U.S. patent application number 11/789761 was filed with the patent office on 2009-01-22 for aib1, a novel steroid receptor co-activator.
This patent application is currently assigned to The Government of the United States of America as represented by the Secretary. Invention is credited to Paul Meltzer, Jeffrey Trent.
Application Number | 20090023645 11/789761 |
Document ID | / |
Family ID | 21961378 |
Filed Date | 2009-01-22 |
United States Patent
Application |
20090023645 |
Kind Code |
A1 |
Meltzer; Paul ; et
al. |
January 22, 2009 |
AIB1, a novel steroid receptor co-activator
Abstract
The invention features a substantially pure DNA which includes a
sequence encoding a novel steroid receptor co-activator which is
overexpressed in breast cancer cells, diagnostic assays for steroid
hormone-responsive cancers, and screening assays to identify
compounds which inhibit an interaction of the co-activator with the
steroid hormone.
Inventors: |
Meltzer; Paul; (Rockville,
MD) ; Trent; Jeffrey; (Rockville, MD) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 S.W. SALMON STREET, SUITE #1600
PORTLAND
OR
97204-2988
US
|
Assignee: |
The Government of the United States
of America as represented by the Secretary
of the Department of Health and Human Services
|
Family ID: |
21961378 |
Appl. No.: |
11/789761 |
Filed: |
April 24, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10379616 |
Mar 4, 2003 |
7232890 |
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11789761 |
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09125635 |
Aug 21, 1998 |
6562589 |
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PCT/US98/12689 |
Jun 17, 1998 |
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10379616 |
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60049728 |
Jun 17, 1997 |
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Current U.S.
Class: |
514/1.1 ;
435/6.14; 514/44R |
Current CPC
Class: |
A61P 35/04 20180101;
C07K 14/4705 20130101; A01K 2217/075 20130101; A61K 38/00 20130101;
A01K 2217/05 20130101 |
Class at
Publication: |
514/12 ; 435/6;
514/44; 514/2 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C12Q 1/68 20060101 C12Q001/68; A61K 31/711 20060101
A61K031/711; A61P 35/04 20060101 A61P035/04; A61K 38/02 20060101
A61K038/02 |
Claims
1. A method of screening a candidate compound which inhibits an
interaction of an AIB1 polypeptide with an ER polypeptide in a cell
comprising (a) providing a GAL4 binding site linked to a reporter
gene; (b) providing a GAL4 binding domain linked to either (i) an
AIB1 polypeptide or (ii) an ER polypeptide; (c) providing a GAL4
transactivation domain II linked to the ER polypeptide if the GAL4
binding domain is linked to the AIB1 polypeptide or linked to the
AIB1 polypeptide if the GAL4 binding domain is linked to the ER
polypeptide; (d) contacting the cell with the compound; and (e)
monitoring expression of the reporter gene, wherein a decrease in
expression in the presence of the compound compared to that in the
absence of the compound indicates that the compound inhibits an
interaction of an AIB1 polypeptide with the ER polypeptide.
2. A method of detecting an aberrantly proliferating cell in a
tissue sample comprising determining the level of AIB1 gene
expression in the sample, wherein an increase in the level of
expression compared to the level in normal control tissue indicates
the presence of an aberrantly proliferating cell.
3. The method of claim 2, wherein the aberrantly proliferating cell
is a steroid hormone-responsive cancer cell.
4. The method of claim 3, wherein the steroid hormone-responsive
cancer cell is a breast cancer cell.
5. The method of claim 3, wherein the cell is a steroid
hormone-responsive cancer cell is an ovarian cancer cell.
6. The method of claim 1, wherein the AIB1 gene expression is
measured using an AIB1 gene-specific polynucleotide probe.
7. The method of claim 1, wherein the AIB1 gene expression is
measured using an antibody that specifically binds AIB1.
8. A method of detecting breast cancer in a tissue sample,
comprising determining the number of cellular copies of an AIB1
gene in the tissue sample, wherein an increase in the number of
copies compared to the number of copies in a normal control tissue
indicates the presence of a breast carcinoma.
9. The method of claim 8, wherein the number of copies in the
tissue is greater than 2.
10. The method of claim 9, wherein the number of copies in the
tissue is greater than 10.
11. The method of claim 10, wherein the number of copies in the
tissue is greater than 20.
12. A method of reducing proliferation of a cancer cell in a mammal
comprising administering to the mammal a compound which inhibits
expression of AIB1.
13. The method of claim 12, wherein the compound reduces
transcription of DNA encoding AIB1 in the cell.
14. The method of claim 12, wherein the compound reduces
translation of an AIB1 mRNA into an AIB1 gene product in the
cell.
15. The method of claim 14, wherein the translation is reduced by
contacting the AIB1 mRNA with an antisense DNA complementary to the
AIB1 mRNA.
16. A method of inhibiting ER-dependent transcription in a breast
cell of an mammal, comprising administering an effective amount of
an AIB1 polypeptide to the mammal.
17. The method of claim 16, wherein the polypeptide comprises a PAS
domain.
18. The method of claim 16, wherein the polypeptide comprises a
bHLH domain.
19. The method of claim 16, wherein the polypeptide comprises an
ER-interacting domain
20. A method of identifying a tamoxifen-sensitive patient,
comprising (a) contacting a patient-derived tissue sample with
tamoxifen; and (b) determining the level of AIB1 gene expression in
the sample, wherein an increase in the level of expression compared
to the level in normal control tissue indicates that the patient is
tamoxifen-sensitive.
21. The method of claim 20, wherein the AIB1 gene expression is
measured using an AIB1 gene-specific polynucleotide probe.
22. The method of claim 20, wherein the AIB1 gene expression is
measured using an antibody that specifically binds AIB1.
23. A method of reducing proliferation of a cancer cell comprising
administering to the mammal a compound which inhibits interaction
of AIB1 with a molecule selected from the group consisting of
steroid receptors and nuclear co-factors.
24. The method of claim 23 wherein the molecule is p300 or CBP.
Description
PRIORITY CLAIM
[0001] This is a divisional of U.S. patent application Ser. No.
10/379,616, filed Mar. 4, 2003, incorporated by reference herein.
U.S. patent application Ser. No. 10/379,616 is a divisional of U.S.
patent application Ser. No. 09/125,635, filed Aug. 21, 1998, now
issued as U.S. Pat. No. 6,562,589. U.S. patent application Ser. No.
09/125,635 is a .sctn.371 U.S. national stage of PCT/US98/12689,
filed Jun. 17, 1998, which was published in English under PCT
Article 21(2), which claims the benefit of U.S. Provisional
Application No. 60/049,728, filed Jun. 17, 1997.
BACKGROUND OF THE INVENTION
[0002] Breast cancer arises from estrogen-responsive breast
epithelial cells. Estrogen activity is thought to promote the
development of breast cancer, and many breast cancers are initially
dependent on estrogen at the time of diagnosis. Anti-estrogen
compositions have therefore been used to treat breast cancer.
[0003] A frequent mechanism of increased gene expression in human
cancers is amplification, i.e., the copy number of a DNA sequence
is increased, in a cancer cell compared to a non-cancerous cell. In
breast cancer, commonly amplified regions are derived from 17q21,
8q24, and 11q13 which encode erbB-2, c-myc, and cyclic DI
respectively (Devilee et al., 1994, Crit. Rev. Oncog. 5:247-270).
Recently, molecular cytogenetic studies have revealed the
occurrence in breast cancers of additional regions of increased DNA
copy number (Isola et al., Am. J. Pathol. 147:905-911, 1995;
Kallioniemi et al., Proc. Natl. Acad. Sci. USA 91:2156-2160, 1994;
Muleris et al., Genes Chromo. Cancer 10:160-170, 1994; Tanner et
al., Cancer Research 54:4257-4260, 1994; Guan et al., Nat. Genet.
8:155-161, 1994).
[0004] Breast cancer is the second leading cause of cancer deaths
in American women, and it is estimated that an American woman has
at least a 10% cumulative lifetime risk of developing this disease.
Early diagnosis is an important factor in breast cancer prognosis
and affects not only survival rate, but the range of therapeutic
options available to the patient. For instance, if diagnosed early,
a "lumpectomy" may be performed, whereas later diagnosis tends to
be associated with more invasive and traumatic surgical treatments
such as radical mastectomy. The treatment of other cancers likewise
is benefitted by early diagnosis, for instance the prognosis in the
treatment of lung cancer, colorectal cancer and prostate cancers is
greatly improved by early diagnosis. There is a need for a simple
and reliable method of diagnosis of cancers in general and of
breast cancer in particular. There is a need for a method of
screening for compounds that inhibit the interaction between an
estrogen receptor ER and an ER-dependent nuclear receptor
co-activator molecule in order to identify molecules useful in
research diagnosis and treatment of cancer. There is also a need
for a method for identifying tamoxifen-sensitive cancer patients in
order to better manage treatment. A solution to these needs would
improve cancer treatment and research and would save lives.
SUMMARY OF THE INVENTION
[0005] The inventors have discovered that the AIB1 protein
(Amplified In Breast Cancer-1) is a member of the Steroid Receptor
Coactivator-1 (SRC-1) family of nuclear receptor co-activators that
interacts with estrogen receptors (ER) to enhance ER-dependent
transcription. The inventors have further discovered that the AIB1
gene is amplified and over-expressed in certain cancers including
breast cancer, and that detection of amplified AIB1 genes can
therefore be used to detect cancerous cells. Importantly, the
inventors have also found that AIB1 amplification is not confined
to breast cancer but is also found in cancers of the lung, ovary,
head and neck, colon, testicles, bladder, prostate, endometrium,
kidney, stomach and also in pheochromocytoma, melanoma, ductal
carcinoma and carcinoid tumor. Such a finding means that AIB1 may
be useful in the detection and treatment of all of the
aforementioned cancers which include some of the most prevalent and
deadly diseases in the western world.
[0006] The inventors have also discovered that AIB1 interacts with
the proteins p300 and CBP, which are nuclear cofactors that
interact with other nuclear factors to promote transcription
(Chacravarti et al., Nature (383) 99-103 1996; Lundblad et al.,
Nature (374) 85-88 1995). The inventors have, furthermore,
determined that in cells with stable over-expression of AIB1, there
is a dramatic increase in steroid receptor activation (almost a
100-fold increase) leading to a corresponding increase in
transcriptional activation. The inventors have also used monoclonal
anti-AIB1 antibodies to demonstrate that AIB1 gene amplification is
directly correlated with increased AIB1 expression, and that these
amplified copies of the gene are expressed in physiological
conditions. The inventors have found that AIB1 is the human
ortholog of the mouse ER-dependent transcriptional activator p/CIP,
with the proteins having an overall amino acid identity of 81.6%.
These finding support the physiological role for AIB1 in cancer
cells as a cofactor involved in transcriptional regulation.
[0007] The invention features a substantially pure DNA which
includes a sequence encoding an AIB1 polypeptide, e.g., a human
AIB1 polypeptide, or a fragment thereof. The DNA may have the
sequence of all or part of the naturally-occurring AIB1-encoding
DNA or a degenerate variant thereof. AIB1-encoding DNA may be
operably linked to regulatory sequences for expression of the
polypeptide. A cell containing AIB1 encoding DNA is also within the
invention.
[0008] The invention also includes a substantially pure DNA
containing a polynucleotides which hybridizes at high stringency to
a AIB1-encoding DNA or the complement thereof. A substantially pure
DNA containing a nucleotide sequence having at least 50% sequence
identity to the full length AIB1 cDNA, e.g., a nucleotide sequence
encoding a polypeptide having the biological activity of a AIB1
polypeptide, is also included.
[0009] The invention also features a substantially pure human AIB1
polypeptide and variants thereof, e.g., polypeptides with
conservative amino acid substitutions or polypeptides with
conservative or non-conservative amino acid substitutions which
retain the biological activity of naturally-occurring AIB1.
[0010] Diagnostic methods, e.g., to identify cells which harbor an
abnormal copy number of the AIB1 DNA, are also encompassed by the
invention. An abnormal copy number, e.g., greater than the normal
diploid copy number, of AIB1 DNA is indicative of an aberrantly
proliferating cell, e.g., a steroid hormone-responsive cancer
cell.
[0011] The invention also includes antibodies, e.g., a monoclonal
antibody or polyclonal antisera, which bind specifically to AIB1
and can be used to detect the level of expression of AIB1 in a cell
or tissue sample. An increase in the level of expression of AIB1 in
a patient-derived tissue sample compared to the level in normal
control tissue indicates the presence of a cell proliferative
disorder such as cancer.
[0012] Screening methods to identify compounds which inhibit an
interaction of AIB1 with a steroid hormone receptor, thus
disrupting a signal transduction pathway which leads to aberrant
cell proliferation, is also within the invention. Proliferation of
a cancer cell can therefore be reduced by administering to an
individual, e.g., a patient diagnosed with a steroid-responsive
cancer, a compound which inhibits expression of AIB1.
[0013] The invention also includes a knockout mutant, for example a
mouse (or other mammal) from which at least one AIB1 gene has been
selectively deleted from its genome. Such a mouse is useful in
research, for instance, the phenotype gives insight into the
physiological role of the deleted gene. For instance the mutant may
be defective in specific biochemical pathways; such a knockout
mutant may be used in complementation experiments to determine the
role of other genes and proteins to determine if any such genes or
proteins complement for the deleted gene. Homozygous and
heterozygous mutants are included in this aspect of the
invention.
[0014] The present invention also includes a mutant organism, for
example a mammal such as a mouse which contains more than the
normal number of AIB1 genes in its genome. Such a mouse may contain
additional copies of the AIB1 gene integrated into its chromosomes,
for instance in the form of a pro-virus, or may carry additional
copies on extra-chromosomal elements such as plasmids. Such a
mutant mouse is useful for research purposes, to elucidate the
physiological or pathological role of AIB1. For instance, the role
of AIB1 expression as cause or effect in cancers may be
investigated by including or transplanting tumors into such
mutants, and comparing such mutants with normal mice having the
same cancer.
[0015] The present invention also includes a mutant organism, for
example a mammal, e.g. a mouse, that contains, either integrated
into a chromosome or on a plasmid, at least one copy of the AIB1
gene driven by a non-native promoter. Such a promoter may be
constitutive or may be inducible. For instance, the AIB1 gene may
be operatively linked to a mouse mammary tumor virus (MMTV)
promoter or other promoter from a mammalian virus allowing
manipulation of AIB1 expression. Such a mutant would be useful for
research purposes to determine the physiological or pathological
role of AIB1. For instance, over or under expression could be
affected and physiological effects observed.
[0016] The invention also includes methods for treatment of cancers
that involve functions of or alterations in the signaling pathways
that use p300 and/or CBP as signal transducing molecules. The
treatments of the invention involve targeting of the AIB1 protein
or AIB1 gene to enhance or reduce interaction with p300 and/or CBP
proteins. For instance, the AIB1 gene sequence as disclosed herein
may be used to construct an anti-sense nucleotide. An anti-sense
RNA may be constructed that is anti-parallel and complementary to
the AIB1 transcript (or part thereof) and which will therefore form
an RNA-RNA duplex with the AIB1 transcript, preventing
transcription and expression of AIB1. Alternatively, treatments may
comprise contacting an AIB1 protein with a molecule that
specifically binds to the AIB1 molecule in vivo, thereby
interfering with AIB1 binding with other factors such as p300 or
CBP. Such processes are designed to inhibit signal transduction
pathways involving AIB1, p300, CBP and other factors and therefore
inhibit cancer cell proliferation that is effected via these
pathways. As explained in more detail below, AIB1 overexpression
results in increased ER-dependent transcriptional activity which
confers a growth advantage upon AIB1 amplification-bearing clones
during the development and progression of estrogen-dependent
cancers.
[0017] Compounds which inhibit or disrupt the interaction of an
AIB1 gene product with a steroid hormone receptor, e.g., ER, are
useful as anti-neoplastic agents for the treatment of patients
suffering from steroid hormone-responsive cancers such as breast
cancer, ovarian cancer, prostate cancer, and colon cancer.
[0018] AIB1 polypeptides or peptide mimetics of such polypeptides,
e.g., those containing domains which interact with steroid hormone
receptors, can be administered to patients to block the interaction
of endogenous intracellular AIB1 and a steroid hormone receptor,
e.g., ER in an aberrantly proliferating cell. It is likely that
AIB1 interacts with a wide range of human transcriptional factors
and that regulation of such interactions will have important
therapeutic applications.
