U.S. patent application number 11/492111 was filed with the patent office on 2007-02-08 for mammalian iap gene family, primers, probes and detection methods.
Invention is credited to Stephen Baird, Robert G. Korneluk, Peter Liston, Alexander E. MacKenzie.
Application Number | 20070031903 11/492111 |
Document ID | / |
Family ID | 27057240 |
Filed Date | 2007-02-08 |
United States Patent
Application |
20070031903 |
Kind Code |
A1 |
Korneluk; Robert G. ; et
al. |
February 8, 2007 |
Mammalian IAP gene family, primers, probes and detection
methods
Abstract
Disclosed is substantially pure DNA encoding mammalian IAP
polypeptides; and methods of using such DNA to express the IAP
polypeptides in cells and animals to inhibit apoptosis. Also
disclosed are conserved regions characteristic of the IAP family
and primer and probes for the identification and isolation of
additional IAP genes. In addition, methods for treating diseases
and disorders involving apoptosis are provided.
Inventors: |
Korneluk; Robert G.;
(Ottawa, CA) ; MacKenzie; Alexander E.; (Ottawa,
CA) ; Baird; Stephen; (Ottawa, CA) ; Liston;
Peter; (Ottawa, CA) |
Correspondence
Address: |
PHILIP SWAIN, PHD;C/O GOWLING LAFLEUR HENDERSON
1 PLACE VILLE MARIE,
37TH FLOOR
MONTREAL
QC
H3B 3P4
CA
|
Family ID: |
27057240 |
Appl. No.: |
11/492111 |
Filed: |
July 25, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11316539 |
Dec 22, 2005 |
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11492111 |
Jul 25, 2006 |
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10600272 |
Jun 20, 2003 |
7067281 |
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11316539 |
Dec 22, 2005 |
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09011356 |
Sep 14, 1998 |
6656704 |
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PCT/IB96/01022 |
Aug 5, 1996 |
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10600272 |
Jun 20, 2003 |
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08576956 |
Dec 22, 1995 |
6156535 |
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09011356 |
Sep 14, 1998 |
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08511485 |
Aug 4, 1995 |
5919912 |
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08576956 |
Dec 22, 1995 |
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Current U.S.
Class: |
435/7.23 ;
435/226 |
Current CPC
Class: |
A61P 9/10 20180101; C07K
14/4747 20130101; A61K 38/00 20130101; A61P 1/00 20180101; A61P
31/18 20180101; A61P 25/00 20180101; A01K 2217/075 20130101; G01N
33/57407 20130101; A61P 43/00 20180101; G01N 2500/00 20130101; C12Q
2600/136 20130101; A01K 2217/05 20130101; C12Q 1/6883 20130101;
G01N 33/502 20130101; A61P 9/00 20180101; C12Q 2600/156 20130101;
G01N 33/6872 20130101; G01N 33/5743 20130101; A61K 48/00 20130101;
A61P 1/16 20180101; G01N 2510/00 20130101; A61P 31/00 20180101;
C12Q 2600/118 20130101; C12Q 2600/158 20130101; G01N 33/57426
20130101; G01N 33/5008 20130101; G01N 33/68 20130101; C12Q 1/6886
20130101; G01N 33/57423 20130101; G01N 33/57419 20130101 |
Class at
Publication: |
435/007.23 ;
435/226 |
International
Class: |
G01N 33/574 20060101
G01N033/574; C12N 9/64 20060101 C12N009/64 |
Claims
1. A substantially pure polypeptide consisting of a sequence having
at least 85% sequence identity to SEQ ID NO:27 or amino acids
269-336 of SEQ ID NO:8, wherein said polypeptide is capable of
inhibiting apoptosis of a mammalian cell when said polypeptide is
expressed in said cell.
2. The polypeptide of claim 1, wherein said polypeptide has at
least 85% sequence identity to SEQ ID NO:27.
3. The polypeptide of claim 1, wherein said polypeptide has at
least 90% sequence identity to SEQ ID NO:27.
4. The polypeptide of claim 1, wherein said polypeptide has at
least 95% sequence identity to SEQ ID NO:27.
5. The polypeptide of claim 1, wherein said polypeptide has at
least 85% sequence identity to amino acids 269-336 of SEQ ID
NO:8.
6. The polypeptide of claim 1, wherein said polypeptide has at
least 90% sequence identity to amino acids 269-336 of SEQ ID
NO:8.
7. The polypeptide of claim 1, wherein said polypeptide ahs at
least 95% sequence identity to amino acids 269-336 of SEQ ID
NO:8.
8. A substantially pure polypeptide consisting of SEQ ID NO:27 or
amino acids 269-336 of SEQ ID NO:8, wherein said polypeptide is
capable of inhibiting apoptosis of a mammalian cell when said
polypeptide is expressed in said cell.
9. The polypeptide of claim 8, wherein said polypeptide has the
sequence of SEQ ID NO:27.
10. The polypeptide of claim 8, wherein said polypeptide has the
sequence of amino acids 269-336 of SEQ ID NO:8.
11. A substantially pure polypeptide comprising SEQ ID NO:27 or
amino acids 269-336 of SEQ ID NO:8, wherein said polypeptide is
capable of inhibiting apoptosis of a mammalian cell when said
polypeptide is expressed in said cell.
12. The polypeptide of claim 11, wherein said polypeptide comprises
the sequence of SEQ ID NO:27.
13. The polypeptide of claim 11, wherein said polypeptide has the
sequence of amino acids 269-336 of SEQ ID NO:8.
14. A method of identifying a compound that modulates apoptosis,
said method comprising: (a) providing a polypeptide of any one of
claims 1-13; (b) contacting said polypeptide with a candidate
compound; and (c) detecting an interaction between said polypeptide
and said candidate compound, wherein an interaction identifies said
candidate compound as a compound that modulates apoptosis.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
09/011,356, filed Feb. 4, 1998 (now pending), which is a U.S.
National Phase application of PCT/IB/96/01022, filed Aug. 5, 1996,
and published in English under PCT article 21(2), which claims
benefit from U.S. Ser. No. 08/576,956, filed Dec. 22, 1995 (now
U.S. Pat. No. 6,156,535), which is a continuation-in-part of U.S.
Ser. No. 08/511,485, filed Aug. 4, 1995 (now U.S. Pat. No.
5,919,912), all of which are hereby incorporated by reference in
their entirety.
BACKGROUND OF THE INVENTION
[0002] The invention relates to apoptosis.
[0003] There are two general ways by which cells die. The most
easily recognized way is by necrosis, which is usually caused by an
injury that is severe enough to disrupt cellular homeostasis.
Typically, the cell's osmotic pressure is disturbed and,
consequently, the cell swells and then ruptures. When the cellular
contents are spilled into the surrounding tissue space, an
inflammatory response often ensues.
[0004] The second general way by which cells die is referred to as
apoptosis, or programmed cell death. Apoptosis often occurs so
rapidly that it is difficult to detect. This may help to explain
why the involvement of apoptosis in a wide spectrum of biological
processes has only recently been recognized.
[0005] The apoptosis pathway has been highly conserved throughout
evolution, and plays a critical role in embryonic development,
viral pathogenesis, cancer, autoimmune disorders, and
neurodegenerative disease. For example, inappropriate apoptosis may
cause or contribute to AIDS, Alzheimer's Disease, Parkinson's
Disease, Amyotrophic Lateral Sclerosis (ALS), retinitis pigmentosa
and other diseases of the retina, myelodysplastic syndrome (e.g.
aplastic anemia), toxin-induced liver disease, including
alcoholism, and ischemic injury (e.g. myocardial infarction,
stroke, and reperfusion injury). Conversely, the failure of an
apoptotic response has been implicated in the development of
cancer, particularly follicular lymphoma, p53-mediated carcinomas,
and hormone-dependent tumors, in autoimmune disorders, such as
lupus erythematosis and multiple sclerosis, and in viral
infections, including those associated with herpes virus, poxvirus,
and adenovirus.
[0006] In patients infected with HIV-1, mature CD4.sup.+ T
lymphocytes respond to stimulation from mitogens or super-antigens
by undergoing apoptosis. However, the great majority of these cells
are not infected with the virus. Thus, inappropriate
antigen-induced apoptosis could be responsible for the destruction
of this vital part of the immune system in the early stages of HIV
infection.
[0007] Baculoviruses encode proteins that are termed inhibitors of
apoptosis proteins (IAPs) because they inhibit the apoptosis that
would otherwise occur when insect cells are infected by the virus.
These proteins are thought to work in a manner that is independent
of other viral proteins. The baculovirus IAP genes include
sequences encoding a ring zinc finger-like motif (RZF), which is
presumed to be directly involved in DNA binding, and two N-terminal
domains that consist of a 70 amino acid repeat motif termed a BIR
domain (Baculovirus IAP Repeat).
SUMMARY OF THE INVENTION
[0008] In general, the invention features a substantially pure DNA
molecule, such as a genomic, cDNA, or synthetic DNA molecule, that
encodes a mammalian IAP polypeptide. This DNA may be incorporated
into a vector, into a cell, which may be a mammalian, yeast, or
bacterial cell, or into a transgenic animal or embryo thereof. In
preferred embodiments, the DNA molecule is a murine gene (e.g.,
m-xiap, m-hiap-1, or m-hiap-2) or a human gene (e.g., xiap, hiap-1,
or hiap-2). In most preferred embodiments the IAP gene is a human
LAP gene. In other various preferred embodiments, the cell is a
transformed cell. In related aspects, the invention features a
transgenic animal containing a transgene that encodes an IAP
polypeptide that is expressed in or delivered to tissue normally
susceptible to apoptosis, i.e., to a tissue that may be harmed by
either the induction or repression of apoptosis. In yet another
aspect, the invention features DNA encoding fragments of LAP
polypeptides including the BIR domains and the RZF domains provided
herein.
[0009] In specific embodiments, the invention features DNA
sequences substantially identical to the DNA sequences shown in
FIGS. 1-6, or fragments thereof. In another aspect, the invention
also features RNA which is encoded by the DNA described herein.
Preferably, the RNA is mRNA. In another embodiment the RNA is
antisense RNA.
[0010] In another aspect, the invention features a substantially
pure polypeptide having a sequence substantially identical to one
of the LAP amino acid sequences shown in FIGS. 1-6.
[0011] In a second aspect, the invention features a substantially
pure DNA which includes a promoter capable of expressing the LAP
gene in a cell susceptible to apoptosis. In preferred embodiments,
the LAP gene is xiap, hiap-1, or hiap-2.
[0012] Most preferably, the genes are human or mouse genes. The
gene encoding HIAP -2 may be the full-length gene, as shown in FIG.
3, or a truncated variant, such as a variant having a deletion of
the sequence boxed in FIG. 3.
[0013] In preferred embodiments, the promoter is the promoter
native to an IAP gene. Additionally, transcriptional and
translational regulatory regions are, preferably, those native to
an IAP gene. In another aspect, the invention provides transgenic
cell lines and transgenic animals. The transgenic cells of the
invention are preferably cells that are altered in their apoptotic
response. In preferred embodiments, the transgenic cell is a
fibroblast, neuronal cell, a lymphocyte cell, a glial cell, an
embryonic stem cell, or an insect cell. Most preferably, the neuron
is a motor neuron and the lymphocyte is a CD4.sup.+ T cell.
[0014] In another aspect, the invention features a method of
inhibiting apoptosis that involves producing a transgenic cell
having a transgene encoding an IAP polypeptide. The transgene is
integrated into the genome of the cell in a way is that allows for
expression. Furthermore, the level of expression in the cell is
sufficient to inhibit apoptosis.
[0015] In a related aspect, the invention features a transgenic
animal, preferably a mammal, more preferably a rodent, and most
preferably a mouse, having either increased copies of at least one
IAP gene inserted into the genome (mutant or wild-type), or a
knockout of at least one IAP gene in the genome. The transgenic
animals will express either an increased or a decreased amount of
IAP polypeptide, depending on the construct used and the nature of
the genomic alteration. For example, utilizing a nucleic acid
molecule that encodes all or part of an IAP to engineer a knockout
mutation in an IAP gene would generate an animal with decreased
expression of either all or part of the corresponding IAP
polypeptide. In contrast, inserting exogenous copies of all or part
of an IAP gene into the genome, preferably under the control of
active regulatory and promoter elements, would lead to increased
expression or the corresponding IAP polypeptide.
[0016] In another aspect, the invention features a method of
detecting an IAP gene in a cell by contacting the IAP gene, or a
portion thereof (which is greater than 9 nucleotides, and
preferably greater than 18 nucleotides in length), with a
preparation of genomic DNA from the cell. The IAP gene and the
genomic DNA are brought into contact under conditions that allow
for hybridization (and therefore, detection) of DNA sequences in
the cell that are at least 50% identical to the DNA encoding
HIAP-1, HIAP-2, or XIAP polypeptides.
[0017] In another aspect, the invention features a method of
producing an IAP polypeptide. This method involves providing a cell
with DNA encoding all or part of an LAP polypeptide (which is
positioned for expression in the cell), culturing the cell under
conditions that allow for expression of the DNA, and isolating the
IAP polypeptide. In preferred embodiments, the IAP polypeptide is
expressed by DNA that is under the control of a constitutive or
inducible promotor. As described herein, the promotor may be a
heterologous promotor.
[0018] In another aspect, the invention features substantially pure
mammalian IAP polypeptide. Preferably, the polypeptide includes an
amino acid sequence that is substantially identical to all, or to a
fragment of, the amino acid sequence shown in any one of FIGS. 1-4.
Most preferably, the polypeptide is the XIAP, HIAP-1, HIAP-2,
M-XIAP, M-HIAP-1, or M-HIAP-2 polypeptide. Fragments including one
or more BIR domains (to the exclusion of the RZF), the RZF domain
(to the exclusion of the BIR domains), and a RZF domain with at
least one BIR domain, as provided herein, are also a part of the
invention.
[0019] In another aspect, the invention features a recombinant
mammalian polypeptide that is capable of modulating apoptosis. The
polypeptide may include at least a RZF domain and a BIR domain as
defined herein. In preferred embodiments, the invention features
(a) a substantially pure polypeptide, and (b) an oligonucleotide
encoding the polypeptide. In instances were the polypeptide
includes a RZF domain, the RZF domain will have a sequence
conforming to:
Glu-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa2-Xaa1-Xaa1-Xaa1-Cys-Lys-Xaa3-Cys-Me-
t-Xaa
1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa3-Xaa1-Phe-Xaa1-Pro-Cys-Gly-His-Xaa1-Xaa1-X-
aa1-Cys-Xaa1-Xaa1-Cys-Ala-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Cys-Pro-Xaa1-Cys,
where Xaa1 is any amino acid, Xaa2 is Glu or Asp, Xaa3 is Val or
Ile (SEQ ID NO: 1); and where the polypeptide includes at least one
BIR domain, the BIR domain will have a sequence conforming to:
Xaa1-Xaa1-Xaa1-Arg-Leu-Xaa1-Thr-Phe-Xaa
1-Xaa1-Trp-Pro-Xaa2-Xaa1-Xaa1-Xaa2-Xaa2-Xaa1-Xaa1-Xaa1-Xaa1-Leu-Ala-Xaa
1-Ala-Gly-Phe-Tyr-Tyr-Xaa1-Gly-Xaa1-Xaa1-Asp-Xaa1-Val-Xaa1-Cys-Phe-Xaa
1-Cys-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Trp-Xaa1-Xaa1-Xaa1-Asp-Xaa1-Xaa1-Xaa1-
-Xaa1-Xaa1-His-Xaa1-Xaa1-Xaa1-Xaa1-Pro-Xaa1-Cys-Xaa1-Phe-Val, where
Xaa1 may be any amino acid and Xaa2 may be any amino acid or may be
absent (SEQ ID NO: 2).
[0020] In various preferred embodiments the polypeptide has at
least two or, more preferably at least three BIR domains, the RZF
domain has one of the IAP sequences shown in FIG. 6, and the BIR
domains are comprised of BIR domains shown in FIG. 5. In other
preferred embodiments the BIR domains are at the amino terminal end
of the protein relative to the RZF domain, which is at or near the
carboxyl terminus of the polypeptide.
[0021] In another aspect, the invention features an IAP gene
isolated according to the method involving: (a) providing a sample
of DNA; (b) providing a pair of oligonucleotides having sequence
homology to a conserved region of an IAP disease-resistance gene;
(c) combining the pair of oligonucleotides with the cell DNA sample
under conditions suitable for polymerase chain reaction-mediated
DNA amplification; and (d) isolating the amplified IAP gene or
fragment thereof.
[0022] In preferred embodiments, the amplification is carried out
using a reverse-transcription polymerase chain reaction, for
example, the RACE method. In another aspect, the invention features
an IAP gene isolated according to the method involving: (a)
providing a preparation of DNA; (b) providing a detectably labelled
DNA sequence having homology to a conserved region of an IAP gene;
(c) contacting the preparation of DNA with the detectably-labelled
DNA sequence under hybridization conditions providing detection of
genes having 50% or greater nucleotide sequence identity; and (d)
identifying an IAP gene by its association with the detectable
label.
[0023] In another aspect, the invention features an IAP gene
isolated according to the method involving: (a) providing a cell
sample; (b) introducing by transformation into the cell sample a
candidate IAP gene; (c) expressing the candidate IAP gene within
the cell sample; and (d) determining whether the cell sample
exhibits an altered apoptotic response, whereby a response
identifies an IAP gene.
[0024] In another aspect, the invention features a method of
identifying an IAP gene in a cell, involving: (a) providing a
preparation of cellular DNA (for example, from the human genome or
a cDNA library (such as a cDNA library isolated from a cell type
which undergoes apoptosis); (b) providing a detectably-labelled DNA
sequence (for example, prepared by the methods of the invention)
having homology to a conserved region of an IAP gene; (c)
contacting the preparation of cellular DNA with the
detectably-labelled DNA sequence under hybridization conditions
providing detection of genes having 50% nucleotide or greater
sequence identity; and (d) identifying an IAP gene by its
association with the detectable label.
[0025] In another aspect, the invention features a method of
isolating an LAP gene from a recombinant library, involving: (a)
providing a recombinant library; (b) contacting the library with a
detectably-labelled gene fragment produced according to the PCR
method of the invention under hybridization conditions providing
detection of genes having 50% or greater nucleotide sequence
identity; and (c) isolating an IAP gene by its association with the
detectable label. In another aspect, the invention features a
method of identifying an IAP gene involving: (a) providing a cell
tissue sample; (b) introducing by transformation into the cell
sample a candidate IAP gene; (c) expressing the candidate IAP gene
within the cell sample; and (d) determining whether the cell sample
exhibits inhibition of apoptosis, whereby a change in (i.e.
modulation of) apoptosis identifies an IAP gene. Preferably, the
cell sample is a cell type that may be assayed for apoptosis (e.g.,
T cells, B cells, neuronal cells, baculovirus-infected insect
cells, glial cells, embryonic stem cells, and fibroblasts). The
candidate LAP gene is obtained, for example, from a cDNA expression
library, and the response assayed is the inhibition of
apoptosis.
[0026] In another aspect, the invention features a method of
inhibiting apoptosis in a mammal wherein the method includes: (a)
providing DNA encoding at least one IAP polypeptide to a cell that
is susceptible to apoptosis; wherein the DNA is integrated into the
genome of the cell and is positioned for expression in the cell;
and the IAP gene is under the control of regulatory sequences
suitable for controlled expression of the gene(s); wherein the IAP
transgene is expressed at a level sufficient to inhibit apoptosis
relative to a cell lacking the IAP transgene. The DNA integrated
into the genome may encode all or part of an IAP polypeptide. It
may, for example, encode a ring zinc finger and one or more BIR
domains. In contrast, it may encode either the ring zinc finger
alone, or one or more BIR domains alone. Skilled artisans will
appreciate that IAP polypeptides may also be administered directly
to inhibit undesirable apoptosis.
[0027] In a related aspect, the invention features a method of
inhibiting apoptosis by producing a cell that has integrated, into
its genome, a transgene that includes the IAP gene, or a fragment
thereof. The IAP gene may be placed under the control of a promoter
providing constitutive expression of the IAP gene. Alternatively,
the IAP transgene may be placed under the control of a promoter
that allows expression of the gene to be regulated by environmental
stimuli. For example, the IAP gene may be expressed using a
tissue-specific or cell type-specific promoter, or by a promoter
that is activated by the introduction of an external signal or
agent, such as a chemical signal or agent. In preferred is
embodiments the cell is a lymphocyte, a neuronal cell, a glial
cell, or a fibroblast. In other embodiments, the cell in an
HIV-infected human, or in a mammal suffering from a
neurodegenerative disease, an ischemic injury, a toxin-induced
liver disease, or a myelodysplastic syndrome.
[0028] In a related aspect, the invention provides a method of
inhibiting apoptosis in a mammal by providing an
apoptosis-inhibiting amount of IAP polypeptide. The IAP polypeptide
may be a full-length polypeptide, or it may be one of the fragments
described herein.
[0029] In another aspect, the invention features a purified
antibody that binds specifically to an IAP family protein. Such an
antibody may be used in any standard immunodetection method for the
identification of an IAP polypeptide. Preferably, the antibody
binds specifically to XIAP, HIAP-1, or HIAP-2. In various
embodiments, the antibody may react with other IAP polypeptides or
may be specific for one or a few LAP polypeptides. The antibody may
be a monoclonal or a polyclonal antibody. Preferably, the antibody
reacts specifically with only one of the IAP polypeptides, for
example, reacts with murine and human XIAP, but not with HIAP-1 or
HIAP-2 from other mammalian species.
[0030] The antibodies of the invention may be prepared by a variety
of methods. For example, the IAP polypeptide, or antigenic
fragments thereof, can be administered to an animal in order to
induce the production of polyclonal antibodies. Alternatively,
antibodies used as described herein may be monoclonal antibodies,
which are prepared using hybridoma technology (see, e.g., Kohler et
al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6:511,
1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et
al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier,
N.Y., 1981). The invention features antibodies that specifically
bind human or murine IAP polypeptides, or fragments thereof. In
particular the invention features "neutralizing" antibodies. By
"neutralizing" antibodies is meant antibodies that interfere with
any of the biological activities of IAP polypeptides, particularly
the ability of IAPs to inhibit apoptosis. The neutralizing antibody
may reduce the ability of IAP polypeptides to inhibit polypeptides
by, preferably 50%, more preferably by 70, and most preferably by
90% or more. Any standard assay of apoptosis, including those
described herein, may be used to assess neutralizing
antibodies.
[0031] In addition to intact monoclonal and polyclonal anti-LAP
antibodies, the invention features various genetically engineered
antibodies, humanized antibodies, and antibody fragments, including
F(ab')2, Fab', Fab, Fv and sFv fragments. Antibodies can be
humanized by methods known in the art, e.g., monoclonal antibodies
with a desired binding specificity can be commercially humanized
(Scotgene, Scotland; Oxford Molecular, Palo Alto, Calif.). Fully
human antibodies, such as those expressed in transgenic animals,
are also features of the invention (Green et al., Nature Genetics
7:13, 1994).
[0032] Ladner (U.S. Pat. Nos. 4,946,778 and 4,704,692) describes
methods for preparing single polypeptide chain antibodies. Ward et
al. (Nature 341:544, 1989) describe the preparation of heavy chain
variable domains, which they term "single domain antibodies," which
have high antigen-binding affinities. McCafferty et al. (Nature
348:552, 1990) show that complete antibody V domains can be
displayed on the surface of fd bacteriophage, that the phage bind
specifically to antigen, and that rare phage (one in a million) can
be isolated after affinity chromatography. Boss et al. (U.S. Pat.
