAG mutant-2 (D4-2), as well as the neo control
(D1-3)
CA 02303483 2000-03-08
WO 99/32514 PCT/US98l26705
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-I cells as
reflected by
75% reduction of soft agar colony number when compared to the neo control (Dl-
3), 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 (laconic 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 DI-
6 cells, and SAG (sense) expressing D12-1 cells (Figure 3). 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 1?), 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), 6401 (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?/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
(pJMl7,
circularized Ad5 genome) and a recombinant plasmid (pEC-SAG; a CMV driven
plasmid
containing left arm of Ad5 genome) into 293 cells.
31
CA 02303483 2000-03-08
WO 99/32514 PCTNS98/26705
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 N,m) for enzymatic staining (e.g., 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 10%
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 Fe(++).
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 (a)
Cu, Zn-SOD,
(b) Mn-SOD, (c) both Cu, Zn-SOD and Mn-SOD, and (d) 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
32
CA 02303483 2000-03-08
WO 99/32514 PCTNS98/26705
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 radical-
induced cell killing indicates that SAG is an effective oxygen radical
scavenger.
Example 20. Prevention of IL-1 /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-1 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
stereotacdcally 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
et al., 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
33
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
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 SAGKanMX4-5:
5'-TTCTCCAGTGGCAGAGAACTTTAAAGAGAAATAGTTCAAC
CGTACGCTGCAGGTCGAC-3' (SEQ ID 17), and SAGKanMX4-3: 5'-ACCTCGGTA
TGATTTAAATGTTTACGGGCAATTCATTI"'I"T
ATCGATGAATTCGAGCTCG-3' (SEQ ID 18). The primer SAGKanMX4-S consists of
yeast SAG DNA sequence (ATCC Accession number 274876) 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°C 2 min, followed
by 25 cycles at 94°C, 1 min, 56°C, 1.5 min, 72°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 6418 (200 ~.g/mL, BRL) to
select
transfectants containing the kanamycin cassette, which have had the yeast SAG
deleted by
homologous recombination.
Several 6418-resistant clones were selected and assayed to determine whether
heterozygous or homozygous deletions had been produced. The primers used are
SAGPCR-
5: 5'-TTCTCCAGTGGCAGAGAAC-3' (SEQ ID 19) and SAGPCR-3: 5'-
ATGATTTAAATGTTTACGGGC-3' (SEQ ID 20). These primers constitute fragments of
SAGKanMX4-5 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
34
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
mutants give rise to I.Skb band, consisting of the kanamycin cassette. Both
the 0.35 kb and
I.5 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 (InvSC I from In Vitrogen) and no 6418-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 (y21-
ySAG/ySAG::Kan) were sponilated 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°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 p.L of 1 M glycerol containing 0.5 mg/mL zymolase T20. After 30 min at
37°C, the
suspension was diluted with 800 ~,L 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
I S 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 6418, 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 if ySAG 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 (S-FOA; used to
select against
the UR.A3-containing centromere plasmid (Boeke et aL, Mol. Gen. Genet, 1984;
I97:345).
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
CA 02303483 2000-03-08
WO 99/32514 PCT/US98I26705
to grow, indicating that ySAG is essential for normal vegetative growth and
not simply for
sporulation.
Example 23: Human SAG rescue of yeast 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; MM10,
sequence ID 39; and MM14, sequence ID 47, Figure lA) 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/G4I8-
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 (m/z) 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 SO ~t.M ZnCl2 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
36
CA 02303483 2000-03-08
WO 99/32514 PCTNS981~6705
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
2.5
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 N,M CuS04 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 ~,M
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
pmtein. Adding copper to higher concentrations resulted in precipitation of
the protein.
Example 2S: SAG minimizes or prevents LDL oxidation induced by copper ion or a
free
radical generator
Due to its H202 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 (2,2-
azobis-2-amidinopropane hydrochloride), was used as a model to test potential
protection
activity of SAG against lipid peroxidation.
Lipoproteins (100 ~,g of protein/mL, Intraocel) were incubated with 10 ~t.M
CuS04 or
with 5 mM AAPH for 4 hours at 37°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 ~.M butylated hydrozytoluence (BHT) and
refrigeration at
4°C. The extent of lipoprotein oxidation was measured by the TBARS
assay, using
malondialdehyde (MDA) for the standard curve, as described (Buege & Aust,
1978).
37
CA 02303483 2000-03-08
WO 99/32514 PGTNS98/26705
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°C for 1 S 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
25%
inhibition at a concentration of 100 ~.M. 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
1 S by binding and chelating copper ion through its free SH groups, SAG
prevents copper-
initiated 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 p,M (750 p,g/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 ZnS04 induces a time-dependent release of cytochrome C in cytoplasm.
Compared to
the vector control cell (D 1-6), the SAG overexpressing cell (D 12-1 ) 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
38
CA 02303483 2000-03-08
WO 99/32514 PCTNS98/Z6705
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 CuS04 (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 HYSY human neuroblastoma cells and a few stable lines
were selected which expressed exogenous SAG as determined by Western blot. One
SAG-
transfectant (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 CuS04 or 200 ~tM ZnS04
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 TLJNEL assay, a
fluorescein
labelling assay of free 3'-OH termini generated from cleavage of genomic DNA
during
apoptosis.
In Situ cell death assay (TLJNEL assay) was performed according to the
manufactwer's instructions (Boehringer Mannheim). Briefly, 5 x 104 cells were
plated into
the 8-well glass slides. After treatment with 1.25 mM copper (CuS04) or 200
~,M zinc
(ZnS04) for 16 hours, cells were fixed with 0.5% glutaraldehyde for 10 min,
then washed
39
CA 02303483 2000-03-08
WO 99/32514 PCTNS98/26705
with PBS twice. The fixed cells were incubated in permeabilization solution
(0.1 % Triton
X-100, 0.1 % sodium citrate) for 2 min on ice. The TUNEL reaction mixture (50
~.L) was
added to samples and incubated for 1 hour at 37°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 CuS04, or 200 ~M ZnS04. 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 ~.g/mL or 25
~.g/mL),
along with the control ~-galactosidase (25 ~g/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 [3H]thymidine (5 ~,Ci/mL, Amersham)
was performed
10 to 24 hours after injection. Cultures were washed with isotonic phosphate-
buffered saline
and fixed in 3.7% (vo11vo1) formaldehyde. Induction of [3H]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 ~-galactosidase does
not induce
DNA synthesis and no [3H]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
(SYSY), overexpressing hSAG protein by hSAG cDNA transfection. Both the vector-
expressing 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 proliferationlgrowth.
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
CA 02303483 2000-03-08
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.