[0019] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
Sequence Listing
[0020] The nucleic acid and amino acid sequences listed in the
accompanying Sequence Listing are shown using standard letter
abbreviations for nucleotide bases and three-letter code for amino
acids. Only one strand of each nucleic acid sequence is shown, but
the complementary strand is understood to be included by any
reference to the displayed strand.
[0021] SEQ ID NO: 1 shows the nucleic acid sequence of the human
AIB1 cDNA.
[0022] SEQ ID NO: 2 shows the amino acid sequence of the
Per/Arnt/Sim (PAS) domain of AIB1.
[0023] SEQ ID NO: 3 shows the amino acid sequence of the basic
helix-loop-helix domain (bHLH) of AIB1.
[0024] SEQ ID NO: 4 shows the amino acid sequence of the human AIB1
protein.
[0025] SEQ ID NO: 5 shows the nucleic acid sequence of primer
N8F1.
[0026] SEQ ID NO: 6 shows the nucleic acid sequence of the forward
primer designed from the 5' sequence of pCMVSPORT-B11, PM-U2.
[0027] SEQ ID NO: 7 shows the nucleic acid sequence of the reverse
primer designed from the 5' sequence of pCMVSPORT-B11, PM-U2.
[0028] SEQ ID NO: 8 shows the amino acid sequence of the
ER-interacting domain of AIB1.
[0029] SEQ ID NO: 9 shows the nucleic acid sequence of pCIP, the
mouse ortholog of AIB1 and the amino acid sequence for this
gene.
[0030] SEQ ID NO: 10 shows the nucleic acid sequence of the forward
primer AIB1/mESTF1 used to screen mouse BAC.
[0031] SEQ ID NO: 11 shows the nucleic acid sequence of the reverse
primer AIB1/mESTR1 used to screen mouse BAC.
[0032] SEQ ID NO: 12 shows the amino acid sequence of pCIP, the
mouse ortholog of AIB1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1A is a diagram of an amino acid sequence of full
length AIB1 (SEQ ID NO:4) in which residues highlighted in black
are identical in AIB1, TIF2, and SRC1. Residues identical with TIF2
(GenBank Accession No. X97674) or SRC-1 (GenBank Accession No.
U59302) are highlighted in grey or boxed, respectively.
[0034] FIG. 1B is a diagram showing the structural features of
AIB1. The following domains are indicated: bHLH domain, PAS domains
(with the highly conserved PAS A and B regions shown in dark gray),
S/T (serine/threonine)-rich regions, and a group of charged
residues (+/-). A glutamine-rich region and polyglutamine tract are
also indicated. The numbers beneath the diagram indicate the
location (approximate residue number) of the domain with respect to
the amino acid sequence shown in FIG. 1A. The alignment was
generated using DNASTAR software.
[0035] FIG. 2 is a photograph of a Northern blot analysis showing
increased expression of AIB1 in the cell lines BT-474, ZR-75-1,
MCF7, and BG-1.
[0036] FIG. 3 is a bar graph showing that the addition of full
length AIB1 DNA to a cell resulted in an increase of
estrogen-dependent transcription from an ER reporter plasmid. COS-1
cells were transiently transfected with 250 ng ER expression vector
(pHEGO-hyg), 10 ng of luciferase reporter plasmid (pGL3.luc.3ERE or
10 ng pGL3 lacking ERE) and increasing amounts of pcDNA3.1-AIB1 and
incubated in the absence (open bars) or presence of 10 nM
17.beta.-stradiol (E2, solid bars) or 100 nM 4-hydroxytamoxifen
(hatched bars). Luciferase activity was expressed in relative
luminescence units (RLU). The data are the mean of three
determinations from one of four replicate experiments. Error bars
indicate one standard deviation.
[0037] FIG. 4 is a schematic diagram comparing the DNA and protein
structures of pCIP (the mouse ortholog of AIB1) and the human AIB1;
exons are shown as black boxes.
[0038] FIGS. 5A and-5B are a table showing the introns and exons of
the mouse AIB1 gene (pCIP) (SEQ ID NO:9). The "Exon" column refers
to the number of the exon; "cDNA bp 5'-exon" refers to the
nucleotide position in the mouse cDNA sequence for the 5' exon;
"cDNA bp 3' exon" refers to the last few nucleotides of the 3'
position of the intron. "Exon sequence" refers to the exon itself.
"5' intron" refers to the adjacent intron reading from the exon
into the splice donor nucleotides (usually GT).
[0039] FIG. 6A and FIG. 6B are a table showing the introns and
exons of the human AIB1 gene (SEQ ID NO:1). The "Exon" column
refers to the number of the exon; "cDNA bp 5'-exon" refers to the
nucleotide position in the mouse cDNA sequence for the 5' exon;
"cDNA bp 3' exon" refers to the last few nucleotides of the 3'
position of the intron. "Exon sequence" refers to the exon itself.
"5' intron" refers to the adjacent intron reading from the exon
into the splice donor nucleotides (usually GT).
DETAILED DESCRIPTION
[0040] The invention is based on the discovery of a novel gene,
amplified in breast cancer-1 (AIB1), which is overexpressed in
breast cancer. AIB1 has the structural features of a co-activator
of the steroid hormone receptor family. The steroid hormone
estrogen and other related steroid hormones act on cells through
specific steroid receptors.
[0041] Members of the steroid receptor coactivator (SRC) family of
transcriptional co-activators interact with nuclear hormone
receptors to enhance ligand-dependent transcription. AIB1 is a
novel member of the SRC family which was found to be overexpressed
in breast cancers. The AIB1 gene is located at human chromosome
20q. High-level AIB1 amplification and overexpression were observed
in several estrogen receptor (ER) positive breast and ovarian
cancer cell lines, as well as in uncultured breast cancer
specimens. AIB1 amplification is not confined to breast cancer but
is also found in cancers of the lung, ovary, head and neck, colon,
testicles, bladder, prostate, endometrium, kidney, stomach and also
in pheochromocytoma, melanoma, ductal carcinoma and carcinoid
tumor.
[0042] Transfection of AIB1 into cells resulted in marked
enhancement of estrogen-dependent transcription. These observations
indicated that AIB1 functions as a co-activator of steroid hormone
receptors such as ER (including estrogen receptor a (ER.alpha.) and
estrogen receptor .beta. (ER.beta.)), androgen receptor (e.g.,
expressed in prostate cells), retinoid receptor (e.g., isoforms
.alpha., .gamma., and retinoid X receptor (RXR)), progesterone
receptor (e.g., expressed in breast cells), mineralocorticoid
receptor (implicated in salt metabolism disorders), vitamin D
receptor (implicated in calcium metabolism disorders), thyroid
hormone receptor (e.g, thyroid hormone receptor .alpha.), or
glucocorticoid receptor (e.g., expressed in spleen and thymus
cells). The altered expression of AIB1 contributes to the
initiation and progression of steroid hormone-responsive cancers by
increasing the transcriptional activity of the steroid
receptor.
[0043] A substantially pure DNA which includes an AIB1-encoding
polynucleotides (or the complement thereof) is claimed. 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 AIB1 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 at a site other than its natural site; 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 an additional polypeptide sequence.
Preferably, the polypeptide includes a Per/Arnt/Sim (PAS) domain
(LLQALDGFLFVVNRDGNIVFVSENVTQYLQYKQEDLVNTSVYNILHEEDRKDFLKNLPKSTVN
GVSWTNETQRQKSHTFNCRMLMKTPHDILEDINASPEMRQRYETMQCFALSQPRAMMEEGED
LQSCMICVARRITTGERTFPSNPESFITRHDLSGKVVNIDTNSLRSSMRPGFEDIIRRCIQ; SEQ.
I.D. NO. 2) and/or a basic helix-loop-helix
(bHLH) domain
(RKRKLPCDTPGQGLTCSGEKRRREQESKYIEELAELISANLSDIDNFNVKPDKCA
ILKETVRQIRQIKEQGKT; SEQ. I.D. NO. 3); more preferably, the AIB1
polypeptide includes the amino acid sequence of the entire
naturally-occurring AIB1 protein (FIG. 1; SEQ. I.D. NO. 4).
Preferably, the peptide includes an ER-interacting domain of AIB1
(e.g., a domain comprising approximately amino acids 300 to
1250:
CIQRFFSLNDGQSWSQKRHYQEAYLNGHAETPVYRFSLADGTIVTAQTKSKLF
RNPVTNDRHGFVSTHFLQREQNGYRPNPNPVGQGIRPPMAGCNSSVGGMSMS
[0044]
PNQGLQMPSSRAYGLADPSTTGQMSGARYGGSSNIASLTPGPGMQSPSSYQNNNYGLNMSSPPH
GSPGLAPNQQNIMISPRNRGSPKIASHQFSPVAGVHSPMASSGNTGNHSFSSSSLSALQAISEGVGT
SLLSTLSSPGPKLDNSPNMNITQPSKVSNQDSKSPLGFYCDQNPVESSMCQSNSRDHLSDKESKES
SVEGAENQRGPLESKGHKKLLQLLTCSSDDRGHSSLTNSPLDSSCKESSVSVTSPSGVSSSTSGGV
SSTSNMHGSLLQEKHRILHKLLQNGNSPAEVAKITAEATGKDTSSITSCGDGNVVKQEQLSPKKK
ENNALLRYLLDRDDPSDALSKELQPQVEGVDNKMSQCTSSTIPSSSQEKDPKIKTETSEEGSGDLD
NLDAILGDLTSSDFYNNSISSNGSHLGTKQQVFQGTNSLGLKSSQSVQSIRPPYNRAVSLDSPVSV
GSSPPVKNISAFPMLPKQPMLGGNPRMMDSQENYGSSMGGPNRNVTVTQTPSSGDWGLPNSKA
GRMEPMNSNSMGRPGGDYNTSLPRPALGGSIPTLPLRSNSIPGARPVLQQQQQMLQMRPGEIPM
GMGANPYGQAAASNQLGSWPDGMLSMEQVSHGTQNRPLLRNSLDDLVGPPSNLEGQSDERAL
LDQLHTLLSNTDATGLEEIDRALGIPELVNQGQALEPKQDAFQGQEAAVMMDQKAGLYGQTYP
AQGPPMQGGFHLQGQSPSFNSMMNQMNQQGNFPLQGMHPRANIMRPRTNTPKQLRMQLQQR
LQGQQFLNQSRQALELKMENPTAGGAAVMRPMMQPQQGFLNAQMVAQRSRELLSHHFRQQR
VAMMMQQQQQQQ (SEQ. I.D. NO. 8). A cell containing substantially
purified AIB1-encoding DNA is also within the invention.
[0045] The invention also includes a substantially pure DNA which
contains a polynucleotide which hybridizes at high stringency to an
AIB1 cDNA having the sequence of SEQ. I.D. NO. 1, or the complement
thereof and a substantially pure DNA which contains a nucleotide
sequence having at least 50% (for example at least 75%, 90%, 95%,
or 98-100%) sequence identity to SEQ. I.D. NO. 1, provided the
nucleotide sequence encodes a polypeptide having the biological
activity of a AIB1 polypeptide. By "biological activity" is meant
steroid receptor co-activator activity. For example, allelic
variations of the naturally-occurring AIB1-encoding sequence (SEQ.
I.D. NO. 1) are encompassed by the invention. Sequence identity can
be determined by comparing the nucleotide sequences of two nucleic
acids using the BLAST sequence analysis software, for instance, the
NCBI gapped BLAST 2.0 program set to default parameters. This
software is available from The National Center for Biotechnology
Information (www.ncbi.nlm,nih.gov/BLAST).
[0046] Hybridization is carried out using standard techniques such
as those described in Ausubel et al., Current Protocols in
Molecular Biology, John Wiley & Sons, (1989). "High stringency"
refers to DNA hybridization and wash conditions characterized by
high temperature and low salt concentration, e.g., wash conditions
of 65.degree. C. at a salt concentration of approximately
0.1.times.SSC. "Low" to "moderate" stringency refers to DNA
hybridization and wash conditions characterized by low temperature
and high salt concentration, e.g. wash conditions of less than
60.degree. C. at a salt concentration of at least 1.0.times.SSC.
For example, high stringency conditions may include hybridization
at about 42.degree. C., and about 50% formamide; a first wash at
about 65.degree. C., about 2.times.SSC, and 1% SDS; followed by a
second wash at about 65.degree. C. and about 0.1%.times.SSC. Lower
stringency conditions suitable for detecting DNA sequences having
about 50% sequence identity to an AIB1 gene are detected by, for
example, hybridization at about 42.degree. C. in the absence of
formamide; a first wash at about 42.degree. C., about 6.times.SSC,
and about 1% SDS; and a second wash at about 50.degree. C., about
6.times.SSC, and about 1% SDS.
[0047] A substantially pure DNA including (a) the sequence of SEQ
ID NO. 1 or (b) a degenerate variant thereof is also within the
invention. The AIB1-encoding DNA is preferably operably linked to
regulatory sequences (including, e.g., a promoter) for expression
of the polypeptide.
[0048] By "operably linked" is meant that a coding sequence and a
regulatory sequence(s) 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
sequence(s).
[0049] The invention also includes a substantially pure human AIB1
polypeptide or fragment thereof. The AIB1 fragment may include an
ER-interaction domain such as one having the amino acid sequence of
SEQ. I.D. NO. 8. Alternatively, the fragment may contain the amino
acid sequence of SEQ. I.D. NOS. 2, 3, or 4.
[0050] Screening methods to identify candidate compounds which
inhibit estrogen-dependent transcription, AIB1 expression, or an
AIB1/ER interaction (and as a result, proliferation of steroid
hormone-responsive cancer cells) are within the scope of the
invention. For example, a method of identifying a candidate
compound which inhibits ER-dependent transcription is carried out
by contacting the compound with an AIB1 polypeptide and determining
whether the compound binds to the polypeptide. Binding of the
compound to the polypeptide indicates that the compound inhibits
ER-dependent transcription, and in turn, proliferation of steroid
hormone-responsive cancer cells. Preferably, the AIB1 polypeptide
contains a PAS domain or a bHLH domain. Alternatively, the method
is carried out by contacting the compound with an AIB1 polypeptide
and an ER polypeptide and determining the ability of the compound
to interfere with the binding of the ER polypeptide with the AIB1
polypeptide. A compound which interferes with an AIB1/ER
interaction inhibits ER-dependent transcription.
[0051] A method of screening a candidate compound which inhibits an
interaction of an AIB1 polypeptide with an ER polypeptide in a cell
includes the steps of (a) providing a GAL4 binding site linked to a
reporter gene; (b) providing a GAL4 binding domain linked to either
(i) an AIB1 polypeptide or (ii) an ER polypeptide; (c) providing a
GAL4 transactivation domain II linked to the ER polypeptide if the
GAL4 binding domain is linked to the AIB1 polypeptide or linked to
the AIB1 polypeptide if the GAL4 binding domain is linked to the ER
polypeptide; (d) contacting the cell with the compound; and (e)
monitoring expression of the reporter gene. A decrease in
expression in the presence of the compound compared to that in the
absence of the compound indicates that the compound inhibits an
interaction of an AIB1 polypeptide with the ER polypeptide.
[0052] Diagnostic methods to identify an aberrantly proliferating
cell, e.g., a steroid hormone-responsive cancer cell such as a
breast cancer cell, ovarian cancer cell, or prostate cancer cell,
are also included in the invention. For example, a method of
detecting an aberrantly proliferating cell in a tissue sample is
carried out by determining the level of AIB1 gene expression in the
sample. An increase in the level of gene expression compared to
that in a normal control tissue indicates the presence of an
aberrantly proliferating cell. AIB1 gene expression is measured
using an AIB1 gene-specific polynucleotides probe, e.g. in a
Northern assay or polymerase chain reaction (PCR)-based assay, to
detect AIB1 mRNA transcripts. AIB1 gene expression can also be
measured using an antibody specific for an AIB1 gene product, e.g.,
by immunohistochemistry or Western blotting.