No. 4,816,397) describe various methods for producing
immunoglobulins, and immunologically functional fragments thereof,
which include at least the variable domains of the heavy and light
chain in a single host cell. Cabilly et al. (U.S. Pat. No.
4,816,567) describe methods for preparing chimeric antibodies.
[0033] In another aspect, the invention features a method of
identifying a compound that modulates apoptosis. The method
includes providing a cell expressing an IAP polypeptide, contacting
the cell with a candidate compound, and monitoring the expression
of an IAP gene. An alteration in the level of expression of the IAP
gene indicates the presence of a compound which modulates
apoptosis. The compound may be an inhibitor or an enhancer of
apoptosis. In various preferred embodiments, the cell is a
fibroblast, a neuronal cell, a glial cell, a lymphocyte (T cell or
B cell), or an insect cell; the polypeptide expression being
monitored is XIAP, HIAP-1, HIAP-2, M-XIAP, M-HIAP-1, or M-HIAP-2
(i.e., human or murine).
[0034] In a related aspect, the invention features methods of
detecting compounds that modulate apoptosis using the interaction
trap technology and LAP polypeptides, or fragments thereof, as a
component of the bait. In preferred embodiments, the compound being
tested as a modulator of apoptosis is also a polypeptide.
[0035] In another aspect, the invention features a method for
diagnosing a cell proliferation disease, or an increased likelihood
of such a disease, using an IAP nucleic acid probe or antibody.
Preferably, the disease is a cancer. Most preferably, the disease
is selected from the group consisting of promyelocytic leukemia, a
HeLa-type carcinoma, chronic myelogenous leukemia (preferably using
xiap or hiap-2 related probes), lymphoblastic leukemia (preferably
using a xiap related probe), Burkitt's lymphoma (preferably using
an hiap-1 related probe), colorectal adenocarcinoma, lung
carcinoma, and melanoma (preferably using a xiap probe).
Preferably, a diagnosis is indicated by a 2-fold increase in
expression or activity, more preferably, at least a 10-fold
increase in expression or activity.
[0036] Skilled artisans will recognize that a mammalian IAP, or a
fragment thereof (as described herein), may serve as an active
ingredient in a therapeutic composition. This composition,
depending on the IAP or fragment included, may be used to modulate
apoptosis and thereby treat any condition that is caused by a
disturbance in apoptosis.
[0037] In addition, apoptosis may be induced in a cell by
administering to the cell a negative regulator of the IAP-dependent
anti-apoptotic pathway. The negative regulator may be, but is not
limited to, an IAP polypeptide that includes a ring zinc finger,
and an IAP polypeptide that includes a ring zinc finger and lacks
at least one BIR domain. Alternatively, apoptosis may be induced in
the cell by administering a gene encoding an IAP polypeptide, such
as these two polypeptides. In yet another method, the negative
regulator may be a purified antibody, or a fragment thereof, that
binds specifically to an IAP polypeptide. For example, the antibody
may bind to an approximately 26 kDa cleavage product of an IAP
polypeptide that includes at least one BIR domain but lacks a ring
zinc finger domain. The negative regulator may also be an IAP
antisense mRNA molecule.
[0038] As summarized above, an IAP nucleic acid, or an IAP
polypeptide may be used to modulate apoptosis. Furthermore, an IAP
nucleic acid, or an IAP polypeptide, may be used in the manufacture
of a medicament for the modulation of apoptosis.
[0039] By "IAP gene" is meant a gene encoding a polypeptide having
at least one BIR domain and a ring zinc finger domain which is
capable of modulating (inhibiting or enhancing) apoptosis in a cell
or tissue when provided by other intracellular or extracellular
delivery methods. In preferred embodiments the IAP gene is a gene
having about 50% or greater nucleotide sequence identity to at
least one of the IAP amino acid encoding sequences of FIGS. 1-4 or
portions thereof. Preferably, the region of sequence over which
identity is measured is a region encoding at least one BIR domain
and a ring zinc finger domain. Mammalian IAP genes include
nucleotide sequences isolated from any mammalian source.
Preferably, the mammal is a human.
[0040] The term "IAP gene" is meant to encompass any member of the
family of apoptosis inhibitory genes, which are characterized by
their ability to modulate apoptosis. An IAP gene may encode a
polypeptide that has at least 20%, preferably at least 30%, and
most preferably at least 50% amino acid sequence identity with at
least one of the conserved regions of one of the IAP members
described herein (i.e., either the BIR or ring zinc finger domains
from the human or murine xiap, hiap-1 and hiap-2). Representative
members of the IAP gene family include, without limitation, the
human and murine xiap, hiap-1, and hiap-2 genes.
[0041] By "IAP protein" or "IAP polypeptide" is meant a
polypeptide, or fragment thereof, encoded by an IAP gene.
[0042] By "BIR domain" is meant a domain having the amino acid
sequence of the consensus sequence:
Xaa1-Xaa1-Xaa1-Arg-Leu-Xaa1-Thr-Phe-Xaa1-Xaa1-Trp-Pro-Xaa2-Xaa1-Xaa1-Xaa2-
-Xaa2-Xaa1-Xaa1-Xaa1-Xaa1-Leu-Ala-Xaa1-Ala-Gly-Phe-Tyr-Tyr-Xaa1-Gly-Xaa1-X-
aa1-Asp-Xaa1-Val-Xaa1-Cys-Phe-Xaa1-Cys-Xaa
1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Trp-Xaa1-Xaa1-Xaa1-Asp-Xaa1-Xaa1-Xaa1-Xaa1-Xaa-
1-His-Xaa1-Xaa1-Xaa1-Xaa1-Pro-Xaa1-Cys-Xaa1-Phe-Val, wherein Xaa1
is any amino acid and Xaa2 is any amino acid or is absent (SEQ ID
NO: 2). Preferably, the sequence is substantially identical to one
of the BIR domain sequences provided herein for XIAP, HIAP-1, or
HIAP-2.
[0043] By "ring zinc finger" or "RZF" is meant a domain having the
amino acid sequence of the consensus sequence:
Glu-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa2-Xaa1-Xaa1-Xaa1-Cys-Lys-Xaa3-Cys-Me-
t-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa3-Xaa1-Phe-Xaa1-Pro-Cys-Gly-His-Xaa1-Xaa1-Xa-
a1-Cys-Xaa1-Xaa1-Cys-Ala-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Cys-Pro-Xaa1-Cys,
wherein Xaa1 is any amino acid, Xaa2 is Glu or Asp, and Xaa3 is Val
or Ile (SEQ ID NO: 1).
[0044] Preferably, the sequence is substantially identical to the
RZF domains provided herein for the human or murine XIAP, HIAP-1,
or HIAP-2.
[0045] By "modulating apoptosis" or "altering apoptosis" is meant
increasing or decreasing the number of cells that would otherwise
undergo apoptosis in a given cell population. Preferably, the cell
population is selected from a group including T cells, neuronal
cells, fibroblasts, or any other cell line known to undergo
apoptosis in a laboratory setting (e.g., the baculovirus infected
insect cells). It will be appreciated that the degree of modulation
provided by an IAP or modulating compound in a given assay will
vary, but that one skilled in the art can determine the
statistically significant change in the level of apoptosis which
identifies an LAP or a compound which modulates an IAP.
[0046] By "inhibiting apoptosis" is meant any decrease in the
number of cells which undergo apoptosis relative to an untreated
control. Preferably, the decrease is at least 25%, more preferably
the decrease is 50%, and most preferably the decrease is at least
one-fold.
[0047] By "polypeptide" is meant any chain of more than two amino
acids, regardless of post-translational modification such as
glycosylation or phosphorylation.
[0048] By "substantially identical" is meant a polypeptide or
nucleic acid is exhibiting at least 50%, preferably 85%, more
preferably 90%, and most preferably 95% homology to a reference
amino acid or nucleic acid sequence. For polypeptides, the length
of comparison sequences will generally be at least 16 amino acids,
preferably at least 20 amino acids, more preferably at least 25
amino acids, and most preferably 35 amino acids. For nucleic acids,
the length of comparison sequences will generally be at least 50
nucleotides, preferably at least 60 nucleotides, more preferably at
least 75 nucleotides, and most preferably 110 nucleotides.
[0049] Sequence identity is typically measured using sequence
analysis software with the default parameters specified therein
(e.g., Sequence Analysis Software Package of the Genetics Computer
Group, University of Wisconsin Biotechnology Center, 1710
University Avenue, Madison, Wis. 53705). This software program
matches similar sequences by assigning degrees of homology to
various substitutions, deletions, and other modifications.
Conservative substitutions typically include substitutions within
the following groups: glycine, alanine, valine, isoleucine,
leucine; aspartic acid, glutamic acid, asparagine, glutamine;
serine, threonine; lysine, arginine; and phenylalanine,
tyrosine.
[0050] By "substantially pure polypeptide" is meant a polypeptide
that has been separated from the components that naturally
accompany it. Typically, the polypeptide is substantially pure when
it is at least 60%, by weight, free from the proteins and
naturally-occurring organic molecules with which it is naturally
associated. Preferably, the polypeptide is an IAP polypeptide that
is at least 75%, more preferably at least 90%, and most preferably
at least 99%, by weight, pure. A substantially pure IAP polypeptide
may be obtained, for example, by extraction from a natural source
(e.g. a fibroblast, neuronal cell, or lymphocyte) by expression of
a recombinant nucleic acid encoding an IAP polypeptide, or by
chemically synthesizing the protein. Purity can be measured by any
appropriate method, e.g., by column chromatography, polyacrylamide
gel electrophoresis, or HPLC analysis.
[0051] A protein is substantially free of naturally associated
components when it is separated from those contaminants which
accompany it in its natural state. Thus, a protein which is
chemically synthesized or produced in a cellular system different
from the cell from which it naturally originates will be
substantially free from its naturally associated components.
Accordingly, substantially pure polypeptides include those derived
from eukaryotic organisms but synthesized in E. coli or other
prokaryotes. By "substantially pure DNA" is meant DNA that is free
of the genes which, in the naturally-occurring genome of the
organism from which the DNA of the invention is derived, flank the
gene. The term therefore includes, for example, a recombinant DNA
which is incorporated into a vector; into an autonomously
replicating plasmid or virus; or into the genomic DNA of a
prokaryote or eukaryote; or which exists as a separate molecule
(e.g., a cDNA or a genomic or cDNA fragment produced by PCR or
restriction endonuclease digestion) independent of other sequences.
It also includes a recombinant DNA which is part of a hybrid gene
encoding additional polypeptide sequence.
[0052] By "transformed cell" is meant a cell into which (or into an
ancestor of which) has been introduced, by means of recombinant DNA
techniques, a DNA molecule encoding (as used herein) an IAP
polypeptide.
[0053] By "transgene" is meant any piece of DNA which is inserted
by artifice into a cell, and becomes part of the genome of the
organism which develops from that cell. Such a transgene may
include a gene which is partly or entirely heterologous (i.e.,
foreign) to the transgenic organism, or may represent a gene
homologous to an endogenous gene of the organism.
[0054] By "transgenic" is meant any cell which includes a DNA
sequence which is inserted by artifice into a cell and becomes part
of the genome of the organism which develops from that cell. As
used herein, the transgenic organisms are generally transgenic
mammalian (e.g., rodents such as rats or mice) and the DNA
(transgene) is inserted by artifice into the nuclear genome.
[0055] By "transformation" is meant any method for introducing
foreign molecules into a cell. Lipofection, calcium phosphate
precipitation, retroviral delivery, electroporation, and biolistic
transformation are just a few of the teachings which may be used.
For example, biolistic transformation is a method for introducing
foreign molecules into a cell using velocity driven
microprojectiles such as tungsten or gold particles. Such
velocity-driven methods originate from pressure bursts which
include, but are not limited to, helium-driven, air-driven, and
gunpowder-driven techniques. Biolistic transformation may be
applied to the transformation or transfection of a wide variety of
cell types and intact tissues including, without limitation,
intracellular organelles (e.g., and mitochondria and chloroplasts),
bacteria, yeast, fungi, algae, animal tissue, and cultured
cells.
[0056] By "positioned for expression" is meant that the DNA
molecule is positioned adjacent to a DNA sequence which directs
transcription and translation of the sequence (i.e., facilitates
the production of, e.g., an IAP polypeptide, a recombinant protein
or a RNA molecule).
[0057] By "reporter gene" is meant a gene whose expression may be
assayed; such genes include, without limitation, glucuronidase
(GUS), luciferase, chloramphenicol transacetylase (CAT), and
lacZ.
[0058] By "promoter" is meant minimal sequence sufficient to direct
transcription. Also included in the invention are those promoter
elements which are sufficient to render promoter-dependent gene
expression controllable for cell type-specific, tissue-specific or
inducible by external signals or agents; such elements may be
located in the 5' or 3' regions of the native gene.
[0059] By "operably linked" is meant that a gene and one or more
regulatory sequences are connected in such a way as to permit gene
expression when the appropriate molecules (e.g., transcriptional
activator proteins are bound to the regulatory sequences).
[0060] By "conserved region" is meant any stretch of six or more
contiguous amino acids exhibiting at least 30%, preferably 50%, and
most preferably 70% amino acid sequence identity between two or
more of the IAP family members, (e.g., between human HIAP-1,
HIAP-2, and XIAP). Examples of preferred conserved regions are
shown (as boxed or designated sequences) in FIGS. 5-7 and Tables 1
and 2, and include, without limitation, BIR domains and ring zinc
finger domains.
[0061] By "detectably-labelled" is meant any means for marking and
identifying the presence of a molecule, e.g., an oligonucleotide
probe or primer, a gene or fragment thereof, or a cDNA molecule.
Methods for detectably-labelling a molecule are well known in the
art and include, without limitation, radioactive labelling (e.g.,
with an isotope such as .sup.32P or .sup.35S) and nonradioactive
labelling (e.g., chemiluminescent labelling, e.g., fluorescein
labelling).
[0062] By "antisense," as used herein in reference to nucleic
acids, is meant a nucleic acid sequence, regardless of length, that
is complementary to the coding strand of a gene.
[0063] By "purified antibody" is meant antibody which is at least
60%, by weight, free from proteins and naturally occurring organic
molecules with which it is naturally associated. Preferably, the
preparation is at least 75%, more is preferably 90%, and most
preferably at least 99%, by weight, antibody, e.g., an IAP specific
antibody. A purified antibody may be obtained, for example, by
affinity chromatography using recombinantly-produced protein or
conserved motif peptides and standard techniques.
[0064] By "specifically binds" is meant an antibody that recognizes
and binds a protein but that does not substantially recognize and
bind other molecules in a sample, e.g., a biological sample, that
naturally includes protein.
[0065] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0066] FIG. 1 is the human xiap cDNA sequence (SEQ ID NO: 3) and
the XIAP polypeptide sequence (SEQ ID NO: 4).
[0067] FIG. 2 is the human hiap-1 cDNA sequence (SEQ ID NO: 5) and
the HIAP-1 polypeptide sequence (SEQ ID NO: 6).
[0068] FIG. 3 is the human hiap-2 cDNA sequence (SEQ ID NO: 7) and
the HIAP-2 polypeptide sequence (SEQ ID NO: 8). The sequence absent
in the hiap-2-.DELTA. variant is boxed.
[0069] FIG. 4 is the murine xiap cDNA sequence (SEQ ID NO: 9) and
encoded murine XIAP polypeptide sequence (SEQ ID NO: 10).
[0070] FIG. 5 is the murine hiap-1 cDNA sequence (SEQ ID NO: 39)
and the encoded murine HIAP-1 polypeptide sequence (SEQ ID NO:
40).
[0071] FIG. 6 is the murine hiap-2 cDNA sequence (SEQ ID NO: 41)
and the encoded murine HLAP-2 polypeptide (SEQ ID NO: 42).
[0072] FIG. 7 is a representation of the alignment of the BIR
domains of LAP proteins (SEQ ID NOs: 11 and 14-31).
[0073] FIG. 8 is a representation of the alignment of human IAP
polypeptides with diap, cp-iap, and the LAP consensus sequence (SEQ
ID NOs: 4, 6, 8, 10, 12, and 13).
[0074] FIG. 9 is a representation of the alignment of the ring zinc
finger domains of IAP proteins (SEQ ID NOs: 32-38).
[0075] FIG. 10 is a photograph of a northern blot illustrating
human hiap-1 and hiap-2 mRNA expression in human tissues.
[0076] FIG. 11 is a photograph of a northern blot illustrating
human hiap-2 mRNA expression in human tissues.
[0077] FIG. 12 is a photograph of a northern blot illustrating
human xiap mRNA expression in human tissues.
[0078] FIGS. 13A and 13B are photographs of agarose gels
illustrating apoptotic DNA ladders and RT-PCR products using hiap-1
and hiap-2 specific probes in HIV-infected T cells.
[0079] FIG. 14A-14D are graphs depicting suppression of apoptosis
by XIAP, HIAP-1, HIAP-2, Bcl-2, smn, and 6-myc.
[0080] FIGS. 15A and 15B are bar graphs depicting the percentage of
viable CHO cells following transient transfection with the cDNA
constructs shown and subsequent serum withdrawal.
[0081] FIGS. 16A and 16B are bar graphs depicting the percentage of
viable CHO cells following transient transfection with the cDNA
constructs shown and subsequent exposure to menadione (FIG. 16A=10
.mu.M menadione; FIG. 16B=20 .mu.M menadione).
[0082] FIG. 17 is a photograph of an agarose gel containing cDNA
fragments that were amplified, with hiap-1-specific primers, from
RNA obtained from Raji, Ramos, EB-3, and Jiyoye cells, and from
normal placenta.
[0083] FIG. 18 is a photograph of a western blot containing protein
extracted from Jurkat and astrocytoma cells stained with an
anti-XIAP antibody. The position and size of a series of marker
proteins is indicated.
[0084] FIG. 19 is a photograph of a western blot containing protein
extracted from Jurkat cells following treatment as described in
Example XII. The blot was stained with a rabbit polyclonal
anti-XIAP antibody. Lane 1, negative control; lane 2, anti-Fas
antibody; lane 3, anti-Fas antibody and cycloheximide; lane 4,
TNF-.alpha.; lane 5, TNF-.alpha. and cycloheximide.
[0085] FIG. 20 is a photograph of a western blot containing protein
extracted from HeLa cells following exposure to anti-Fas
antibodies. The blot was stained with a rabbit polyclonal anti-XIAP
antibody. Lane 1, negative control; lane 2, cycloheximide; lane 3,
anti-Fas antibody; lane 4, anti-Fas antibody and cycloheximide;
lane 5, TNF-.alpha.; lane 6, TNF-.alpha. and cycloheximide.
[0086] FIGS. 21A and 21B are photographs of western blots stained
with rabbit polyclonal anti-XIAP antibody. Protein was extracted
from HeLa cells (FIG. 21A) and Jurkat cells (FIG. 21B) immediately,
1, 2, 3, 5, 10, and 22 hours after exposure to anti-Fas
antibody.
[0087] FIGS. 22A and 22B are photographs of western blots stained
with an anti-CPP32 antibody (FIG. 22A) or a rabbit polyclonal
anti-XIAP antibody (FIG. 22B). Protein was extracted from Jurkat
cells immediately, 3 hours, or 7 hours after exposure to an
anti-Fas antibody. In addition to total protein, cytoplasmic and
nuclear extracts are shown.
[0088] FIG. 23 is a photograph of a polyacrylamide gel following
electrophoresis of the products of an in vitro XMAP cleavage
assay.
DETAILED DESCRIPTION
I. IAP Genes and Polypeptides
[0089] A new class of mammalian proteins that modulate apoptosis
(IAPs) and the genes that encode these proteins have been
discovered. The IAP proteins are characterized by the presence of a
ring zinc finger domain (RZF; FIG. 9) and at least one BIR domain,
as defined by the boxed consensus sequences shown in FIGS. 7 and 8,
and by the sequence domains listed in Tables 1 and 2. As examples
of novel IAP genes and proteins, the cDNA sequences and amino acid
sequences for human IAPs (HIAP-1, HIAP-2, and XIAP) and a new
murine inhibitor of apoptosis, XIAP, are provided. Additional
members of the mammalian IAP family (including homologs from other
species and mutant sequences) may be isolated using standard
cloning techniques and the conserved amino acid sequences, primers,
and probes provided herein and known in the art. Furthermore, IAPs
include those proteins lacking the ring zinc finger, as further
described below. TABLE-US-00001 TABLE 1 NUCLEOTIDE POSITION OF
CONSERVED DOMAINS* Ring Zinc BIR-1 BIR-2 BIR-3 Finger h-xiap
109-312 520-723 826-1023 1348-1485 m-xiap 202-405 613-816 916-1113
1438-1575 h-hiap-1 273-476 693-893 951-1154 1824-1961 m-hiap-1
251-453 670-870 928-1131 1795-1932 h-hiap-2 373-576 787-987
1042-1245 1915-2052 m-hiap-2 215-418 608-808 863-1066 1763-1876
*Positions indicated correspond to those shown in FIGS. 1-4.
[0090] TABLE-US-00002 TABLE 2 AMINO ACID POSITION OF CONSERVED
DOMAINS* Ring Zinc BIR-1 BIR-2 BIR-3 Finger h-XAIP 26-93 163-230
265-330 439-484 m-XIAP 26-93 163-230 264-329 438-483 h-HIAP1 29-96
169-235 255-322 546-591 m-HIAP1 29-96 169-235 255-322 544-589
h-HIAP2 46-113 184-250 269-336 560-605 m-HIAP2 25-92 156-222
241-308 541-578 *Positions indicated correspond to those shown in
FIGS. 1-4.
[0091] Recognition of the mammalian IAP family has provided an
emergent pattern of protein structure. Recognition of this pattern
allows proteins having a known, homologous sequence but unknown
function to be classified as putative inhibitors of apoptosis. A
Drosophila gene, now termed diap, was classified in this way (for
sequence information see Genbank Accession Number M96581 and FIG.
6). The conservation of these proteins across species indicates
that the apoptosis signalling pathway has been conserved throughout
evolution.
[0092] The IAP proteins may be used to inhibit the apoptosis that
occurs as part of numerous disease processes or disorders. For
example, IAP polypeptides or nucleic acid encoding IAP polypeptides
may be administered for the treatment or prevention of apoptosis
that occurs as a part of AIDS, neurodegenerative diseases, ischemic
injury, toxin-induced liver disease and myelodysplastic syndromes.
Nucleic acid encoding the LAP polypeptide may also be provided to
inhibit apoptosis.
II. Cloning of IAP Genes
A. Human Xiap
[0093] The search for human genes involved in apoptosis resulted in
the identification of an X-linked sequence tag site (STS) in the
GenBank database, which demonstrated strong homology with the
conserved RZF domain of CpIAP and OpIAP, the two baculovirus genes
known to inhibit apoptosis (Clem et al., Mol. Cell Biol. 14:5212,
1994; Birnbaum et al., J. Virol. 68:2521, 1994). Screening a human
fetal brain ZapII cDNA library (Stratagene, La Jolla, Calif.) with
this STS resulted in the identification and cloning of xiap (for
X-linked Inhibitor of Apoptosis Protein gene). The human gene has a
1.5 kb coding sequence that includes three BIR domains (Crook et
al., J. Virol. 67:2168, 1993; Clem et al., Science 254:1388, 1991;
Birnbaum et al., J. Virol. 68:2521, 1994) and a zinc finger.