41
CA 02303483 2000-03-08
WO 99/32514 PCT/U898/26705
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Yi Sun
(B) STREET: 4841 Hillway Court
(C) CITY: Ann Arbor
(D) STATE: Michigan
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 48105
(G) TELEPHONE: (313) 996-1959
(H) TELEFAX: (313) 996-7158
(ii) TITLE OF INVENTION: Sensitive to Apoptosis Gene (SAG)
(iii) NUMBER OF SEQUENCES: 50
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID N0: l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1140 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/ICEY: CDS
(B) LOCATION:17.:355
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION:17..355
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION:1..1140
(D) OTHER INFORMATION:/note= "Mouse SAG"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 1:
GTTCTGCGCC GCCGCC ATG GCG GAC GTG GAG GAC GGC GAG GAA CCC TGC 49
Met Ala Asp Val Glu Asp Gly Glu Glu Pro Cys
1 5 10
GTC CTT TCT TCG CAC TCC GGG AGC GCA GGC TCC AAG TCG GGA GGC GAC 97
Val Leu Ser Ser His Ser Gly Ser Ala Gly Ser Lys Ser Gly Gly Asp
15 20 25
1
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
AAG TTCTCTCTC AAGAAGTGG GCGGTA GCC TGG AGCTGG I45
ATG AAC ATG
LysMet PheSerLeu LysLysTrp AlaVal Ala Trp SerTrp
Asn Met
30 35 40
GACGTT GAGTGCGAT ACCTGTGCC TGCAGG GTC GTG ATGGAT 193
ATC CAG
AspVal GluCysAsp ThrCysAla CysArg Val Val MetAsp
Ile Gln
45 50 55
GCCTGC CTTCGATGT CAAGCTGAA AAGCAA GAG TGT GTTGTG 241
AAC GAC
AlaCys LeuArgCys GlnAlaGlu LysGln Glu Cys ValVal
Asn Asp
60 65 ?0 75
GTCTGG GGAGAGTGT AACCATTCC CACAAC TGC ATG TCCCTG 289
TTC TGC
ValTrp GlyGluCys AsnHisSer HisAsn Cys Met SerLeu
Phe Cys
80 85 90
TGGGTG AAACAGAAC AATCGCTGC CTGTGC CAG GAC TGGGTA 337
CCT CAG
TrpVal LysGlnAsn AsnArgCys LeuCys Gln Asp TrpVal
Pro Gln
95 100 105
GTCCAA AGAATCGGC AAATGAGAGGTGG 385
CCCAGGCGCT
CCTGGTGTGG
ValGln ArgIleGly Lys
110
TTGCTGACCC TGGACAAAGACTAAACACTG CAGGGGATTCATCCTTGAGA GAGAGAGGAT445
GCTGTGCGCC TTTGAGACTCACCAAAGGCT TGCTTTATTAATTTGTCTGT TTAGTTTTGG505
GAAATTCTCT ACAATTAAGATAATTTGTTA AAAATGGCCTTTCCTACCTC TGGTGTGTGT565
GTGTGATACG AATGCATAGAAGAGCGAGAA CACCAGAAAATGATCTTTGT TTATCTGTAC625
CCACGACTGG AACATTGTGTTCACAGAAGA ACATTGTTTGTGTTTATGCT TGAGGGTTAA685
AAAATAGATA AACGAATGTTACAGTAACAA ATAAAATGCATTGAAAAGCC GACTCCTCCT745
AATCCTTTTT GTGTTGGGAGAGAGGCAAGC GAGGCCACCCTGCTGTCTTC ATTTGCTGTG805
AATGAGGATT TTAACCTGCACTCAGTGAAG AGGCGTAACTGTCGGGTAAA CTGTAATATG865
GCGTAACTGT CGGGTAAACGGCTTTGTCTC CTGACTTCTCCATCTTTGAC TTGGCCAGGA925
AGCCTGGATT GTTCAACCACTTAGTTCTAA AGAACTGTTTTCTGTTTTTG CCGAAGGTTG985
TATTGTATGT TTTAGTCAAAAATATTAGTA GGAAAATGGCTTACTAGTAT AACACTGAAG1045
TTCATTATGC AATGTTTTAATAAAATATTG TGCTTTGAGTTATTAAAGTT TGATATATAC1105
TCTTAAAATC ATTAAACTAATTCATCAATT AAATG 1140
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
2
CA 02303483 2000-03-08
WO 99/32514 PCT/ITS98~6705
Met Ala Asp Val Glu Asp Gly Glu Glu Pro Cys Val Leu Ser Ser His
1 5 10 15
Ser Gly Ser Ala Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys
50 55 60
Gln Ala Glu Asn Lys Gln~Glu Asp Cys Val Val Val Trp Gly Glu Cys
65 70 ~ 75 80
Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
Lys
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 754 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/REY: CDS
(B) LOCATION:1..339
(ix) FEATURE:
(A) NAME/KEY: mat-peptide
(B) LOCATION:1..339 .
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION:1..754
(D) OTHER INFORMATION:/note= "Human SAG"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144
3
CA 02303483 2000-03-08
WO 99/32514 PGT/US98/26705
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192
Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys
50 55 60
CAA GAA AAA GAC GTT GTCTGG GGA TGT 240
GCT AAC CAA TGT GTG GAA
GAG
GlnAla GluAsnLys GluAsp ValVal ValTrp Gly Cys
Gln Cys Glu
65 70 75 80
AATCAT TCCTTCCAC TGCTGC TCCCTG TGGGTG AAA AAC 288
AAC ATG CAG
AsnHis SerPheHis CysCys'MetSerLeu TrpVal Lys Asn
Asn Gln
85 90 95
AATCGC TGCCCTCTC CAGCAG TGGGTG GTCCAA AGA GGC 336
TGC GAC ATC
AsnArg CysProLeu GlnGln TrpVal ValGln Arg Gly
Cys Asp Ile
100 105 110
AAATGAGAGTGGT CCCTGGTGGA 389
TAGAAGGCTT
CTTAGCGCAG
TTGTTCAGAG
Lys
TCTTGTAATCCAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG449
GAGCCGATGGATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT509
ATCTTCAGAAATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC569
TGTGGTGTGTGTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT629
GAATCACCTTATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT689
TTTCGATGCTTATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT749
TTAAA 754
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys
50 55 60
4
CA 02303483 2000-03-08
WO 99/32514 PCTNS98/26705
Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys
65 70 75 80
Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
Lys
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide P1
downstream primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
AAGCTTTTTT TTTTTTTR 18
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide: P2
upstream primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
AAGCTTNNNN NNN 13
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide SAG TA. O1"
S
CA 0230348312000-03-08
WO 99/32514 PCT/US98/26705
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
CGGGATCCCC ATGGCCGACG TGAGG 25
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide SAG T.02"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
CGGGATCCTC ATTTGCCGAT TCTTTG 26
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide P.01"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 9:
TATGGCTAGC ATGGCCGACG TGGAGG 26
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 10:
Gln Asn Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg
1 5 10 15
6
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 747 base pairs
(B} TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/REY: CDS
(B) LOCATION:1..270
(ix) FEATURE:
(A) NAME/KEY: mat~eptide
(B) LOCATION:1..270
(xi} SEQUENCE DESCRIPTION: SEQ ID NO: 11:
ATG GACGTGGAA GAC GAA TGC GCC GCCTCTCAC 48
GCC GGA GAG ACC CTG
Met AspValGlu Asp Glu Glu Cys Ala AlaSerHis
Ala Gly Thr Leu
1 5 10 15
TCC AGCTCAGGC TCC TCG GGA GAC AAG TTCTCCCTC 96
GGG AAG GGC ATG
Ser SerSerGly Ser Ser Gly Asp Lys PheSerLeu
Gly Lys Gly Met
20 25 30
AAG TGGAACGCG GTG ATG TGG TGG GAC GAGTGCGAT 144
AAG GCC AGC GTG
Lys TrpAsnAla Val Met Trp Trp Asp GluCysAsp
Lys Ala Ser Val
35 40 45
ACG GCCATCTGC AGG CAG ATG GTC TTA GTCAAGCTG 192
TGC GTC CCT GAT
Thr AlaIleCys Arg Gln Met Val Leu ValLysLeu
Cys Val Pro Asp
50 55 60
AAA AACAAGAGG ACT TTG TGG GGG GAG GTAATCATT 240
ACA GTG TCT AAT
Lys AsnLysArg Thr Leu Trp Gly Glu ValIleIle
Thr Val Ser Asn
65 70 75 80
CCT ACAACTGCT GCA CCC TGT TGAAACAGAA 290
TCC TGT GGG CAATCGCTGC
Pro ThrThrAla Ala Pro Cys
Ser Cys Gly
85 90
CCTCTCTGCC AGCAGGACTG GGTGGTCCAA AGAATCGGCA AATGAGAGTG GTTAGAAGGC 350
TTCTTAGCGC AGTTGTTCAG AGCCCTGGTG GATCTTGTAA TCCAGTGCCC TACAAAGGCT 410
AGAACACTAC AGGGGATGAA TTCTTCAAAT AGGAGCCGAT GGATCTGTGG TCTTTGGACT 470
CATCAAAGCC TTGGTTAGCA TTTGTCAGTT TTATCTTCAG AAATTCTCTG TGATTAAGAA 530
GATAATTTAT TAAAGGTGGT CCTTCCTACC TCTGTGGTGT GTGTCGCGCA CACAGCTTAG 590
AAGTGCTATA AAAAAGGAAA GAGCTCCAAA TTGAATCACC TTATAATTTA CCCATTTCTA 650
TACAACAGGC AGTGGAAGCA GTTTCGAGAC TTTTTCGATG CTTATGGTTG ATCAGTTAAA 710
7.
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/Z6705
AAAGAATGTT ACAGTAACAA ATAAAGTGCA GTTTAAA 747
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 90 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Cys Ala Ile Cys Arg Val Gln Met Pro Val Leu Asp Val Lys Leu
50 55 60
Lys Thr Asn Lys Arg Thr Val Leu Trp Ser Gly Glu Asn Val Ile Ile
65 70 75 80
Pro Ser Thr Thr Ala Ala Cys Pro Cys Gly
85 90
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 706 base pairs ,
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..291
(ix} FEATURE:
(A) NAME/KEY: mat~eptide
(B) LOCATION:1..291
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
ATGGCC GAC GTG GAA GAC GGA GAA ACC TGC GCC GCCTCTCAC 48
GAG CTG
MetAla Asp Val Glu Asp Gly Glu Thr Cys Ala AlaSerHis
Glu Leu
1 5 10 15
TCCGGG AGC TCA GGC TCC AAG GGA GGC GAC AAG TTCTCCCTC 96
TCG ATG
SerGly Ser Ser Gly Ser Lys Gly Gly Asp Lys PheSerLeu
Ser Met
20 25 30
g
CA 02303483 2000-03-08
WO 99/32514 PCT/US98n6705
AAGAAGTGG GCG GCC ATG TGGGACGTG GAG GAT 144
AAC GTG TGG TGC
AGC
LysLysTrp Ala Ala Met Ser TrpAspVal Glu Asp
Asn Val Trp Cys
35 40 45
ACGTGCGCC TGC GTC CAG ATG GTGGTCTGG GGA TGT 192
ATC AGG GTG GAA
ThrCysAla Cys Val Gln Met ValValTrp Gly Cys
Ile Arg Val Glu
50 55 60
AATCATTCC CAC TGC TGC TCC CTGTGGGTG AAA AAC 240
TTC AAC ATG CAG
AsnHisSer His Cys Cys Ser LeuTrpVal Lys Asn
Phe Asn Met Gln
65 70 75 80
AATCGCTGC CTC CAG CAG TGG GTGGTCCAA AGA GGC 288
CCT TGC GAC ATC
AsnArgCys Leu Gln Gln Trp ValValGln Arg Gly
Pro Cys Asp Ile
85 90 95
AAATGAGAGTGGT CCCTGGTGGA 341
TAGAAGGCTT
CTTAGCGCAG
TTGTTCAGAG
Lys
TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 401
GAGCCGATGGATCTGTGGTCTTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT461
ATCTTCAGAAATTCTCTGTGATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC521
TGTGGTGTGTGTCGCGCACACAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT581
GAATCACCTTATAATTTACCCATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT641
TTTCGATGCTTATGGTTGATCAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT701
TTAAA 706
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Cys Ala Ile Cys Arg Val Gln Val Met Val Val Trp Gly Glu Cys
50 55 60
Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
65 70 75 80
9
CA 02303483 2000-03-08
WO 99132514 PCT/US98/26705
Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
85 90 95
Lys
(2} INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide hSAG. Ml"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
GCCATCTGCA GGGTCCAG 18
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide SAG T.02L"
(xi} SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GGATCCTCAT TTGCCGATTC TTTGGAC 27
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide
SAGKanMX4-5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
TTCTCCAGTG GCAGAGAACT TTAAAGAGAA ATAGTTCAAC CGTACGCTGC AGGTCGAC 58
1~
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
(2) INFORMATION FOR SEQ ID N0: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide SAGKan MX
4-3"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
ACCTCGGTAT GATTTAAATG TTTACGGGCA ATTCATTTTT ATCGATGAAT TCGAGCTCG 59
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide SAG pcr 5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
TTCTCCAGTG GCAGAGAAC 19
(2) INFORMATION FOR SEQ ID N0: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide SAG pcr 3"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
ATGATTTAAA TGTTTACGGG C 21
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 754 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
I1
CA 02303483 2000-03-08
WO 99132514 PCTNS98/26705
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix)
FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..339
(ix)
FEATURE:
(A) NAME/KEY: mat~eptide
(B) LOCATION:1..339
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
ATG GCC GTG GAA GGA GAA TGC GCCCTGGCC CAC 48
GAC GAC GAG ACC TCT
Met Ala Val Glu Gly Glu Cys AlaLeuAla His
Asp Asp Glu Thr Ser
1 5 10 15
TCC GGG TCA GGC AAG GGA GAC AAGATGTTC CTC 96