[0053] Aberrantly proliferating cells, e.g., cancer cells, in a
tissue sample may be detected by determining the number of cellular
copies of an AIB1 gene in the tissue. An increase in the number of
gene copies in a cell of a patient-derived tissue, compared to that
in normal control tissue indicates the presence of a cancer. A copy
number greater than 2 (the normal diploid copy number) is
indicative of an aberrantly proliferative cell. Preferably, the
copy number is greater than 5 copies per diploid genome, more
preferably 10 copies, more preferably greater than 20, and most
preferably greater than 25 copies. An increase in copy number
compared to the normal diploid copy number indicates that the
tissue sample contains aberrantly proliferating steroid
hormone-responsive cancer cells. AIB1 copy number is measured by
fluorescent in situ hybridization (FISH), Southern hybridization
techniques, and other methods well known in the art (Kallioniemi et
al., PNAS 91: 2156-2160 (1994); Guan et al., Nature Genetics 8:
155-161 (1994); Tanner et al., Clin. Cancer Res. 1: 1455-1461
(1995); Guan et al., Cancer Res. 56: 3446-3450 (August 1996);
Anzick et al., Science 277: 965-968 (August 1997)).
[0054] Aberrantly proliferating cells can also be identified by
genetic polymorphisms in the polyglutamine tract of AIB1, e.g.,
variations in the size of this domain which alter AIB1 co-activator
activity.
[0055] The invention also includes methods of treating a mammal,
e.g., a human patient. For example, a method of reducing
proliferation of a steroid hormone-responsive cancer cell, e.g., an
estrogen-responsive breast cancer cell, in a mammal is carried out
by administering to the mammal a compound which inhibits expression
of AIB1. The compound reduces transcription of AIB1-encoding DNA in
the cell. Alternatively, the compound reduces translation of an
AIB1 mRNA into an AIB1 gene product in the cell. For example,
translation of AIB1 mRNA into an AIB1 gene product is inhibited by
contacting the mRNA with antisense polynucleotides complementary to
the AIB1 mRNA.
[0056] A method of inhibiting ER-dependent transcription in a
breast cell of a mammal is carried out by administering an
effective amount of an AIB1 polypeptide or a peptide mimetic
thereof to the mammal. Preferably, the polypeptide inhibits an
AIB1/ER interaction; more preferably, the polypeptide contains an
ER-interacting domain; a PAS domain or a bHLH domain of AIB1. By
binding to ER, such a polypeptide inhibits binding of AIB1 to ER,
thereby inhibiting ER-dependent transcription.
[0057] The invention also includes antibodies, e.g., a monoclonal
antibody or polyclonal antisera, which bind specifically to AIB1.
The term "antibody" as used in this invention includes whole
antibodies as well as fragments thereof, such as Fab, Fab',
F(ab').sub.2, and Fv which bind to an AIB1 epitope. These antibody
fragments are defined as follows: (1) Fab, the fragment which
contains a monovalent antigen-binding fragment of an antibody
molecule produced by digestion of whole antibody with the enzyme
papain to yield an intact light chain and a portion of one heavy
chain; (2) Fab', the fragment of an antibody molecule obtained by
treating whole antibody with pepsin, followed by reduction, to
yield an intact light chain and a portion of the heavy chain; two
Fab' fragments are obtained per antibody molecule; (3)
(Fab').sub.2, the fragment of the antibody obtained by treating
whole antibody with the enzyme pepsin without subsequent reduction;
F(ab').sub.2, a dimer of two Fab' fragments held together by two
disulfide bonds; (4) Fv, a genetically engineered fragment
containing the variable region of the light chain and the variable
region of the heavy chain expressed as two chains; and (5) single
chain antibody ("SCA"), a genetically engineered molecule
containing the variable region of the light chain, the variable
region of the heavy chain, linked by a suitable polypeptide linker
as a genetically fused single chain molecule. Methods of making
these fragments are routine.
[0058] Also within the invention is a method of identifying a
tamoxifen-sensitive patient (one who is likely to respond to
tamoxifen treatment by a reduction in rate of tumor growth) wherein
the method includes the steps of (a) contacting a patient-derived
tissue sample with tamoxifen; and (b) determining the level of AIB1
gene expression or amplification in the sample. An increase in the
level of expression or gene copy number compared to the level or
cellular copy number in normal control tissue indicates that the
patient is tamoxifen-sensitive.
[0059] AIB1 gene expression is measured using an AIB1 gene-specific
polynucleotide probe, e.g., in a Northern blot or PCR-based assay
to detect AIB1 mRNA transcripts or in a Southern blot or FISH assay
to detect amplification of the gene (which correlates directly with
AIB1 gene expression). Alternatively, AIB1 gene expression is
measured by detecting an AIB1 gene product, e.g., using an
AIB1-specific antibody.
[0060] Transgenic mammals, e.g., mice, which overexpress an AIB1
gene product, e.g., by virtue of harboring multiple copies of
AIB1-encoding DNA, are also within the invention.
[0061] "Transgenic" as used herein means a mammal which bears a
transgene, a DNA sequence which is inserted by artifice into an
embryo, and which then becomes part of the genome of the mammal
that develops from that embryo. Any non-human mammal which may be
produced by transgenic technology is included in the invention;
preferred mammals include, mice, rats, cows, pigs, sheep, goats,
rabbits, guinea pigs, hamsters, and horses.
[0062] By "transgene" is meant DNA which is partly or entirely
heterologous (i.e., foreign) to the transgenic mammal, or DNA
homologous to an endogenous gene of the transgenic mammal, but
which is inserted into the mammal's genome at a location which
differs from that of the natural gene.
[0063] Also within the invention is a knockout mutant, for instance
a knockout mouse wherein the mouse has had at least one copy of the
AIB1 gene (also called the pCIP gene in mice) deleted from its
genome. Such a knockout mutant would be useful in research, for
instance the phenotype gives insight into the physiological role of
AIB1. Complementation experiments using such a knockout mutant can
be used to identify other genes and proteins that make up for the
lack of AIB1 in the mutant to restore wild-type phenotype.
[0064] Also within the invention is a mutant, such as a mouse,
which contains more than the normal number of copies of the AIB1
(pCIP) gene, either integrated into a chromosome, for instance as a
pro-virus, or in an extra-chromosomal element, such as on a
plasmid.
[0065] Also within the invention is a mutant, for example, a mouse,
which contains the AIB1 (pCIP) gene driven by a non-native
promoter, such as a constitutive or an inducible promoter, such as
the mouse mammary tumor virus (MMTV) promoter.
[0066] The invention also includes methods of treatment for cancers
the growth of which involves alternations of signaling pathways
involving p300 and/or CBP. For example, AIB1 (pCIP) may be
contacted with a molecule that binds to AIB1 and inhibits AIB1's
interaction with p300, thereby disrupting signaling of this pathway
and reducing transcription of molecules whose transcription is
positively regulated by this pathway; thereby reducing tumor
growth.
EXAMPLE 1
Cloning and Expression of AIB1
A. Cloning of AIB1
[0067] Chromosome microdissection and hybrid selection techniques
were used to isolate probes and clone gene sequences which map to
chromosome 20q, one of the recurrent sites of DNA amplification in
breast cancer cells identified by molecular cytogenetics
(Kallioniemi et al., PNAS 91: 2156-2160 (1994); Guan et al., Nature
Genetics 8: 155-161 (1994); Tanner et al., Clin. Cancer Res. 1:
1455-1461 (1995); Guan et al., Cancer Res. 56: 3446-3450 (August
1996); Anzick et al., Science 277: 965-968 (August 1997)). AIB1 is
a member of the SRC-1 family of nuclear receptor (NR)
co-activators. AIB1 functions to enhance ER-dependent
transcription. SRC-1 and the closely related TIF2 are steroid
receptor co-activators with an affinity for NRs. The mouse ortholog
of human AIB1 is called pCIP. In this application pCIP and AIB1
will be used synonymously unless the contrary is clearly
expressed.
[0068] To characterize AIB1, the full length cDNA was cloned and
sequenced. An AIB1 specific primer N8F1
(5'-TCATCACTTCCGACAACAGAGG-3'; SEQ. I.D. NO. 5) was biotinylated
and used to capture cDNA clones from a human lung cDNA library
(Gibco, BRL) using the GENETRAPPER cDNA Positive Selection System
(Gibco, BRL). The largest clone (5.8 kb), designated pCMVSPORT-B11,
was selected for sequence analysis. To obtain full-length
AIB1-encoding DNA, a random-primed library from BT474 was
constructed in bacteriophage .lamda.-Zap (Stratagene) and
hybridized with a 372 bp .sup.32P-labeled PCR product amplified
from a human spleen cDNA library using primers designed form the 5'
sequence of pCMVSPORT-B11, PM-U2 (5'-CCAGAAACGTCACTATCAAG-3',
forward primer; SEQ. I.D. NO. 6) and B11-11RA
(5'-TTACTGGAACCCCCATACC-3', reverse primer; SEQ. I.D. NO. 7).
Plasmid rescue of 19 positive clones yielded a clone,
pBluescript-R22, which overlapped pCMVSPORT-B11 and contained the
5' end of the coding region. To generate a full length AIB1 clone,
the 4.85 kb HindIII/XhoI fragment of pCMVSPORT-B11 was subcloned
into HindIII/XhoI sites of pBluescript-R22. The 4.84 kb NotI/NheI
fragment of the full length clone containing the entire coding
region was then subcloned into the NotI/XbaI sites of the
expression vector, pcDNA3.1 (Invitrogen), generating
pcDNA3.1-AIB1.
[0069] The cloned DNA sequence (SEQ. I.D. No. 1) revealed an open
reading frame (beginning at the underlined "ATG") encoding a
protein of 1420 amino acids with a predicted molecular weight of
155 kDa (FIG. 1A). Database searches with BLASTP identified a
similarity of AIB1 with TIF2 (45% protein identity) and SRC-1 (33%
protein identity). Like TIF2 and SRC-1, AIB1 contains a bHLH domain
preceding a PAS domain, serine/threonine-rich regions, and a
charged cluster (FIG. 1B). There is also a glutamine-rich region
which, unlike SRC-1 and TIF2, contains a polyglutamine tract (FIG.
1B). The polyglutamine tract of AIB1 is subject to genetic
polymorphism. Variations in the size of this domain alter AIB1
co-activator activity.
B. Expression of AIB1
[0070] Amplification and expression of AIB1 in several ER positive
and negative breast and ovarian cancer cell lines was examined.
Established breast cancer cell lines used in the experiments
described below (see, e.g., FIG. 2) were obtained from the American
Type Culture Collection (ATCC): BT-474, MCF-7, T47D, MDA-MB-361,
MDA-MB-468, BT-20, MDA-MB-436, and MDA-MB-453; the Arizona Cancer
Center (ACC): UACC-812; or the National Cancer Institute (NCI):
ZR75-1.
[0071] AIB1 gene copy number was determined by FISH. For FISH
analysis, interphase nuclei were fixed in methanol:acetic acid
(3:1) and dropped onto microscope slides. AIB1 amplification was
detected in the breast cancer cell line ZR75-1, the ovarian cancer
cell line BG-1, and two uncultured breast cancer samples.
Intra-chromosomal amplification of AIB1 was apparent in metaphase
chromosomes of ZR75-1 and BG1. Numerous copies of AIB1 were
resolved in the adjacent interphase nuclei. Extrachromosomal copies
(e.g., in episomes or double minute chromosomes) of AIB1 have also
been detected. The Spectrum-Orange (Vysis) labeled AIB1 P1 probe
was hybridized with a biotinylated reference probe for 20q11
(RMC20P037) or a fluorescein labeled probe for 20p (RMC20C039).
[0072] High level amplification of AIB1 (greater than 20 fold),
similar to that observed in BT-474 and MCF-7, was seen in two
additional ER-positive cell lines, breast carcinoma ZR75-1, and
ovarian carcinoma BG-1 (see FIG. 2). Interphase FISH studies
demonstrated that amplification of chromosome 20q in breast cancer
is complex, involving several distinct variably co-amplified
chromosomal segments derived from 20q11, 20q12, and 20q13. Probes
for the 20q11 and 20q13 regions of amplification did not detect
amplification in ZR75-1 and BG-1, suggesting that amplification of
AIB1 (which maps to 20q12) occurred independently in these cell
lines.
[0073] To determine if AIB1 amplification also occurred in
uncultured cells from patient biopsies, breast cancer specimens
were screened for AIB1 amplification by interphase FISH. In two of
16 specimens analyzed, high AIB1 copy number (up to 25 copies/cell)
was detected. Both tumor specimens tested came from post-menopausal
patients and were ER/PR positive. One of the specimens was obtained
from a metastatic tumor of a patient who subsequently responded
favorably to tamoxifen treatment.
[0074] AIB1 expression was also examined in cells with and without
AIB1 amplification and compared to expression of ER, SRC-1 and TIF2
by Northern blotting. In accordance with its amplification status,
AIB1 was highly overexpressed in BT-474, MCF-7, ZR75-1, and BG-1
(FIG. 2). Three of the four cell lines exhibiting AIB1
overexpression also demonstrated prominent ER expression, while two
others displayed lower but detectable ER expression (BT-474 and
BT-20). FIG. 2 also shows that the expression of TIF2 and SRC-1
remained relatively constant in all cell lines tested. Taken
together, these observations demonstrate that AIB1 amplification is
associated with significant overexpression of AIB1 gene product.
The correlation of elevated AIB1 expression with ER positivity in
tumors indicates that AIB1 is a component of the estrogen signaling
pathway, the amplification of which is selected during cancer
development and progression.
[0075] To determine whether expression of AIB1 increases ER
ligand-dependent transactivation, transient transfection assays
were performed. The effect of increasing levels of AIB1 on
transcription of an ER dependent reporter was measured. The results
demonstrated that co-transfection of AIB1 led to a dose dependent
increase in estrogen-dependent transcription (FIG. 3). This effect
was not observed when the estrogen antagonist, 4-hydroxytamoxifen
(4-OHT), was substituted for 17.beta.-estradiol or when the
estrogen response element (ERE) was removed from the reporter
plasmid (FIG. 3). A modest increase in basal transcription levels
was observed with higher concentrations of AIB1 even in the absence
of an ERE suggesting that AIB1 may have an intrinsic
transactivation function. These results demonstrate that, like the
closely related TIF2 and SRC-1, AIB1 functions as an ER
co-activator.
EXAMPLE 2
Characterization of AIB1
A. Functional Domains of AIB1
[0076] TIF-2, SRC-1, and AIB1 are characterized by highly conserved
N-terminal bHLH and PAS domains. The PAS region functions as a
protein dimerization interface in the mammalian aryl hydrocarbon
receptor and the aryl hydrocarbon receptor nuclear transporter
proteins, as well as the Drosophila transcription factors sim and
per. The PAS region (SEQ. I.D. NO. 2) of AIB1 functions as a
protein interaction domain, mediating binding between AIB1 and
other proteins. However, steroid hormone activators lacking the PAS
domain are capable of interacting with nuclear steroid hormone
receptors. The highly conserved bHLH domain (SEQ. I.D. NO. 3)
participates in protein interactions which mediate or modulate
transmission of the hormone signal to the transcriptional
apparatus. The ER-interacting domain (SEQ. I.D. NO. 8) mediates
binding of AIB1 with a steroid hormone receptor protein.
[0077] AIB1 also interacts with the transcriptional integrators
CREB binding protein (CBP) and p300. These transcriptional
integrators interact directly with the basal transcriptional
machinery. The CBP/p300 receptor association domain of AIB1 does
not encompass the bHLH/PAS regions.
B. Purification of Gene Products
[0078] DNA containing a sequence that encodes part or all of the
amino acid sequence of AIB1 can be subcloned into an expression
vector, using a variety of methods known in the art. The
recombinant protein can then be purified using standard methods.
For example, a recombinant polypeptide can be expressed as a fusion
protein in procaryotic cells such as E. coli. Using the maltose
binding protein fusion and purification system (New England
Biolabs), the cloned human cDNA sequence is inserted downstream and
in frame of the gene encoding maltose binding protein (malE). The
malE fusion protein is overexpressed in E. coli and can be readily
purified in quantity. In the absence of convenient restriction
sites in the human cDNA sequence, PCR can be used to introduce
restriction sites compatible with the pMalE vector at the 5' and 3'
end of the cDNA fragment to facilitate insertion of the cDNA
fragment into the vector. Following expression of the fusion
protein, it can be purified by affinity chromatography. For
example, the fusion protein can be purified by virtue of the
ability of the maltose binding protein portion of the fusion
protein to bind to amylase immobilized on a column.