Northern blot analysis with xiap revealed message greater than 7
kb, which is expressed in various tissues, particularly liver and
kidney (FIG. 12). The large size of the transcript reflects large
5' and 3' untranslated regions.
B. Human Hiap-1 and Hiap-2
[0094] The hiap-1 and hiap-2 genes were cloned by screening a human
liver library (Stratagene Inc., LaJolla, Calif.) with a probe
including the entire xiap coding region at low stringency (the
final wash was performed at 40.degree. C. with 2.times.SSC, 10%
SDS; FIGS. 2 and 3). The hiap-1 and hiap-2 genes were also detected
independently using a probe derived from an expressed sequence tag
(EST; GenBank Accession No. T96284), which includes a portion of a
BIR domain. The EST sequence was originally isolated by the
polymerase chain reaction; a cDNA library was used as a template
and amplified with EST-specific primers. The is DNA amplified probe
was then used to screen the human liver cDNA library for
full-length hiap coding sequences. A third DNA was subsequently
detected that includes the hiap-2 sequence but that appears to lack
one exon, presumably due to alternative mRNA splicing (see boxed
region in FIG. 3). The expression of hiap-1 and hiap-2 in human
tissues as assayed by northern blot analysis is shown in FIGS. 8
and 9.
C. M-Xiap
[0095] Fourteen cDNA and two genomic clones were identified by
screening a mouse embryo .lamda.gt11 cDNA library (Clontech, Palo
Alto, Calif.) and a mouse FIX II genomic library with a xiap cDNA
probe, respectively. A cDNA contig spanning 8.0 kb was constructed
using 12 overlapping mouse clones. Sequence analysis revealed a
coding sequence of approximately 1.5 kb. The mouse gene, m-xiap,
encodes a polypeptide with striking homology to human XIAP at and
around the initiation methionine, the stop codon, the three BIR
domains, and the RZF domain. As with the human gene, the mouse
homologue contains large 5' and 3' UTRs, which could produce a
transcript as large as 7-8 kb.
[0096] Analysis of the sequence and restriction map of m-xiap
further delineate the structure and genomic organization of m-xiap.
Southern blot analysis and inverse PCR techniques (Groden et al.,
Cell 66:589, 1991) can be employed to map exons and define
exon-intron boundaries.
[0097] Antisera can be raised against a M-XLAP fusion protein that
was obtained from, for example, E. coli using a bacterial
expression system. The resulting antisera can be used along with
northern blot analysis to analyze the spatial and temporal
expression of m-xiap in the mouse.
D. M-Hiap-4 and M-Hiap-2
[0098] The murine homologs of hiap-1 and hiap-2 were cloned and
sequenced in the same general manner as m-xiap using the human
hiap-1 and hiap-2 sequences as probes. Cloning of m-hiap-1 and
m-hiap-2 further demonstrate that homologs from different species
may be isolated using the techniques provided herein and those
generally known to artisans skilled in molecular biology.
III. Identification of Additional IAP Genes
[0099] Standard techniques, such as the polymerase chain reaction
(PCR) and DNA hybridization, may be used to clone additional human
IAP genes and their homologues in other species. Southern blots of
human genomic DNA hybridized at low stringency with probes specific
for xiap, hiap-1 and hiap-2 reveal bands that correspond to other
known human LAP sequences as well as additional bands that do not
correspond to known IAP sequences. Thus, additional IAP sequences
may be readily identified using low stringency hybridization.
Examples of murine and human xiap, hiap-1, and hiap-2 specific
primers, which may be used to clone additional genes by RT-PCR, are
shown in Table 5.
IV. Characterization of IAP Activity and Intracellular Localization
Studies
[0100] The ability of putative IAPs to modulate apoptosis can be
defined in in vitro systems in which alterations of apoptosis can
be detected. Mammalian expression constructs carrying IAP cDNAs,
which are either full-length or truncated, can be introduced into
cell lines such as CHO, NIH 3T3, HL60, Rat-1, or Jurkat cells. In
addition, Sf21 insect cells may be used, in which case the IAP gene
is preferentially expressed using an insect heat shock promotor.
Following transfection, apoptosis can be induced by standard
methods, which include serum withdrawal, or application of
staurosporine, menadione (which induces apoptosis via free radial
formation), or anti-Fas antibodies. As a control, cells are
cultured under the same conditions as those induced to undergo
apoptosis, but either not transfected, or transfected with a vector
that lacks an LAP insert. The ability of each IAP construct to
inhibit apoptosis upon expression can be quantified by calculating
the survival index of the cells, i.e., the ratio of surviving
transfected cells to surviving control cells. These experiments can
confirm the presence of apoptosis inhibiting activity and, as
discussed below, can also be used to determine the functional
region(s) of an IAP. These assays may also be performed in
combination with the application of additional compounds in order
to identify compounds that modulate apoptosis via IAP
expression.
A. Cell Survival Following Transfection with Full-Length IAP
Constructs and Induction of Apoptosis
[0101] Specific examples of the results obtained by performing
various apoptosis suppression assays are shown in FIGS. 14A to 14D.
For example, CHO cell survival following transfection with one of
six constructs and subsequent serum withdrawal is shown in FIG.
14A. The cells were transfected using Lipofectace.TM. with 2 .mu.g
of one of the following recombinant plasmids: pCDNA3-6myc-xiap
(xiap), pCDNA3-6myc-hiap-1 (hiap-1), pCDNA3-6myc-hiap-2 (hiap-2),
pCDNA3-bcl-2 (bcl-2), pCDNA3-HA-smn (smn), and pCDNA3-6myc (6-myc).
Oligonucleotide primers were synthesized to allow PCR amplification
and cloning of the xiap, hiap-1, and hiap-2 ORFs in pCDNA3
(Invitrogen). Each construct was modified to incorporate a
synthetic myc tag encoding six repeats of the peptide sequence
MEQKLISEEDL (SEQ ID NO: 43), thus allowing detection of myc-IAP
fusion proteins via monoclonal anti-myc antiserum (Egan et al.,
Nature 363:45, 1993). Triplicate samples of cell lines in 24-well
dishes were washed 5 times with serum free media and maintained in
serum free conditions during the course of the experiment. Cells
that excluded trypan blue, and that were therefore viable, were
counted with a hemocytometer immediately, 24 hours, 48 hours, and
72 hours after serum withdrawal. Survival was calculated as a
percentage of the initial number of viable cells. In this
experiment, as well as those presented in FIGS. 14B and 14D, the
percentage of viable cells shown represents the average of three
separate experiments performed in triplicate, .+-.standard
deviation.
[0102] The survival of CHO cells following transfection (with each
one of the six constructs described above) and exposure to
menadione is shown in FIG. 14B. The cells were plated in 24-well
dishes, allowed to grow overnight, and then exposed to 20 .mu.M
menadione (Sigma Chemical Co., St. Louis, Mo.) for 1.5 hours.
Triplicate samples were harvested at the time of exposure to
menadione and 24 hours afterward, and survival was assessed by
trypan blue exclusion.
[0103] The survival of Rat-1 cells following transfection (with
each one of the six constructs described above) and exposure to
staurosporine is shown in FIG. 14C. Rat-1 cells were transfected
and then selected in medium containing 800 .mu.g/ml G418 for two
weeks. The cell line was assessed for resistance to
staurosporine-induced apoptosis (1 .mu.M) for 5 hours. Viable cells
were counted 24 hours after exposure to staurosporine by trypan
blue exclusion. The percentage of viable cells shown represents the
average of two experiments, .+-.standard deviation.
[0104] The Rat-1 cell line was also used to test the resistance of
these cells to menadione (FIG. 14D) following transfection with
each of the six constructs described above. The cells were exposed
to 10 .mu.M menadione for 1.5 hours, and the number of viable cells
was counted 18 hours later.
B. Comparison of Cell Survival Following Transfection with
Full-Length vs. Partial IAP Constructs
[0105] In order to investigate the mechanism whereby human IAPs,
including XIAP, HIAP-1, and HIAP-2, afford protection against cell
death, expression vectors were constructed that contained either:
(1) full-length IAP cDNA (as described above), (2) a portion of an
IAP gene that encodes the BIR domains, but not the RZF, or (3) a
portion of an IAP gene that encodes the RZF, but not the BIR
domains. Human and murine xiap or m-xiap cDNAs were tested by
transient or stable expression in HeLa, Jurkat, and CHO cell lines.
Following transfection, apoptosis was induced by serum withdrawal,
application of menadione, or application of an anti-Fas antibody.
Cell death was then assessed, as described above, by trypan blue
exclusion. As a control for transfection efficiency, the cells were
co-transfected with a .beta.-gal expression construct. Typically,
approximately 20% of the cells were successfully transfected.
[0106] When CHO cells were transiently transfected, constructs
containing full-length xiap or m-xiap cDNAs conferred modest
protection against cell death (FIG. 15A). In contrast, the survival
of CHO cells transfected with constructs encoding only the BIR
domains (i.e., lacking the RZF domain; see FIG. 15A) was markedly
enhanced 72 hours after serum deprivation. Furthermore, a large
percentage of cells expressing the BIR domains were still viable
after 96 hours, at which time no viable cells remained in the
control, i.e. non-transfected, cell cultures (see "CHO" in FIG.
15A), and less than 5% of the cells transfected with the vector
only, i.e., lacking a cDNA insert, remained viable (see "pcDNA3" in
FIG. 15A). Deletion of any of the BIR domains results in the
complete loss of apoptotic suppression, which is reflected by a
decrease in the percentage of surviving CHO cells to control levels
within 72 hours of serum withdrawal (FIG. 15B; see "xiap.DELTA.1"
(which encodes amino acids 89-497 of XIAP (SEQ ID NO.:4)),
"xiap.DELTA.2" (which encodes amino acids 246-497 of XIAP (SEQ ID
NO.:4)), and "xiap.DELTA.3" (which encodes amino acids 342-497 of
IAP (SEQ ID NO.:4)) at 72 hours).
[0107] Stable pools of transfected CHO cells, which were maintained
for several months under G418 selection, were induced to undergo
apoptosis by exposure to 10 .mu.M menadione for 2 hours. Among the
CHO cells tested were those that were stably transfected with: (1)
full-length m-xiap cDNA (miap), (2) full-length xiap cDNA (xiap),
(3) full-length bcl-2 cDNA (Bcl-2), (4) cDNA encoding the three BIR
domains (but not the RZF) of M-XIAP (BIR), and (5) cDNA encoding
the RZF (but not BIR domains) of M-XIAP (RZF). Cells that were
non-transfected (CHO) or transfected with the vector only (pcDNA3),
served as controls for this experiment. Following exposure to 10
.mu.M menadione, the transfected cells were washed with phosphate
buffered saline (PBS) and cultured for an additional 24 hours in
menadione-free medium. Cell death was assessed, as described above,
by trypan blue exclusion. Less than 10% of the non-transfected or
vector-only transfected cells remained viable at the end of the 24
hour survival period. Cells expressing the RZF did not fare
significantly better. However, expression of full-length m-xiap,
xiap, or bcl-2, and expression of the BIR domains, enhanced cell
survival (FIG. 16A). When the concentration of menadione was
increased from 10 .mu.M to 20 .mu.M (with all other conditions of
the experiment being the same as when 10 .mu.M menadione was
applied), the percentage of viable CHO cells that expressed the BIR
domain cDNA construct was higher than the percentage of viable
cells that expressed either full-length m-xiap or bcl-2 (FIG.
16B).
C. Analysis of the Subcellular Location of Expressed RZF and BIR
Domains
[0108] The assays of cell death described above indicate that the
RZF may act as a negative regulator of the anti-apoptotic function
of IAPs. One way in which the RZF, and possibly other IAP domains,
may exert their regulatory influence is by altering the expression
of genes, whose products function in the apoptotic pathway.
[0109] In order to determine whether the subcellular locations of
expressed RZF and BIR domains are consistent with roles as nuclear
regulatory factors, COS cells were transiently transfected with the
following four constructs, and the expressed polypeptide was
localized by immunofluorescence microscopy: (1) pcDNA3-6myc-xiap,
which encodes all 497 amino acids of SEQ ID NO: 4, (2)
pcDNA3-6myc-m-xiap, which encodes all 497 amino acids of mouse xiap
(SEQ ID NO: 10), (3) pcDNA3-6myc-mxiap-BIR, which encodes amino
acids 1 to 341 of m-xiap (SEQ ID NO: 10), and (4)
pcDNA3-6myc-mxiap-RZF, which encodes amino acids 342-497 of m-xiap
(SEQ ID NO: 10). The cells were grown on multi-well tissue culture
slides for 12 hours, and then fixed and permeabilized with
methanol. The constructs used (here and in the cell death assays)
were tagged with a human Myc epitope tag at the N-terminus.
Therefore, a monoclonal anti-Myc antibody and a secondary goat
anti-mouse antibody, which was conjugated to FITC, could be used to
localize the expressed products in transiently transfected COS
cells. Full-length XIAP and MIAP were located in the cytoplasm,
with accentuated expression in the peri-nuclear zone. The same
pattern of localization was observed when the cells expressed a
construct encoding the RZF domain (but not the BIR domains).
However, cells expressing the BIR domains (without the RZF)
exhibited, primarily, nuclear staining. The protein expressed by
the BIR domain construct appeared to be in various stages of
transfer to the nucleus.
[0110] These observations are consistent with the fact that, as
described below, XIAP is cleaved within T cells that are treated
with anti-Fas antibodies (which are potent inducers of apoptosis),
and its N-terminal domain is translocated to the nucleus.
D. Examples of Additional Apoptosis Assays
[0111] Specific examples of apoptosis assays are also provided in
the following references. Assays for apoptosis in lymphocytes are
disclosed by: Li et al., Science 268:429, 1995; Gibellini et al.,
Br. J. Haematol. 89:24, 1995; Martin et al., J. Immunol. 152:330,
1994; Terai et al., J. Clin. Invest. 87:1710, 1991; Dhein et al.,
Nature 373:438, 1995; Katsikis et al., J. Exp. Med. 1815:2029,
1995; Westendorp et al., Nature 375:497, 1995; DeRossi et al.,
Virology 198:234, 1994.
[0112] Assays for apoptosis in fibroblasts are disclosed by:
Vossbeck et al., Int. J. Cancer 61:92, 1995; Goruppi et al.,
Oncogene 9:1537, 1994; Fernandez et al., Oncogene 9:2009, 1994;
Harrington et al., EMBO J., 13:3286, 1994; Itoh et al., J. Biol.
Chem. 268:10932, 1993.
[0113] Assays for apoptosis in neuronal cells are disclosed by:
Melino et al., Ann. Neurol. 36:864, 1994; Sato et al., J.
Neurobiol. 25:1227, 1994; Ferrari et al., J. Neurosci. 1516:2857,
1995; Talley et al., Mol. Cell Biol. 1585:2359, 1995; Talley et
al., Mol. Cell. Biol. 15:2359, 1995; Walkinshaw et al., J. Clin.
Invest. 95:2458, 1995.
[0114] Assays for apoptosis in insect cells are disclosed by: Clem
et al., Science 254:1388, 1991; Crook et al., J. Virol. 67:2168,
1993; Rabizadeh et al., J. Neurochem. 61:2318, 1993; Birnbaum et
al., J. Virol. 68:2521, 1994; Clem et al., Mol. Cell. Biol.
14:5212, 1994.
V. Construction of a Transgenic Animal
[0115] Characterization of IAP genes provides information that is
necessary for an LAP knockout animal model to be developed by
homologous recombination. Preferably, the model is a mammalian
animal, most preferably a mouse. Similarly, an animal model of IAP
overproduction may be generated by integrating one or more IAP
sequences into the genome, according to standard transgenic
techniques.
[0116] A replacement-type targeting vector, which would be used to
create a knockout model, can be constructed using an isogenic
genomic clone, for example, from a mouse strain such as 129/Sv
(Stratagene Inc., LaJolla, Calif.). The targeting vector is
introduced into a suitably-derived line of embryonic stem (ES)
cells by electroporation to generate ES cell lines that carry a
profoundly truncated form of an IAP. To generate chimeric founder
mice, the targeted cell lines are injected into a mouse blastula
stage embryo. Heterozygous offspring are interbred to homozygosity.
Knockout mice would provide the means, in vivo, to screen for
therapeutic compounds that modulate apoptosis via an IAP-dependent
pathway.
VI. IAP Protein Expression
[0117] IAP genes may be expressed in both prokaryotic and
eukaryotic cell types. If an IAP modulates apoptosis by
exacerbating it, it may be desirable to express that protein under
control of an inducible promotor.
[0118] In general, IAPs according to the invention may be produced
by transforming a suitable host cell with all or part of an
IAP-encoding cDNA fragment that has been placed into a suitable
expression vector.
[0119] Those skilled in the art of molecular biology will
understand that a wide variety of expression systems may be used to
produce the recombinant protein. The precise host cell used is not
critical to the invention. The IAP protein may be produced in a
prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., S.
cerevisiae, insect cells such as Sf21 cells, or mammalian cells
such as COS-1, NIH 3T3, or HeLa cells). These cells are publicly
available, for example, from the American Type Culture Collection
(ATCC), Rockville, Md.; see also Ausubel et al., Current Protocols
in Molecular Biology, John Wiley & Sons, New York, N.Y., 1994.
The method of transduction and the choice of expression vehicle
will depend on the host system selected. Transformation and
transfection methods are described, e.g., in Ausubel et al.
(supra), and expression vehicles may be chosen from those provided,
e.g. in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al.,
1985, Supp. 1987).
[0120] A preferred expression system is the baculovirus system
using, for example, the vector pBacPAK9, which is available from
Clontech (Palo Alto, Calif.). If desired, this system may be used
in conjunction with other protein expression techniques, for
example, the myc tag approach described by Evan et al. (Mol. Cell
Biol. 5:3610, 1985).
[0121] Alternatively, an IAP may be produced by a
stably-transfected mammalian cell line. A number of vectors
suitable for stable transfection of mammalian cells are available
to the public, (e.g., see Pouwels et al., supra), as are methods
for constructing such cell lines (e.g., see Ausubel et al., supra).
In one example, cDNA encoding an IAP is cloned into an expression
vector that includes the dihydrofolate reductase (DHFR) gene.
Integration of the plasmid and, therefore, integration of the
IAP-encoding gene into the host cell chromosome is selected for by
inclusion of 0.01-300 .mu.M methotrexate in the cell culture medium
(as described in Ausubel et al., supra). This dominant selection
can be accomplished in most cell types. Recombinant protein
expression can be increased by DHFR-mediated amplification of the
transfected gene.
[0122] Methods for selecting cell lines bearing gene amplifications
are described in Ausubel et al. (supra). These methods generally
involve extended culture in medium containing gradually increasing
levels of methotrexate. The most commonly used DHFR-containing
expression vectors are pCVSEII-DHFR and pAdD26SV(A) (described in
Ausubel et al., supra). The host cells described above or,
preferably, a DHFR-deficient CHO cell line (e.g., CHO DHFR.sup.-
cells, ATCC Accession No. CRL 9096) are among those most preferred
for DHFR selection of a stably-transfected cell line or
DHFR-mediated gene amplification.
[0123] Once the recombinant protein is expressed, it is isolated
by, for example, affinity chromatography. In one example, an
anti-IAP antibody, which may be produced by the methods described
herein, can be attached to a column and used to isolate the IAP
protein. Lysis and fractionation of IAP-harboring cells prior to
affinity chromatography may be performed by standard methods (see
e.g., Ausubel et al., supra). Once isolated, the recombinant
protein can, if desired, be purified further by e.g., by high
performance liquid chromatography (HPLC; e.g., see Fisher,
Laboratory Techniques In Biochemistry And Molecular Biology, Work
and Burdon, Eds., Elsevier, 1980).
[0124] Polypeptides of the invention, particularly short IAP
fragments, can also be produced by chemical synthesis (e.g., by the
methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984
The Pierce Chemical Co., Rockford, Ill.). These general techniques
of polypeptide expression and purification can also be used to
produce and isolate useful IAP fragments or analogs, as described
herein.
VII. Anti-IAP Antibodies
[0125] In order to generate IAP-specific antibodies, an IAP coding
sequence (i.e., amino acids 180-276) can be expressed as a
C-terminal fusion with glutathione S-transferase (GST; Smith et
al., Gene 67:31, 1988). The fusion protein can be purified on
glutathione-Sepharose beads, eluted with glutathione, and cleaved
with thrombin (at the engineered cleavage site), and purified to
the degree required to successfully immunize rabbits. Primary
immunizations can be carried out with Freund's complete adjuvant
and subsequent immunizations performed with Freund's incomplete
adjuvant. Antibody titres are monitored by western blot and
immunoprecipitation analyses using the thrombin-cleaved IAP
fragment of the GST-IAP fusion protein. Immune sera are affinity
purified using CNBr-Sepharose-coupled IAP protein. Antiserum
specificity is determined using a panel of unrelated GST proteins
(including GSTp53, Rb, HPV-16 E6, and E6-AP) and GST-trypsin (which
was generated by PCR using known sequences).
[0126] As an alternate or adjunct immunogen to GST fusion proteins,
peptides corresponding to relatively unique hydrophilic regions of
LAP may be generated and coupled to keyhole limpet hemocyanin (KLH)
through an introduced C-terminal lysine. Antiserum to each of these
peptides is similarly affinity purified on peptides conjugated to
BSA, and specificity is tested by ELISA and western blotting using
peptide conjugates, and by western blotting and immunoprecipitation
using IAP expressed as a GST fusion protein.
[0127] Alternatively, monoclonal antibodies may be prepared using
the IAP proteins described above and standard hybridoma technology
(see, e.g., Kohler et al., Nature 256:495, 1975; Kohler et al.,
Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol.
6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell
Hybridomas, Elsevier, New York, N.Y., 1981; Ausubel et al., supra).
Once produced, monoclonal antibodies are also tested for specific
IAP recognition by western blot or immunoprecipitation analysis (by
the methods described in Ausubel et al., supra).
[0128] Antibodies that specifically recognize IAPs or fragments of
IAPs, such as those described herein containing one or more BIR
domains (but not a ring zinc finger domain), or that contain a ring
zinc finger domain (but not a BIR domain) are considered useful in
the invention. They may, for example, be used in an immunoassay to
monitor IAP expression levels or to determine the subcellular
location of an LAP or IAP fragment produced by a mammal. Antibodies
that inhibit the 26 kDa IAP cleavage product described herein
(which contains at least one BIR domain) may be especially useful
in inducing apoptosis in cells undergoing undesirable
proliferation.
[0129] Preferably, antibodies of the invention are produced using
IAP sequence that does not reside within highly conserved regions,
and that appears likely to be antigenic, as analyzed by criteria
such as those provided by the Peptide structure program (Genetics
Computer Group Sequence Analysis Package, Program Manual for the
GCG Package, Version 7, 1991) using the algorithm of Jameson and
Wolf (CABIOS 4:181, 1988). Specifically, these regions, which are
found between BIR1 and BIR2 of all IAPs, are: amino acid 99 to
amino acid 170 of HIAP-1, amino acid 123 to amino acid 184 of
HIAP-2, and amino acid 116 to amino acid 133 of either XIAP or
M-XIAP. These fragments can be generated by standard techniques,
e.g., by PCR, and cloned into the pGEX expression vector (Ausubel
et al., supra). Fusion proteins are expressed in E. coli and
purified using a glutathione agarose affinity matrix as described
in Ausubel et al. (supra). In order to minimize the potential for
obtaining antisera that is non-specific, or exhibits low-affinity
binding to IAP, two or three fusions are generated for each
protein, and each fusion is injected into at least two rabbits.