AGC TCC TCG GGC TCC
Ser Gly Ser Gly Lys Gly Asp LysMetPhe Leu
Ser Ser Ser Gly Ser
20 25 30
AAG AAG AAC GCG GCC TGG TGG GACGTGGAG GAT 144
TGG GTG ATG AGC TGC
Lys Lys Asn Ala Ala Trp Trp AspValGlu Asp
Trp Val Met Ser Cys
35 40 45
ACG AGC ATC TGC GTC GTG GAT GCCTGTCTT TGT 192
GCC AGG CAG ATG AGA
Thr Ser Ile Cys Val Val Asp AlaCysLeu Cys
Ala Arg Gln Met Arg
50 55 60
CAA GCT AAC AAA GAG TGT GTG GTCTGGGGA TGT 240
GAA CAA GAC GTT GAA
Gln Ala Asn Lys Glu Cys Val VaITrpGly Cys
GIu Gln Asp Val Glu
65 70 75 80
AAT CAT TTC CAC TGC ATG CTG TGGGTGAAA AAC 288
TCC AAC TGC TCC CAG
Asn His Phe His Cys Met Leu TrpValLys Asn
Ser Asn Cys Ser Gln
85 90 95
AAT CGC CCT CTC CAG GAC GTG GTCCAAAGA GGC 336
TGC TGC CAG TGG ATC
Asn Arg Pro Leu Gln Asp Val ValGlnArg Gly
Cys Cys Gln Trp Ile
100 105 110
AAA TGAGAGTGGT CCCTGGTGGA 389
TAGAAGGCTT
CTTAGCGCAG
TTGTTCAGAG
Lys
TCTTGTAATC CAGTGCCCTACAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG449
GAGCCGATGG ATCTGTGGTCTTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT509
ATCTTCAGAA ATTCTCTGTGATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC569
TGTGGTGTGT GTCGCGCACACAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT629
GAATCACCTT ATAATTTACCCATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT689
TTTCGATGCT TATGGTTGATCAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT749
TTAAA 754
12
CA 02303483 2000-03-08
WO 99/32514 PCTNS98/Z6705
(2) INFORMATION FOR SEQ ID NO': 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Ser Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys
50 55 60
Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys
65 70 75 80
Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
g5 90 95
Asr~ Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
Lys
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 754 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..339
(ix)
FEATURE:
(A) NAME/KEY: mat~eptide
(B) LOCATION:1..339
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
13
CA 02303483 2000-03-08
WO PCTNS98/26705
99/32514
TCC AGC TCA TCC AAG GGA GAC AAGATG TTC CTC 96
GGG GGC TCG GGC TCC
Ser Ser Ser Ser Lys Gly Asp LysMet Phe Leu
Gly Gly Ser Gly Ser
20 25 30
AAG TGG AAC GTG GCC TGG TGG GACGTG GAG GAT 144
AAG GCG ATG AGC TGC
Lys Trp Asn Val Ala Trp Trp AspVal Glu Asp
Lys Ala Met Ser Cys
35 40 45
ACG GCC ATC AGG GTC GTG GAT GCCTGT CTT TGT 192
TGC AGC CAG ATG AGA
Thr Ala Ile Arg Val Val Asp AlaCys Leu Cys
Cys Ser Gln Met Arg
50 55 60
CAA GAA AAC CAA GAG TGT GTG GTCTGG GGA TGT 240
GCT AAA GAC GTT GAA
Gln Glu Asn Gln Glu Cys Val ValTrp Gly Cys
Ala Lys Asp Val Glu
65 70 75 80
AAT TCC TTC AAC TGC ATG CTG TGGGTG AAA AAC 288
CAT CAC TGC TCC CAG
Asn Ser Phe Asn Cys Met Leu TrpVal Lys Asn
His His Cys Ser Gln
85 90 95
AAT TGC CCT TGC CAG GAC GTG GTCCAA AGA GGC 336
CGC CTC CAG TGG ATC
Asn Cys Pro Cys Gln Asp Val ValGln Arg Gly
Arg Leu Gln Trp Ile
100 105 110
AAA CCCTGGTGGA 389
TGAGAGTGGT
TAGAAGGCTT
CTTAGCGCAG
TTGTTCAGAG
Lys
TCTTGTAATC CAGTGCCCTACAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG449
GAGCCGATGG ATCTGTGGTCTTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT509
ATCTTCAGAA ATTCTCTGTGATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC569
TGTGGTGTGT GTCGCGCACACAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT629
GAATCACCTT ATAATTTACCCATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT689
TTTCGATGCT TATGGTTGATCAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT749
Z"r~ 7
54
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
I4
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Cys Ala Ile Ser Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys
50 55 60
Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys
65 70 75 80
Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
Lys
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 754 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..339
(ix)
FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION:1..339
(xi) SEQUENCE
DESCRIPTION:
SEQ ID NO:
25:
ATG GCC GAC GAAGAC GGA GAA TGC CTGGCC CAC 48
GTG GAG ACC GCC TCT
Met AIa Asp GluAsp Gly Glu Cys LeuAla His
Val Glu Thr Ala Ser
1 5 10 15
TCC GGG AGC GGCTCC AAG GGA GAC ATGTTC CTC 96
TCA TCG GGC AAG TCC
Ser Gly Ser GlySer Lys Gly Asp MetPhe Leu
Ser Ser Gly Lys Ser
20 25 30
AAG AAG TGG GCGGTG GCC TGG TGG GTGGAG GAT 144
AAC ATG AGC GAC TGC
Lys Lys Trp AlaVal Ala Trp Trp Va1Glu Asp
Asn Met Ser Asp Cys
35 40 45
ACG AGC GCC AGCAGG GTC GTG GAT TGTCTT TGT 192
ATC CAG ATG GCC AGA
Thr Ser Ala SerArg Val Val Asp CysLeu Cys
Ile Gln Met Ala Arg
50 55 60
CAA GCT GAA AAACAA GAG TGT GTG TGGGGA TGT 240
AAC GAC GTT GTC GAA
Gln Ala Glu LysGln Glu Cys Val TrpGly Cys
Asn Asp Val Val Glu
65 70 75 80
AAT CAT TCC CACAAC TGC ATG CTG GTGAAA AAC 288
TTC TGC TCC TGG CAG
IS
CA 0230348312000-03-08
WO 99132514 PCT/US98/26705
Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
AAT CGC TGC CCT CTC TGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336
Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389
Lys
TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449
GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509
ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569
TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629
GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689
TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749
754
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Ser Ala Ile Ser Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys
50 55 60
Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys
65 70 75 80
Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
Lys
(2) INFORMATION FOR SEQ ID N0: 27:
16
CA 02303483 2000-03-08