[0079] To facilitate protein purification, the pMalE plasmid
contains a factor Xa cleavage site upstream of the site into which
the cDNA is inserted into the vector. Thus, the fusion protein
purified as described above can be cleaved with factor Xa to
separate the maltose binding protein portion of the fusion protein
from recombinant human cDNA gene product. The cleavage products can
be subjected to further chromatography to purify recombinant
polypeptide from the maltose binding protein. Alternatively, an
antibody specific for the desired recombinant gene product can be
used to purify the fusion protein and/or the gene product cleaved
from the fusion protein. Many comparable commercially available
fusion protein expression systems can be utilized similarly.
[0080] AIB1 polypeptides can also be expressed in eucaryotic cells,
e.g., yeast cells, either alone or as a fusion protein. For
example, a fusion protein containing the GAL4 DNA-binding domain or
activation domain fused to a functional domain of AIB1, e.g., the
PAS domain, the bHLH domain, or the ER-interacting domain, can be
expressed in yeast cells using standard methods such as the yeast
two hybrid system described below. Alternatively, AIB1 polypeptides
can be expressed in COS-1 cells using methods well known in the
art, e.g., by transfecting a DNA encoding an AIB1 polypeptide into
COS-1 cells using, e.g., the Lipofectamine transfection protocol
described below, and culturing the cells under conditions suitable
for protein expression.
EXAMPLE 3
Detection of AIB1
A. Detection of Nucleotides Encoding AIB1
[0081] Determination of gene copy number in cells of a
patient-derived sample is known in the art. For example, AIB1
amplification in cancer-derived cell lines as well as uncultured
breast cancer cells was carried out using bicolor FISH analysis as
follows. A genomic P1 clone containing AIB1 was labeled with
Spectrum Orange-dUTP (Vysis) using the BioPrime DNA Labeling System
(Gibco BRL). A 20q11 P1 clone was labeled with Biotin-16-dUTP (BMB)
using nick translation. Fluorescent images were captured using a
Zeiss axiophot microscope equipped with a CCD camera and IP Lab
Spectrum software (Signal Analytics). Interphase FISH analysis of
uncultured breast cancer samples was performed using known methods
(Kallioniemi et al., PNAS 91: 2156-2160 (1994); Guan et al., Nature
Genetics 8: 155-161 (1994); Tanner et al., Clin. Cancer Res. 1:
1455-1461 (1995); Guan et al., Cancer Res. 56: 3446-3450 (August
1996); Anzick et al., Science 277: 965-968 (August 1997)).
Alternatively, standard Southern hybridization techniques can be
employed to evaluate gene amplification. For example, Southern
analysis is carried out using a non-repetitive fragment of genomic
AIB1 DNA, e.g., derived from the 20q11 P1 clone described above or
another AIB1 gene-containing genomic clone, as a probe.
[0082] The level of gene expression may be measured using methods
known in the art, e.g., in situ hybridization, Northern blot
analysis, or Western blot analysis using AIB1-specific monoclonal
or polyclonal antibodies. AIB1 gene transcription was measured
using Northern analysis. For example, the data shown in FIG. 2 was
obtained as follows. The blot was hybridized sequentially with a
probe (ER, AIB1, TIF2, SRC-1, or .beta.-actin as indicated to the
left of the photograph). AIB1 expression was compared to that of
ER, TIF2, and SRC-1. cDNA clones were obtained from Research
Genetics [TIF2 (clone 132364, GenBank accession no. R25318); SRC-1
(clone 418064, GenBank accession no. W90426)], the American Type
Culture Collection (pHEGO-hyg, ATCC number 79995), or Clontech
(.beta. actin). The AIB1 probe was a 2.2 kb NotI/SacI fragment of
pCMVSPORT-B11. The .beta.-actin probe was used as a control for
loading error. To avoid cross-hybridization between these related
genes and to match signal intensities, similar sized probes from
the 3'UTRs of AIB1, TIF2, and SRC-1 were utilized. Each of these
probes detected a signal in normal mammary RNA on longer exposure.
Electrophoresis, transfer and hybridization of 15 .mu.g total RNA
was performed by standard methods.
B. Detection of AIB1 Gene Products
[0083] AIB1 polypeptides to be used as antigens to raise
AIB1-specific antibodies can be generated by methods known in the
art, e.g., proteolytic cleavage, de novo synthesis, or expression
of a recombinant polypeptide from the cloned AIB1 gene or a
fragment thereof. AIB1-specific antibodies are then produced using
standard methodologies for raising polyclonal antisera and making
monoclonal antibody-producing hybridoma cell lines (see Coligan et
al., eds., Current Protocols in Immunology, 1992, Greene Publishing
Associates and Wiley-Interscience). To generate monoclonal
antibodies, a mouse is immunized with an AIB1 polypeptide,
antibody-secreting B cells isolated from the mouse, and the B cells
immortalized with a non-secretory myeloma cell fusion partner.
Hybridomas are then screened for production of an AIB1-specific
antibody and cloned to obtain a homogenous cell population which
produces a monoclonal antibody.
[0084] For administration to human patients, antibodies, e.g., AIB1
specific monoclonal antibodies, can be humanized by methods known
in the art. Antibodies with a desired binding specificity can be
commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo
Alto, Calif.).
EXAMPLE 4
Detection of AIB1-Related Cell Proliferative Disorders
A. Diagnostic and Prognostic Methods
[0085] The invention includes a method of detecting an aberrantly
proliferating cell, e.g., a steroid hormone-responsive cancer cell
such as a breast cancer cell, an ovarian cancer cell, colon cancer
cell, or prostate cancer cell, by detecting the number of AIB1 gene
copies in the cell and/or the level of expression of the AIB1 gene
product. AIB1 gene amplification or gene expression in a
patient-derived tissue sample is measured as described above and
compared to the level of amplification or gene expression in normal
non-cancerous cells. An increase in the level of amplification or
gene expression detected in the patient-derived biopsy sample
compared to the normal control is diagnostic of a diseased state,
i.e., the presence of a steroid hormone responsive cancer.
[0086] Because of the importance of estrogen exposure to mammary
carcinogenesis and of anti-estrogen treatment in breast cancer
therapy, such assays are also useful to determine the frequency of
alterations of AIB1 expression in pre-malignant breast lesions
(e.g. ductal carcinoma in situ) and during the progression from
hormone dependent to hormone independent tumor growth.
[0087] The diagnostic methods of the invention are useful to
determine the prognosis of a patient and estrogen responsive status
of a steroid hormone-responsive cancer.
[0088] AIB1 expression can also be measured at the protein level by
detecting an AIB1 gene products with an AIB1-specific monoclonal or
polyclonal antibody preparation.
B. Diagnosis of Tamoxifen-Sensitivity
[0089] Overexpression of AIB1, e.g., as a result of AIB1 gene
amplification, in steroid hormone-responsive cancers can predict
whether the cancer is treatable with anti-endocrine compositions,
e.g., tamoxifen. AIB1 amplification or overexpression in a
patient-derived tissue sample compared to a normal (non-cancerous)
tissue indicates tumor progression.
[0090] Absence of AIB1, e.g., loss of all or part of the AIB1 gene,
but retention of ER-positivity in steroid hormone-responsive
cancers predicts failure or poor responsiveness to anti-endocrine
therapy, e.g., administration of anti-estrogen compositions such as
tamoxifen. Since loss of AIB1 expression in a cancer cell may
indicate a disruption of the ER signal transduction pathway,
anti-estrogen therapy may be ineffective to treat such cancers.
Patients identified in this manner (who would otherwise be treated
with anti-estrogens) would be treated with alternative
therapies.
[0091] Loss of estrogen receptor in recurrent breast caner is also
associated with poor response to endocrine therapy. Up to 30% to
40% of metastases from hormone receptor-positive primary breast
cancer do not respond to endocrine therapy. The frequency of
hormone receptor status changes between primary and recurrent
tumors and whether such a change might explain unresponsiveness to
endocrine therapy was examined. Primary breast cancer samples and
matched asynchronous recurrences were studied from 50 patients who
had not received any adjuvant therapy. ER and progesterone receptor
(PR) status was determined immunohistochemically from
histologically representative formalin-fixed paraffin-embedded
tumor samples. ER status was ascertained by mRNA in situ
hybridization. Thirty-five (70%) of 50 primary tumors were positive
for ER and 30 (60%) for PR. Hormone receptor status of the
recurrent tumor differed from that of the primary tumor in 18 cases
(36%). Discordant cases were due to the loss of ER (n=6), loss of
PR (n=6), or loss of both receptors (n=6). Receptor-negative
primary tumors were always accompanied by receptor-negative
recurrences. Among 27 patients with ER-positive primary tumors,
loss of ER was a significant predictor (P=0.0085) of poor response
to subsequent endocrine therapy. Only one of eight patients (12.5%)
with lost ER expression responded to tamoxifen therapy, whereas the
response rate was 74% (14 of 19) for patients whose recurrent
tumors retained ER expression. Loss of ER expression in recurrent
breast cancer predicts poor response to endocrine therapy in
primarily ER-positive patients. Evaluation of ER expression and/or
AIB1 expression (or gene copy number) is useful to determine the
most effective approach to treatment of steroid-responsive
cancers.
EXAMPLE 5
Screening of Candidate Compounds
A. In Vitro Assays
[0092] The invention includes methods of screening to identify
compounds which inhibit the interaction of AIB1 with ER, thereby
decreasing estrogen dependent transcription which leads to aberrant
cell proliferation. A transcription assay is carried out in the
presence and absence of the candidate compound. A decrease in
transcription in the presence of the compound compared to that in
its absence indicates that the compound blocks an AIB1/ER
interaction and inhibits estrogen dependent transcription.
[0093] To determine the effect of AIB1 on estrogen-dependent
transcription, an ER reporter plasmid can be used. The
transcription assays described herein were conducted as follows.
COS-1 cells were grown and maintained in phenol-red free DMEM
medium supplemented with 10% charcoal-stripped fetal bovine serum.
Cells were plated into 6-well culture dishes at 1.5.times.10.sup.5
cells/well and allowed to grow overnight. Transfection of cells
with the ER reporter plasmid was performed with Lipofectamine
(Gibco, BRL) following the manufacturer's protocol. Three ng
pRL-CMV were used as an internal control for transfection
efficiency. Ligand or ethanol vehicle was added 234 hours
post-transfection and cell lysates were harvested 48 hours
post-transfection. Reporter activities were determined using the
Dual-Luciferase Reporter Assay System (Promega) and the results
expressed in relative luminescence units (RLU; luciferase/Renilla
luciferase). pRL-CMV and pGL3-promoter were obtained from Promega.
pHEGO-hyg was obtained from ATCC. The ER reporter pGL3.luc.3ERE
contains three tandem copies of the ERE upstream from the SV40
promoter driving the luciferase gene. Standard mammalian expression
vectors were utilized. Empty pcDNA3 vector was added to each of the
pcDNA3.1-AIB1 dilutions to maintain constant amounts of plasmid
DNA.
[0094] Compounds which inhibit the interaction of AIB1 with ER are
also identified using a standard co-precipitation assay. AIB1/ER
co-precipitation assays are carried out as follows. An AIB1
polypeptide and an ER polypeptide are incubated together to allow
complex formation. One of the polypeptides is typically a fusion
protein, e.g., GST-AIB1, and the other is tagged with a detectable
label, e.g., .sup.32P-labeled ER). After incubation, the complex is
precipitated, e.g., using glutathione-Sepharose beads. The beads
are washed, filtered through a glass fiber filter, and collected.
The amount of co-precipitated .sup.32P-label is measured. A
reduction in the amount of co-precipitated label in the presence of
a candidate compound compared to that in the absence of the
candidate compound indicates that the compound inhibits an AIB1/ER
interaction
[0095] Alternatively, a standard in vitro binding assay can be
used. For example, one polypeptide, e.g., AIB1, can be bound to a
solid support and contacted with the second polypeptide, e.g., ER.
The amount of the second polypeptide which is retained on the solid
support is then measured. A reduction in the amount of retained
(second) polypeptide in the presence of a candidate compound
compared to that in its absence indicates that the compound
inhibits an AIB1/ER interaction. Techniques for column
chromatography and coprecipitation of polypeptides are well known
in the art.
[0096] An evaluation of AIB1/ER interaction and identification of
compounds that blocks or reduces the interaction can also be
carried out in vivo using a yeast two-hybrid expression system in
which the activity of a transcriptional activator is reconstituted
when the two proteins or polypeptides of interest closely interact
or bind to one another.
[0097] The yeast GAL4 protein consists of functionally
distinguishable domains. One domain is responsible for DNA-binding
and the other for transcriptional activation. In the two-hybrid
expression system, plasmids encoding two hybrid proteins, a first
fusion protein containing the GAL4 DNA-binding domain fused to a
first protein, e.g., AIB1, and the second fusion protein containing
the GAL4 activation domain fused to a second protein, e.g., ER, are
introduced into yeast. If the two proteins are able to interact
with one another, the ability to activate transcription from
promoters containing Gal4-binding sites upstream from an activating
sequence from GALL (UAS.sub.G) is reconstituted leading to the
expression of a reporter gene. A reduction in the expression of the
reporter gene in the presence of a candidate compound compared to
that in the absence of the compound indicates that the compound
reduces an AIB1/ER interaction.
[0098] A method of identifying a DNA-binding protein which
regulates AIB1 transcription can be carried out as follows:
A DNA containing a cis-acting regulatory element can be immobilized
on polymeric beads, such as agarose or acrylamide. A mixture of
proteins, such as a cell lysate, is allowed to come in contact with
and bind to the DNA. Following removal of non-binding proteins,
specifically-bound proteins, are eluted with a competing DNA
sequence which may be identical to the immobilized sequence.
Specific binding of a protein to the DNA regulatory element
indicates that the protein may regulate AIB1 transcription.
Functional activity of the identified trans-acting factor can be
confirmed with an appropriate functional assay, such as one which
measures the level of transcription of a reporter gene having the
cis-acting regulatory gene 5' to the transcription start site of
AIB1.
[0099] A method of identifying a compound which decreases the level
of AIB1 transcription can be accomplished by contacting an
immobilized AIB1-derived cis-acting regulatory element with a
trans-acting regulatory factor in the presence and absence of
candidate compound. A detectable change, i.e., a reduction, in
specific binding of the trans-acting factor to its DNA target
indicates that the candidate compound inhibits AIB1
transcription.
[0100] In addition to interacting with ER, AIB1 also interacts with
the transcriptional integrators CBP and p300. CBP and p300
participate in the basal transcriptional apparatus in a cell. Thus,
another approach to inhibit signal transduction through AIB1 is to
prevent the formation of or disrupt an interaction of AIB1 with CBP
and/or p300. Compounds which inhibit signal transduction (and
therefore cell proliferation) can be identified by contacting AIB1
(or a fragment thereof which interacts with CBP or p300) with CBP
or p300 (or a fragment thereof containing an AIB1-interacting
domain, e.g., a C-terminal fragment) in the presence and absence of
a candidate compound. For example, a C-terminal fragment of CBP
involved in steroid receptor co-activator interaction contains 105
amino acids in the Q-rich region of CBP (Kamei et al., 1996, Cell
85:403-414; Yao et al., 1996, Proc. Natl. Acad. Sci. USA
93:10626-10631; Hanstein et al., 1996, Proc. Natl. Acad. Sci. USA
93:11540-11545). A decrease in AIB1 interaction with CBP or p300 in
the presence of a candidate compound compared to that its absence
indicates that the compound inhibits AIB1 interaction with these
transcriptional integrators, and as a result, AIB1-mediated signal
transduction leading to DNA transcription and cell proliferation.
Compounds which inhibit AIB1 interaction with transcriptional
integrators can also be identified using a co-precipitation assay
and the yeast two-hybrid expression system described above.
B. In Vivo Assays
[0101] Transgenic mice are made by standard methods, e.g., as
described in Leder et al., U.S. Pat. No. 4,736,866, herein
incorporated by reference, or Hogan et al., 1986 Manipulating the
Mouse Embryo. Cold Spring Harbor Laboratory" New York.