Antisera are raised by injections in series, preferably including
at least three booster injections.
VIII. Identification of Molecules that Modulate IAP Protein
Expression
[0130] Isolation of IAP cDNAs also facilitates the identification
of molecules that increase or decrease IAP expression. In one
approach, candidate molecules are added, in varying concentration,
to the culture medium of cells expressing IAP mRNA. IAP expression
is then measured, for example, by northern blot analysis (Ausubel
et al., supra) using an IAP cDNA, or cDNA fragment, as a
hybridization probe (see also Table 5). The level of IAP expression
in the presence of the candidate molecule is compared to the level
of IAP expression in the absence of the candidate molecule, all
other factors (e.g. cell type and culture conditions) being
equal.
[0131] The effect of candidate molecules on IAP-mediated apoptosis
may, instead, be measured at the level of translation by using the
general approach described above with standard protein detection
techniques, such as western blotting or immunoprecipitation with an
IAP-specific antibody (for example, the LAP antibody described
herein).
[0132] Compounds that modulate the level of IAP may be purified, or
substantially purified, or may be one component of a mixture of
compounds such as an extract or supernatant obtained from cells
(Ausubel et al., supra). In an assay of a mixture of compounds, IAP
expression is tested against progressively smaller subsets of the
compound pool (e.g., produced by standard purification techniques
such as HPLC or FPLC) until a single compound or minimal number of
effective compounds is demonstrated to modulate IAP expression.
[0133] Compounds may also be screened for their ability to modulate
IAP apoptosis inhibiting activity. In this approach, the degree of
apoptosis in the presence of a candidate compound is compared to
the degree of apoptosis in its absence, under equivalent
conditions. Again, the screen may begin with a pool of candidate
compounds, from which one or more useful modulator compounds are
isolated in a step-wise fashion. Apoptosis activity may be measured
by any standard assay, for example, those described herein.
[0134] Another method for detecting compounds that modulate the
activity of IAPs is to screen for compounds that interact
physically with a given IAP polypeptide. These compounds may be
detected by adapting interaction trap expression systems known in
the art. These systems detect protein interactions using a
transcriptional activation assay and are generally described by
Gyuris et al. (Cell 75:791, 1993) and Field et al. (Nature 340:245,
1989), and are commercially available from Clontech (Palo Alto,
Calif.). In addition, PCT Publication WO 95/28497 describes an
interaction trap assay in which proteins involved in apoptosis, by
virtue of their interaction with Bcl-2, are detected. A similar
method may be used to identify proteins and other compounds that
interact with IAPs.
[0135] Compounds or molecules that function as modulators of
IAP-mediated cell death may include peptide and non-peptide
molecules such as those present in cell extracts, mammalian serum,
or growth medium in which mammalian cells have been cultured.
[0136] A molecule that promotes an increase in IAP expression or
LAP activity is considered particularly useful in the invention;
such a molecule may be used, for example, as a therapeutic to
increase cellular levels of IAP and thereby exploit the ability of
IAP polypeptides to inhibit apoptosis.
[0137] A molecule that decreases IAP activity (e.g., by decreasing
IAP gene expression or polypeptide activity) may be used to
decrease cellular proliferation. This would be advantageous in the
treatment of neoplasms (see Table 3, below), or other cell
proliferative diseases. TABLE-US-00003 TABLE 3 NORTHERN BLOT IAP
RNA LEVELS IN CANCER CELLS* xiap hiap1 hiap2 Promyelocytic Leukemia
HL-60 + + + Hela S-3 + + + Chronic Myelogenous Leukemia K-562 +++ +
+++ Lymphoblastic Leukemia MOLT-4 +++ + + Burkitt's Lymphoma Raji +
+(.times.10) + Colorectal Adenocarcinoma SW-480 +++ +++ +++ Lung
Carcinoma A-549 + + + Melanoma G-361 +++ + + *Levels are indicated
by a (+) and are the approximate increase in RNA levels relative to
northern blots of RNA from non-cancerous control cell lines. A
single plus indicates an estimated increase of at least 1-fold
[0138] Molecules that are found, by the methods described above, to
effectively modulate IAP gene expression or polypeptide activity
may be tested further in animal models. If they continue to
function successfully in an in vivo setting, they may be used as
therapeutics to either inhibit or enhance apoptosis, as
appropriate.
IX. IAP Therapy
[0139] The level of IAP gene expression correlates with the level
of apoptosis. Thus, IAP genes also find use in anti-apoptosis gene
therapy. In particular, a functional IAP gene may be used to
sustain neuronal cells that undergo apoptosis in the course of a
neurodegenerative disease, lymphocytes (i.e., T cells and B cells),
or cells that have been injured by ischemia.
[0140] Retroviral vectors, adenoviral vectors, adeno-associated
viral vectors, or other viral vectors with the appropriate tropism
for cells likely to be involved in apoptosis (for example,
epithelial cells) may be used as a gene transfer delivery system
for a therapeutic IAP gene construct. Numerous vectors useful for
this purpose are generally known (Miller, Human Gene Therapy 15,
1990; Friedman, Science 244:1275, 1989; Eglitis and Anderson,
Biotechniques 6:608, 1988; Tolstoshev and Anderson, Curr. Opin.
Biotechnol. 1:55, 1990; Sharp, Lancet 337:1277, 1991; Cometta et
al., Nucleic Acid Research and Molecular Biology 36:311, 1987;
Anderson, Science 226:401, 1984; Moen, Blood Cells 17:407, 1991;
Miller et al., Biotechniques 7:980, 1989; La Salle et al., Science
259:988, 1993; Johnson, Chest 107:77S, 1995). Retroviral vectors
are particularly well developed and have been used in clinical
settings (Rosenberg et al., N. Engl. J. Med. 323:370, 1990;
Anderson et al., U.S. Pat. No. 5,399,346). Non-viral approaches may
also be employed for the introduction of therapeutic DNA into cells
otherwise predicted to undergo apoptosis. For example, IAP may be
introduced into a neuron or a T cell by lipofection (Felgner et
al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al.,
Neurosci. Lett. 117:259, 1990; Brigham et al., Am. J. Med. Sci.
298:278, 1989; Staubinger et al., Meth. Enzymol. 101:512, 1983),
asialorosonucoid-polylysine conjugation (Wu et al., J. Biol. Chem.
263:14621, 1988; Wu et al., J. Biol. Chem. 264:16985, 1989); or,
less preferably, microinjection under surgical conditions (Wolff et
al., Science 247:1465, 1990).
[0141] For any of the methods of application described above, the
therapeutic IAP DNA construct is preferably applied to the site of
the predicted apoptosis event (for example, by injection). However,
it may also be applied to tissue in the vicinity of the predicted
apoptosis event or to a blood vessel supplying the cells predicted
to undergo apoptosis.
[0142] In the constructs described, IAP cDNA expression can be
directed from any suitable promoter (e.g., the human
cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein
promoters), and regulated by any appropriate mammalian regulatory
element. For example, if desired, enhancers known to preferentially
direct gene expression in neural cells, T cells, or B cells may be
used to direct LAP expression. The enhancers used could include,
without limitation, those that are characterized as tissue- or
cell-specific in their expression. Alternatively, if an IAP genomic
clone is used as a therapeutic construct (for example, following
its isolation by hybridization with the IAP cDNA described above),
regulation may be mediated by the cognate regulatory sequences or,
if desired, by regulatory sequences derived from a heterologous
source, including any of the promoters or regulatory elements
described above.
[0143] Less preferably, IAP gene therapy is accomplished by direct
administration of the IAP mRNA or antisense IAP mRNA to a cell that
is expected to undergo apoptosis. The mRNA may be produced and
isolated by any standard technique, but is most readily produced by
in vitro transcription using an IAP cDNA under the control of a
high efficiency promoter (e.g., the T7 promoter). Administration of
IAP mRNA to malignant cells can be carried out by any of the
methods for direct nucleic acid administration described above.
[0144] Ideally, the production of LAP protein by any gene therapy
approach will result in cellular levels of IAP that are at least
equivalent to the normal, cellular level of IAP in an unaffected
cell. Treatment by any IAP-mediated gene therapy approach may be
combined with more traditional therapies.
[0145] Another therapeutic approach within the invention involves
administration of recombinant IAP protein, either directly to the
site of a predicted apoptosis event (for example, by injection) or
systemically (for example, by any conventional recombinant protein
administration technique). The dosage of IAP depends on a number of
factors, including the size and health of the individual patient,
but, generally, between 0.1 mg and 100 mg inclusive are
administered per day to an adult in any pharmaceutically acceptable
formulation.
X. Administration of IAP Polypeptides, IAP Genes, or Modulators of
IAP Synthesis or Function
[0146] An IAP protein, gene, or modulator may be administered
within a pharmaceutically-acceptable diluent, carrier, or
excipient, in unit dosage form. Conventional pharmaceutical
practice may be employed to provide suitable formulations or
compositions to administer LAP to patients suffering from a disease
that is caused by excessive apoptosis. Administration may begin
before the patient is symptomatic. Any appropriate route of
administration may be employed, for example, administration may be
parenteral, intravenous, intraarterial, subcutaneous,
intramuscular, intracranial, intraorbital, ophthalmic,
intraventricular, intracapsular, intraspinal, intracisternal,
intraperitoneal, intranasal, aerosol, or oral administration.
Therapeutic formulations may be in the form of liquid solutions or
suspensions; for oral administration, formulations may be in the
form of tablets or capsules; and for intranasal formulations, in
the form of powders, nasal drops, or aerosols.
[0147] Methods well known in the art for making formulations are
found, for example, in "Remington's Pharmaceutical Sciences."
Formulations for parenteral administration may, for example,
contain excipients, sterile water, or saline, polyalkylene glycols
such as polyethylene glycol, oils of vegetable origin, or
hydrogenated napthalenes. Biocompatible, biodegradable lactide
polymer, lactide/glycolide copolymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control
the release of the compounds. Other potentially useful parenteral
delivery systems for IAP modulatory compounds include
ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, and liposomes. Formulations for
inhalation may contain excipients, for example, lactose, or may be
aqueous solutions containing, for example, polyoxyethylene-9-lauryl
ether, glycocholate and deoxycholate, or may be oily solutions for
administration in the form of nasal drops, or as a gel.
[0148] If desired, treatment with an IA protein, gene, or
modulatory compound may be combined with more traditional therapies
for the disease such as surgery, steroid therapy, or chemotherapy
for autoimmune disease; antiviral therapy for AIDS; and tissue
plasminogen activator (TPA) for ischemic injury.
XI. Detection of Conditions Involving Altered Apoptosis
[0149] IAP polypeptides and nucleic acid sequences find diagnostic
use in the detection or monitoring of conditions involving aberrant
levels of apoptosis. For example, decrease expression of LAP may be
correlated with enhanced apoptosis in humans (see section XII,
below). Accordingly, a decrease or increase in the level of IAP
production may provide an indication of a deleterious condition.
Levels of IAP expression may be assayed by any standard technique.
For example, IAP expression in a biological sample (e.g., a biopsy)
may be monitored by standard northern blot analysis or may be aided
by PCR (see, e.g., Ausubel et al., supra; PCR Technology:
Principles and Applications for DNA Amplification, H. A. Ehrlich,
Ed. Stockton Press, NY; Yap et al., Nucl. Acids. Res. 19:4294,
1991).
[0150] Alternatively, a biological sample obtained from a patient
may be analyzed for one or more mutations in the LAP sequences
using a mismatch detection approach. Generally, these techniques
involve PCR amplification of nucleic acid from the patient sample,
followed by identification of the mutation (i.e., mismatch) by
either altered hybridization, aberrant electrophoretic gel
migration, binding or cleavage mediated by mismatch binding
proteins, or direct nucleic acid sequencing. Any of these
techniques may be used to facilitate mutant IAP detection, and each
is well known in the art; examples of particular techniques are
described, without limitation, in Orita et al., Proc. Natl. Acad.
Sci. USA 86:2766, 1989; Sheffield et al., Proc. Natl. Acad. Sci.
USA 86:232, 1989).
[0151] In yet another approach, immunoassays are used to detect or
monitor IAP protein in a biological sample. IAP-specific polyclonal
or monoclonal antibodies (produced as described above) may be used
in any standard immunoassay format (e.g., ELISA, western blot, or
RIA) to measure IAP polypeptide levels. These levels would be
compared to wild-type IAP levels, with a decrease in IAP production
indicating a condition involving increased apoptosis. Examples of
immunoassays are described, e.g., in Ausubel et al., supra.
Immunohistochemical techniques may also be utilized for IAP
detection. For is example, a tissue sample may be obtained from a
patient, sectioned, and stained for the presence of LAP using an
anti-LAP antibody and any standard detection system (e.g., one
which includes a secondary antibody conjugated to horseradish
peroxidase). General guidance regarding such techniques can be
found in, e.g., Bancroft and Stevens (Theory and Practice of
Histological Techniques, Churchill Livingstone, 1982) and Ausubel
et al. (supra).
[0152] In one preferred example, a combined diagnostic method may
be employed that begins with an evaluation of IAP protein
production (for example, by immunological techniques or the protein
truncation test (Hogerrorst et al., Nat. Gen. 10:208, 1995)) and
also includes a nucleic acid-based detection technique designed to
identify more subtle LAP mutations (for example, point mutations).
As described above, a number of mismatch detection assays are
available to those skilled in the art, and any preferred technique
may be used. Mutations in IAP may be detected that either result in
loss of IAP expression or loss of IAP biological activity. In a
variation of this combined diagnostic method, IAP biological
activity is measured as protease activity using any appropriate
protease assay system (for example, those described above).
[0153] Mismatch detection assays also provide an opportunity to
diagnose an IAP-mediated predisposition to diseases caused by
inappropriate apoptosis. For example, a patient heterozygous for an
IAP mutation may show no clinical symptoms and yet possess a higher
than normal probability of developing one or more types of
neurodegenerative, myelodysplastic or ischemic diseases. Given this
diagnosis, a patient may take precautions to minimize their
exposure to adverse environmental factors (for example, UV exposure
or chemical mutagens) and to carefully monitor their medical
condition (for example, through frequent physical examinations).
This type of LAP diagnostic approach may also be used to detect IAP
mutations in prenatal screens. The IAP diagnostic assays described
above may be carried out using any biological sample (for example,
any biopsy sample or bodily fluid or tissue) in which LAP is
normally expressed. Identification of a mutant LAP gene may also be
assayed using these sources for test samples.
[0154] Alternatively, an IAP mutation, particularly as part of a
diagnosis for predisposition to IAP-associated degenerative
disease, may be tested using a DNA sample from any cell, for
example, by mismatch detection techniques. Preferably, the DNA
sample is subjected to PCR amplification prior to analysis.
[0155] In order to demonstrate the utility of IAP gene sequences as
diagnostics and prognostics for cancer, a Human Cancer Cell Line
Multiple Tissue Northern Blot (Clontech, Palo Alto, Calif.;
#7757-1) was probed. This northern blot contained approximately 2
.mu.g of poly A.sup.+ RNA per lane from eight different human cell
lines: (1) promyelocytic leukemia HL-60, (2) HeLa cell S3, (3)
chronic myelogenous leukemia K-562, (4) lymphoblastic leukemia
MOLT-4, (5) Burkitt's lymphoma Raji, (6) colorectal adenocarcinoma
SW480, (7) lung carcinoma A549, and (8) melanoma G361. As a
control, a Human Multiple Tissue Northern Blot (Clontech, Palo
Alto, Calif.; #7759-1) was probed. This northern blot contained
approximately 2 .mu.g of poly A.sup.+ RNA from eight different
human tissues: (1) spleen, (2) thymus, (3) prostate, (4) testis,
(5) ovary, (6) small intestine, (7) colon, and (8) peripheral blood
leukocytes.
[0156] The northern blots were hybridized sequentially with: (1) a
1.6 kb probe to the xiap coding region, (2) a 375 bp hiap-2
specific probe corresponding to the 3' untranslated region, (3) a
1.3 kb probe to the coding region of hiap-1, which cross-reacts
with hiap-2, (4) a 1.0 kb probe derived from the coding region of
bcl-2, and (5) a probe to .beta.-actin, which was provided by the
manufacturer. Hybridization was carried out at 50.degree. C.
overnight, according to the manufacturer's suggestion. The blot was
washed twice with 2.times.SSC, 0.1% SDS at room temperature for 15
minutes and then with 2.times.SSC, 0.1% SDS at 50.degree. C.
[0157] All cancer lines tested showed increased IAP expression
relative to samples from non-cancerous control tissues (Table 3).
Expression of xiap was particularly high in HeLa (S-3), chronic
myelogenous leukemia (K-562), colorectal adenocarcinoma (SW480),
and melanoma (G-361) lines. Expression of hiap-1 was extremely high
in Burkitt's lymphoma, and was also elevated in colorectal
adenocarcinoma. Expression of hiap-2 was particularly high in
chronic myelogenous leukemia (K-562) and colorectal adenocarcinoma
(SW480). Expression of bcl-2 was upregulated only in HL-60 leukemia
cells.
[0158] These observations suggest that upregulation of the
anti-apoptotic IAP genes may be a widespread phenomenon, perhaps
occurring much more frequently than upregulation of bcl-2.
Furthermore, upregulation may be necessary for the establishment or
maintenance of the transformed state of cancerous cells.
[0159] In order to pursue the observation described above, i.e.,
that hiap-1 is overexpressed in the Raji Burkitt's lymphoma cell
line, RT-PCR analysis was performed in multiple Burkitt's lymphoma
cell lines. Total RNA was extracted from cells of the Raji, Ramos,
EB-3, and Jiyoye cell lines, and as a positive control, from normal
placental tissue. The RNA was reverse transcribed, and amplified by
PCR with the following set of oligonucleotide primers:
5'-AGTGCGGGTTTTTATTATGTG-3' (SEQ ID NO: 44) and
5'-AGATGACCACAAGGAATAAACACTA-3' (SEQ ID NO: 45), which selectively
amplify a hiap-1 cDNA fragment. RT-PCR was conducted using a Perkin
Elmer 480 Thermocycler to carry out 35 cycles of the following
program: 94.degree. C. for 1 minute, 50.degree. C. for 1.5 minutes,
and 72.degree. C. for a minute. The PCR reaction product was
electrophoresed on an agarose gel and stained with ethidium
bromide. Amplified cDNA fragments of the appropriate size were
clearly visible in all lanes containing Burkitt's lymphoma samples,
but absent in the lanes containing the normal placental tissue
sample, and absent in lanes containing negative control samples,
where template DNA was omitted from the reaction (FIG. 17).
XII. Accumulation of a 26 kDa Cleavage Protein in Astrocytoma
Cells
A. Identification of a 26 kDa Cleavage Protein
[0160] A total protein extract was prepared from Jurkat and
astrocytoma cells by sonicating them (X3 for 15 seconds at
4.degree. C.) in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM PMSF, 1
.mu.g/ml aprotinin, and 5 mM benzamidine. Following sonication, the
samples were centrifuged (14,000 RPM in a microfuge) for five
minutes. Twenty .mu.g of protein was loaded per well on a 10%
SDS-polyacrylamide gel, electrophoresed, and electroblotted by
standard methods to PVDF membranes. Western blot analysis,
performed as described previously, revealed that the astrocytoma
cell line (CCF-STTG1) abundantly expressed an anti-xiap reactive
band of approximately 26 kDa, despite the lack of an apoptotic
trigger event (FIG. 18). In fact, this cell line has been
previously characterized as being particularly resistant to
standard apoptotic triggers.
[0161] A 26 kDa XIAP-reactive band was also observed under the
following experimental conditions. Jurkat cells (a transformed
human T cell line) were induced to undergo apoptosis by exposure to
an anti-Fas antibody (1 .mu.g/ml). Identical cultures of Jurkat
cells were exposed either to: (1) anti-Fas antibody and
cycloheximide (20 .mu.g/ml), (2) tumor necrosis factor alpha
(TNF-.alpha., at 1,000 U/ml), or (3) TNF-.alpha. and cycloheximide
(20 .mu.g/ml). All cells were harvested 6 hours after treatment
began. In addition, as a negative control, anti-Fas antibody was
added to an extract after the cells were harvested. The cells were
harvested in SDS sample buffer, electrophoresed on a 12.5% SDS
polyacrylamide gel, and electroblotted onto PVDF membranes using
standard methods. The membranes were immunostained with a rabbit
polyclonal anti-XIAP antibody at 1:1000 for 1 hour at room
temperature. Following four 15 minute washes, a goat anti-rabbit
antibody conjugated to horse-radish peroxidase was applied at room
temperature for 1 hour. Unbound secondary antibody was washed away,
and chemiluminescent detection of XIAP protein was performed. The
western blot revealed the presence of the full-length, 55 kDa XIAP
protein, both in untreated and treated cells. In addition, a novel,
approximately 26 kDa XIAP-reactive band was also observed in
apoptotic cell extracts, but not in the control, untreated cell
extracts (FIG. 19).
[0162] Cleavage of XIAP occurs in a variety of cell types,
including other cancer cell lines such as HeLa. The expression of
the 26 kDa XIAP cleavage product was demonstrated in HeLa cells as
follows. HeLa cells were treated with either: (1) cyclohexamide (20
.mu.g/ml), (2) anti-Fas antibody (1 .mu.g/ml), (3) anti-Fas
antibody (1 .mu.g/ml) and cyclohexamide (20 .mu.g/ml), (4)
TNF.alpha. (1,000 U/ml), or (5) TNF.alpha. (1,000 U/ml) and
cyclohexamide (20 .mu.g/ml). All cells were harvested 18 hours
after treatment began. As above, anti-Fas antibody was added to an
extract after the cells were harvested. HeLa cells were harvested,
and the western blot was probed under the same conditions as used
to visualize xiap-reactive bands from Jurkat cell samples. A 26 kDa
XIAP band was again seen in the apoptotic cell preparations (FIG.
20). Furthermore, the degree of XIAP cleavage correlated positively
with the extent of apoptosis. Treatment of HeLa cells with
cycloheximide or TNF.alpha. alone caused only minor apoptosis, and
little cleavage product was observed. If the cells were treated
with the anti-Fas antibody, a greater amount of cleavage product
was apparent. These data indicate that XIAP is cleaved in more than
one cell type and in response to more than one type of apoptotic
trigger.
B. Time Course of Expression
[0163] The time course over which the 26 kDa cleavage product
accumulates was examined by treating HeLa and Jurkat cells with
anti-Fas antibody (1 .mu.g/ml) and harvesting them either
immediately, or 1, 2, 3, 5, 10, or 22 hours after treatment.
Protein extracts were prepared and western blot analysis was
performed as described above. Both types of cells accumulated
increasing quantities of the 26 kDa cleavage product over the time
course examined (FIGS. 21A and 21B).
C. Subcellular Localization of the 26 kDa XIAP Cleavage Product
[0164] In order to determine the subcellular location of the 26 kDa
cleavage product, Jurkat cells were induced to undergo apoptosis by
exposure to anti-Fas antibody (1 .mu.g/ml) and were then harvested
either immediately, 3 hours, or 7 hours later. Total protein
extracts were prepared, as described above, from cells is harvested
at each time point. In order to prepare nuclear and cytoplasmic
cell extracts, apoptotic Jurkat cells were washed with isotonic
Tris buffered saline (pH 7.0) and lysed by freezing and thawing
five times in cell extraction buffer (50 mM PIPES, 50 mM KCl, 5 mM
EGTA, 2 mM MgCl.sub.2, 1 mM DTT, and 20 .mu.M cytochalasin B).