WO 99132514 PCT/US98IZ6705
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 754 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..339
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION:1..339
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
ATG GCCGAC GAA GAC GGA GAG GAA ACC CTG GCC CAC 48
GTG TGC GCC TCT
Met AlaAsp Glu Asp Gly Glu Glu Thr Leu Ala His
Val Cys Ala Ser
1 5 10 15
TCC GGGAGC GGC TCC AAG TCG GGA GGC ATG TTC CTC 96
TCA GAC AAG TCC
Ser GlySer Gly Ser Lys Ser Gly Gly Met Phe Leu
Ser Asp Lys Ser
20 25 30
AAG AAGTGG GCG GTG GCC ATG TGG AGC GTG GAG GAT 144
AAC TGG GAC TGC
Lys LysTrp Ala Val Ala Met Trp Ser Val Glu Asp
Asn Trp Asp Cys
35 40 45
ACG TGCGCC TGC AGG GTC CAG GTG ATG AGT CTT TGT 192
ATC GAT GCC AGA
Thr CysAla Cys Arg Val Gln Val Met Ser Leu Cys
Ile Asp Ala Arg
50 55 60
CAA GCTGAA AAA CAA GAG GAC TGT GTT TGG GGA TGT 240
AAC GTG GTC GAA
Gln AlaGlu Lys Gln Glu Asp Cys Val Trp Gly Cys
Asn Val Val Glu
65 70 75 80
AAT CATTCC CAC AAC TGC TGC ATG TCC GTG AAA AAC 288
TTC CTG TGG CAG
Asn HisSer His Asn Cys Cys Met Ser Val Lys Asn
Phe Leu Trp Gln
85 90 95
AAT CGCTGC CTC TGC CAG CAG GAC TGG CAA AGA GGC 336
CCT GTG GTC ATC
Asn ArgCys Leu Cys Gln Gln Asp Trp Gln Arg Gly
Pro Val Val Ile
100 105 110
AAA TGAGAGTGGT CCCTGGTGGA 389
TAGAAGGCTT
CTTAGCGCAG
TTGTTCAGAG
Lys
TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449
GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509
ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569
TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629
17
CA 0230348312000-03-08
WO 99/32514 ~ PCT/US98/Z6705
GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689
TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749
I"r~ 7 5 4
(2) INFORMATION FOR SEQ ID N0: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Ser Leu Arg Cys
50 55 60
Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys
65 70 75 80
Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
Lys
(2) INFORMATION FOR SEQ ID N0: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 754 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..339
(ix)
FEATURE:
(A) NAME/KEY: mat~eptide
(B) LOCATION:1..339
Ig
CA 02303483 2000-03-08
WO 99/32514 PGTNS98/Z6705
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 1 96
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
AAG TGG GCG GTG ATG GTG GAG TGC GAT 144
AAG AAC GCC TGG
AGC
TGG
GAC
LysLysTrp Ala Val Met Ser Trp Val Glu Cys Asp
Asn Ala Trp Asp
35 40 45
ACGTGCGCC TGC AGG CAG ATG GAT TGT CTT AGA TGT 192
ATC GTC GTG GCC
ThrCysAla Cys Arg Gln Met Asp Cys Leu Arg Cys
Ile Val Val Ala
50 55 60
CAAGCTGAA AAA CAA GAC GTT GTG TGG GGA GAA AGT 240
AAC GAG TGT GTC
GlnAlaGlu Lys Gln Asp Val Val Trp Gly Glu Ser
Asn Glu Cys Val
65 70 75 80
AATCATTCC CAC AAC TGC TCC CTG GTG AAA CAG AAC 288
TTC TGC ATG TGG
AsnHisSer His Asn Cys Ser Leu Val Lys Gln Asn
Phe Cys Met Trp
85 90 95
AATCGCTGC CTC TGC CAG TGG GTG CAA AGA ATC GGC 336
CCT CAG GAC GTC
AsnArgCys Leu Cys Gln Trp Val Gln Arg Ile Gly
Pro Gln Asp Val
100 105 110
AAATGAGAGTGGT CCCTGGTGGA 389
TAGAAGGCTT
CTTAGCGCAG
TTGTTCAGAG
Lys
TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449
GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509
ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569
TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629
GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689
TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749
TTAAA 754
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
19
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys
50 55 60
Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Ser
65 70 75 80
Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
Lys
(2) INFORMATION FOR SEQ ID N0: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 754 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..339
(ix) FEATURE:
(A) NAME/KEY: mat-peptide
(B) LOCATION:1..339
(xi) SEQUENCE DESCRIPTION:
SEQ ID NO: 31:
ATG GAC GTG GAA GAC GAG GAA TGC CTG GCCTCTCAC 48
GCC GGA ACC GCC
~Iet Asp Val Glu Asp Glu Glu Cys Leu AlaSerHis
Ala Gly Thr Ala
1 5 10 15
TCC AGC TCA GGC TCC TCG GGA GAC ATG TTCTCCCTC 96
GGG AAG GGC AAG
Ser Ser Ser Gly Ser Ser Gly Asp Met PheSerLeu
Gly Lys Gly Lys
20 25 30
AAG TGG AAC GCG GTG ATG TGG TGG GTG GAGTGCGAT 144
AAG GCC AGC GAC
Lys Trp Asn Ala Val Met Trp Trp Val GluCysAsp
Lys Ala Ser Asp
35 40 45
ACG GCC ATC TGC AGG CAG GTG GAT TGT CTTAGATGT 192
TGC GTC ATG GCC
Thr Ala Ile Cys Arg Gln Val Asp Cys LeuArgCys
Cys Val Met Ala
50 55 60
CAA GAA AAC AAA CAA GAC TGT GTG TGG GGAGAATGT 240
GCT GAG GTT GTC
CA 02303483 2000-03-08
WO 99/32514 PCTNS98/26705
Gln Glu Gln Glu CysVal ValVal Trp Gly Cys
Ala Asn Asp Glu
Lys
65 70 75 80
AAT TCC CACAAC TGC ATGTCC CTGTGG GTG AAA AAC 288
AAA TTC TGC CAG
Asn Ser HisAsn Cys MetSer LeuTrp Val Lys Asn
Lys Phe Cys Gln
85 90 95
AAT TGC CTCTGC CAG GACTGG GTGGTC CAA AGA GGC . 336
CGC CCT CAG ATC
Asn Cys LeuCys Gln AspTrp ValVal Gln Arg Gly
Arg Pro Gln Ile
100 105 110
AAA 389
TGAGAGTGGT
TAGAAGGCTT
CTTAGCGCAG
TTGTTCAGAG
CCCTGGTGGA
Lys
TCTTGTAATC CAGTGCCCTACAAAGGCTAG AACACTACAG GGGATGAATTCTTCAAATAG 449
GAGCCGATGG ATCTGTGGTCTTTGGACTCA TCAAAGCCTT GGTTAGCATTTGTCAGTTTT 509
ATCTTCAGAA ATTCTCTGTGATTAAGAAGA TAATTTATTA AAGGTGGTCCTTCCTACCTC 569
TGTGGTGTGT GTCGCGCACACAGCTTAGAA GTGCTATAAA AAAGGAAAGAGCTCCAAATT 629
GAATCACCTT ATAATTTACCCATTTCTATA CAACAGGCAG TGGAAGCAGTTTCGAGACTT 689
TTTCGATGCT TATGGTTGATCAGTTAAAAA AGAATGTTAC AGTAACAAATAAAGTGCAGT 749
TTAAA 754
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys
50 55 60
Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys
65 70 75 80
Asn Lys Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
21