[0102] Briefly, a vector containing a promoter operably linked to
AIB1-encoding cDNA is injected into murine zygotes, e.g.,
C57BL/6J.times.DBA/2F2 zygotes. Incorporation of the transgene into
murine genomic DNA is monitored using methods well known in the art
of molecular biology, e.g., dot blotting tail DNA with a probe
complimentary to the 3' region of the gene contained in the AIB1
transgene construct. Mice thus confirmed to harbor the transgene
can then be used as founders. Animal lines are created by crossing
founders with C57BL/6J mice (The Jackson Laboratory, Bar Harbor,
Me.). AIB1 transgenic mice can be used to screen candidate
compounds in vivo to identify compounds which inhibit aberrant cell
proliferation, e.g., as measured by reduction tumor growth or
metastasis. AIB1 transgenic mice are also useful to identify other
genes involved in steroid hormone receptor-dependent cancers and to
establish mouse cell lines which overexpress AIB1.
AIB1-overexpressing cell lines are useful to screen for compounds
that interfere with AIB1 function, e.g, by blocking the interaction
of AIB1 with a ligand.
EXAMPLE 6
AIB1 Therapy
[0103] As discussed above, AIB1 is a novel member of the SRC-1
family of transcriptional co-activators. Amplification and
overexpression of AIB1 in ER-positive breast and ovarian cancer
cells and in breast cancer biopsies implicate this protein as a
critical component of the estrogen response pathway. AIB1
overexpression results in increased ER-dependent transcriptional
activity which confers a growth advantage of AIB1
amplification-bearing clones during the development and progression
of estrogen-dependent cancers.
[0104] Compounds which inhibit or disrupt the interaction of an
AIB1 gene product with a steroid hormone receptor, e.g., ER, are
useful as anti-neoplastic agents for the treatment of patients
suffering from steroid hormone-responsive cancers such as breast
cancer, ovarian cancer, prostate cancer, and colon cancer.
Likewise, compounds which disrupt interaction between AIB1 and p300
and/or CBP are also useful as anti-neoplastic agents.
[0105] AIB1 polypeptides or peptide mimetics of such polypeptides,
e.g., those containing domains which interact with steroid hormone
receptors, can be administered to patients to block the interaction
of endogenous intracellular AIB1 and a steroid hormone receptor,
e.g., ER in an aberrantly proliferating cell. A mimetic may be made
by introducing conservative amino acid substitutions into the
peptide. Certain amino acid substitutions are conservative since
the old and the new amino acid share a similar hydrophobicity or
hydrophylicity or are similarly acidic, basic or neutrally charged
(Stryer "Biochemistry" 1975, Ch. 2, Freeman and Company, New York).
Conservative substitutions replace one amino acid with another
amino acid that is similar in size, hydrophobicity, etc. Examples
of conservative substitutions are shown in the table below (Table
1).
TABLE-US-00001 TABLE 1 Original Residue Conservative Substitutions
Ala ser Arg lys Asn gln, his Asp glu Cys ser Gln asn Glu asp Gly
pro His asn; gln Ile leu, val Leu ile; val Lys arg; gln; glu Met
leu; ile Phe met; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val
ile; leu
[0106] Variations in the cDNA sequence that result in amino acid
changes, whether conservative or not, should be minimized in order
to preserve the functional and immunologic identity of the encoded
protein.
[0107] Compositions administered therapeutically include
polypeptide mimetics in which one or more peptide bonds have been
replaced with an alternative type of covalent bond which is not
susceptible to cleavage by peptidases. Where proteolytic
degradation of the peptides following injection into the subject is
a problem, replacement of a particularly sensitive peptide bond
with a noncleavable peptide mimetic yields a more stable and thus
more useful therapeutic polypeptide. Such mimetics, and methods of
incorporating them into polypeptides, are well known in the art.
Similarly, the replacement of an L-amino acid residue with a
D-amino acid residue is a standard way of rendering the polypeptide
less sensitive to proteolysis. Also useful are amino-terminal
blocking groups such as t-butyloxycarbonyl, acetyl, theyl,
succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl,
benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl,
methoxyadipyl, methoxysuberyl, and 2,4,-dinitrophenyl.
[0108] AIB1 polypeptides or related peptide mimetics may be
administered to a patient intravenously in a pharmaceutically
acceptable carrier such as physiological saline. Standard methods
for intracellular delivery of peptides can be used, e.g. packaged
in liposomes. Such methods are well known to those of ordinary
skill in the art. It is expected that an intravenous dosage of
approximately 1 to 100 .mu.moles of the polypeptide of the
invention would be administered per kg of body weight per day. The
compositions of the invention are useful for parenteral
administration, such as intravenous, subcutaneous, intramuscular,
and intraperitoneal.
[0109] The therapeutic compositions of this invention may also be
administered by the use of surgical implants which release the
compounds of the invention. These devices could be readily
implanted into the target tissue, e.g., a solid tumor mass, and
could be mechanical or passive. Mechanical devices, such as pumps,
are well known in the art, as are passive devices (e.g., consisting
of a polymer matrix which contains therapeutic formulations; these
polymers may slowly dissolve or degrade to release the compound, or
may be porous and allow release via pores).
[0110] Antisense therapy in which a DNA sequence complementary to
an AIB1 mRNA transcript is either produced in the cell or
administered to the cell can be used to decrease AIB1 gene
expression thereby inhibiting undesired cell proliferation, e.g.,
proliferation of steroid hormone-responsive cancer cells. An
antisense polynucleotide, i.e., one which is complementary of the
coding sequence of the AIB1 gene, is introduced into the cells in
which the gene is overproduced. The antisense strand (either RNA or
DNA) may be directly introduced into the cells in a form that is
capable of binding to the transcripts. Alternatively, a vector
containing a DNA sequence which, once within the target cells, is
transcribed into the appropriate antisense mRNA, may be
administered. An antisense nucleic acid which hybridizes to the
coding strand of AIB1 DNA can decrease or inhibit production of an
AIB1 gene product by associating with the normally single-stranded
mRNA transcript, and thereby interfering with translation.
[0111] DNA is introduced into target cells of the patient with or
without a vector or using standard vectors and/or gene delivery
systems. Suitable gene delivery systems may include liposomes,
receptor-mediated delivery systems, naked DNA, and viral vectors
such as herpes viruses, retroviruses, and adenoviruses, among
others. The DNA of the invention may be administered in a
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers are biologically compatible vehicles which are suitable
for administration to an animal e.g., physiological saline. A
therapeutically effective amount is an amount of the nucleic acid
of the invention which is capable of producing a medically
desirable result in a patient. As is well known in the medical
arts, dosage for any given patient depends upon many factors,
including the patient's size, body surface area, age, the
particular compound to be administered, sex, time and route of
administration, general health, and other drugs being administered
concurrently. Dosages will vary, but a preferred dosage for
intravenous administration of a nucleic acid is from approximately
10.sup.6 to 10.sup.22 copies of the nucleic acid molecule.
[0112] Determination of optimal dosage is well within the abilities
of a pharmacologist of ordinary skill.
EXAMPLE 7
AIB1 Knockout and Overexpression Mouse Mutants
[0113] Mutants organism that underexpress or overexpress AIB1 are
useful for research. Such mutants allow insight into the
physiological and/or pathological role of AIB1 in a healthy and/or
pathological organism. These mutants are said to be "genetically
engineered," meaning that information in the form of nucleotides
has been transferred into the mutant's genome at a location, or in
a combination, in which it would not normally exist. Nucleotides
transferred in this way are said to be "non-native." For example, a
WAP promoter inserted upstream of a native AIB1 gene would be
non-native. An extra copy of a mouse AIB1 gene present on a plasmid
and transformed into a mouse cell would be non-native. Mutants may
be, for example, produced from mammals, such as mice, that either
overexpress AIB1 or underexpress AIB1 or that do not express AIB1
at all. Overexpression mutants are made by increasing the number of
AIB1 genes in the organism, or by introducing an AIB1 gene into the
organism under the control of a constitutive or inducible or viral
promoter such as the mouse mammary tumor virus (MMTV) promoter or
the whey acidic protein (WAP) promoter or the metallothionein
promoter. Mutants that underexpress AIB1 may be made by using an
inducible or repressible promoter, or by deleting the AIB1 gene, or
by destroying or limiting the function of the AIB1 gene, for
instance by disrupting the gene by transposon insertion.
[0114] Anti-sense genes may be engineered into the organism, under
a constitutive or inducible promoter, to decrease or prevent AIB1
expression. A gene is said to be "functionally deleted" when
genetic engineering has been used to negate or reduce gene
expression to negligible levels. When a mutant is referred to in
this application as having the AIB1 gene altered or functionally
deleted, this reference refers to the AIB1 gene and to any ortholog
of this gene, for instance "a transgenic animal wherein at least
one AIB1 gene has been functionally deleted" would encompass the
mouse ortholog of the AIB1 gene, pCIP. When a mutant is referred to
as having "more than the normal copy number" of a gene, this means
that it has more than the usual number of genes found in the
wild-type organism, eg: in the diploid mouse or human.
[0115] A mutant mouse overexpressing AIB1 may be made by
constructing a plasmid having the AIB1 gene driven by a promoter,
such as the mouse mammary tumor virus (MMTV) promoter or the whey
acidic protein (WAP) promoter. This plasmid may be introduced into
mouse oocytes by microinjection. The oocytes are implanted into
pseudopregnant females, and the litters are assayed for insertion
of the transgene. Multiple strains containing the transgene are
then available for study.
[0116] WAP is quite specific for mammary gland expression during
lactation, and MMTV is expressed in a variety of tissues including
mammary gland, salivary gland and lymphoid tissues. Many other
promoters might be used to achieve various patterns of expression,
e.g., the metallothionein promoter.
[0117] An inducible system may be created in which AIB1 is driven
by a promoter regulated by an agent which can be fed to the mouse
such as tetracycline. Such techniques are well known in the
art.
[0118] A mutant knockout mouse from which the AIB1 (also called
pCIP) gene is deleted was made by removing coding regions of the
AIB1 gene from mouse embryonic stem cells. FIG. 5 shows the
intron/exon structure for pCIP. Using this table, mutations can be
targeted to coding sequences, avoiding silent mutations caused by
deletion of non-coding sequences. (FIG. 6 shows the intron/exon
structure for the human AIB1 gene). These cells were microinjected
into mouse embryos leading to the deletion of the mouse AIB1 gene
in the germ line of a transgenic mouse. The mouse AIB1 gene was
mapped and isolated by the following method: The primers AIB/mEST
F1 (5'-TCCTTTTCCCAGCAGCAGTTTG-3'; SEQ. I.D. 10) and AIB1/mEST R1
(5'ATGCCAGACATGGGCATGGG-3' SEQ. I.D. 11) were used to screen a
mouse Bacterial Artificial Chromosome (BAC) library and to isolate
a mouse BAC (designated 195H10). This BAC was assigned to mouse
chromosome 2 by fluorescence in situ hybridization (FISH). This
region is the mouse equivalent of the portion of human chromosome
20 which carries AIB1.
[0119] To map the structure of the gene, first the structure of the
human AIB1 gene was determined by polymerase chain reaction of a
human genomic DNA clone containing AIB1 using standard methods
(Genomics 1995 Jan. 20; 25(2):501-506) and then the sequences of
the intron exon boundaries were determined (FIG. 4). Based on this
information, the corresponding regions of the mouse BAC were
sequenced. The structure of the mouse gene corresponds closely to
that of the human gene (FIG. 4). This information localizes the
coding regions of the mouse AIB1 gene so that a targeting vector
can be constructed to remove these regions from mouse embryonic
stem cells. These cells can be then injected into mouse embryos
leading to deletion of the mouse AIB1 gene in the germ line of a
transgenic mouse. The methods of creating deletion mutations by
using a targeting vector have been described in Cell (Thomas and
Capecch, Cell 51(3):503-512, 1987).
[0120] References and patents referred to herein are incorporated
by reference.
[0121] The above examples are provided by way of illustration only
and are in no way intended to limit the scope of the invention. One
of skill in the art will see that the invention may be modified in
various ways without departing from the spirit or principle of the
invention. We claim all such modifications.