Nuclei were pelleted by centrifugation and resuspended in isotonic
Tris (pH 7.0) and frozen at -80.degree. C. The cytoplasmic fraction
of the extract was processed further by centrifugation at 60,000
RPM in a TA 100.3 rotor for 30 minutes. Supernatants were removed
and frozen at -80.degree. C. Samples of both nuclear and
cytoplasmic fractions were loaded on a 12.5% SDS-polyacrylamide
gel, and electroblotted onto PVDF membranes. Western blot analysis
was then performed using either an anti-CPP32 antibody
(Transduction Laboratories Lexington, Ky.; FIG. 22A) or the rabbit
anti-XIAP antibody described above (FIG. 22B).
[0165] The anti-CPP32 antibody, which recognizes the CPP32 protease
(also known as YAMA or apopain) partitioned almost exclusively in
the cytoplasmic fraction. The 55 kDa XIAP protein localized
exclusively in the cytoplasm of apoptotic cells, in agreement with
the studies presented above, where XIAP protein in normal, healthy
COS cells was seen to localize, by immunofluorescence microscopy,
to the cytoplasm. In contrast, the 26 kDa cleavage product
localized exclusively to the nuclear fraction of apoptotic Jurkat
cells. Taken together, these observations suggest that the
anti-apoptotic component of XIAP could be the 26 kDa cleavage
product, which exerts its influence within the nucleus.
D. In Vitro Cleavage of XIAP Protein and Characterization of the
Cleavage Product
[0166] For this series of experiments, XIAP protein was labeled
with .sup.35S using the plasmid pcDNA3-6myc-xiap, T7 RNA
polymerase, and a coupled transcription/translation kit (Promega)
according to the manufacturer's instructions. Radioactively labeled
XIAP protein was separated from unincorporated methionine by column
chromatography using Sephadex G-50.TM.. In addition, extracts of
apoptotic Jurkat cells were prepared following treatment with
anti-Fas antibody (1 .mu.g/ml) for three hours. To prepare the
extracts, the cells were lysed in Triton X-100 buffer (1% Triton
X-100, 25 mM Tris HCl) on ice for two hours and then
microcentrifuged for 5 minutes. The soluble extract was retained
(and was labelled "TX100"). Cells were lysed in cell extraction
buffer with freeze/thawing. The soluble cytoplasmic fraction was
set aside (and labelled "CEB"). Nuclear pellets from the
preparation of the CEB cytoplasmic fraction were solubilized with
Triton X-100 buffer, microcentrifuged, and the soluble fractions,
which contains primarily nuclear DNA, was retained (and labelled
"CEB-TX100"). Soluble cell extract was prepared by lysing cells
with NP-40 buffer, followed by microcentrifugation for 5 minutes
(and was labeled NP-40). In vitro cleavage was performed by
incubating 16 .mu.l of each extract (CEB, TX-100, CEB-TX100, and
NP40) with 4 .mu.l of in vitro translated XIAP protein at
37.degree. C. for 7 hours. Negative controls, containing only TX100
buffer or CEB buffer were also included. The proteins were
separated on a 10% SDS-polyacrylamide gel, which was then dried and
exposed to X-ray film overnight.
[0167] In vitro cleavage of XIAP was apparent in the CEB extract.
The observed molecular weight of the cleavage product was
approximately 36 kDa (FIG. 23). The 10 kDa shift in the size of the
cleavage product indicates that the observed product is derived
from the amino-terminus of the recombinant protein, which contains
six copies of the myc epitope (10 kDa). It thus appears that the
cleavage product possesses at least two of the BIR domains, and
that it is localized to the nucleus.
XIII. Treatment of HIV Infected Individuals
[0168] The expression of hiap-1 and hiap-2 is decreased
significantly in HIV-infected human cells. Furthermore, this
decrease precedes apoptosis. Therefore, administration of HLAP-1,
HIAP-2, genes encoding these proteins, or compounds that upregulate
these genes can be used to prevent T cell attrition in HIV-infected
patients. The following assay may also be used to screen for
compounds that alter hiap-1 and hiap-2 expression, and which also
prevent apoptosis.
[0169] Cultured mature lymphocyte CD-4.sup.+ T cell lines (H9,
labelled "a"; CEM/CM-3, labelled "b"; 6T-CEM, labelled "c"; and
Jurkat, labelled "d" in FIGS. 13A and 13B), were examined for signs
of apoptosis (FIG. 13A) and hiap gene expression (FIG. 13B) after
exposure to mitogens or HIV infection. Apoptosis was demonstrated
by the appearance of DNA "laddering" upon gel electrophoresis and
gene expression was assessed by PCR. The results obtained from
normal (non-infected, non-mitogen stimulated) cells are shown in
each lane labelled "1" in FIGS. 13A and 13B. The results obtained
24 hours after PHA/PMA (phytohemagglutinin/phorbol ester)
stimulation are shown in each lane labelled "2". The results
obtained 24 hours after HIV strain III.sub.B infection are shown in
each lane labelled "3". The "M" refers to standard DNA markers (the
123 bp ladder in FIG. 13B, and the lambda HindIII ladder in FIG.
13A (both from Gibco-BRL)). DNA ladders (Prigent et al., J.
Immunol. Meth., 160:139, 1993), which indicate apoptosis, are
evident when DNA from the samples described above are
electrophoresed on an ethidium bromide-stained agarose gel (FIG.
13A). The sensitivity and degree of apoptosis of the four T cell
lines tested varies following mitogen stimulation and HIV
infection.
[0170] In order to examine hiap gene expression, total RNA was
prepared from the cultured cells and reverse transcribed using
oligo-dT priming. The RT cDNA products were amplified by PCR using
specific primers (as shown in Table 5) for the detection of
hiap-2a, hiap-2b and hiap-1. The PCR was conducted using a Perkin
Elmer 480 thermocycler with 35 cycles of the following program:
94.degree. C. for one minute, 55.degree. C. for 2 minutes and
72.degree. C. for 1.5 minutes. The RT-PCR reaction products were
electrophoresed on a 1% agarose gel, which was stained with
ethidium bromide. Absence of hiap-2 transcripts is noted in all
four cell lines 24 hours after HIV infection. In three of four cell
lines (all except H9), the hiap-1 gene is also dramatically
down-regulated after HIV infection. PHA/PMA mitogen stimulation
also appears to decrease hiap gene expression; particularly of
hiap-2 and to a lesser extent, of hiap-1. The data from these
experiments is summarized in Table 5. The expression of
.beta.-actin was consistent in all cell lines tested, indicating
that there is not a flaw in the RT-PCR assay that could account for
the decrease in hiap gene expression. TABLE-US-00004 TABLE 4
OLIGONUCLEOTIDE PRIMERS FOR THE SPECIFIC RT-PCR AMPLIFICATION OF
UNIQUE IAP GENES Forward Primer Reverse Primer (nucleotide
(nucleotide Size of Product IAP Gene position*) position*) (bp)
h-xiap p2415 (876-896) P2449 (1291-1311) 435 m-xiap P2566 (458-478)
p2490 (994-1013) 555 h-hiap1 P2465 (827-847) p2464 (1008-1038) 211
m-hiap1 P2687 (747-767) p2684 (1177-1197) 450 hiap2 p2595
(1562-1585) p2578 (2339-2363) 801.sup.a 618.sup.b m-hiap2 p2693
(1751-1772) p2734 (2078-2100) 349 *Nucleotide position as
determined from FIGS. 1-4 for each IAP gene .sup.aPCR product size
of hiap2a .sup.bPCR product size of hiap2b
[0171] TABLE-US-00005 TABLE 5 APOPTOSIS AND HIAP GENE EXPRESSION IN
CULTURED T-CELLS FOLLOWING MITOGEN STIMULATION OR HIV INFECTION
Cell Line Condition Apoptosis hiap1 hiap2 H9 not stimulated - +
.+-. PHA/PMA stimulated +++ + .+-. HIV infected ++ + - CEM/CM-3 not
stimulated - + .+-. PHA/PMA stimulated .+-. + - HIV infected .+-. -
- 6T-CEM not stimulated - + + PHA/PMA stimulated .+-. - - HIV
infected + - - Jurkat not stimulated - + ++ PHA/PMA stimulated + +
+ HIV infected .+-. - -
XIV. Assignment of Xiap, Hiap-1, and Hiap-2 to Chromosomes Xq25 and
11q22-23 by Fluorescence in Situ Hybridization (FISH)
[0172] Fluorescence in situ hybridization (FISH) was used to
identify the chromosomal location of xiap, hiap-1 and hiap-2. The
probes used were cDNAs cloned in plasmid vectors: the 2.4 kb xiap
clone included 1493 bp of coding sequence, 34 bp of 5' UTR
(untranslated region) and 913 bp of 3'UTR; the hiap-1 cDNA was 3.1
kb long and included 1812 bp coding and 1300 bp of 3' UTR; and the
hiap-2 clone consisted of 1856 bp of coding and 1200 bp of 5' UTR.
A total of 1 .mu.g of probe DNA was labelled with biotin by nick
translation (BRL). Chromosome spreads prepared from a normal
peripheral blood culture were denatured for 2 minutes at 70.degree.
C. in 50% formamide/2.times.SSC and subsequently hybridized with
the biotin labelled DNA probe for 18 hours at 37.degree. C. in a
solution consisting of 2.times.SSC/70% formamide/10% dextran
sulfate. After hybridization, the spreads were washed in
2.times.SSC/50% formamide, followed by a wash in 2.times.SSC at
42.degree. C. The biotin labelled DNA was detected by fluorescein
isothiocyanate (FITC) conjugated avidin antibodies and anti-avidin
antibodies (ONCOR detection kit), according to the manufacturer's
instructions. Chromosomes were counterstained with propidium iodide
and examined with a Olympus BX60 epifluorescence microscope. For
chromosome identification, the slides with recorded labelled
metaphase spreads were destained, dehydrated, dried, digested with
trypsin for 30 seconds and stained with 4% Giemsa stain for 2
minutes. The chromosome spreads were relocated and the images were
compared.
[0173] A total of 101 metaphase spreads were examined with the xiap
probe, as described above. Symmetrical fluorescent signals on
either one or both homologs of chromosome Xq25 were observed in 74%
of the cells analyzed. Following staining with hiap-1 and hiap-2
probes, 56 cells were analyzed and doublet signals in the region
11q22-23 were observed in 83% of cells examined. The xiap gene was
mapped to Xq25 while the hiap-1 and hiap-2 genes were mapped at the
border of 11q22 and 11q23 bands.
[0174] These experiments confirmed the location of the xiap gene on
chromosome Xq25. No highly consistent chromosomal abnormalities
involving band Xq25 have been reported so far in any malignancies.
However, deletions within this region are associated with a number
of immune system defects including X-linked lymphoproliferative
disease (Wu et al., Genomics 17:163, 1993).
[0175] Cytogenetic abnormalities of band 11q23 have been identified
in more than 50% of infant leukemias regardless of the phenotype
(Martinez-Climet et al., Leukaemia 9:1299, 1995). Rearrangements of
the MLL Gene (mixed lineage leukemia or myeloid lymphoid leukemia;
Ziemin Van der Poel et al., Proc. Natl. Acad. Sci. USA 88:10735,
1991) have been detected in 80% of cases with 11q23 translocation,
however patients whose rearrangements clearly involved regions
other than the MLL gene were also reported (Kobayashi et al., Blood
82:547, 1993). Thus, the IAP genes may follow the Bcl-2 paradigm,
and would therefore play an important role in cancer
transformation.
XV. Preventive Anti-Apoptotic Therapy
[0176] In a patient diagnosed to be heterozygous for an IAP
mutation or to be susceptible to IAP mutations (even if those
mutations do not yet result in alteration or loss of IAP biological
activity), or a patient diagnosed as HIV positive, any of the above
therapies may be administered before the occurrence of the disease
phenotype. For example, the therapies may be provided to a patient
who is HIV positive but does not yet show a diminished T cell count
or other overt signs of AIDS. In particular, compounds shown to
increase IAP expression or IAP biological activity may be
administered by any standard dosage and route of administration
(see above). Alternatively, gene therapy using an IAP expression
construct may be undertaken to reverse or prevent the cell defect
prior to the development of the degenerative disease.
[0177] The methods of the instant invention may be used to reduce
or diagnose the disorders described herein in any mammal, for
example, humans, domestic pets, or livestock. Where a non-human
mammal is treated or diagnosed, the IAP polypeptide, nucleic acid,
or antibody employed is preferably specific for that species.
Other Embodiments
[0178] In other embodiments, the invention includes any protein
which is substantially identical to a mammalian IAP polypeptides
(FIGS. 1-6; SEQ ID NOs: 1-42); such homologs include other
substantially pure naturally-occurring mammalian IAP proteins as
well as allelic variants; natural mutants; induced mutants; DNA
sequences which encode proteins and also hybridize to the IAP DNA
sequences of FIGS. 1-6 (SEQ ID NOS: 1-42) under high stringency
conditions or, less preferably, under low stringency conditions
(e.g., washing at 2.times.SSC at 40.degree. C. with a probe length
of at least 40 nucleotides); and proteins specifically bound by
antisera directed to a IAP polypeptide. The term also includes
chimeric polypeptides that include a IAP portion.
[0179] The invention further includes analogs of any
naturally-occurring IAP polypeptide. Analogs can differ from the
naturally-occurring IAP protein by amino acid sequence differences,
by post-translational modifications, or by both. Analogs of the
invention will generally exhibit at least 85%, more preferably 90%,
and most preferably 95% or even 99% identity with all or part of a
naturally occurring IAP amino acid sequence. The length of sequence
comparison is at least 15 amino acid residues, preferably at least
25 amino acid residues, and more preferably more than 35 amino acid
residues. Modifications include in vivo and in vitro chemical
derivatization of polypeptides, e.g., acetylation, carboxylation,
phosphorylation, or glycosylation; such modifications may occur
during polypeptide synthesis or processing or following treatment
with isolated modifying enzymes. Analogs can also differ from the
naturally-occurring IAP polypeptide by alterations in primary
sequence. These include genetic variants, both natural and induced
(for example, resulting from random mutagenesis by irradiation or
exposure to ethanemethylsulfate or by site-specific mutagenesis as
described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A
Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al.,
supra). Also included are cyclized peptides, molecules, and analogs
which contain residues other than L-amino acids, e.g., D-amino
acids or nonnaturally occurring or synthetic amino acids, e.g.,
.beta. or .gamma. amino acids. In addition to full-length
polypeptides, the invention also includes LAP polypeptide
fragments. As used herein, the term "fragment," means at least 20
contiguous amino acids, preferably at least 30 contiguous amino
acids, more preferably at least 50 contiguous amino acids, and most
preferably at least 60 to 80 or more contiguous amino acids.
Fragments of IAP polypeptides can be generated by methods known to
those skilled in the art or may result from normal protein
processing (e.g., removal of amino acids from the nascent
polypeptide that are not required for biological activity or
removal of amino acids by alternative mRNA splicing or alternative
protein processing events).
[0180] Preferable fragments or analogs according to the invention
are those which facilitate specific detection of a LAP nucleic acid
or amino acid sequence in a sample to be diagnosed. Particularly
useful LAP fragments for this purpose include, without limitation,
the amino acid fragments shown in Table 2.
Sequence CWU 1
1
45 1 46 PRT Artificial Sequence Synthetic based on Homo sapiens,
Mus musculus, Drosophila melanogaster, Cydia pomonella, and Orgyia
pseudotsugata VARIANT (2)...(45) Xaa at positions 2, 3, 4, 5, 6, 7,
9, 10, 11, 17, 18, 19, 20, 21, 23, 25, 30, 31, 32, 34, 35, 38, 39,
40, 41, 42, and 45 may be any amino acid. VARIANT (8)...(8) Xaa at
position 8 is Glu or Asp. VARIANT (14)...(14) Xaa at position 14 is
Val or Ile. VARIANT (22)...(22) Xaa at position 22 is Val or Ile. 1
Glu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Lys Xaa Cys Met 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Pro Cys Gly His Xaa Xaa