CA 02303483 2000-03-08
WO 99/32514 PCTNS98/26705
Lys
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 754 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..339
(ix) FEATURE:
(A) NAME/KEY: mat~eptide
(B) LOCATION:1..339
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
ATGGCC GTG GAA GGA GAG ACCTGCGCC CTGGCC 48
GAC GAC GAA TCT
CAC
MetAla Val Glu Gly Glu ThrCysAla LeuAla His
Asp Asp Glu Ser
1 5 10 15
TCCGGG TCA GGC AAG TCG GGCGACAAG ATGTTC CTC 96
AGC TCC GGA TCC
SerGly Ser Gly Lys Ser GlyAspLys MetPhe Leu
Ser Ser Gly Ser
20 25 30
AAGAAG AAC GCG GCC ATG AGCTGGGAC GTGGAG GAT 144
TGG GTG TGG TGC
LysLys Asn Ala Ala Met SerTrpAsp ValGlu Asp
Trp Val Trp Cys
35 40 45
ACGTGC ATC TGC GTC CAG ATGGATGCC TGTCTT TGT 192
GCC AGG GTG AGA
ThrCys Ile Cys Val Gln MetAspAla CysLeu Cys
Ala Arg Val Arg
50 55 60
CAAGCT AAC AAA GAG GAC GTTGTGGTC TGGGGA TGT 240
GAA CAA TGT GAA
GlnAla Asn Lys Glu Asp ValValVal TrpGly Cys
Glu Gln Cys Glu
65 70 75 80
AATCAT TTC AAG TGC TGC TCCCTGTGG GTGAAA AAC 288
TCC AAC ATG CAG
AsnHis Phe Lys Cys Cys SerLeuTrp ValLys Asn
Ser Asn Met Gln
85 90 95
AATCGC CCT CTC CAG CAG TGGGTGGTC CAAAGA GGC 336
TGC TGC GAC ATC
AsnArg Pro Leu Gln Gln TrpValVal GlnArg Gly
Cys Cys Asp Ile
100 105 110
AAATGAGAGTGGT CCCTGGTGGA 389
TAGAAGGCTT
CTTAGCGCAG
TTGTTCAGAG
Lys
TCTTGTAATC GGGATGAATT 449
CAGTGCCCTA CTTCAAATAG
CAAAGGCTAG
AACACTACAG
GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509
22
CA 0230348312000-03-08
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
Z'T~ 754
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys
50 55 60
Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys
65 70 75 80
Asn His Ser Phe Lys Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
Lys
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 754 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..339
(ix) FEATURE:
23
CA 02303483 2000-03-08
WO 99/32514 PCTNS98/26705
(A) NAME/KEY: mat~eptide
(B) LOCATION:1..339
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
ATG GCC GAC GTG. GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
ACGTGC ATCTGCAGG GTC GTG GCC TGT CTT TGT 192
GCC CAG ATG AGA
GAT
ThrCys IleCysArg Val Val MetAspAla Cys Leu Cys
Ala Gln Arg
50 55 60
CAAGCT AACAAACAA GAG TGT GTTGTGGTC TGG GGA TGT 240
GAA GAC GAA
GlnAla AsnLysGln Glu Cys ValValVal Trp Gly Cys
Glu Asp Glu
65 70 75 80
AATCAT TTCCACAAC TGC ATG TCCCTGTGG GTG AAA AAC 288
TCC AGC CAG
AsnHis PheHisAsn Cys Met SerLeuTrp Val Lys Asn
Ser Ser Gln
85 90 95
AATCGC CCTCTCTGC CAG GAC TGGGTGGTC CAA AGA GGC 336
TGC CAG ATC
AsnArg ProLeuCys Gln Asp TrpValVal Gln Arg Gly
Cys Gln Ile
100 105 110
AAATGAGAGTGGT CCCTGGTGGA 389
TAGAAGGCTT
CTTAGCGCAG
TTGTTCAGAG
Lys
TCTTGTAATC CAGTGCCCTACAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG449
GAGCCGATGG ATCTGTGGTCTTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT509
ATCTTCAGAA ATTCTCTGTGATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC569
TGTGGTGTGT GTCGCGCACACAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT629
GAATCACCTT ATAATTTACCCATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT689
TTTCGATGCT TATGGTTGATCAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT749
TTAAA 754
(2} INFORMATION FOR SEQ ID N0: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
24
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys
50 55 60
Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys
65 70 75 80
Asn His Ser Phe His Asn Cys Ser Met Ser Leu Trp Val Lys Gln Asn
85 90 95
Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
Lys
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 754 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/REY: CDS
(B) LOCATION:1..339
(ix) FEATURE:
(A) NAME/KEY: mat~eptide
(B) LOCATION:1..339
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192
CA 02303483 2000-03-08
WO 99/32514 PCT/US98126705
Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys
50 55 60
CAAGCT AACAAA CAAGAGGAC GTTGTG GTCTGG GGA TGT 240
GAA TGT GAA
GlnAla AsnLys GlnGluAsp ValVal ValTrp Gly Cys
Glu Cys Glu
65 70 75 80
AATCAT TTCCAC AACTGCTGC TCCCTG TGGGTG AAA AAC 288
TCC ATG CAG
AsnHis PheHis AsnCysCys SerLeu TrpVal Lys Asn
Ser Met Gln
85 90 95
AATCGC CCTCTC TGCCAGCAG TGGGTG GTCCAA AGA GGC 336
AGC GAC ATC
AsnArg ProLeu CysGlnGln TrpVal ValGln Arg Gly
Ser Asp Ile
100 105 210
AAATGAGAGTGGT CCCTGGTGGA 3$9
TAGAAGGCTT
CTTAGCGCAG
TTGTTCAGAG
Lys
TCTTGTAATCCAGTGCCCTACAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG449
GAGCCGATGGATCTGTGGTCTTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT509
ATCTTCAGAAATTCTCTGTGATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC569
TGTGGTGTGTGTCGCGCACACAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT629
GAATCACCTTATAATTTACCCATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT689
TTTCGATGCTTATGGTTGATCAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT749
TTAAA 754
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 38:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys
50 S5 60
Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys
65 70 75 80
Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
26
CA 02303483 2000-03-08
WO 99/32514 PCT/IJS98/2G705
Asn Arg Ser Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
Lys
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 754 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..339
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION:1..339
(xi) SEQUENCE DESCRIPTION:
SEQ ID NO: 39:
ATG GAC GTG GAA GAC GAG GAA TGC CTG GCC CAC 48
GCC GGA ACC GCC TCT
Met Asp Val Glu Asp Glu Glu Cys Leu Ala His
Ala Gly Thr Ala Ser
1 5 10 15
TCC AGC TCA GGC TCC TCG GGA GAC ATG TTC CTC 96
GGG AAG GGC AAG TCC