Sequence CWU 1
1
1216835DNAHomo sapiensCDS(201)..(4463) 1cggcggcggc tgcggcttag
tcggtggcgg ccggcggcgg ctgcgggctg agcggcgagt 60ttccgattta aagctgagct
gcgaggaaaa tggcggcggg aggatcaaaa tacttgctgg 120atggtggact
cagagaccaa taaaaataaa ctgcttgaac atcctttgac tggttagcca
180gttgctgatg tatattcaag atg agt gga tta gga gaa aac ttg gat cca
ctg 233Met Ser Gly Leu Gly Glu Asn Leu Asp Pro Leu1 5 10gcc agt gat
tca cga aaa cgc aaa ttg cca tgt gat act cca gga caa 281Ala Ser Asp
Ser Arg Lys Arg Lys Leu Pro Cys Asp Thr Pro Gly Gln15 20 25ggt ctt
acc tgc agt ggt gaa aaa cgg aga cgg gag cag gaa agt aaa 329Gly Leu
Thr Cys Ser Gly Glu Lys Arg Arg Arg Glu Gln Glu Ser Lys30 35 40tat
att gaa gaa ttg gct gag ctg ata tct gcc aat ctt agt gat att 377Tyr
Ile Glu Glu Leu Ala Glu Leu Ile Ser Ala Asn Leu Ser Asp Ile45 50
55gac aat ttc aat gtc aaa cca gat aaa tgt gcg att tta aag gaa aca
425Asp Asn Phe Asn Val Lys Pro Asp Lys Cys Ala Ile Leu Lys Glu
Thr60 65 70 75gta aga cag ata cgt caa ata aaa gag caa gga aaa act
att tcc aat 473Val Arg Gln Ile Arg Gln Ile Lys Glu Gln Gly Lys Thr
Ile Ser Asn80 85 90gat gat gat gtt caa aaa gcc gat gta tct tct aca
ggg cag gga gtt 521Asp Asp Asp Val Gln Lys Ala Asp Val Ser Ser Thr
Gly Gln Gly Val95 100 105att gat aaa gac tcc tta gga ccg ctt tta
ctt cag gca ttg gat ggt 569Ile Asp Lys Asp Ser Leu Gly Pro Leu Leu
Leu Gln Ala Leu Asp Gly110 115 120ttc cta ttt gtg gtg aat cga gac
gga aac att gta ttt gta tca gaa 617Phe Leu Phe Val Val Asn Arg Asp
Gly Asn Ile Val Phe Val Ser Glu125 130 135aat gtc aca caa tac ctg
caa tat aag caa gag gac ctg gtt aac aca 665Asn Val Thr Gln Tyr Leu
Gln Tyr Lys Gln Glu Asp Leu Val Asn Thr140 145 150 155agt gtt tac
aat atc tta cat gaa gaa gac aga aag gat ttt ctt aag 713Ser Val Tyr
Asn Ile Leu His Glu Glu Asp Arg Lys Asp Phe Leu Lys160 165 170aat
tta cca aaa tct aca gtt aat gga gtt tcc tgg aca aat gag acc 761Asn
Leu Pro Lys Ser Thr Val Asn Gly Val Ser Trp Thr Asn Glu Thr175 180
185caa aga caa aaa agc cat aca ttt aat tgc cgt atg ttg atg aaa aca
809Gln Arg Gln Lys Ser His Thr Phe Asn Cys Arg Met Leu Met Lys
Thr190 195 200cca cat gat att ctg gaa gac ata aac gcc agt cct gaa
atg cgc cag 857Pro His Asp Ile Leu Glu Asp Ile Asn Ala Ser Pro Glu
Met Arg Gln205 210 215aga tat gaa aca atg cag tgc ttt gcc ctg tct
cag cca cga gct atg 905Arg Tyr Glu Thr Met Gln Cys Phe Ala Leu Ser
Gln Pro Arg Ala Met220 225 230 235atg gag gaa ggg gaa gat ttg caa
tct tgt atg atc tgt gtg gca cgc 953Met Glu Glu Gly Glu Asp Leu Gln
Ser Cys Met Ile Cys Val Ala Arg240 245 250cgc att act aca gga gaa
aga aca ttt cca tca aac cct gag agc ttt 1001Arg Ile Thr Thr Gly Glu
Arg Thr Phe Pro Ser Asn Pro Glu Ser Phe255 260 265att acc aga cat
gat ctt tca gga aag gtt gtc aat ata gat aca aat 1049Ile Thr Arg His
Asp Leu Ser Gly Lys Val Val Asn Ile Asp Thr Asn270 275 280tca ctg
aga tcc tcc atg agg cct ggc ttt gaa gat ata atc cga agg 1097Ser Leu
Arg Ser Ser Met Arg Pro Gly Phe Glu Asp Ile Ile Arg Arg285 290
295tgt att cag aga ttt ttt agt cta aat gat ggg cag tca tgg tcc cag
1145Cys Ile Gln Arg Phe Phe Ser Leu Asn Asp Gly Gln Ser Trp Ser
Gln300 305 310 315aaa cgt cac tat caa gaa gct tat ctt aat ggc cat
gca gaa acc cca 1193Lys Arg His Tyr Gln Glu Ala Tyr Leu Asn Gly His
Ala Glu Thr Pro320 325 330gta tat cga ttc tcg ttg gct gat gga act
ata gtg act gca cag aca 1241Val Tyr Arg Phe Ser Leu Ala Asp Gly Thr
Ile Val Thr Ala Gln Thr335 340 345aaa agc aaa ctc ttc cga aat cct
gta aca aat gat cga cat ggc ttt 1289Lys Ser Lys Leu Phe Arg Asn Pro
Val Thr Asn Asp Arg His Gly Phe350 355 360gtc tca acc cac ttc ctt
cag aga gaa cag aat gga tat aga cca aac 1337Val Ser Thr His Phe Leu
Gln Arg Glu Gln Asn Gly Tyr Arg Pro Asn365 370 375cca aat cct gtt
gga caa ggg att aga cca cct atg gct gga tgc aac 1385Pro Asn Pro Val
Gly Gln Gly Ile Arg Pro Pro Met Ala Gly Cys Asn380 385 390 395agt
tcg gta ggc ggc atg agt atg tcg cca aac caa ggc tta cag atg 1433Ser
Ser Val Gly Gly Met Ser Met Ser Pro Asn Gln Gly Leu Gln Met400 405
410ccg agc agc agg gcc tat ggc ttg gca gac cct agc acc aca ggg cag
1481Pro Ser Ser Arg Ala Tyr Gly Leu Ala Asp Pro Ser Thr Thr Gly
Gln415 420 425atg agt gga gct agg tat ggg ggt tcc agt aac ata gct
tca ttg acc 1529Met Ser Gly Ala Arg Tyr Gly Gly Ser Ser Asn Ile Ala
Ser Leu Thr430 435 440cct ggg cca ggc atg caa tca cca tct tcc tac
cag aac aac aac tat 1577Pro Gly Pro Gly Met Gln Ser Pro Ser Ser Tyr
Gln Asn Asn Asn Tyr445 450 455ggg ctc aac atg agt agc ccc cca cat
ggg agt cct ggt ctt gcc cca 1625Gly Leu Asn Met Ser Ser Pro Pro His
Gly Ser Pro Gly Leu Ala Pro460 465 470 475aac cag cag aat atc atg
att tct cct cgt aat cgt ggg agt cca aag 1673Asn Gln Gln Asn Ile Met
Ile Ser Pro Arg Asn Arg Gly Ser Pro Lys480 485 490ata gcc tca cat
cag ttt tct cct gtt gca ggt gtg cac tct ccc atg 1721Ile Ala Ser His
Gln Phe Ser Pro Val Ala Gly Val His Ser Pro Met495 500 505gca tct
tct ggc aat act ggg aac cac agc ttt tcc agc agc tct ctc 1769Ala Ser
Ser Gly Asn Thr Gly Asn His Ser Phe Ser Ser Ser Ser Leu510 515
520agt gcc ctg caa gcc atc agt gaa ggt gtg ggg act tcc ctt tta tct
1817Ser Ala Leu Gln Ala Ile Ser Glu Gly Val Gly Thr Ser Leu Leu
Ser525 530 535act ctg tca tca cca ggc ccc aaa ttg gat aac tct ccc
aat atg aat 1865Thr Leu Ser Ser Pro Gly Pro Lys Leu Asp Asn Ser Pro
Asn Met Asn540 545 550 555att acc caa cca agt aaa gta agc aat cag
gat tcc aag agt cct ctg 1913Ile Thr Gln Pro Ser Lys Val Ser Asn Gln
Asp Ser Lys Ser Pro Leu560 565 570ggc ttt tat tgc gac caa aat cca
gtg gag agt tca atg tgt cag tca 1961Gly Phe Tyr Cys Asp Gln Asn Pro
Val Glu Ser Ser Met Cys Gln Ser575 580 585aat agc aga gat cac ctc
agt gac aaa gaa agt aag gag agc agt gtt 2009Asn Ser Arg Asp His Leu
Ser Asp Lys Glu Ser Lys Glu Ser Ser Val590 595 600gag ggg gca gag
aat caa agg ggt cct ttg gaa agc aaa ggt cat aaa 2057Glu Gly Ala Glu
Asn Gln Arg Gly Pro Leu Glu Ser Lys Gly His Lys605 610 615aaa tta
ctg cag tta ctt acc tgt tct tct gat gac cgg ggt cat tcc 2105Lys Leu
Leu Gln Leu Leu Thr Cys Ser Ser Asp Asp Arg Gly His Ser620 625 630
635tcc ttg acc aac tcc ccc cta gat tca agt tgt aaa gaa tct tct gtt
2153Ser Leu Thr Asn Ser Pro Leu Asp Ser Ser Cys Lys Glu Ser Ser
Val640 645 650agt gtc acc agc ccc tct gga gtc tcc tcc tct aca tct
gga gga gta 2201Ser Val Thr Ser Pro Ser Gly Val Ser Ser Ser Thr Ser
Gly Gly Val655 660 665tcc tct aca tcc aat atg cat ggg tca ctg tta
caa gag aag cac cgg 2249Ser Ser Thr Ser Asn Met His Gly Ser Leu Leu
Gln Glu Lys His Arg670 675 680att ttg cac aag ttg ctg cag aat ggg
aat tca cca gct gag gta gcc 2297Ile Leu His Lys Leu Leu Gln Asn Gly
Asn Ser Pro Ala Glu Val Ala685 690 695aag att act gca gaa gcc act
ggg aaa gac acc agc agt ata act tct 2345Lys Ile Thr Ala Glu Ala Thr
Gly Lys Asp Thr Ser Ser Ile Thr Ser700 705 710 715tgt ggg gac gga
aat gtt gtc aag cag gag cag cta agt cct aag aag 2393Cys Gly Asp Gly
Asn Val Val Lys Gln Glu Gln Leu Ser Pro Lys Lys720 725 730aag gag
aat aat gca ctt ctt aga tac ctg ctg gac agg gat gat cct 2441Lys Glu
Asn Asn Ala Leu Leu Arg Tyr Leu Leu Asp Arg Asp Asp Pro735 740
745agt gat gca ctc tct aaa gaa cta cag ccc caa gtg gaa gga gtg gat
2489Ser Asp Ala Leu Ser Lys Glu Leu Gln Pro Gln Val Glu Gly Val
Asp750 755 760aat aaa atg agt cag tgc acc agc tcc acc att cct agc
tca agt caa 2537Asn Lys Met Ser Gln Cys Thr Ser Ser Thr Ile Pro Ser
Ser Ser Gln765 770 775gag aaa gac cct aaa att aag aca gag aca agt
gaa gag gga tct gga 2585Glu Lys Asp Pro Lys Ile Lys Thr Glu Thr Ser
Glu Glu Gly Ser Gly780 785 790 795gac ttg gat aat cta gat gct att
ctt ggt gat ctg act agt tct gac 2633Asp Leu Asp Asn Leu Asp Ala Ile
Leu Gly Asp Leu Thr Ser Ser Asp800 805 810ttt tac aat aat tcc ata
tcc tca aat ggt agt cat ctg ggg act aag 2681Phe Tyr Asn Asn Ser Ile
Ser Ser Asn Gly Ser His Leu Gly Thr Lys815 820 825caa cag gtg ttt
caa gga act aat tct ctg ggt ttg aaa agt tca cag 2729Gln Gln Val Phe
Gln Gly Thr Asn Ser Leu Gly Leu Lys Ser Ser Gln830 835 840tct gtg
cag tct att cgt cct cca tat aac cga gca gtg tct ctg gat 2777Ser Val
Gln Ser Ile Arg Pro Pro Tyr Asn Arg Ala Val Ser Leu Asp845 850
855agc cct gtt tct gtt ggc tca agt cct cca gta aaa aat atc agt gct
2825Ser Pro Val Ser Val Gly Ser Ser Pro Pro Val Lys Asn Ile Ser
Ala860 865 870 875ttc ccc atg tta cca aag caa ccc atg ttg ggt ggg
aat cca aga atg 2873Phe Pro Met Leu Pro Lys Gln Pro Met Leu Gly Gly
Asn Pro Arg Met880 885 890atg gat agt cag gaa aat tat ggc tca agt
atg ggt ggg cca aac cga 2921Met Asp Ser Gln Glu Asn Tyr Gly Ser Ser
Met Gly Gly Pro Asn Arg895 900 905aat gtg act gtg act cag act cct
tcc tca gga gac tgg ggc tta cca 2969Asn Val Thr Val Thr Gln Thr Pro
Ser Ser Gly Asp Trp Gly Leu Pro910 915 920aac tca aag gcc ggc aga
atg gaa cct atg aat tca aac tcc atg gga 3017Asn Ser Lys Ala Gly Arg
Met Glu Pro Met Asn Ser Asn Ser Met Gly925 930 935aga cca gga gga
gat tat aat act tct tta ccc aga cct gca ctg ggt 3065Arg Pro Gly Gly
Asp Tyr Asn Thr Ser Leu Pro Arg Pro Ala Leu Gly940 945 950 955ggc
tct att ccc aca ttg cct ctt cgg tct aat agc ata cca ggt gcg 3113Gly
Ser Ile Pro Thr Leu Pro Leu Arg Ser Asn Ser Ile Pro Gly Ala960 965
970aga cca gta ttg caa cag cag cag cag atg ctt caa atg agg cct ggt
3161Arg Pro Val Leu Gln Gln Gln Gln Gln Met Leu Gln Met Arg Pro
Gly975 980 985gaa atc ccc atg gga atg ggg gct aat ccc tat ggc caa
gca gca gca 3209Glu Ile Pro Met Gly Met Gly Ala Asn Pro Tyr Gly Gln
Ala Ala Ala990 995 1000tct aac caa ctg ggt tcc tgg ccc gat ggc atg
ttg tcc atg gaa caa 3257Ser Asn Gln Leu Gly Ser Trp Pro Asp Gly Met
Leu Ser Met Glu Gln1005 1010 1015gtt tct cat ggc act caa aat agg
cct ctt ctt agg aat tcc ctg gat 3305Val Ser His Gly Thr Gln Asn Arg
Pro Leu Leu Arg Asn Ser Leu Asp1020 1025 1030 1035gat ctt gtt ggg
cca cct tcc aac ctg gaa ggc cag agt gac gaa aga 3353Asp Leu Val Gly
Pro Pro Ser Asn Leu Glu Gly Gln Ser Asp Glu Arg1040 1045 1050gca
tta ttg gac cag ctg cac act ctt ctc agc aac aca gat gcc aca 3401Ala
Leu Leu Asp Gln Leu His Thr Leu Leu Ser Asn Thr Asp Ala Thr1055
1060 1065ggc ctg gaa gaa att gac aga gct ttg ggc att cct gaa ctt
gtc aat 3449Gly Leu Glu Glu Ile Asp Arg Ala Leu Gly Ile Pro Glu Leu
Val Asn1070 1075 1080cag gga cag gca tta gag ccc aaa cag gat gct
ttc caa ggc caa gaa 3497Gln Gly Gln Ala Leu Glu Pro Lys Gln Asp Ala
Phe Gln Gly Gln Glu1085 1090 1095gca gca gta atg atg gat cag aag
gca gga tta tat gga cag aca tac 3545Ala Ala Val Met Met Asp Gln Lys
Ala Gly Leu Tyr Gly Gln Thr Tyr1100 1105 1110 1115cca gca cag ggg
cct cca atg caa gga ggc ttt cat ctt cag gga caa 3593Pro Ala Gln Gly
Pro Pro Met Gln Gly Gly Phe His Leu Gln Gly Gln1120 1125 1130tca
cca tct ttt aac tct atg atg aat cag atg aac cag caa ggc aat 3641Ser
Pro Ser Phe Asn Ser Met Met Asn Gln Met Asn Gln Gln Gly Asn1135
1140 1145ttt cct ctc caa gga atg cac cca cga gcc aac atc atg aga
ccc cgg 3689Phe Pro Leu Gln Gly Met His Pro Arg Ala Asn Ile Met Arg
Pro Arg1150 1155 1160aca aac acc ccc aag caa ctt aga atg cag ctt
cag cag agg ctg cag 3737Thr Asn Thr Pro Lys Gln Leu Arg Met Gln Leu
Gln Gln Arg Leu Gln1165 1170 1175ggc cag cag ttt ttg aat cag agc
cga cag gca ctt gaa ttg aaa atg 3785Gly Gln Gln Phe Leu Asn Gln Ser