Xaa 20 25 30 Cys Xaa Xaa Cys Ala Xaa Xaa Xaa Xaa Xaa Cys Pro Xaa
Cys 35 40 45 2 68 PRT Artificial Sequence VARIANT (1)...(66) Xaa at
positions 1, 2, 3, 6, 9, 10, 14, 15, 18, 19, 20, 21, 24, 30, 32,
33, 35, 37, 40, 42, 43, 44, 45, 46, 47, 49, 50, 51, 53, 54, 55, 56,
57, 59, 60, 61, 62, 64 and 66 may be any amino acid. VARIANT
(13)...(17) Xaa at positions 13, 16 and 17 may be any amino acid or
may be absent. Synthetic based on Homo sapiens, Mus musculus,
Drosophila melanogaster, Cydia pomonella, and Orgyia pseudotsugata
2 Xaa Xaa Xaa Arg Leu Xaa Thr Phe Xaa Xaa Trp Pro Xaa Xaa Xaa Xaa 1
5 10 15 Xaa Xaa Xaa Xaa Xaa Leu Ala Xaa Ala Gly Phe Tyr Tyr Xaa Gly
Xaa 20 25 30 Xaa Asp Xaa Val Xaa Cys Phe Xaa Cys Xaa Xaa Xaa Xaa
Xaa Xaa Trp 35 40 45 Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa His Xaa
Xaa Xaa Xaa Pro Xaa 50 55 60 Cys Xaa Phe Val 65 3 2540 DNA Homo
sapiens variation (2540)...(2540) N may be any nucleotide 3
gaaaaggtgg acaagtccta ttttcaagag aagatgactt ttaacagttt tgaaggatct
60 aaaacttgtg tacctgcaga catcaataag gaagaagaat ttgtagaaga
gtttaataga 120 ttaaaaactt ttgctaattt tccaagtggt agtcctgttt
cagcatcaac actggcacga 180 gcagggtttc tttatactgg tgaaggagat
accgtgcggt gctttagttg tcatgcagct 240 gtagatagat ggcaatatgg
agactcagca gttggaagac acaggaaagt atccccaaat 300 tgcagattta
tcaacggctt ttatcttgaa aatagtgcca cgcagtctac aaattctggt 360
atccagaatg gtcagtacaa agttgaaaac tatctgggaa gcagagatca ttttgcctta
420 gacaggccat ctgagacaca tgcagactat cttttgagaa ctgggcaggt
tgtagatata 480 tcagacacca tatacccgag gaaccctgcc atgtattgtg
aagaagctag attaaagtcc 540 tttcagaact ggccagacta tgctcaccta
accccaagag agttagcaag tgctggactc 600 tactacacag gtattggtga
ccaagtgcag tgcttttgtt gtggtggaaa actgaaaaat 660 tgggaacctt
gtgatcgtgc ctggtcagaa cacaggcgac actttcctaa ttgcttcttt 720
gttttgggcc ggaatcttaa tattcgaagt gaatctgatg ctgtgagttc tgataggaat
780 ttcccaaatt caacaaatct tccaagaaat ccatccatgg cagattatga
agcacggatc 840 tttacttttg ggacatggat atactcagtt aacaaggagc
agcttgcaag agctggattt 900 tatgctttag gtgaaggtga taaagtaaag
tgctttcact gtggaggagg gctaactgat 960 tggaagccca gtgaagaccc
ttgggaacaa catgctaaat ggtatccagg gtgcaaatat 1020 ctgttagaac
agaagggaca agaatatata aacaatattc atttaactca ttcacttgag 1080
gagtgtctgg taagaactac tgagaaaaca ccatcactaa ctagaagaat tgatgatacc
1140 atcttccaaa atcctatggt acaagaagct atacgaatgg ggttcagttt
caaggacatt 1200 aagaaaataa tggaggaaaa aattcagata tctgggagca
actataaatc acttgaggtt 1260 ctggttgcag atctagtgaa tgctcagaaa
gacagtatgc aagatgagtc aagtcagact 1320 tcattacaga aagagattag
tactgaagag cagctaaggc gcctgcaaga ggagaagctt 1380 tgcaaaatct
gtatggatag aaatattgct atcgtttttg ttccttgtgg acatctagtc 1440
acttgtaaac aatgtgctga agcagttgac aagtgtccca tgtgctacac agtcattact
1500 ttcaagcaaa aaatttttat gtcttaatct aactctatag taggcatgtt
atgttgttct 1560 tattaccctg attgaatgtg tgatgtgaac tgactttaag
taatcaggat tgaattccat 1620 tagcatttgc taccaagtag gaaaaaaaat
gtacatggca gtgttttagt tggcaatata 1680 atctttgaat ttcttgattt
ttcagggtat tagctgtatt atccattttt tttactgtta 1740 tttaattgaa
accatagact aagaataaga agcatcatac tataactgaa cacaatgtgt 1800
attcatagta tactgattta atttctaagt gtaagtgaat taatcatctg gattttttat
1860 tcttttcaga taggcttaac aaatggagct ttctgtatat aaatgtggag
attagagtta 1920 atctccccaa tcacataatt tgttttgtgt gaaaaaggaa
taaattgttc catgctggtg 1980 gaaagataga gattgttttt agaggttggt
tgttgtgttt taggattctg tccattttct 2040 tgtaaaggga taaacacgga
cgtgtgcgaa atatgtttgt aaagtgattt gccattgttg 2100 aaagcgtatt
taatgataga atactatcga gccaacatgt actgacatgg aaagatgtca 2160
gagatatgtt aagtgtaaaa tgcaagtggc gggacactat gtatagtctg agccagatca
2220 aagtatgtat gttgttaata tgcatagaac gagagatttg gaaagatata
caccaaactg 2280 ttaaatgtgg tttctcttcg gggagggggg gattggggga
ggggccccag aggggtttta 2340 gaggggcctt ttcactttcg acttttttca
ttttgttctg ttcggatttt ttataagtat 2400 gtagaccccg aagggtttta
tgggaactaa catcagtaac ctaacccccg tgactatcct 2460 gtgctcttcc
tagggagctg tgttgtttcc cacccaccac ccttccctct gaacaaatgc 2520
ctgagtgctg gggcactttn 2540 4 497 PRT Homo sapiens 4 Met Thr Phe Asn
Ser Phe Glu Gly Ser Lys Thr Cys Val Pro Ala Asp 1 5 10 15 Ile Asn
Lys Glu Glu Glu Phe Val Glu Glu Phe Asn Arg Leu Lys Thr 20 25 30
Phe Ala Asn Phe Pro Ser Gly Ser Pro Val Ser Ala Ser Thr Leu Ala 35
40 45 Arg Ala Gly Phe Leu Tyr Thr Gly Glu Gly Asp Thr Val Arg Cys
Phe 50 55 60 Ser Cys His Ala Ala Val Asp Arg Trp Gln Tyr Gly Asp
Ser Ala Val 65 70 75 80 Gly Arg His Arg Lys Val Ser Pro Asn Cys Arg
Phe Ile Asn Gly Phe 85 90 95 Tyr Leu Glu Asn Ser Ala Thr Gln Ser
Thr Asn Ser Gly Ile Gln Asn 100 105 110 Gly Gln Tyr Lys Val Glu Asn
Tyr Leu Gly Ser Arg Asp His Phe Ala 115 120 125 Leu Asp Arg Pro Ser
Glu Thr His Ala Asp Tyr Leu Leu Arg Thr Gly 130 135 140 Gln Val Val
Asp Ile Ser Asp Thr Ile Tyr Pro Arg Asn Pro Ala Met 145 150 155 160
Tyr Cys Glu Glu Ala Arg Leu Lys Ser Phe Gln Asn Trp Pro Asp Tyr 165
170 175 Ala His Leu Thr Pro Arg Glu Leu Ala Ser Ala Gly Leu Tyr Tyr
Thr 180 185 190 Gly Ile Gly Asp Gln Val Gln Cys Phe Cys Cys Gly Gly
Lys Leu Lys 195 200 205 Asn Trp Glu Pro Cys Asp Arg Ala Trp Ser Glu
His Arg Arg His Phe 210 215 220 Pro Asn Cys Phe Phe Val Leu Gly Arg
Asn Leu Asn Ile Arg Ser Glu 225 230 235 240 Ser Asp Ala Val Ser Ser
Asp Arg Asn Phe Pro Asn Ser Thr Asn Leu 245 250 255 Pro Arg Asn Pro
Ser Met Ala Asp Tyr Glu Ala Arg Ile Phe Thr Phe 260 265 270 Gly Thr
Trp Ile Tyr Ser Val Asn Lys Glu Gln Leu Ala Arg Ala Gly 275 280 285
Phe Tyr Ala Leu Gly Glu Gly Asp Lys Val Lys Cys Phe His Cys Gly 290
295 300 Gly Gly Leu Thr Asp Trp Lys Pro Ser Glu Asp Pro Trp Glu Gln
His 305 310 315 320 Ala Lys Trp Tyr Pro Gly Cys Lys Tyr Leu Leu Glu
Gln Lys Gly Gln 325 330 335 Glu Tyr Ile Asn Asn Ile His Leu Thr His
Ser Leu Glu Glu Cys Leu 340 345 350 Val Arg Thr Thr Glu Lys Thr Pro
Ser Leu Thr Arg Arg Ile Asp Asp 355 360 365 Thr Ile Phe Gln Asn Pro
Met Val Gln Glu Ala Ile Arg Met Gly Phe 370 375 380 Ser Phe Lys Asp
Ile Lys Lys Ile Met Glu Glu Lys Ile Gln Ile Ser 385 390 395 400 Gly
Ser Asn Tyr Lys Ser Leu Glu Val Leu Val Ala Asp Leu Val Asn 405 410
415 Ala Gln Lys Asp Ser Met Gln Asp Glu Ser Ser Gln Thr Ser Leu Gln
420 425 430 Lys Glu Ile Ser Thr Glu Glu Gln Leu Arg Arg Leu Gln Glu
Glu Lys 435 440 445 Leu Cys Lys Ile Cys Met Asp Arg Asn Ile Ala Ile
Val Phe Val Pro 450 455 460 Cys Gly His Leu Val Thr Cys Lys Gln Cys
Ala Glu Ala Val Asp Lys 465 470 475 480 Cys Pro Met Cys Tyr Thr Val
Ile Thr Phe Lys Gln Lys Ile Phe Met 485 490 495 Ser 5 2676 DNA Homo
sapiens variation (2470)...(2470) N may be any nucleotide variation
(2476)...(2476) N may be any nucleotide variation (2483)...(2483) N
may be any nucleotide variation (2602)...(2602) N may be any
nucleotide 5 tccttgagat gtatcagtat aggatttagg atctccatgt tggaactcta
aatgcataga 60 aatggaaata atggaaattt ttcattttgg cttttcagcc
tagtattaaa actgataaaa 120 gcaaagccat gcacaaaact acctccctag
agaaaggcta gtcccttttc ttccccattc 180 atttcattat gaacatagta
gaaaacagca tattcttatc aaatttgatg aaaagcgcca 240 acacgtttga
actgaaatac gacttgtcat gtgaactgta ccgaatgtct acgtattcca 300
cttttcctgc tggggttcct gtctcagaaa ggagtcttgc tcgtgctggt ttctattaca
360 ctggtgtgaa tgacaaggtc aaatgcttct gttgtggcct gatgctggat
aactggaaaa 420 gaggagacag tcctactgaa aagcataaaa agttgtatcc
tagctgcaga ttcgttcaga 480 gtctaaattc cgttaacaac ttggaagcta
cctctcagcc tacttttcct tcttcagtaa 540 cacattccac acactcatta
cttccgggta cagaaaacag tggatatttc cgtggctctt 600 attcaaactc
tccatcaaat cctgtaaact ccagagcaaa tcaagaattt tctgccttga 660
tgagaagttc ctacccctgt ccaatgaata acgaaaatgc cagattactt acttttcaga
720 catggccatt gacttttctg tcgccaacag atctggcacg agcaggcttt
tactacatag 780 gacctggaga cagagtggct tgctttgcct gtggtggaaa
attgagcaat tgggaaccga 840 aggataatgc tatgtcagaa cacctgagac
attttcccaa atgcccattt atagaaaatc 900 agcttcaaga cacttcaaga
tacacagttt ctaatctgag catgcagaca catgcagccc 960 gctttaaaac
attctttaac tggccctcta gtgttctagt taatcctgag cagcttgcaa 1020
gtgcgggttt ttattatgtg ggtaacagtg atgatgtcaa atgcttttgc tgtgatggtg
1080 gactcaggtg ttgggaatct ggagatgatc catgggttca acatgccaag
tggtttccaa 1140 ggtgtgagta cttgataaga attaaaggac aggagttcat
ccgtcaagtt caagccagtt 1200 accctcatct acttgaacag ctgctatcca
catcagacag cccaggagat gaaaatgcag 1260 agtcatcaat tatccatttg
gaacctggag aagaccattc agaagatgca atcatgatga 1320 atactcctgt
gattaatgct gccgtggaaa tgggctttag tagaagcctg gtaaaacaga 1380
cagttcagag aaaaatccta gcaactggag agaattatag actagtcaat gatcttgtgt
1440 tagacttact caatgcagaa gatgaaataa gggaagagga gagagaaaga
gcaactgagg 1500 aaaaagaatc aaatgattta ttattaatcc ggaagaatag
aatggcactt tttcaacatt 1560 tgacttgtgt aattccaatc ctggatagtc
tactaactgc cggaattatt aatgaacaag 1620 aacatgatgt tattaaacag
aagacacaga cgtctttaca agcaagagaa ctgattgata 1680 cgattttagt
aaaaggaaat attgcagcca ctgtattcag aaactctctg caagaagctg 1740
aagctgtgtt atatgagcat ttatttgtgc aacaggacat aaaatatatt cccacagaag
1800 atgtttcaga tctaccagtg gaagaacaat tgcggagact accagaagaa
agaacatgta 1860 aagtgtgtat ggacaaagaa gtgtccatag tgtttattcc
ttgtggtcat ctagtagtat 1920 gcaaagattg tgctccttct ttaagaaagt
gtcctatttg taggagtaca atcaagggta 1980 cagttcgtac atttctttca
tgaagaagaa ccaaaacatc gtctaaactt tagaattaat 2040 ttattaaatg
tattataact ttaactttta tcctaatttg gtttccttaa aatttttatt 2100
tatttacaac tcaaaaaaca ttgttttgtg taacatattt atatatgtat ctaaaccata
2160 tgaacatata ttttttagaa actaagagaa tgataggctt ttgttcttat
gaacgaaaaa 2220 gaggtagcac tacaaacaca atattcaatc caaatttcag
cattattgaa attgtaagtg 2280 aagtaaaact taagatattt gagttaacct
ttaagaattt taaatatttt ggcattgtac 2340 taataccggg aacatgaagc
caggtgtggt ggtatgtacc tgtagtccca ggctgaggca 2400 agagaattac
ttgagcccag gagtttgaat ccatcctggg cagcatactg agaccctgcc 2460
tttaaaaacn aacagnacca aanccaaaca ccagggacac atttctctgt cttttttgat
2520 cagtgtccta tacatcgaag gtgtgcatat atgttgaatc acattttagg
gacatggtgt 2580 ttttataaag aattctgtga gnaaaaattt aataaagcaa
ccaaattact cttaaaaaaa 2640 aaaaaaaaaa aaaaaactcg aggggcccgt accaat
2676 6 604 PRT Homo sapiens 6 Met Asn Ile Val Glu Asn Ser Ile Phe
Leu Ser Asn Leu Met Lys Ser 1 5 10 15 Ala Asn Thr Phe Glu Leu Lys
Tyr Asp Leu Ser Cys Glu Leu Tyr Arg 20 25 30 Met Ser Thr Tyr Ser
Thr Phe Pro Ala Gly Val Pro Val Ser Glu Arg 35 40 45 Ser Leu Ala
Arg Ala Gly Phe Tyr Tyr Thr Gly Val Asn Asp Lys Val 50 55 60 Lys
Cys Phe Cys Cys Gly Leu Met Leu Asp Asn Trp Lys Arg Gly Asp 65 70
75 80 Ser Pro Thr Glu Lys His Lys Lys Leu Tyr Pro Ser Cys Arg Phe
Val 85 90 95 Gln Ser Leu Asn Ser Val Asn Asn Leu Glu Ala Thr Ser
Gln Pro Thr 100 105 110 Phe Pro Ser Ser Val Thr His Ser Thr His Ser
Leu Leu Pro Gly Thr 115 120 125 Glu Asn Ser Gly Tyr Phe Arg Gly Ser
Tyr Ser Asn Ser Pro Ser Asn 130 135 140 Pro Val Asn Ser Arg Ala Asn
Gln Glu Phe Ser Ala Leu Met Arg Ser 145 150 155 160 Ser Tyr Pro Cys
Pro Met Asn Asn Glu Asn Ala Arg Leu Leu Thr Phe 165 170 175 Gln Thr
Trp Pro Leu Thr Phe Leu Ser Pro Thr Asp Leu Ala Arg Ala 180 185 190
Gly Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val Ala Cys Phe Ala Cys 195
200 205 Gly Gly Lys Leu Ser Asn Trp Glu Pro Lys Asp Asn Ala Met Ser
Glu 210 215 220 His Leu Arg His Phe Pro Lys Cys Pro Phe Ile Glu Asn
Gln Leu Gln 225 230 235 240 Asp Thr Ser Arg Tyr Thr Val Ser Asn Leu
Ser Met Gln Thr His Ala 245 250 255 Ala Arg Phe Lys Thr Phe Phe Asn
Trp Pro Ser Ser Val Leu Val Asn 260 265 270 Pro Glu Gln Leu Ala Ser
Ala Gly Phe Tyr Tyr Val Gly Asn Ser Asp 275 280 285 Asp Val Lys Cys
Phe Cys Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser 290 295 300 Gly Asp
Asp Pro Trp Val Gln His Ala Lys Trp Phe Pro Arg Cys Glu 305 310 315
320 Tyr Leu Ile Arg Ile Lys Gly Gln Glu Phe Ile Arg Gln Val Gln Ala
325 330 335 Ser Tyr Pro His Leu Leu Glu Gln Leu Leu Ser Thr Ser Asp
Ser Pro 340 345 350 Gly Asp Glu Asn Ala Glu Ser Ser Ile Ile His Leu
Glu Pro Gly Glu 355 360 365 Asp His Ser Glu Asp Ala Ile Met Met Asn
Thr Pro Val Ile Asn Ala 370 375 380 Ala Val Glu Met Gly Phe Ser Arg
Ser Leu Val Lys Gln Thr Val Gln 385 390 395 400 Arg Lys Ile Leu Ala
Thr Gly Glu Asn Tyr Arg Leu Val Asn Asp Leu 405 410 415 Val Leu Asp
Leu Leu Asn Ala Glu Asp Glu Ile Arg Glu Glu Glu Arg 420 425 430 Glu
Arg Ala Thr Glu Glu Lys Glu Ser Asn Asp Leu Leu Leu Ile Arg 435 440
445 Lys Asn Arg Met Ala Leu Phe Gln His Leu Thr Cys Val Ile Pro Ile
450 455 460 Leu Asp Ser Leu Leu Thr Ala Gly Ile Ile Asn Glu Gln Glu
His Asp 465 470 475 480 Val Ile Lys Gln Lys Thr Gln Thr Ser Leu Gln
Ala Arg Glu Leu Ile 485 490 495 Asp Thr Ile Leu Val Lys Gly Asn Ile
Ala Ala Thr Val Phe Arg Asn 500 505 510 Ser Leu Gln Glu Ala Glu Ala
Val Leu Tyr Glu His Leu Phe Val Gln 515 520 525 Gln Asp Ile Lys Tyr
Ile Pro Thr Glu Asp Val Ser Asp Leu Pro Val 530 535 540 Glu Glu Gln
Leu Arg Arg Leu Pro Glu Glu Arg Thr Cys Lys Val Cys 545 550 555 560
Met Asp Lys Glu Val Ser Ile Val Phe Ile Pro Cys Gly His Leu Val 565
570 575 Val Cys Lys Asp Cys Ala Pro Ser Leu Arg Lys Cys Pro Ile Cys
Arg 580 585 590 Ser Thr Ile Lys Gly Thr Val Arg Thr Phe Leu Ser 595
600 7 2580 DNA Homo sapiens variation (2412)...(2412) N may be any
nucleotide 7 ttaggttacc tgaaagagtt actacaaccc caaagagttg tgttctaagt
agtatcttgg 60 taattcagag agatactcat cctacctgaa tataaactga
gataaatcca gtaaagaaag 120 tgtagtaaat tctacataag agtctatcat
tgatttcttt ttgtggtgga aatcttagtt 180 catgtgaaga aatttcatgt
gaatgtttta gctatcaaac agtactgtca cctactcatg 240 cacaaaactg
cctcccaaag acttttccca ggtccctcgt atcaaaacat taagagtata 300
atggaagata gcacgatctt gtcagattgg acaaacagca acaaacaaaa aatgaagtat
360 gacttttcct gtgaactcta cagaatgtct acatattcaa ctttccccgc
cggggtgcct 420 gtctcagaaa ggagtcttgc tcgtgctggt ttttattata
ctggtgtgaa tgacaaggtc 480 aaatgcttct gttgtggcct gatgctggat
aactggaaac taggagacag tcctattcaa 540 aagcataaac agctatatcc
tagctgtagc tttattcaga atctggtttc agctagtctg 600 ggatccacct
ctaagaatac gtctccaatg agaaacagtt ttgcacattc attatctccc 660
accttggaac atagtagctt gttcagtggt tcttactcca gccttcctcc aaaccctctt
720 aattctagag cagttgaaga catctcttca tcgaggacta acccctacag
ttatgcaatg 780 agtactgaag aagccagatt tcttacctac catatgtggc
cattaacttt tttgtcacca 840 tcagaattgg caagagctgg tttttattat
ataggacctg gagatagggt agcctgcttt 900 gcctgtggtg ggaagctcag
taactgggaa ccaaaggatg atgctatgtc agaacaccgg 960 aggcattttc
ccaactgtcc atttttggaa aattctctag aaactctgag gtttagcatt 1020
tcaaatctga gcatgcagac acatgcagct cgaatgagaa catttatgta ctggccatct
1080 agtgttccag ttcagcctga gcagcttgca agtgctggtt tttattatgt
gggtcgcaat 1140 gatgatgtca aatgctttgg ttgtgatggt ggcttgaggt
gttgggaatc tggagatgat 1200 ccatgggtag aacatgccaa gtggtttcca
aggtgtgagt tcttgatacg aatgaaaggc 1260 caagagtttg ttgatgagat
tcaaggtaga tatcctcatc ttcttgaaca gctgttgtca 1320 acttcagata
ccactggaga agaaaatgct gacccaccaa ttattcattt tggacctgga 1380
gaaagttctt cagaagatgc tgtcatgatg aatacacctg tggttaaatc tgccttggaa
1440 atgggcttta atagagacct ggtgaaacaa acagttctaa gtaaaatcct
gacaactgga 1500 gagaactata aaacagttaa tgatattgtg tcagcacttc
ttaatgctga agatgaaaaa 1560 agagaagagg agaaggaaaa acaagctgaa
gaaatggcat cagatgattt gtcattaatt 1620 cggaagaaca gaatggctct
ctttcaacaa ttgacatgtg tgcttcctat cctggataat 1680 cttttaaagg
ccaatgtaat taataaacag gaacatgata ttattaaaca aaaaacacag 1740
atacctttac aagcgagaga actgattgat accatttggg ttaaaggaaa tgctgcggcc
1800 aacatcttca aaaactgtct aaaagaaatt gactctacat tgtataagaa
cttatttgtg 1860 gataagaata tgaagtatat tccaacagaa gatgtttcag
gtctgtcact ggaagaacaa 1920 ttgaggaggt tgcaagaaga acgaacttgt
aaagtgtgta tggacaaaga agtttctgtt 1980 gtatttattc cttgtggtca
tctggtagta tgccaggaat gtgccccttc tctaagaaaa 2040 tgccctattt
gcaggggtat aatcaagggt actgttcgta catttctctc ttaaagaaaa 2100
atagtctata ttttaacctg cataaaaagg tctttaaaat attgttgaac acttgaagcc
2160 atctaaagta aaaagggaat tatgagtttt tcaattagta acattcatgt
tctagtctgc 2220 tttggtacta ataatcttgt ttctgaaaag atggtatcat
atatttaatc ttaatctgtt 2280 tatttacaag ggaagattta tgtttggtga
actatattag tatgtatgtg tacctaaggg 2340 agtagcgtcn ctgcttgtta
tgcatcattt caggagttac tggatttgtt gttctttcag 2400 aaagctttga
anactaaatt atagtgtaga aaagaactgg aaaccaggaa ctctggagtt 2460
catcagagtt atggtgccga attgtctttg gtgcttttca cttgtgtttt aaaataagga
2520 tttttctctt atttctcccc ctagtttgtg agaaacatct caataaagtg
ctttaaaaag 2580 8 618 PRT Homo sapiens 8 Met His Lys Thr Ala Ser