Ser Ser Ser Gly Ser Ser Gly Asp Met Phe Leu
Gly Lys Gly Lys Ser
20 25 30
AAG TGG AAC GCG GTG ATG TGG TGG GTG GAG GAT 144
AAG GCC AGC GAC TGC
Lys Trp Asn Ala Val Met Trp Trp Val Glu Asp
Lys Ala Ser Asp Cys
35 40 45
ACG GCC ATC TGC AGG CAG GTG GAT TGT CTT TGT 192
TGC GTC ATG GCC AGA
Thr Ala Ile Cys Arg Gln Val Asp Cys Leu Cys
Cys Val Met Ala Arg
50 55 60
CAA GAA AAC AAA CAA GAC TGT GTG TGG GGA TGT 240
GCT GAG GTT GTC GAA
Gln Glu Asn Lys Gln Asp Cys Val Trp Gly Cys
Ala Glu Val Val Glu
65 70 75 80
AAT TCC TTC AAG AAC TGC ATG CTG GTG AAA AAC 288
AAA TGC TCC TGG CAG
Asn Ser Phe Lys Asn Cys Met Leu Val Lys Asn
Lys Cys Ser Trp Gln
85 90 95
AAT TGC CCT CTC TGC CAG GAC GTG CAA AGA GGC 336
CGC CAG TGG GTC ATC
Asn Cys Pro Leu Cys Gln Asp Val Gln Arg Gly
Arg Gln Trp Val Ile
100 105 110
AAA CCCTGGTGGA 389
TGAGAGTGGT
TAGAAGGCTT
CTTAGCGCAG
TTGTTCAGAG
Lys
27
i I
CA 02303483 2000-03-08
WO 99/32514 PCT/IJS98/26705
TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT 449
CTTCAAATAG
GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT 509
TGTCAGTTTT
ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC 569
TTCCTACCTC
TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA 629
GCTCCAAATT
GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT 689
TTCGAGACTT
TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT 749
AAAGTGCAGT
TTAAA 754
(2) INFORMATION FOR SEQ ID N0: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 40:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys
50 55 60
Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys
65 70 75 80
Asn Lys Ser Phe Lys Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
Lys
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 754 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
28
CA 02303483 2000-03-08
WO 99/32514 PCTIUS98/26705
(A) NAME/KEY: CDS
(B) LOCATION:1..339
(ix) FEATURE:
(A) NAME/KEY: mat~eptide
(B) LOCATION:1..339
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 41:
ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
AAGAAGTGG GCG GCC ATG GTGGAG GAT 144
AAC GTG TGG AGC TGC
TGG GAC
LysLysTrp AsnAlaVal Ala Met Ser AspValGlu Asp
Trp Trp Cys
35 40 45
ACGTGCGCC ATCTGCAGG GTC CAG ATG GCCTGTCTT AGT 192
GTG GAT AGA
ThrCysAla IleCysArg Val Gln Met AlaCysLeu Ser
Val Asp Arg
50 55 60
CAAGCTGAA AACAAACAA GAG GAC GTT GTCTGGGGA TGT 240
TGT GTG GAA
GlnAlaGlu AsnLysGln Glu Asp Val ValTrpGly Cys
Cys Val Glu
65 70 75 80
AATCATTCC TTCCACAAC TGC TGC TCC TGGGTGAAA AAC 288
ATG CTG CAG
AsnHisSer PheHisAsn ~Cys Cys Ser TrpValLys Asn
Met Leu Gln
85 90 95
AATCGCTGC CCTCTCTGC CAG CAG TGG GTCCAAAGA GGC 336
GAC GTG ATC
AsnArgCys ProLeuCys Gln Gln Trp ValGlnArg Gly
Asp Val Ile
100 105 110
AAATGAGAGTGGT CCCTGGTGGA 389
TAGAAGGCTT
CTTAGCGCAG
TTGTTCAGAG
Lys
TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449
GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509
ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569
TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629
GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689
TTTCGATGGT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749
TTAAA 754
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
29
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/Z6705
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Ser
50 55 60
Gln Ala Glu Asn Lys Gln Glu Asp Gys Val Val Val Trp Gly Glu Cys
65 70 75 80
Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
Lys
(2) INFORMATION F'OR SEQ ID N0: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 754 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..339
(ix) FEATURE:
(A) NAME/KEY: mat~eptide
(B) LOCATION:1..339
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
Lys Lys AsnAla Ala TrpSerTrp AspVal Glu Asp
Trp Val Met Cys
35 40 45
ACG TGC ATCTGC GTC GTGATGGAT GCCTGT CTT TGT 192
GCC AGG CAG AGA
Thr Cys IleCys Val ValMetAsp AlaCys Leu Cys
Ala Arg Gln Arg
50 55 60
CAA GCT AACAAA GAG AGTGTTGTG GTCTGG GGA TGT 240
GAA CAA GAC GAA
Gln Ala AsnLys Glu SerValVal ValTrp Gly Cys
Glu Gln Asp Glu
65 70 75 80
AAT CAT TTCCAC TGC ATGTCCCTG TGGGTG AAA AAC 288
TCC AAC TGC CAG
Asn His PheHis Cys MetSerLeu TrpVal Lys Asn
Ser Asn Cys Gln
85 90 95
AAT CGC CCTCTC CAG GACTGGGTG GTCCAA AGA GGC 336
TGC TGC CAG ATC
Asn Arg ProLeu Gln AspTrpVal ValGln Arg Gly
Cys Cys Gln Ile
100 105 110
AAA TGAGAGTGGT CCCTGGTGGA 389
TAGAAGGCTT
CTTAGCGCAG
TTGTTCAGAG
Lys
TCTTGTAATCCAGTGCCCTACAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG449
GAGCCGATGGATCTGTGGTCTTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT509
ATCTTCAGAAATTCTCTGTGATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC569
TGTGGTGTGTGTCGCGCACACAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT629
GAATCACCTTATAATTTACCCATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT689
TTTCGATGCTTATGGTTGATCAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT749
TTAAA 754
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Cys Ala Ile Cys Arg Val Gln VaI Met Asp Ala Cys Leu Arg Cys
50 55 60
31
CA 02303483 2000-03-08
WO 99/32514 PGT/I1S98/Z6705
Gln Ala Glu Asn Lys Gln Glu Asp Ser Val Val Val Trp Gly Glu Cys
65 70 75 80
Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
Lys
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 754 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..339
(ix) FEATURE:
(A) NAME/KEY: mat~eptide
(B) LOCATION:1..339
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
ATG GTG GGA GAG ACC TGCGCCCTG GCCTCT 48
GCC GAA GAA CAC
GAC GAC
MetAla Val Glu Gly Glu Thr CysAIaLeu AlaSerHis
Asp Asp Glu
1 5 10 15
TCCGGG TCA GGC AAG TCG GGC GACAAGATG TTCTCCCTC 96
AGC TCC GGA
SerGly Ser Gly Lys Ser Gly AspLysMet PheSerLeu
Ser Ser Gly