Arg Gln Ala Leu Glu Leu Lys Met1180 1185 1190 1195gaa aac cct act
gct ggt ggt gct gcg gtg atg agg cct atg atg cag 3833Glu Asn Pro Thr
Ala Gly Gly Ala Ala Val Met Arg Pro Met Met Gln1200 1205 1210ccc
cag cag ggt ttt ctt aat gct caa atg gtc gcc caa cgc agc aga 3881Pro
Gln Gln Gly Phe Leu Asn Ala Gln Met Val Ala Gln Arg Ser Arg1215
1220 1225gag ctg cta agt cat cac ttc cga caa cag agg gtg gct atg
atg atg 3929Glu Leu Leu Ser His His Phe Arg Gln Gln Arg Val Ala Met
Met Met1230 1235 1240cag cag cag cag cag cag caa cag cag cag cag
cag cag cag cag cag 3977Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln Gln Gln Gln Gln1245 1250 1255caa cag caa cag caa cag caa cag
cag caa cag cag caa acc cag gcc 4025Gln Gln Gln Gln Gln Gln Gln Gln
Gln Gln Gln Gln Gln Thr Gln Ala1260 1265 1270 1275ttc agc cca cct
cct aat gtg act gct tcc ccc agc atg gat ggg ctt 4073Phe Ser Pro Pro
Pro Asn Val Thr Ala Ser Pro Ser Met Asp Gly Leu1280 1285 1290ttg
gca gga ccc aca atg cca caa gct cct ccg caa cag ttt cca tat 4121Leu
Ala Gly Pro Thr Met Pro Gln Ala Pro Pro Gln Gln Phe Pro Tyr1295
1300 1305caa cca aat tat gga atg gga caa caa cca gat cca gcc ttt
ggt cga 4169Gln Pro Asn Tyr Gly Met Gly Gln Gln Pro Asp Pro Ala Phe
Gly Arg1310 1315 1320gtg tct agt cct ccc aat gca atg atg tcg tca
aga atg ggt ccc tcc 4217Val Ser Ser Pro Pro Asn Ala Met Met Ser Ser
Arg Met Gly Pro Ser1325 1330 1335cag aat ccc atg atg caa cac ccg
cag gct gca tcc atc tat cag tcc 4265Gln Asn Pro Met Met Gln His Pro
Gln Ala Ala Ser Ile Tyr Gln Ser1340 1345 1350 1355tca gaa atg aag
ggc tgg cca tca gga aat ttg gcc agg aac agc tcc 4313Ser Glu Met Lys
Gly Trp Pro Ser Gly Asn Leu Ala Arg Asn Ser Ser1360 1365 1370ttt
tcc cag cag cag ttt gcc cac cag ggg aat cct gca gtg tat agt 4361Phe
Ser Gln Gln Gln Phe Ala His Gln Gly Asn Pro Ala Val Tyr Ser1375
1380 1385atg gtg cac atg aat ggc agc agt ggt cac atg gga cag atg
aac atg 4409Met Val His Met Asn Gly Ser Ser Gly His Met Gly Gln Met
Asn Met1390 1395 1400aac ccc atg ccc atg tct ggc atg cct atg ggt
cct gat cag aaa tac 4457Asn Pro Met Pro Met Ser Gly Met Pro Met Gly
Pro Asp Gln Lys Tyr1405 1410 1415tgc tga catctctgca ccaggacctc
ttaaggaaac cactgtacaa atgacactgc 4513Cys1420actaggatta ttgggaagga
atcattgttc caggcatcca tcttggaaga aaggaccagc 4573tttgagctcc
atcaagggta ttttaagtga tgtcatttga gcaggactgg attttaagcc
4633gaagggcaat atctacgtgt ttttcccccc tccttctgct gtgtatcatg
gtgttcaaaa 4693cagaaatgtt ttttggcatt ccacctccta gggatataat
tctggagaca tggagtgtta 4753ctgatcataa aacttttgtg tcactttttt
ctgccttgct agccaaaatc tcttaaatac 4813acgtaggtgg gccagagaac
attggaagaa tcaagagaga ttagaatatc tggtttctct 4873agttgcagta
ttggacaaag agcatagtcc cagccttcag gtgtagtagt tctgtgttga
4933ccctttgtcc agtggaattg gtgattctga attgtccttt actaatggtg
ttgagttgct 4993ctgtccctat tatttgccct aggctttctc ctaatgaagg
ttttcatttg ccattcatgt 5053cctgtaatac ttcacctcca ggaactgtca
tggatgtcca aatggctttg cagaaaggaa 5113atgagatgac agtatttaat
cgcagcagta gcaaactttt cacatgctaa tgtgcagctg 5173agtgcacttt
atttaaaaag aatggataaa tgcaatattc ttgaggtctt gagggaatag
5233tgaaacacat tcctggtttt tgcctacact tacgtgttag acaagaacta
tgattttttt 5293tttaaagtac tggtgtcacc ctttgcctat atggtagagc
aataatgctt tttaaaaata 5353aacttctgaa aacccaaggc caggtactgc
attctgaatc agaatctcgc agtgtttctg 5413tgaatagatt tttttgtaaa
tatgaccttt aagatattgt attatgtaaa atatgtatat 5473accttttttt
gtaggtcaca acaactcatt tttacagagt ttgtgaagct aaatatttaa
5533cattgttgat ttcagtaagc tgtgtggtga ggctaccagt ggaagagaca
tcccttgact 5593tttgtggcct gggggagggg tagtgctcca cagcttttcc
ttccccaccc cccagcctta 5653gatgcctcgc tcttttcaat ctcttaatct
aaatgctttt taaagagatt atttgtttag 5713atgtaggcat tttaattttt
taaaaattcc tctaccagaa ctaagcactt tgttaatttg 5773gggggaaaga
atagatatgg ggaaataaac ttaaaaaaaa atcaggaatt taaaaaaacg
5833agcaatttga agagaatctt ttggatttta agcagtccga aataatagca
attcatgggc 5893tgtgtgtgtg tgtgtatgtg tgtgtgtgtg tgtgtatgtt
taattatgtt
accttttcat 5953cccctttagg agcgttttca gattttggtt gctaagacct
gaatcccata ttgagatctc 6013gagtagaatc cttggtgtgg tttctggtgt
ctgctcagct gtcccctcat tctactaatg 6073tgatgctttc attatgtccc
tgtggattag aatagtgtca gttatttctt aagtaactca 6133gtacccagaa
cagccagttt tactgtgatt cagagccaca gtctaactga gcacctttta
6193aacccctccc tcttctgccc cctaccactt ttctgctgtt gcctctcttt
gacacctgtt 6253ttagtcagtt gggaggaagg gaaaaatcaa gtttaattcc
ctttatctgg gttaattcat 6313ttggttcaaa tagttgacgg aattgggttt
ctgaatgtct gtgaatttca gaggtctctg 6373ctagccttgg tatcattttc
tagcaataac tgagagccag ttaattttaa gaatttcaca 6433catttagcca
atctttctag atgtctctga aggtaagatc atttaatatc tttgatatgc
6493ttacgagtaa gtgaatcctg attatttcca gacccaccac cagagtggat
cttattttca 6553aagcagtata gacaattatg agtttgccct ctttccccta
ccaagttcaa aatatatcta 6613agaaagattg taaatccgaa aacttccatt
gtagtggcct gtgcttttca gatagtatac 6673tctcctgttt ggagacagag
gaagaaccag gtcagtctgt ctctttttca gctcaattgt 6733atctgaccct
tctttaagtt atgtgtgtgg ggagaaatag aatggtgctc ttatctttct
6793tgactttaaa aaaattatta aaaacaaaaa aaaaaaaaaa aa 68352186PRTHomo
sapiens 2Leu Leu Gln Ala Leu Asp Gly Phe Leu Phe Val Val Asn Arg
Asp Gly1 5 10 15Asn Ile Val Phe Val Ser Glu Asn Val Thr Gln Tyr Leu
Gln Tyr Lys20 25 30Gln Glu Asp Leu Val Asn Thr Ser Val Tyr Asn Ile
Leu His Glu Glu35 40 45Asp Arg Lys Asp Phe Leu Lys Asn Leu Pro Lys
Ser Thr Val Asn Gly50 55 60Val Ser Trp Thr Asn Glu Thr Gln Arg Gln
Lys Ser His Thr Phe Asn65 70 75 80Cys Arg Met Leu Met Lys Thr Pro
His Asp Ile Leu Glu Asp Ile Asn85 90 95Ala Ser Pro Glu Met Arg Gln
Arg Tyr Glu Thr Met Gln Cys Phe Ala100 105 110Leu Ser Gln Pro Arg
Ala Met Met Glu Glu Gly Glu Asp Leu Gln Ser115 120 125Cys Met Ile
Cys Val Ala Arg Arg Ile Thr Thr Gly Glu Arg Thr Phe130 135 140Pro
Ser Asn Pro Glu Ser Phe Ile Thr Arg His Asp Leu Ser Gly Lys145 150
155 160Val Val Asn Ile Asp Thr Asn Ser Leu Arg Ser Ser Met Arg Pro
Gly165 170 175Phe Glu Asp Ile Ile Arg Arg Cys Ile Gln180
185373PRTHomo sapiens 3Arg Lys Arg Lys Leu Pro Cys Asp Thr Pro Gly
Gln Gly Leu Thr Cys1 5 10 15Ser Gly Glu Lys Arg Arg Arg Glu Gln Glu
Ser Lys Tyr Ile Glu Glu20 25 30Leu Ala Glu Leu Ile Ser Ala Asn Leu
Ser Asp Ile Asp Asn Phe Asn35 40 45Val Lys Pro Asp Lys Cys Ala Ile
Leu Lys Glu Thr Val Arg Gln Ile50 55 60Arg Gln Ile Lys Glu Gln Gly
Lys Thr65 7041420PRTHomo sapiens 4Met Ser Gly Leu Gly Glu Asn Leu
Asp Pro Leu Ala Ser Asp Ser Arg1 5 10 15Lys Arg Lys Leu Pro Cys Asp
Thr Pro Gly Gln Gly Leu Thr Cys Ser20 25 30Gly Glu Lys Arg Arg Arg
Glu Gln Glu Ser Lys Tyr Ile Glu Glu Leu35 40 45Ala Glu Leu Ile Ser
Ala Asn Leu Ser Asp Ile Asp Asn Phe Asn Val50 55 60Lys Pro Asp Lys
Cys Ala Ile Leu Lys Glu Thr Val Arg Gln Ile Arg65 70 75 80Gln Ile
Lys Glu Gln Gly Lys Thr Ile Ser Asn Asp Asp Asp Val Gln85 90 95Lys
Ala Asp Val Ser Ser Thr Gly Gln Gly Val Ile Asp Lys Asp Ser100 105
110Leu Gly Pro Leu Leu Leu Gln Ala Leu Asp Gly Phe Leu Phe Val
Val115 120 125Asn Arg Asp Gly Asn Ile Val Phe Val Ser Glu Asn Val
Thr Gln Tyr130 135 140Leu Gln Tyr Lys Gln Glu Asp Leu Val Asn Thr
Ser Val Tyr Asn Ile145 150 155 160Leu His Glu Glu Asp Arg Lys Asp
Phe Leu Lys Asn Leu Pro Lys Ser165 170 175Thr Val Asn Gly Val Ser
Trp Thr Asn Glu Thr Gln Arg Gln Lys Ser180 185 190His Thr Phe Asn
Cys Arg Met Leu Met Lys Thr Pro His Asp Ile Leu195 200 205Glu Asp
Ile Asn Ala Ser Pro Glu Met Arg Gln Arg Tyr Glu Thr Met210 215
220Gln Cys Phe Ala Leu Ser Gln Pro Arg Ala Met Met Glu Glu Gly
Glu225 230 235 240Asp Leu Gln Ser Cys Met Ile Cys Val Ala Arg Arg
Ile Thr Thr Gly245 250 255Glu Arg Thr Phe Pro Ser Asn Pro Glu Ser
Phe Ile Thr Arg His Asp260 265 270Leu Ser Gly Lys Val Val Asn Ile
Asp Thr Asn Ser Leu Arg Ser Ser275 280 285Met Arg Pro Gly Phe Glu
Asp Ile Ile Arg Arg Cys Ile Gln Arg Phe290 295 300Phe Ser Leu Asn
Asp Gly Gln Ser Trp Ser Gln Lys Arg His Tyr Gln305 310 315 320Glu
Ala Tyr Leu Asn Gly His Ala Glu Thr Pro Val Tyr Arg Phe Ser325 330
335Leu Ala Asp Gly Thr Ile Val Thr Ala Gln Thr Lys Ser Lys Leu
Phe340 345 350Arg Asn Pro Val Thr Asn Asp Arg His Gly Phe Val Ser
Thr His Phe355 360 365Leu Gln Arg Glu Gln Asn Gly Tyr Arg Pro Asn
Pro Asn Pro Val Gly370 375 380Gln Gly Ile Arg Pro Pro Met Ala Gly
Cys Asn Ser Ser Val Gly Gly385 390 395 400Met Ser Met Ser Pro Asn
Gln Gly Leu Gln Met Pro Ser Ser Arg Ala405 410 415Tyr Gly Leu Ala
Asp Pro Ser Thr Thr Gly Gln Met Ser Gly Ala Arg420 425 430Tyr Gly
Gly Ser Ser Asn Ile Ala Ser Leu Thr Pro Gly Pro Gly Met435 440
445Gln Ser Pro Ser Ser Tyr Gln Asn Asn Asn Tyr Gly Leu Asn Met
Ser450 455 460Ser Pro Pro His Gly Ser Pro Gly Leu Ala Pro Asn Gln
Gln Asn Ile465 470 475 480Met Ile Ser Pro Arg Asn Arg Gly Ser Pro
Lys Ile Ala Ser His Gln485 490 495Phe Ser Pro Val Ala Gly Val His
Ser Pro Met Ala Ser Ser Gly Asn500 505 510Thr Gly Asn His Ser Phe
Ser Ser Ser Ser Leu Ser Ala Leu Gln Ala515 520 525Ile Ser Glu Gly
Val Gly Thr Ser Leu Leu Ser Thr Leu Ser Ser Pro530 535 540Gly Pro
Lys Leu Asp Asn Ser Pro Asn Met Asn Ile Thr Gln Pro Ser545 550 555
560Lys Val Ser Asn Gln Asp Ser Lys Ser Pro Leu Gly Phe Tyr Cys
Asp565 570 575Gln Asn Pro Val Glu Ser Ser Met Cys Gln Ser Asn Ser
Arg Asp His580 585 590Leu Ser Asp Lys Glu Ser Lys Glu Ser Ser Val
Glu Gly Ala Glu Asn595 600 605Gln Arg Gly Pro Leu Glu Ser Lys Gly
His Lys Lys Leu Leu Gln Leu610 615 620Leu Thr Cys Ser Ser Asp Asp
Arg Gly His Ser Ser Leu Thr Asn Ser625 630 635 640Pro Leu Asp Ser
Ser Cys Lys Glu Ser Ser Val Ser Val Thr Ser Pro645 650 655Ser Gly
Val Ser Ser Ser Thr Ser Gly Gly Val Ser Ser Thr Ser Asn660 665
670Met His Gly Ser Leu Leu Gln Glu Lys His Arg Ile Leu His Lys
Leu675 680 685Leu Gln Asn Gly Asn Ser Pro Ala Glu Val Ala Lys Ile
Thr Ala Glu690 695 700Ala Thr Gly Lys Asp Thr Ser Ser Ile Thr Ser
Cys Gly Asp Gly Asn705 710 715 720Val Val Lys Gln Glu Gln Leu Ser
Pro Lys Lys Lys Glu Asn Asn Ala725 730 735Leu Leu Arg Tyr Leu Leu
Asp Arg Asp Asp Pro Ser Asp Ala Leu Ser740 745 750Lys Glu Leu Gln
Pro Gln Val Glu Gly Val Asp Asn Lys Met Ser Gln755 760 765Cys Thr
Ser Ser Thr Ile Pro Ser Ser Ser Gln Glu Lys Asp Pro Lys770 775
780Ile Lys Thr Glu Thr Ser Glu Glu Gly Ser Gly Asp Leu Asp Asn
Leu785 790 795 800Asp Ala Ile Leu Gly Asp Leu Thr Ser Ser Asp Phe
Tyr Asn Asn Ser805 810 815Ile Ser Ser Asn Gly Ser His Leu Gly Thr
Lys Gln Gln Val Phe Gln820 825 830Gly Thr Asn Ser Leu Gly Leu Lys
Ser Ser Gln Ser Val Gln Ser Ile835 840 845Arg Pro Pro Tyr Asn Arg
Ala Val Ser Leu Asp Ser Pro Val Ser Val850 855 860Gly Ser Ser Pro
Pro Val Lys Asn Ile Ser Ala Phe Pro Met Leu Pro865 870 875 880Lys
Gln Pro Met Leu Gly Gly Asn Pro Arg Met Met Asp Ser Gln Glu885 890
895Asn Tyr Gly Ser Ser Met Gly Gly Pro Asn Arg Asn Val Thr Val
Thr900 905 910Gln Thr Pro Ser Ser Gly Asp Trp Gly Leu Pro Asn Ser
Lys Ala Gly915 920 925Arg Met Glu Pro Met Asn Ser Asn Ser Met Gly
Arg Pro Gly Gly Asp930 935 940Tyr Asn Thr Ser Leu Pro Arg Pro Ala
Leu Gly Gly Ser Ile Pro Thr945 950 955 960Leu Pro Leu Arg Ser Asn
Ser Ile Pro Gly Ala Arg Pro Val Leu Gln965 970 975Gln Gln Gln Gln
Met Leu Gln Met Arg Pro Gly Glu Ile Pro Met Gly980 985 990Met Gly
Ala Asn Pro Tyr Gly Gln Ala Ala Ala Ser Asn Gln Leu Gly995 1000
1005Ser Trp Pro Asp Gly Met Leu Ser Met Glu Gln Val Ser His Gly
Thr1010 1015 1020Gln Asn Arg Pro Leu Leu Arg Asn Ser Leu Asp Asp