Gln Arg Leu Phe Pro Gly Pro Ser Tyr Gln 1 5 10 15 Asn Ile Lys Ser
Ile Met Glu Asp Ser Thr Ile Leu Ser Asp Trp Thr 20 25 30 Asn Ser
Asn Lys Gln Lys Met Lys Tyr Asp Phe Ser Cys Glu Leu Tyr 35 40 45
Arg Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val Pro Val Ser Glu 50
55 60 Arg Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val Asn Asp
Lys 65 70 75 80 Val Lys Cys Phe Cys Cys Gly Leu Met Leu Asp Asn Trp
Lys Leu Gly 85 90 95 Asp Ser Pro Ile Gln Lys His Lys Gln Leu Tyr
Pro Ser Cys Ser Phe 100 105 110 Ile Gln Asn Leu Val Ser Ala Ser Leu
Gly Ser Thr Ser Lys Asn Thr 115 120 125 Ser Pro Met Arg Asn Ser Phe
Ala His Ser Leu Ser Pro Thr Leu Glu 130 135 140 His Ser Ser Leu Phe
Ser Gly Ser Tyr Ser Ser Leu Pro Pro Asn Pro 145 150 155 160 Leu Asn
Ser Arg Ala Val Glu Asp Ile Ser Ser Ser Arg Thr Asn Pro 165 170 175
Tyr Ser Tyr Ala Met Ser Thr Glu Glu Ala Arg Phe Leu Thr Tyr His 180
185 190 Met Trp Pro Leu Thr Phe Leu Ser Pro Ser Glu Leu Ala Arg Ala
Gly 195 200 205 Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val Ala Cys Phe
Ala Cys Gly 210 215 220 Gly Lys Leu Ser Asn Trp Glu Pro Lys Asp Asp
Ala Met Ser Glu His 225 230 235 240 Arg Arg His Phe Pro Asn Cys Pro
Phe Leu Glu Asn Ser Leu Glu Thr 245 250 255 Leu Arg Phe Ser Ile Ser
Asn Leu Ser Met Gln Thr His Ala Ala Arg 260 265 270 Met Arg Thr Phe
Met Tyr Trp Pro Ser Ser Val Pro Val Gln Pro Glu 275 280 285 Gln Leu
Ala Ser Ala Gly Phe Tyr Tyr Val Gly Arg Asn Asp Asp Val 290 295 300
Lys Cys Phe Gly Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser Gly Asp 305
310 315 320 Asp Pro Trp Val Glu His Ala Lys Trp Phe Pro Arg Cys Glu
Phe Leu 325 330 335 Ile Arg Met Lys Gly Gln Glu Phe Val Asp Glu Ile
Gln Gly Arg Tyr 340 345 350 Pro His Leu Leu Glu Gln Leu Leu Ser Thr
Ser Asp Thr Thr Gly Glu 355 360 365 Glu Asn Ala Asp Pro Pro Ile Ile
His Phe Gly Pro Gly Glu Ser Ser 370 375 380 Ser Glu Asp Ala Val Met
Met Asn Thr Pro Val Val Lys Ser Ala Leu 385 390 395 400 Glu Met Gly
Phe Asn Arg Asp Leu Val Lys Gln Thr Val Leu Ser Lys 405 410 415 Ile
Leu Thr Thr Gly Glu Asn Tyr Lys Thr Val Asn Asp Ile Val Ser 420 425
430 Ala Leu Leu Asn Ala Glu Asp Glu Lys Arg Glu Glu Glu Lys Glu Lys
435 440 445 Gln Ala Glu Glu Met Ala Ser Asp Asp Leu Ser Leu Ile Arg
Lys Asn 450 455 460 Arg Met Ala Leu Phe Gln Gln Leu Thr Cys Val Leu
Pro Ile Leu Asp 465 470 475 480 Asn Leu Leu Lys Ala Asn Val Ile Asn
Lys Gln Glu His Asp Ile Ile 485 490 495 Lys Gln Lys Thr Gln Ile Pro
Leu Gln Ala Arg Glu Leu Ile Asp Thr 500 505 510 Ile Trp Val Lys Gly
Asn Ala Ala Ala Asn Ile Phe Lys Asn Cys Leu 515 520 525 Lys Glu Ile
Asp Ser Thr Leu Tyr Lys Asn Leu Phe Val Asp Lys Asn 530 535 540 Met
Lys Tyr Ile Pro Thr Glu Asp Val Ser Gly Leu Ser Leu Glu Glu 545 550
555 560 Gln Leu Arg Arg Leu Gln Glu Glu Arg Thr Cys Lys Val Cys Met
Asp 565 570 575 Lys Glu Val Ser Val Val Phe Ile Pro Cys Gly His Leu
Val Val Cys 580 585 590 Gln Glu Cys Ala Pro Ser Leu Arg Lys Cys Pro
Ile Cys Arg Gly Ile 595 600 605 Ile Lys Gly Thr Val Arg Thr Phe Leu
Ser 610 615 9 2100 DNA Mus musculus 9 gacactctgc tgggcggcgg
gccgccctcc tccgggacct cccctcggga accgtcgccc 60 gcggcgctta
gttaggactg gagtgcttgg cgcgaaaagg tggacaagtc ctattttcca 120
gagaagatga cttttaacag ttttgaagga actagaactt ttgtacttgc agacaccaat
180 aaggatgaag aatttgtaga agagtttaat agattaaaaa catttgctaa
cttcccaagt 240 agtagtcctg tttcagcatc aacattggcg cgagctgggt
ttctttatac cggtgaagga 300 gacaccgtgc aatgtttcag ttgtcatgcg
gcaatagata gatggcagta tggagactca 360 gctgttggaa gacacaggag
aatatcccca aattgcagat ttatcaatgg tttttatttt 420 gaaaatggtg
ctgcacagtc tacaaatcct ggtatccaaa atggccagta caaatctgaa 480
aactgtgtgg gaaatagaaa tccttttgcc cctgacaggc cacctgagac tcatgctgat
540 tatctcttga gaactggaca ggttgtagat atttcagaca ccatataccc
gaggaaccct 600 gccatgtgta gtgaagaagc cagattgaag tcatttcaga
actggccgga ctatgctcat 660 ttaaccccca gagagttagc tagtgctggc
ctctactaca caggggctga tgatcaagtg 720 caatgctttt gttgtggggg
aaaactgaaa aattgggaac cctgtgatcg tgcctggtca 780 gaacacagga
gacactttcc caattgcttt tttgttttgg gccggaacgt taatgttcga 840
agtgaatctg gtgtgagttc tgataggaat ttcccaaatt caacaaactc tccaagaaat
900 ccagccatgg cagaatatga agcacggatc gttacttttg gaacatggat
atactcagtt 960 aacaaggagc agcttgcaag agctggattt tatgctttag
gtgaaggcga taaagtgaag 1020 tgcttccact gtggaggagg gctcacggat
tggaagccaa gtgaagaccc ctgggaccag 1080 catgctaagt gctacccagg
gtgcaaatac ctattggatg agaaggggca agaatatata 1140 aataatattc
atttaaccca tccacttgag gaatctttgg gaagaactgc tgaaaaaaca 1200
ccaccgctaa ctaaaaaaat cgatgatacc atcttccaga atcctatggt gcaagaagct
1260 atacgaatgg gatttagctt caaggacctt aagaaaacaa tggaagaaaa
aatccaaaca 1320 tccgggagca gctatctatc acttgaggtc ctgattgcag
atcttgtgag tgctcagaaa 1380 gataatacgg aggatgagtc aagtcaaact
tcattgcaga aagacattag tactgaagag 1440 cagctaaggc gcctacaaga
ggagaagctt tccaaaatct gtatggatag aaatattgct 1500 atcgtttttt
ttccttgtgg acatctggcc acttgtaaac agtgtgcaga agcagttgac 1560
aaatgtccca tgtgctacac cgtcattacg ttcaaccaaa aaatttttat gtcttagtgg
1620 ggcaccacat gttatgttct tcttgctcta attgaatgtg taatgggagc
gaactttaag 1680 taatcctgca tttgcattcc attagcatcc tgctgtttcc
aaatggagac caatgctaac 1740 agcactgttt ccgtctaaac attcaatttc
tggatctttc gagttatcag ctgtatcatt 1800 tagccagtgt tttactcgat
tgaaacctta gacagagaag cattttatag cttttcacat 1860 gtatattggt
agtacactga cttgatttct atatgtaagt gaattcatca cctgcatgtt 1920
tcatgccttt tgcataagct taacaaatgg agtgttctgt ataagcatgg agatgtgatg
1980 gaatctgccc aatgacttta attggcttat tgtaaacacg gaaagaactg
ccccacgctg 2040 ctgggaggat aaagattgtt ttagatgctc acttctgtgt
tttaggattc tgcccattta 2100 10 496 PRT Mus musculus 10 Met Thr Phe
Asn Ser Phe Glu Gly Thr Arg Thr Phe Val Leu Ala Asp 1 5 10 15 Thr
Asn Lys Asp Glu Glu Phe Val Glu Glu Phe Asn Arg Leu Lys Thr 20 25
30 Phe Ala Asn Phe Pro Ser Ser Ser Pro Val Ser Ala Ser Thr Leu Ala
35 40 45 Arg Ala Gly Phe Leu Tyr Thr Gly Glu Gly Asp Thr Val Gln
Cys Phe 50 55 60 Ser Cys His Ala Ala Ile Asp Arg Trp Gln Tyr Gly
Asp Ser Ala Val 65 70 75 80 Gly Arg His Arg Arg Ile Ser Pro Asn Cys
Arg Phe Ile Asn Gly Phe 85 90 95 Tyr Phe Glu Asn Gly Ala Ala Gln
Ser Thr Asn Pro Gly Ile Gln Asn 100 105 110 Gly Gln Tyr Lys Ser Glu
Asn Cys Val Gly Asn Arg Asn Pro Phe Ala 115 120 125 Pro Asp Arg Pro
Pro Glu Thr His Ala Asp Tyr Leu Leu Arg Thr Gly 130 135 140 Gln Val
Val Asp Ile Ser Asp Thr Ile Tyr Pro Arg Asn Pro Ala Met 145 150 155
160 Cys Ser Glu Glu Ala Arg Leu Lys Ser Phe Gln Asn Trp Pro Asp Tyr
165 170 175 Ala His Leu Thr Pro Arg Glu Leu Ala Ser Ala Gly Leu Tyr
Tyr Thr 180 185 190 Gly Ala Asp Asp Gln Val Gln Cys Phe Cys Cys Gly
Gly Lys Leu Lys 195 200 205 Asn Trp Glu Pro Cys Asp Arg Ala Trp Ser
Glu His Arg Arg His Phe 210 215 220 Pro Asn Cys Phe Phe Val Leu Gly
Arg Asn Val Asn Val Arg Ser Glu 225 230 235 240 Ser Gly Val Ser Ser
Asp Arg Asn Phe Pro Asn Ser Thr Asn Ser Pro 245 250 255 Arg Asn Pro
Ala Met Ala Glu Tyr Glu Ala Arg Ile Val Thr Phe Gly 260 265 270 Thr
Trp Ile Tyr Ser Val Asn Lys Glu Gln Leu Ala Arg Ala Gly Phe 275 280
285 Tyr Ala Leu Gly Glu Gly Asp Lys Val Lys Cys Phe His Cys Gly Gly
290 295 300 Gly Leu Thr Asp Trp Lys Pro Ser Glu Asp Pro Trp Asp Gln
His Ala 305 310 315 320 Lys Cys Tyr Pro Gly Cys Lys Tyr Leu Leu Asp
Glu Lys Gly Gln Glu 325 330 335 Tyr Ile Asn Asn Ile His Leu Thr His
Pro Leu Glu Glu Ser Leu Gly 340 345 350 Arg Thr Ala Glu Lys Thr Pro
Pro Leu Thr Lys Lys Ile Asp Asp Thr 355 360 365 Ile Phe Gln Asn Pro
Met Val Gln Glu Ala Ile Arg Met Gly Phe Ser 370 375 380 Phe Lys Asp
Leu Lys Lys Thr Met Glu Glu Lys Ile Gln Thr Ser Gly 385 390 395 400
Ser Ser Tyr Leu Ser Leu Glu Val Leu Ile Ala Asp Leu Val Ser Ala 405
410 415 Gln Lys Asp Asn Thr Glu Asp Glu Ser Ser Gln Thr Ser Leu Gln
Lys 420 425 430 Asp Ile Ser Thr Glu Glu Gln Leu Arg Arg Leu Gln Glu
Glu Lys Leu 435 440 445 Ser Lys Ile Cys Met Asp Arg Asn Ile Ala Ile
Val Phe Phe Pro Cys 450 455 460 Gly His Leu Ala Thr Cys Lys Gln Cys
Ala Glu Ala Val Asp Lys Cys 465 470 475 480 Pro Met Cys Tyr Thr Val
Ile Thr Phe Asn Gln Lys Ile Phe Met Ser 485 490 495 11 67 PRT
Orgyia pseudotsugata 11 Lys Ala Ala Arg Leu Gly Thr Tyr Thr Asn Trp
Pro Val Gln Phe Leu 1 5 10 15 Glu Pro Ser Arg Met Ala Ala Ser Gly
Phe Tyr Tyr Leu Gly Arg Gly 20 25 30 Asp Glu Val Arg Cys Ala Phe
Cys Lys Val Glu Ile Thr Asn Trp Val 35 40 45 Arg Gly Asp Asp Pro
Glu Thr Asp His Lys Arg Trp Ala Pro Gln Cys 50 55 60 Pro Phe Val 65
12 275 PRT Cydia pomonella 12 Met Ser Asp Leu Arg Leu Glu Glu Val
Arg Leu Asn Thr Phe Glu Lys 1 5 10 15 Trp Pro Val Ser Phe Leu Ser
Pro Glu Thr Met Ala Lys Asn Gly Phe 20 25 30 Tyr Tyr Leu Gly Arg
Ser Asp Glu Val Arg Cys Ala Phe Cys Lys Val 35 40 45 Glu Ile Met
Arg Trp Lys Glu Gly Glu Asp Pro Ala Ala Asp His Lys 50 55 60 Lys
Trp Ala Pro Gln Cys Pro Phe Val Lys Gly Ile Asp Val Cys Gly 65 70
75 80 Ser Ile Val Thr Thr Asn Asn Ile Gln Asn Thr Thr Thr His Asp
Thr 85 90 95 Ile Ile Gly Pro Ala His Pro Lys Tyr Ala His Glu Ala
Ala Arg Val 100 105 110 Lys Ser Phe His Asn Trp Pro Arg Cys Met Lys
Gln Arg Pro Glu Gln 115 120 125 Met Ala Asp Ala Gly Phe Phe Tyr Thr
Gly Tyr Gly Asp Asn Thr Lys 130 135 140 Cys Phe Tyr Cys Asp Gly Gly
Leu Lys Asp Trp Glu Pro Glu Asp Val 145 150 155 160 Pro Trp Glu Gln
His Val Arg Trp Phe Asp Arg Cys Ala Tyr Val Gln 165 170 175 Leu Val
Lys Gly Arg Asp Tyr Val Gln Lys Val Ile Thr Glu Ala Cys 180 185 190
Val Leu Pro Gly Glu Asn Thr Thr Val Ser Thr Ala Ala Pro Val Ser 195
200 205 Glu Pro Ile Pro Glu Thr Lys Ile Glu Lys Glu Pro Gln Val Glu
Asp 210 215 220 Ser Lys Leu Cys Lys Ile Cys Tyr Val Glu Glu Cys Ile
Val Cys Phe 225 230 235 240 Val Pro Cys Gly His Val Val Ala Cys Ala
Lys Cys Ala Leu Ser Val 245 250 255 Asp Lys Cys Pro Met Cys Arg Lys
Ile Val Thr Ser Val Leu Lys Val 260 265 270 Tyr Phe Ser 275 13 498
PRT Drosophila melanogaster 13 Met Thr Glu Leu Gly Met Glu Leu Glu
Ser Val Arg Leu Ala Thr Phe 1 5 10 15 Gly Glu Trp Pro Leu Asn Ala
Pro Val Ser Ala Glu Asp Leu Val Ala 20 25 30 Asn Gly Phe Phe Ala
Thr Gly Lys Trp Leu Glu Ala Glu Cys His Phe 35 40 45 Cys His Val
Arg Ile Asp Arg Trp Glu Tyr Gly Asp Gln Val Ala Glu 50 55 60 Arg
His Arg Arg Ser Ser Pro Ile Cys Ser Met Val Leu Ala Pro Asn 65 70
75 80 His Cys Gly Asn Val Pro Arg Ser Gln Glu Ser Asp Asn Glu Gly
Asn 85 90 95 Ser Val Val Asp Ser Pro Glu Ser Cys Ser Cys Pro Asp
Leu Leu Leu 100 105 110 Glu Ala Asn Arg Leu Val Thr Phe Lys Asp Trp
Pro Asn Pro Asn Ile 115 120 125 Thr Pro Gln Ala Leu Ala Lys Ala Gly
Phe Tyr Tyr Leu Asn Arg Leu 130 135 140 Asp His Val Lys Cys Val Trp
Cys Asn Gly Val Ile Ala Lys Trp Glu 145 150 155 160 Lys Asn Asp Asn
Ala Phe Glu Glu His Lys Arg Phe Phe Pro Gln Cys 165 170 175 Pro Arg
Val Gln Met Gly Pro Leu Ile Glu Phe Ala Thr Gly Lys Asn 180 185 190
Leu Asp Glu Leu Gly Ile Gln Pro Thr Thr Leu Pro Leu Arg Pro Lys 195
200 205 Tyr Ala Cys Val Asp Ala Arg Leu Arg Thr Phe Thr Asp Trp Pro
Ile 210 215 220 Ser Asn Ile Gln Pro Ala Ser Ala Leu Ala Gln Ala Gly
Leu Tyr Tyr 225 230 235 240 Gln Lys Ile Gly Asp Gln Val Arg Cys Phe
His Cys Asn Ile Gly Leu 245 250 255 Arg Ser Trp Gln Lys Glu Asp Glu
Pro Trp Phe Glu His Ala Lys Trp 260 265 270 Ser Pro Lys Cys Gln Phe
Val Leu Leu Ala Lys Gly Pro Ala Tyr Val 275 280 285 Ser Glu Val Leu
Ala Thr Thr Ala Ala Asn Ala Ser Ser Gln Pro Ala 290 295 300 Thr Ala
Pro Ala Pro Thr Leu Gln Ala Asp Val Leu Met Asp Glu Ala 305 310 315
320 Pro Ala Lys Glu Ala Leu Thr Leu Gly Ile Asp Gly Gly Val Val Arg
325 330 335 Asn Ala Ile Gln Arg Lys Leu Leu Ser Ser Gly Cys Ala Phe
Ser Thr 340 345 350 Leu Asp Glu Leu Leu His Asp Ile Phe Asp Asp Ala
Gly Ala Gly Ala 355 360 365 Ala Leu Glu Val Arg Glu Pro Pro Glu Pro
Ser Ala Pro Phe Ile Glu 370 375 380 Pro Cys Gln Ala Thr Thr Ser Lys
Ala
Ala Ser Val Pro Ile Pro Val 385 390 395 400 Ala Asp Ser Ile Pro Ala
Lys Pro Gln Ala Ala Glu Ala Val Ser Asn 405 410 415 Ile Ser Lys Ile
Thr Asp Glu Ile Gln Lys Met Ser Val Ser Thr Pro 420 425 430 Asn Gly
Asn Leu Ser Leu Glu Glu Glu Asn Arg Gln Leu Lys Asp Ala 435 440 445
Arg Leu Cys Lys Val Cys Leu Asp Glu Glu Val Gly Val Val Phe Leu 450
455 460 Pro Cys Gly His Leu Ala Thr Cys Asn Gln Cys Ala Pro Ser Val
Ala 465 470 475 480 Asn Cys Pro Met Cys Arg Ala Asp Ile Lys Gly Phe
Val Arg Thr Phe 485 490 495 Leu Ser 14 67 PRT Cydia pomonella 14
Glu Glu Val Arg Leu Asn Thr Phe Glu Lys Trp Pro Val Ser Phe Leu 1 5
10 15 Ser Pro Glu Thr Met Ala Lys Asn Gly Phe Tyr Tyr Leu Gly Arg
Ser 20 25 30 Asp Glu Val Arg Cys Ala Phe Cys Lys Val Glu Ile Met
Arg Trp Lys 35 40 45 Glu Gly Glu Asp Pro Ala Ala Asp His Lys Lys
Trp Ala Pro Gln Cys 50 55 60 Pro Phe Val 65 15 67 PRT Drosophila
melanogaster 15 Glu Ala Asn Arg Leu Val Thr Phe Lys Asp Trp Pro Asn
Pro Asn Ile 1 5 10 15 Thr Pro Gln Ala Leu Ala Lys Ala Gly Phe Tyr
Tyr Leu Asn Arg Leu 20 25 30 Asp His Val Lys Cys Val Trp Cys Asn
Gly Val Ile Ala Lys Trp Glu 35 40 45 Lys Asn Asp Asn Ala Phe Glu
Glu His Lys Arg Phe Phe Pro Gln Cys 50 55 60 Pro Arg Val 65 16 68
PRT Mus musculus 16 Glu Phe Asn Arg Leu Lys Thr Phe Ala Asn Phe Pro
Ser Ser Ser Pro 1 5 10 15 Val Ser Ala Ser Thr Leu Ala Arg Ala Gly
Phe Leu Tyr Thr Gly Glu 20 25 30 Gly Asp Thr Val Gln Cys Phe Ser
Cys His Ala Ala Ile Asp Arg Trp 35 40 45 Gln Tyr Gly Asp Ser Ala
Val Gly Arg His Arg Arg Ile Ser Pro Asn 50 55 60 Cys Arg Phe Ile 65
17 68 PRT Homo sapiens 17 Glu Phe Asn Arg Leu Lys Thr Phe Ala Asn
Phe Pro Ser Gly Ser Pro 1 5 10 15 Val Ser Ala Ser Thr Leu Ala Arg
Ala Gly Phe Leu Tyr Thr Gly Glu 20 25 30 Gly Asp Thr Val Arg Cys
Phe Ser Cys His Ala Ala Val Asp Arg Trp 35 40 45 Gln Tyr Gly Asp
Ser Ala Val Gly Arg His Arg Lys Val Ser Pro Asn 50 55 60 Cys Arg
Phe Ile 65 18 68 PRT Homo sapiens 18 Glu Leu Tyr Arg Met Ser Thr
Tyr Ser Thr Phe Pro Ala Gly Val Pro 1 5 10 15 Val Ser Glu Arg Ser
Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val 20 25 30 Asn Asp Lys
Val Lys Cys Phe Cys Cys Gly Leu Met Leu Asp Asn Trp 35 40 45 Lys
Arg Gly Asp Ser Pro Thr Glu Lys His Lys Lys Leu Tyr Pro Ser 50 55
60 Cys Arg Phe Val 65 19 68 PRT Homo sapiens 19 Glu Leu Tyr Arg Met
Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val Pro 1 5 10 15 Val Ser Glu
Arg Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val 20 25 30 Asn
Asp Lys Val Lys Cys Phe Cys Cys Gly Leu Met Leu Asp Asn Trp 35 40
45 Lys Leu Gly Asp Ser Pro Ile Gln Lys His Lys Gln Leu Tyr Pro Ser
50 55 60 Cys Ser Phe Ile 65 20 68 PRT Mus musculus 20 Glu Glu Ala
Arg Leu Lys Ser Phe Gln Asn Trp Pro Asp Tyr Ala His 1 5 10 15 Leu
Thr Pro Arg Glu Leu Ala Ser Ala Gly Leu Tyr Tyr Thr Gly Ala 20 25
30 Asp Asp Gln Val Gln Cys Phe Cys Cys Gly Gly Lys Leu Lys Asn Trp
35 40 45 Glu Pro Cys Asp Arg Ala Trp Ser Glu His Arg Arg His Phe
Pro Asn 50 55 60 Cys Phe Phe Val 65 21 68 PRT Homo sapiens 21 Glu
Glu Ala Arg Leu Lys Ser Phe Gln Asn Trp Pro Asp Tyr Ala His 1 5 10
15 Leu Thr Pro Arg Glu Leu Ala Ser Ala Gly Leu Tyr Tyr Thr Gly Ile
20 25 30 Gly Asp Gln Val Gln Cys Phe Cys Cys Gly Gly Lys Leu Lys
Asn Trp 35 40 45 Glu Pro Cys Asp Arg Ala Trp Ser Glu His Arg Arg
His Phe Pro Asn 50 55 60 Cys Phe Phe Val 65 22 67 PRT Homo sapiens
22 Glu Asn Ala Arg Leu Leu Thr Phe Gln Thr Trp Pro Leu Thr Phe Leu
1 5 10 15 Ser Pro Thr Asp Leu Ala Arg Ala Gly Phe Tyr Tyr Ile Gly
Pro Gly 20 25 30 Asp Arg Val Ala Cys Phe Ala Cys Gly Gly Lys Leu
Ser Asn Trp Glu 35 40 45 Pro Lys Asp Asn Ala Met Ser Glu His Leu
Arg His Phe Pro Lys Cys 50 55 60 Pro Phe Ile 65 23 67 PRT Homo
sapiens 23 Glu Glu Ala Arg Phe Leu Thr Tyr His Met Trp Pro Leu Thr
Phe Leu 1 5 10 15 Ser Pro Ser Glu Leu Ala Arg Ala Gly Phe Tyr Tyr
Ile Gly Pro Gly 20 25 30 Asp Arg Val Ala Cys Phe Ala Cys Gly Gly
Lys Leu Ser Asn Trp Glu 35 40 45 Pro Lys Asp Asp Ala Met Ser Glu
His Arg Arg His Phe Pro Asn Cys 50 55 60 Pro Phe Leu 65 24 66 PRT
Mus musculus 24 Tyr Glu Ala Arg Ile Val Thr Phe Gly Thr Trp Ile Tyr
Ser Val Asn 1 5 10 15 Lys Glu Gln Leu Ala Arg Ala Gly Phe Tyr Ala
Leu Gly Glu Gly Asp 20 25 30 Lys Val Lys Cys Phe His Cys Gly Gly
Gly Leu Thr Asp Trp Lys Pro 35 40 45 Ser Glu Asp Pro Trp Asp Gln
His Ala Lys Cys Tyr Pro Gly Cys Lys 50 55 60 Tyr Leu 65 25 66 PRT
Homo sapiens 25 Tyr Glu Ala Arg Ile Phe Thr Phe Gly Thr Trp Ile Tyr
Ser Val Asn 1 5 10 15 Lys Glu Gln Leu Ala Arg Ala Gly Phe Tyr Ala
Leu Gly Glu Gly Asp 20 25 30 Lys Val Lys Cys Phe His Cys Gly Gly