20 25 30
AAGAAG AAC GCG GCC ATG AGC TGGGACGTG GAGTGCGAT 144
TGG GTG TGG
LysLys Asn Ala Ala Met Ser TrpAspVal GluCysAsp
Trp Val Trp
35 40 45
ACGTGC ATC TGC GTC CAG ATG GATGCCAGT CTTAGAAGT 192
GCC AGG GTG
ThrCys Ile Cys Val Gln Met AspAlaSer LeuArgSer
Ala Arg Val
50 55 60
CAAGCT AAC AAA GAG GAC GTT GTGGTCTGG GGAGAATGT 240
GAA CAA TGT
GlnAla Asn Lys Glu Asp Val ValValTrp GlyGluCys
Glu Gln Cys
65 70 75 80
AATCAT TTC CAC TGC TGC TCC CTGTGGGTG AAACAGAAC 288
TCC AAC ATG
AsnHis Phe His Cys Cys Ser LeuTrpVal LysGlnAsn
Ser Asn Met
85 90 95
AATCGC CCT CTC CAG CAG TGG GTGGTCCAA AGAATCGGC 336
TGC TGC GAC
AsnArg Pro Leu Gln Gln Trp ValValGln ArgIleGly
Cys Cys Asp
100 105 110
32
CA 0230348312000-03-08
WO 99/32514 PCTIUS98/26705
AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389
Lys
TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449
GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509
ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569
TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629
GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689
TTTCGATGCT TATGGTTGAT CAGTTA.AAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749
TTAAA 754
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Ser Leu Arg Ser
50 55 60
Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys
65 70 75 80
Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
Lys
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 754 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
33
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..339
(ix) FEATURE:
(A) NAME/KEY: mat~eptide
(B) LOCATION:1..339
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 1S
TCC GGG AGC TCA GGC TCC AAG TCG GGA GGC GAC AAG ATG TTC TCC CTC 96
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
AAG AAG TGG AAC GCG GTG GCC ATG TGG AGC TGG GAC GTG GAG TGC GAT 144
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
ACG TGC GCC ATC TGC AGG GTC CAG GTG ATG GAT GCC TGT CTT AGA TGT 192
Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys
50 55 60
CAA GCT GAA AAC AAA CAA GAG GAC TGT GTT GTG GTC TGG GGA GAA TGT 240
Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys
65 70 75 80
AAT CAT TCC TTC CAC AAC TGC TGC ATG TCG CTG TGG GTG AAA CAG AAC 288
Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
AAT CGC AGC CCT CTC AGC CAG CAG GAC TGG GTG GTC CAA AGA ATC GGC 336
Asn Arg Ser Pro Leu Ser Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389
Lys
TCTTGTAATC CAGTGCCCTA CAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG 449
GAGCCGATGG ATCTGTGGTC TTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT 509
ATCTTCAGAA ATTCTCTGTG ATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC 569
TGTGGTGTGT GTCGCGCACA CAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT 629
GAATCACCTT ATAATTTACC CATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT 689
TTTCGATGCT TATGGTTGAT CAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT 749
TTAAA 754
34
CA 02303483 2000-03-08
WO 99/32514 PCT/US98I26705
(2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE bESCRIPTION: SEQ ID NO: 48:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp
35 40 45
Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys
50 55 60
Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys
65 70 75 80
Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
Asn Arg Ser Pro Leu Ser Gln Gln Asp Trp Val VaI Gln Arg Ile Gly
100 105 110
Lys
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 754 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..339
(ix) FEATURE:
(A) NAME/KEY: mat~eptide
(B) LOCATION:1..339
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
ATG GCC GAC GTG GAA GAC GGA GAG GAA ACC TGC GCC CTG GCC TCT CAC 48
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
CA 02303483 2000-03-08
WO 99/32514 PCT/US98/26705
TCCGGG TCA TCCAAGTCGGGA GAC AAG TTC CTC 96
AGC GGC GGC ATG TCC
SerGly SerSerGly SerLysSerGly GlyAsp LysMetPhe Leu
Ser
2U 25 30
AAGAAG TGGAACGCG GTGGCCATGTGG AGCTGG GACGTGGAG GAT 144
AGC
LysLys TrpAsnAla ValAlaMetTrp SerTrp AspValGlu Asp
Ser
35 40 45
ACGTGC GCCATCTGC AGGGTCCAGGTG ATGGAT GCCTGTCTT TGT 192
AGA
ThrCys AlaIleCys ArgValGlnVal MetAsp AlaCysLeu Cys
Arg
50 55 60
CAAGCT GAAAACAAA CAAGAGGACTGT GTTGTG GTCTGGGGA TGT 240
GAA
GlnAla GluAsnLys GlnGluAspCys ValVal ValTrpGly Cys'
Glu
65 70 75 80
AATCAT TCCTTCCAC AACTGCTGCATG TCCCTG TGGGTGAAA AAC 288
CAG
AsnHis SerPheHis AsnCysCysMet SerLeu TrpValLys Asn
Gln
85 90 95
AATCGC TGCCCTCTC TGCCAGCAGGAC TGGGTG GTCCAAAGA GGC 336
ATC
AsnArg CysProLeu CysGlnGlnAsp TrpVal ValGlnArg Gly
Ile
100 105 110
AAA TGAGAGTGGT TAGAAGGCTT CTTAGCGCAG TTGTTCAGAG CCCTGGTGGA 389
Lys
TCTTGTAATC CAGTGCCCTACAAAGGCTAG AACACTACAG GGGATGAATT CTTCAAATAG449
GAGCCGATGG ATCTGTGGTCTTTGGACTCA TCAAAGCCTT GGTTAGCATT TGTCAGTTTT509
ATCTTCAGAA ATTCTCTGTGATTAAGAAGA TAATTTATTA AAGGTGGTCC TTCCTACCTC569
TGTGGTGTGT GTCGCGCACACAGCTTAGAA GTGCTATAAA AAAGGAAAGA GCTCCAAATT629
GAATCACCTT ATAATTTACCCATTTCTATA CAACAGGCAG TGGAAGCAGT TTCGAGACTT689
TTTCGATGCT TATGGTTGATCAGTTAAAAA AGAATGTTAC AGTAACAAAT AAAGTGCAGT749
TTAAA 754
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His
1 5 10 15
Ser Gly Ser Ser Gly Ser Lys Ser Gly Gly Asp Lys Met Phe Ser Leu
20 25 30
Lys Lys Trp Asn Ala Val Ala Met Trp Ser Trp Asp Val Glu Ser Asp
35 40 45
36
CA 02303483 2000-03-08
WO 99132514 PCT/US98/26705
Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys
50 55 60
Gln Ala Glu Asn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys
65 70 75 80
Asn His Ser Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn
85 90 95
Asn Arg Cys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly
100 105 110
Lys
37