Gly Leu Thr Asp Trp Lys Pro 35 40 45 Ser Glu Asp Pro Trp Glu Gln
His Ala Lys Trp Tyr Pro Gly Cys Lys 50 55 60 Tyr Leu 65 26 68 PRT
Homo sapiens 26 His Ala Ala Arg Phe Lys Thr Phe Phe Asn Trp Pro Ser
Ser Val Leu 1 5 10 15 Val Asn Pro Glu Gln Leu Ala Ser Ala Gly Phe
Tyr Tyr Val Gly Asn 20 25 30 Ser Asp Asp Val Lys Cys Phe Cys Cys
Asp Gly Gly Leu Arg Cys Trp 35 40 45 Glu Ser Gly Asp Asp Pro Trp
Val Gln His Ala Lys Trp Phe Pro Arg 50 55 60 Cys Glu Tyr Leu 65 27
68 PRT Homo sapiens 27 His Ala Ala Arg Met Arg Thr Phe Met Tyr Trp
Pro Ser Ser Val Pro 1 5 10 15 Val Gln Pro Glu Gln Leu Ala Ser Ala
Gly Phe Tyr Tyr Val Gly Arg 20 25 30 Asn Asp Asp Val Lys Cys Phe
Gly Cys Asp Gly Gly Leu Arg Cys Trp 35 40 45 Glu Ser Gly Asp Asp
Pro Trp Val Glu His Ala Lys Trp Phe Pro Arg 50 55 60 Cys Glu Phe
Leu 65 28 68 PRT Orgyia pseudotsugata 28 Glu Ala Ala Arg Leu Arg
Thr Phe Ala Glu Trp Pro Arg Gly Leu Lys 1 5 10 15 Gln Arg Pro Glu
Glu Leu Ala Glu Ala Gly Phe Phe Tyr Thr Gly Gln 20 25 30 Gly Asp
Lys Thr Arg Cys Phe Cys Cys Asp Gly Gly Leu Lys Asp Trp 35 40 45
Glu Pro Asp Asp Ala Pro Trp Gln Gln His Ala Arg Trp Tyr Asp Arg 50
55 60 Cys Glu Tyr Val 65 29 68 PRT Cydia pomonella 29 Glu Ala Ala
Arg Val Lys Ser Phe His Asn Trp Pro Arg Cys Met Lys 1 5 10 15 Gln
Arg Pro Glu Gln Met Ala Asp Ala Gly Phe Phe Tyr Thr Gly Tyr 20 25
30 Gly Asp Asn Thr Lys Cys Phe Tyr Cys Asp Gly Gly Leu Lys Asp Trp
35 40 45 Glu Pro Glu Asp Val Pro Trp Glu Gln His Val Arg Trp Phe
Asp Arg 50 55 60 Cys Ala Tyr Val 65 30 68 PRT Drosophila
melanogaster 30 Val Asp Ala Arg Leu Arg Thr Phe Thr Asp Trp Pro Ile
Ser Asn Ile 1 5 10 15 Gln Pro Ala Ser Ala Leu Ala Gln Ala Gly Leu
Tyr Tyr Gln Lys Ile 20 25 30 Gly Asp Gln Val Arg Cys Phe His Cys
Asn Ile Gly Leu Arg Ser Trp 35 40 45 Gln Lys Glu Asp Glu Pro Trp
Phe Glu His Ala Lys Trp Ser Pro Lys 50 55 60 Cys Gln Phe Val 65 31
66 PRT Drosophila melanogaster 31 Glu Ser Val Arg Leu Ala Thr Phe
Gly Glu Trp Pro Leu Asn Ala Pro 1 5 10 15 Val Ser Ala Glu Asp Leu
Val Ala Asn Gly Phe Phe Gly Thr Trp Met 20 25 30 Glu Ala Glu Cys
Asp Phe Cys His Val Arg Ile Asp Arg Trp Glu Tyr 35 40 45 Gly Asp
Leu Val Ala Glu Arg His Arg Arg Ser Ser Pro Ile Cys Ser 50 55 60
Met Val 65 32 46 PRT Homo sapiens 32 Glu Gln Leu Arg Arg Leu Gln
Glu Glu Arg Thr Cys Lys Val Cys Met 1 5 10 15 Asp Lys Glu Val Ser
Val Val Phe Ile Pro Cys Gly His Leu Val Val 20 25 30 Cys Gln Glu
Cys Ala Pro Ser Leu Arg Lys Cys Pro Ile Cys 35 40 45 33 46 PRT Homo
sapiens 33 Glu Gln Leu Arg Arg Leu Pro Glu Glu Arg Thr Cys Lys Val
Cys Met 1 5 10 15 Asp Lys Glu Val Ser Ile Val Phe Ile Pro Cys Gly
His Leu Val Val 20 25 30 Cys Lys Asp Cys Ala Pro Ser Leu Arg Lys
Cys Pro Ile Cys 35 40 45 34 46 PRT Mus musculus 34 Glu Gln Leu Arg
Arg Leu Gln Glu Glu Lys Leu Ser Lys Ile Cys Met 1 5 10 15 Asp Arg
Asn Ile Ala Ile Val Phe Phe Pro Cys Gly His Leu Ala Thr 20 25 30
Cys Lys Gln Cys Ala Glu Ala Val Asp Lys Cys Pro Met Cys 35 40 45 35
46 PRT Homo sapiens 35 Glu Gln Leu Arg Arg Leu Gln Glu Glu Lys Leu
Cys Lys Ile Cys Met 1 5 10 15 Asp Arg Asn Ile Ala Ile Val Phe Val
Pro Cys Gly His Leu Val Thr 20 25 30 Cys Lys Gln Cys Ala Glu Ala
Val Asp Lys Cys Pro Met Cys 35 40 45 36 46 PRT Drosophila
melanogaster 36 Glu Glu Asn Arg Gln Leu Lys Asp Ala Arg Leu Cys Lys
Val Cys Leu 1 5 10 15 Asp Glu Glu Val Gly Val Val Phe Leu Pro Cys
Gly His Leu Ala Thr 20 25 30 Cys Asn Gln Cys Ala Pro Ser Val Ala
Asn Cys Pro Met Cys 35 40 45 37 46 PRT Cydia pomonella 37 Glu Lys
Glu Pro Gln Val Glu Asp Ser Lys Leu Cys Lys Ile Cys Tyr 1 5 10 15
Val Glu Glu Cys Ile Val Cys Phe Val Pro Cys Gly His Val Val Ala 20
25 30 Cys Ala Lys Cys Ala Leu Ser Val Asp Lys Cys Pro Met Cys 35 40
45 38 46 PRT Orgyia pseudotsugata 38 Ala Val Glu Ala Glu Val Ala
Asp Asp Arg Leu Cys Lys Ile Cys Leu 1 5 10 15 Gly Ala Glu Lys Thr
Val Cys Phe Val Pro Cys Gly His Val Val Ala 20 25 30 Cys Gly Lys
Cys Ala Ala Gly Val Thr Thr Cys Pro Val Cys 35 40 45 39 2474 DNA
Mus musculus 39 gaattccggg agacctacac ccccggagat cagaggtcat
tgctggcgtt cagagcctag 60 gaagtgggct gcggtatcag cctagcagta
aaaccgacca gaagccatgc acaaaactac 120 atccccagag aaagacttgt
cccttcccct ccctgtcatc tcaccatgaa catggttcaa 180 gacagcgcct
ttctagccaa gctgatgaag agtgctgaca cctttgagtt gaagtatgac 240
ttttcctgtg agctgtaccg attgtccacg tattcagctt ttcccagggg agttcctgtg
300 tcagaaagga gtctggctcg tgctggcttt tactacactg gtgccaatga
caaggtcaag 360 tgcttctgct gtggcctgat gctagacaac tggaaacaag
gggacagtcc catggagaag 420 cacagaaagt tgtaccccag ctgcaacttt
gtacagactt tgaatccagc caacagtctg 480 gaagctagtc ctcggccttc
tcttccttcc acggcgatga gcaccatgcc tttgagcttt 540 gcaagttctg
agaatactgg ctatttcagt ggctcttact cgagctttcc ctcagaccct 600
gtgaacttcc gagcaaatca agattgtcct gctttgagca caagtcccta ccactttgca
660 atgaacacag agaaggccag attactcacc tatgaaacat ggccattgtc
ttttctgtca 720 ccagcaaagc tggccaaagc aggcttctac tacataggac
ctggagatag agtggcctgc 780 tttgcgtgcg atgggaaact gagcaactgg
gaacgtaagg atgatgctat gtcagagcac 840 cagaggcatt tccccagctg
tccgttctta aaagacttgg gtcagtctgc ttcgagatac 900 actgtctcta
acctgagcat gcagacacac gcagcccgta ttagaacatt ctctaactgg 960
ccttctagtg cactagttca ttcccaggaa cttgcaagtg cgggctttta ttatacagga
1020 cacagtgatg atgtcaagtg tttatgctgt gatggtgggc tgaggtgctg
ggaatctgga 1080 gatgacccct gggtggaaca tgccaagtgg tttccaaggt
gtgagtactt gctcagaatc 1140 aaaggccaag aatttgtcag ccaagttcaa
gctggctatc ctcatctact tgagcagcta 1200 ttatctacgt cagactcccc
agaagatgag aatgcagacg cagcaatcgt gcattttggc 1260 cctggagaaa
gttcggaaga tgtcgtcatg atgagcacgc ctgtggttaa agcagccttg 1320
gaaatgggct tcagtaggag cctggtgaga cagacggttc agtggcagat cctggccact
1380 ggtgagaact acaggaccgt cagtgacctc gttataggct tactcgatgc
agaagacgag 1440 atgagagagg agcagatgga gcaggcggcc gaggaggagg
agtcagatga tctagcacta 1500 atccggaaga acaaaatggt gcttttccaa
catttgacgt gtgtgacacc aatgctgtat 1560 tgcctcctaa gtgcaagggc
catcactgaa caggagtgca atgctgtgaa acagaaacca 1620 cacaccttac
aagcaagcac actgattgat actgtgttag caaaaggaaa cactgcagca 1680
acctcattca gaaactccct tcgggaaatt gaccctgcgt tatacagaga tatatttgtg
1740 caacaggaca ttaggagtct tcccacagat gacattgcag ctctaccaat
ggaagaacag 1800 ttgcggcccc tcccggagga cagaatgtgt aaagtgtgta
tggaccgaga ggtatccatc 1860 gtgttcattc cctgtggcca tctggtcgtg
tgcaaagact gcgctccctc tctgaggaag 1920 tgtcccatct gtagagggac
catcaagggc acagtgcgca catttctctc ctgaacaaga 1980 ctaatggtcc
atggctgcaa cttcagccag gaggaagttc actgtcactc ccagttccat 2040
tcggaacttg aggccagcct ggatagcacg agacaccgcc aaacacacaa atataaacat
2100 gaaaaacttt tgtctgaagt caagaatgaa tgaattactt atataataat
tttaattggt 2160 ttccttaaaa gtgctatttg ttcccaactc agaaaattgt
tttctgtaaa catatttaca 2220 tactacctgc atctaaagta ttcatatatt
catatattca gatgtcatga gagagggttt 2280 tgttcttgtt cctgaaaagc
tggtttatca tctgatcagc atatactgcg caacgggcag 2340 ggctagaatc
catgaaccaa gctgcaaaga tctcacgcta aataaggcgg aaagatttgg 2400
agaaacgaaa ggaaattctt tcctgtccaa tgtatactct tcagactaat gacctcttcc
2460 tatcaagcct tcta 2474 40 602 PRT Mus musculus 40 Met Asn Met
Val Gln Asp Ser Ala Phe Leu Ala Lys Leu Met Lys Ser 1 5 10 15 Ala
Asp Thr Phe Glu Leu Lys Tyr Asp Phe Ser Cys Glu Leu Tyr Arg 20 25
30 Leu Ser Thr Tyr Ser Ala Phe Pro Arg Gly Val Pro Val Ser Glu Arg
35 40 45 Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Ala Asn Asp
Lys Val 50 55 60 Lys Cys Phe Cys Cys Gly Leu Met Leu Asp Asn Trp
Lys Gln Gly Asp 65 70 75 80 Ser Pro Met Glu Lys His Arg Lys Leu Tyr
Pro Ser Cys Asn Phe Val 85 90 95 Gln Thr Leu Asn Pro Ala Asn Ser
Leu Glu Ala Ser Pro Arg Pro Ser 100 105 110 Leu Pro Ser Thr Ala Met
Ser Thr Met Pro Leu Ser Phe Ala Ser Ser 115 120 125 Glu Asn Thr Gly
Tyr Phe Ser Gly Ser Tyr Ser Ser Phe Pro Ser Asp 130 135 140 Pro Val
Asn Phe Arg Ala Asn Gln Asp Cys Pro Ala Leu Ser Thr Ser 145 150 155
160 Pro Tyr His Phe Ala Met Asn Thr Glu Lys Ala Arg Leu Leu Thr Tyr
165 170 175 Glu Thr Trp Pro Leu Ser Phe Leu Ser Pro Ala Lys Leu Ala
Lys Ala 180 185 190 Gly Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val Ala
Cys Phe Ala Cys 195 200 205 Asp Gly Lys Leu Ser Asn Trp Glu Arg Lys
Asp Asp Ala Met Ser Glu 210 215 220 His Gln Arg His Phe Pro Ser Cys
Pro Phe Leu Lys Asp Leu Gly Gln 225 230 235 240 Ser Ala Ser Arg Tyr
Thr Val Ser Asn Leu Ser Met Gln Thr His Ala 245 250 255 Ala Arg Ile
Arg Thr Phe Ser Asn Trp Pro Ser Ser Ala Leu Val His 260 265 270 Ser
Gln Glu Leu Ala Ser Ala Gly Phe Tyr Tyr Thr Gly His Ser Asp 275 280
285 Asp Val Lys Cys Leu Cys Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser
290 295 300 Gly Asp Asp Pro Trp Val Glu His Ala Lys Trp Phe Pro Arg
Cys Glu 305 310 315 320 Tyr Leu Leu Arg Ile Lys Gly Gln Glu Phe Val
Ser Gln Val Gln Ala
325 330 335 Gly Tyr Pro His Leu Leu Glu Gln Leu Leu Ser Thr Ser Asp
Ser Pro 340 345 350 Glu Asp Glu Asn Ala Asp Ala Ala Ile Val His Phe
Gly Pro Gly Glu 355 360 365 Ser Ser Glu Asp Val Val Met Met Ser Thr
Pro Val Val Lys Ala Ala 370 375 380 Leu Glu Met Gly Phe Ser Arg Ser
Leu Val Arg Gln Thr Val Gln Trp 385 390 395 400 Gln Ile Leu Ala Thr
Gly Glu Asn Tyr Arg Thr Val Ser Asp Leu Val 405 410 415 Ile Gly Leu
Leu Asp Ala Glu Asp Glu Met Arg Glu Glu Gln Met Glu 420 425 430 Gln
Ala Ala Glu Glu Glu Glu Ser Asp Asp Leu Ala Leu Ile Arg Lys 435 440
445 Asn Lys Met Val Leu Phe Gln His Leu Thr Cys Val Thr Pro Met Leu
450 455 460 Tyr Cys Leu Leu Ser Ala Arg Ala Ile Thr Glu Gln Glu Cys
Asn Ala 465 470 475 480 Val Lys Gln Lys Pro His Thr Leu Gln Ala Ser
Thr Leu Ile Asp Thr 485 490 495 Val Leu Ala Lys Gly Asn Thr Ala Ala
Thr Ser Phe Arg Asn Ser Leu 500 505 510 Arg Glu Ile Asp Pro Ala Leu
Tyr Arg Asp Ile Phe Val Gln Gln Asp 515 520 525 Ile Arg Ser Leu Pro
Thr Asp Asp Ile Ala Ala Leu Pro Met Glu Glu 530 535 540 Gln Leu Arg
Pro Leu Pro Glu Asp Arg Met Cys Lys Val Cys Met Asp 545 550 555 560
Arg Glu Val Ser Ile Val Phe Ile Pro Cys Gly His Leu Val Val Cys 565
570 575 Lys Asp Cys Ala Pro Ser Leu Arg Lys Cys Pro Ile Cys Arg Gly
Thr 580 585 590 Ile Lys Gly Thr Val Arg Thr Phe Leu Ser 595 600 41
2416 DNA Mus musculus 41 ctgtggtgga gatctattgt ccaagtggtg
agaaacttca tctggaagtt taagcggtca 60 gaaatactat tactactcat
ggacaaaact gtctcccaga gactcgccca aggtacctta 120 cacccaaaaa
cttaaacgta taatggagaa gagcacaatc ttgtcaaatt ggacaaagga 180
gagcgaagaa aaaatgaagt ttgacttttc gtgtgaactc taccgaatgt ctacatattc
240 agcttttccc aggggagttc ctgtctcaga gaggagtctg gctcgtgctg
gcttttatta 300 tacaggtgtg aatgacaaag tcaagtgctt ctgctgtggc
ctgatgttgg ataactggaa 360 acaaggggac agtcctgttg aaaagcacag
acagttctat cccagctgca gctttgtaca 420 gactctgctt tcagccagtc
tgcagtctcc atctaagaat atgtctcctg tgaaaagtag 480 atttgcacat
tcgtcacctc tggaacgagg tggcattcac tccaacctgt gctctagccc 540
tcttaattct agagcagtgg aagacttctc atcaaggatg gatccctgca gctatgccat
600 gagtacagaa gaggccagat ttcttactta cagtatgtgg cctttaagtt
ttctgtcacc 660 agcagagctg gccagagctg gcttctatta catagggcct
ggagacaggg tggcctgttt 720 tgcctgtggt gggaaactga gcaactggga
accaaaggat tatgctatgt cagagcaccg 780 cagacatttt ccccactgtc
catttctgga aaatacttca gaaacacaga ggtttagtat 840 atcaaatcta
agtatgcaga cacactctgc tcgattgagg acatttctgt actggccacc 900
tagtgttcct gttcagcccg agcagcttgc aagtgctgga ttctattacg tggatcgcaa
960 tgatgatgtc aagtgccttt gttgtgatgg tggcttgaga tgttgggaac
ctggagatga 1020 cccctggata gaacacgcca aatggtttcc aaggtgtgag
ttcttgatac ggatgaaggg 1080 tcaggagttt gttgatgaga ttcaagctag
atatcctcat cttcttgagc agctgttgtc 1140 cacttcagac accccaggag
aagaaaatgc tgaccctaca gagacagtgg tgcattttgg 1200 ccctggagaa
agttcgaaag atgtcgtcat gatgagcacg cctgtggtta aagcagcctt 1260
ggaaatgggc ttcagtagga gcctggtgag acagacggtt cagcggcaga tcctggccac
1320 tggtgagaac tacaggaccg tcaatgatat tgtctcagta cttttgaatg
ctgaagatga 1380 gagaagagaa gaggagaagg aaagacagac tgaagagatg
gcatcaggtg acttatcact 1440 gattcggaag aatagaatgg ccctctttca
acagttgaca catgtccttc ctatcctgga 1500 taatcttctt gaggccagtg
taattacaaa acaggaacat gatattatta gacagaaaac 1560 acagataccc
ttacaagcaa gagagcttat tgacaccgtt ttagtcaagg gaaatgctgc 1620
agccaacatc ttcaaaaact ctctgaaggg aattgactcc acgttatatg aaaacttatt
1680 tgtggaaaag aatatgaagt atattccaac agaagacgtt tcaggcttgt
cattggaaga 1740 gcagttgcgg agattacaag aagaacgaac ttgcaaagtg
tgtatggaca gagaggtttc 1800 tattgtgttc attccgtgtg gtcatctagt
agtctgccag gaatgtgccc cttctctaag 1860 gaagtgcccc atctgcaggg
ggacaatcaa ggggactgtg cgcacatttc tctcatgagt 1920 gaagaatggt
ctgaaagtat tgttggacat cagaagctgt cagaacaaag aatgaactac 1980
tgatttcagc tcttcagcag gacattctac tctctttcaa gattagtaat cttgctttat
2040 gaagggtagc attgtatatt taagcttagt ctgttgcaag ggaaggtcta
tgctgttgag 2100 ctacaggact gtgtctgttc cagagcagga gttgggatgc
ttgctgtatg tccttcagga 2160 cttcttggga tttgggaatt tggggaaagc
tttggaatcc agtgatgtgg agctcagaaa 2220 tcctggaacc agtgactctg
gtactcagta gatagggtac cctgtacttc ttggtgcttt 2280 tccagtctgg
gaaataagga ggaatctgct gctggtaaaa atttgctgga tgtgagaaat 2340
agatgaaagt gtttcgggtg ggggcgtgca tcagtgtagt gtgtgcaggg atgtatgcag
2400 gccaaacact gtgtag 2416 42 591 PRT Mus musculus 42 Met Glu Lys
Ser Thr Ile Leu Ser Asn Trp Thr Lys Glu Ser Glu Glu 1 5 10 15 Lys
Met Lys Phe Asp Phe Ser Cys Glu Leu Tyr Arg Met Ser Thr Tyr 20 25
30 Ser Ala Phe Pro Arg Gly Val Pro Val Ser Glu Arg Ser Leu Ala Arg
35 40 45 Ala Gly Phe Tyr Tyr Thr Gly Val Asn Asp Lys Val Lys Cys
Phe Cys 50 55 60 Cys Gly Leu Met Leu Asp Asn Trp Lys Gln Gly Asp
Ser Pro Val Glu 65 70 75 80 Lys His Arg Gln Phe Tyr Pro Ser Cys Ser
Phe Val Gln Thr Leu Leu 85 90 95 Ser Ala Ser Leu Gln Ser Pro Ser
Lys Asn Met Ser Pro Val Lys Ser 100 105 110 Arg Phe Ala His Ser Ser
Pro Leu Glu Arg Gly Gly Ile His Ser Asn 115 120 125 Leu Cys Ser Ser
Pro Leu Asn Ser Arg Ala Val Glu Asp Phe Ser Ser 130 135 140 Arg Met
Asp Pro Cys Ser Tyr Ala Met Ser Thr Glu Glu Ala Arg Phe 145 150 155
160 Leu Thr Tyr Ser Met Trp Pro Leu Ser Phe Leu Ser Pro Ala Glu Leu
165 170 175 Ala Arg Ala Gly Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val
Ala Cys 180 185 190 Phe Ala Cys Gly Gly Lys Leu Ser Asn Trp Glu Pro
Lys Asp Tyr Ala 195 200 205 Met Ser Glu His Arg Arg His Phe Pro His
Cys Pro Phe Leu Glu Asn 210 215 220 Thr Ser Glu Thr Gln Arg Phe Ser
Ile Ser Asn Leu Ser Met Gln Thr 225 230 235 240 His Ser Ala Arg Leu
Arg Thr Phe Leu Tyr Trp Pro Pro Ser Val Pro 245 250 255 Val Gln Pro
Glu Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Asp Arg 260 265 270 Asn
Asp Asp Val Lys Cys Leu Cys Cys Asp Gly Gly Leu Arg Cys Trp 275 280
285 Glu Pro Gly Asp Asp Pro Trp Ile Glu His Ala Lys Trp Phe Pro Arg
290 295 300 Cys Glu Phe Leu Ile Arg Met Lys Gly Gln Glu Phe Val Asp
Glu Ile 305 310 315 320 Gln Ala Arg Tyr Pro His Leu Leu Glu Gln Leu
Leu Ser Thr Ser Asp 325 330 335 Thr Pro Gly Glu Glu Asn Ala Asp Pro
Thr Glu Thr Val Val His Phe 340 345 350 Gly Pro Gly Glu Ser Ser Lys
Asp Val Val Met Met Ser Thr Pro Val 355 360 365 Val Lys Ala Ala Leu
Glu Met Gly Phe Ser Arg Ser Leu Val Arg Gln 370 375 380 Thr Val Gln
Arg Gln Ile Leu Ala Thr Gly Glu Asn Tyr Arg Thr Val 385 390 395 400
Asn Asp Ile Val Ser Val Leu Leu Asn Ala Glu Asp Glu Arg Arg Glu 405
410 415 Glu Glu Lys Glu Arg Gln Thr Glu Glu Met Ala Ser Gly Asp Leu
Ser 420 425 430 Leu Ile Arg Lys Asn Arg Met Ala Leu Phe Gln Gln Leu
Thr His Val 435 440 445 Leu Pro Ile Leu Asp Asn Leu Leu Glu Ala Ser
Val Ile Thr Lys Gln 450 455 460 Glu His Asp Ile Ile Arg Gln Lys Thr
Gln Ile Pro Leu Gln Ala Arg 465 470 475 480 Glu Leu Ile Asp Thr Val
Leu Val Lys Gly Asn Ala Ala Ala Asn Ile 485 490 495 Phe Lys Asn Ser
Leu Lys Gly Ile Asp Ser Thr Leu Tyr Glu Asn Leu 500 505 510 Phe Val
Glu Lys Asn Met Lys Tyr Ile Pro Thr Glu Asp Val Ser Gly 515 520 525
Leu Ser Leu Glu Glu Gln Leu Arg Arg Leu Gln Glu Glu Arg Thr Cys 530
535 540 Lys Val Cys Met Asp Arg Glu Val Ser Ile Val Phe Ile Pro Cys
Gly 545 550 555 560 His Leu Val Val Cys Gln Glu Cys Ala Pro Ser Leu
Arg Lys Cys Pro 565 570 575 Ile Cys Arg Gly Thr Ile Lys Gly Thr Val
Arg Thr Phe Leu Ser 580 585 590 43 11 PRT Artificial Sequence
Synthetic based on viral sequence 43 Met Glu Gln Lys Leu Ile Ser
Glu Glu Asp Leu 1 5 10 44 21 DNA Artificial Sequence Synthetic
primer based on Homo sapiens 44 agtgcgggtt tttattatgt g 21 45 25
DNA Artificial Sequence Synthetic primer based on Homo sapiens 45
agatgaccac aaggaataaa cacta 25
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