Leu Val Gly Pro1025 1030 1035 1040Pro Ser Asn Leu Glu Gly Gln Ser
Asp Glu Arg Ala Leu Leu Asp Gln1045 1050 1055Leu His Thr Leu Leu
Ser Asn Thr Asp Ala Thr Gly Leu Glu Glu Ile1060 1065 1070Asp Arg
Ala Leu Gly Ile Pro Glu Leu Val Asn Gln Gly Gln Ala Leu1075 1080
1085Glu Pro Lys Gln Asp Ala Phe Gln Gly Gln Glu Ala Ala Val Met
Met1090 1095 1100Asp Gln Lys Ala Gly Leu Tyr Gly Gln Thr Tyr Pro
Ala Gln Gly Pro1105 1110 1115 1120Pro Met Gln Gly Gly Phe His Leu
Gln Gly Gln Ser Pro Ser Phe Asn1125 1130 1135Ser Met Met Asn Gln
Met Asn Gln Gln Gly Asn Phe Pro Leu Gln Gly1140 1145 1150Met His
Pro Arg Ala Asn Ile Met Arg Pro Arg Thr Asn Thr Pro Lys1155 1160
1165Gln Leu Arg Met Gln Leu Gln Gln Arg Leu Gln Gly Gln Gln Phe
Leu1170 1175 1180Asn Gln Ser Arg Gln Ala Leu Glu Leu Lys Met Glu
Asn Pro Thr Ala1185 1190 1195 1200Gly Gly Ala Ala Val Met Arg Pro
Met Met Gln Pro Gln Gln Gly Phe1205 1210 1215Leu Asn Ala Gln Met
Val Ala Gln Arg Ser Arg Glu Leu Leu Ser His1220 1225 1230His Phe
Arg Gln Gln Arg Val Ala Met Met Met Gln Gln Gln Gln Gln1235 1240
1245Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln1250 1255 1260Gln Gln Gln Gln Gln Gln Gln Gln Thr Gln Ala Phe
Ser Pro Pro Pro1265 1270 1275 1280Asn Val Thr Ala Ser Pro Ser Met
Asp Gly Leu Leu Ala Gly Pro Thr1285 1290 1295Met Pro Gln Ala Pro
Pro Gln Gln Phe Pro Tyr Gln Pro Asn Tyr Gly1300 1305 1310Met Gly
Gln Gln Pro Asp Pro Ala Phe Gly Arg Val Ser Ser Pro Pro1315 1320
1325Asn Ala Met Met Ser Ser Arg Met Gly Pro Ser Gln Asn Pro Met
Met1330 1335 1340Gln His Pro Gln Ala Ala Ser Ile Tyr Gln Ser Ser
Glu Met Lys Gly1345 1350 1355 1360Trp Pro Ser Gly Asn Leu Ala Arg
Asn Ser Ser Phe Ser Gln Gln Gln1365 1370 1375Phe Ala His Gln Gly
Asn Pro Ala Val Tyr Ser Met Val His Met Asn1380 1385 1390Gly Ser
Ser Gly His Met Gly Gln Met Asn Met Asn Pro Met Pro Met1395 1400
1405Ser Gly Met Pro Met Gly Pro Asp Gln Lys Tyr Cys1410 1415
1420522DNAArtificial SequenceDescription of Artificial
SequencePRIMER N8F1 5tcatcacttc cgacaacaga gg 22620DNAArtificial
SequenceDescription of Artificial Sequence forward primer designed
from the 5' sequence of pCMVSPORT-B11, PM-U2 6ccagaaacgt cactatcaag
20719DNAArtificial SequenceDescription of Artificial Sequence
reverse primer designed from the 5' sequence of pCMVSPORT-B11,
PM-U2 7ttactggaac ccccatacc 198951PRTHomo sapiens 8Cys Ile Gln Arg
Phe Phe Ser Leu Asn Asp Gly Gln Ser Trp Ser Gln1 5 10 15Lys Arg His
Tyr Gln Glu Ala Tyr Leu Asn Gly His Ala Glu Thr Pro20 25 30Val Tyr
Arg Phe Ser Leu Ala Asp Gly Thr Ile Val Thr Ala Gln Thr35 40 45Lys
Ser Lys Leu Phe Arg Asn Pro Val Thr Asn Asp Arg His Gly Phe50 55
60Val Ser Thr His Phe Leu Gln Arg Glu Gln Asn Gly Tyr Arg Pro Asn65
70 75 80Pro Asn Pro Val Gly Gln Gly Ile Arg Pro Pro Met Ala Gly Cys
Asn85 90 95Ser Ser Val Gly Gly Met Ser Met Ser Pro Asn Gln Gly Leu
Gln Met100 105 110Pro Ser Ser Arg Ala Tyr Gly Leu Ala Asp Pro Ser
Thr Thr Gly Gln115 120 125Met Ser Gly Ala Arg Tyr Gly Gly Ser Ser
Asn Ile Ala Ser Leu Thr130 135 140Pro Gly Pro Gly Met Gln Ser Pro
Ser Ser Tyr Gln Asn Asn Asn Tyr145 150 155 160Gly Leu Asn Met Ser
Ser Pro Pro His Gly Ser Pro Gly Leu Ala Pro165 170 175Asn Gln Gln
Asn Ile Met Ile Ser Pro Arg Asn Arg Gly Ser Pro Lys180 185 190Ile
Ala Ser His Gln Phe Ser Pro Val Ala Gly Val His Ser Pro Met195 200
205Ala Ser Ser Gly Asn Thr Gly Asn His Ser Phe Ser Ser Ser Ser
Leu210 215 220Ser Ala Leu Gln Ala Ile Ser Glu Gly Val Gly Thr Ser
Leu Leu Ser225 230 235 240Thr Leu Ser Ser Pro Gly Pro Lys Leu Asp
Asn Ser Pro Asn Met Asn245 250 255Ile Thr Gln Pro Ser Lys Val Ser
Asn Gln Asp Ser Lys Ser Pro Leu260 265 270Gly Phe Tyr Cys Asp Gln
Asn Pro Val Glu Ser Ser Met Cys Gln Ser275 280 285Asn Ser Arg Asp
His Leu Ser Asp Lys Glu Ser Lys Glu Ser Ser Val290 295 300Glu Gly
Ala Glu Asn Gln Arg Gly Pro Leu Glu Ser Lys Gly His Lys305 310 315
320Lys Leu Leu Gln Leu Leu Thr Cys Ser Ser Asp Asp Arg Gly His
Ser325 330 335Ser Leu Thr Asn Ser Pro Leu Asp Ser Ser Cys Lys Glu
Ser Ser Val340 345 350Ser Val Thr Ser Pro Ser Gly Val Ser Ser Ser
Thr Ser Gly Gly Val355 360 365Ser Ser Thr Ser Asn Met His Gly Ser
Leu Leu Gln Glu Lys His Arg370 375 380Ile Leu His Lys Leu Leu Gln
Asn Gly Asn Ser Pro Ala Glu Val Ala385 390 395 400Lys Ile Thr Ala
Glu Ala Thr Gly Lys Asp Thr Ser Ser Ile Thr Ser405 410 415Cys Gly
Asp Gly Asn Val Val Lys Gln Glu Gln Leu Ser Pro Lys Lys420 425
430Lys Glu Asn Asn Ala Leu Leu Arg Tyr Leu Leu Asp Arg Asp Asp
Pro435 440 445Ser Asp Ala Leu Ser Lys Glu Leu Gln Pro Gln Val Glu
Gly Val Asp450 455 460Asn Lys Met Ser Gln Cys Thr Ser Ser Thr Ile
Pro Ser Ser Ser Gln465 470 475 480Glu Lys Asp Pro Lys Ile Lys Thr
Glu Thr Ser Glu Glu Gly Ser Gly485 490 495Asp Leu Asp Asn Leu Asp
Ala Ile Leu Gly Asp Leu Thr Ser Ser Asp500 505 510Phe Tyr Asn Asn
Ser Ile Ser Ser Asn Gly Ser His Leu Gly Thr Lys515 520 525Gln Gln
Val Phe Gln Gly Thr Asn Ser Leu Gly Leu Lys Ser Ser Gln530 535
540Ser Val Gln Ser Ile Arg Pro Pro Tyr Asn Arg Ala Val Ser Leu
Asp545 550 555 560Ser Pro Val Ser Val Gly Ser Ser Pro Pro Val Lys
Asn Ile Ser Ala565 570 575Phe Pro Met Leu Pro Lys Gln Pro Met Leu
Gly Gly Asn Pro Arg Met580 585 590Met Asp Ser Gln Glu Asn Tyr Gly
Ser Ser Met Gly Gly Pro Asn Arg595 600 605Asn Val Thr Val Thr Gln
Thr Pro Ser Ser Gly Asp Trp Gly Leu Pro610 615 620Asn Ser Lys Ala
Gly Arg Met Glu Pro Met Asn Ser Asn Ser Met Gly625 630 635 640Arg
Pro Gly Gly Asp Tyr Asn Thr Ser Leu Pro Arg Pro Ala Leu Gly645 650
655Gly Ser Ile Pro Thr Leu Pro Leu Arg Ser Asn Ser Ile Pro Gly
Ala660 665 670Arg Pro Val Leu Gln Gln Gln Gln Gln Met Leu Gln Met
Arg Pro Gly675 680 685Glu Ile Pro Met Gly Met Gly Ala Asn Pro Tyr
Gly Gln Ala Ala Ala690 695 700Ser Asn Gln Leu Gly Ser Trp Pro Asp
Gly Met Leu Ser Met Glu Gln705 710
715 720Val Ser His Gly Thr Gln Asn Arg Pro Leu Leu Arg Asn Ser Leu
Asp725 730 735Asp Leu Val Gly Pro Pro Ser Asn Leu Glu Gly Gln Ser
Asp Glu Arg740 745 750Ala Leu Leu Asp Gln Leu His Thr Leu Leu Ser
Asn Thr Asp Ala Thr755 760 765Gly Leu Glu Glu Ile Asp Arg Ala Leu
Gly Ile Pro Glu Leu Val Asn770 775 780Gln Gly Gln Ala Leu Glu Pro
Lys Gln Asp Ala Phe Gln Gly Gln Glu785 790 795 800Ala Ala Val Met
Met Asp Gln Lys Ala Gly Leu Tyr Gly Gln Thr Tyr805 810 815Pro Ala
Gln Gly Pro Pro Met Gln Gly Gly Phe His Leu Gln Gly Gln820 825
830Ser Pro Ser Phe Asn Ser Met Met Asn Gln Met Asn Gln Gln Gly
Asn835 840 845Phe Pro Leu Gln Gly Met His Pro Arg Ala Asn Ile Met
Arg Pro Arg850 855 860Thr Asn Thr Pro Lys Gln Leu Arg Met Gln Leu
Gln Gln Arg Leu Gln865 870 875 880Gly Gln Gln Phe Leu Asn Gln Ser
Arg Gln Ala Leu Glu Leu Lys Met885 890 895Glu Asn Pro Thr Ala Gly
Gly Ala Ala Val Met Arg Pro Met Met Gln900 905 910Pro Gln Gln Gly
Phe Leu Asn Ala Gln Met Val Ala Gln Arg Ser Arg915 920 925Glu Leu
Leu Ser His His Phe Arg Gln Gln Arg Val Ala Met Met Met930 935
940Gln Gln Gln Gln Gln Gln Gln945 95094621DNAMus
musculusCDS(110)..(4318) 9ggcggcgaac ggatcaaaag aatttgctga
acagtggact ccgagatcgg taaaacgaac 60tcttccctgc ccttcctgaa cagctgtcag
ttgctgatct gtgatcagg atg agt gga 118Met Ser Gly1cta ggc gaa agc tct
ttg gat ccg ctg gcc gct gag tct cgg aaa cgc 166Leu Gly Glu Ser Ser
Leu Asp Pro Leu Ala Ala Glu Ser Arg Lys Arg5 10 15aaa ctg ccc tgt
gat gcc cca gga cag ggg ctt gtc tac agt ggt gag 214Lys Leu Pro Cys
Asp Ala Pro Gly Gln Gly Leu Val Tyr Ser Gly Glu20 25 30 35aag tgg
cga cgg gag cag gag agc aag tac ata gag gag ctg gca gag 262Lys Trp
Arg Arg Glu Gln Glu Ser Lys Tyr Ile Glu Glu Leu Ala Glu40 45 50ctc
atc tct gca aat ctc agc gac atc gac aac ttc aat gtc aag cca 310Leu
Ile Ser Ala Asn Leu Ser Asp Ile Asp Asn Phe Asn Val Lys Pro55 60
65gat aaa tgt gcc atc cta aag gag aca gtg aga cag ata cgg caa ata
358Asp Lys Cys Ala Ile Leu Lys Glu Thr Val Arg Gln Ile Arg Gln
Ile70 75 80aaa gaa caa gga aaa act att tcc agt gat gat gat gtt caa
aaa gct 406Lys Glu Gln Gly Lys Thr Ile Ser Ser Asp Asp Asp Val Gln
Lys Ala85 90 95gat gtg tct tct aca ggg cag gga gtc att gat aaa gac
tct tta gga 454Asp Val Ser Ser Thr Gly Gln Gly Val Ile Asp Lys Asp
Ser Leu Gly100 105 110 115ccg ctt tta cta cag gca ctg gat ggt ttc
ctg ttt gtg gtg aat cga 502Pro Leu Leu Leu Gln Ala Leu Asp Gly Phe
Leu Phe Val Val Asn Arg120 125 130gat gga aac att gta ttc gtg tca
gaa aat gtc aca cag tat ctg cag 550Asp Gly Asn Ile Val Phe Val Ser
Glu Asn Val Thr Gln Tyr Leu Gln135 140 145tac aag cag gag gac ctg
gtt aac aca agt gtc tac agc atc tta cat 598Tyr Lys Gln Glu Asp Leu
Val Asn Thr Ser Val Tyr Ser Ile Leu His150 155 160gag caa gac cgg
aag gat ttt ctt aaa cac tta cca aaa tcc aca gtt 646Glu Gln Asp Arg
Lys Asp Phe Leu Lys His Leu Pro Lys Ser Thr Val165 170 175aat gga
gtt tct tgg act aat gag aac cag aga caa aaa agc cat aca 694Asn Gly
Val Ser Trp Thr Asn Glu Asn Gln Arg Gln Lys Ser His Thr180 185 190
195ttt aat tgt cgt atg ttg atg aaa aca cac gac att ttg gaa gac gtg
742Phe Asn Cys Arg Met Leu Met Lys Thr His Asp Ile Leu Glu Asp
Val200 205 210aat gcc agt ccc gaa aca cgc cag aga tat gaa aca atg
cag tgc ttt 790Asn Ala Ser Pro Glu Thr Arg Gln Arg Tyr Glu Thr Met
Gln Cys Phe215 220 225gcc ctg tct cag cct cgc gct atg ctg gaa gaa
gga gaa gac ttg cag 838Ala Leu Ser Gln Pro Arg Ala Met Leu Glu Glu
Gly Glu Asp Leu Gln230 235 240tgc tgt atg atc tgc gtg gct cgc cgc
gtg act gcg cca ttc cca tcc 886Cys Cys Met Ile Cys Val Ala Arg Arg
Val Thr Ala Pro Phe Pro Ser245 250 255agt cct gag agc ttt att acc
aga cat gac ctt tcc gga aag gtt gtc 934Ser Pro Glu Ser Phe Ile Thr
Arg His Asp Leu Ser Gly Lys Val Val260 265 270 275aat ata gat aca
aac tca ctt aga tct tcc atg agg cct ggc ttt gaa 982Asn Ile Asp Thr
Asn Ser Leu Arg Ser Ser Met Arg Pro Gly Phe Glu280 285 290gac ata
atc cga aga tgt atc cag agg ttc ttc agt ctg aat gat ggg 1030Asp Ile
Ile Arg Arg Cys Ile Gln Arg Phe Phe Ser Leu Asn Asp Gly295 300
305cag tca tgg tcc cag aag cgt cac tat caa gaa gct tat gtt cat ggc
1078Gln Ser Trp Ser Gln Lys Arg His Tyr Gln Glu Ala Tyr Val His
Gly310 315 320cac gca gag acc ccc gtg tat cgt ttc tcc ttg gct gat
gga act att 1126His Ala Glu Thr Pro Val Tyr Arg Phe Ser Leu Ala Asp
Gly Thr Ile325 330 335gtg agt gcg cag aca aaa agc aaa ctc ttc cgc
aat cct gta acg aat 1174Val Ser Ala Gln Thr Lys Ser Lys Leu Phe Arg
Asn Pro Val Thr Asn340 345 350 355gat cgt cac ggc ttc atc tcg acc
cac ttt ctt cag aga gaa cag aat 1222Asp Arg His Gly Phe Ile Ser Thr
His Phe Leu Gln Arg Glu Gln Asn360 365 370gga tac aga cca aac cca
aat ccc gca gga caa ggc atc cga cct cct 1270Gly Tyr Arg Pro Asn Pro
Asn Pro Ala Gly Gln Gly Ile Arg Pro Pro375 380 385gca gca ggg tgt
ggc gtg agc atg tct cca aat cag aat gta cag atg 1318Ala Ala Gly Cys
Gly Val Ser Met Ser Pro Asn Gln Asn Val Gln Met390 395 400atg ggc
agc cgg acc tat ggc gtg cca gac ccc agc aac aca ggg cag 1366Met Gly
Ser Arg Thr Tyr Gly Val Pro Asp Pro Ser Asn Thr Gly Gln405 410
415atg ggt gga gct agg tac ggg gct tct agt agc gta gcc tca ctg acg
1414Met Gly Gly Ala Arg Tyr Gly Ala Ser Ser Ser Val Ala Ser Leu
Thr420 425 430 435cca gga caa agc cta cag tcg cca tct tcc tat cag
aac agc agc tat 1462Pro Gly Gln Ser Leu Gln Ser Pro Ser Ser Tyr Gln
Asn Ser Ser Tyr440 445 450ggg ctc agc atg agc agt ccc ccc cac ggc
agt cct ggt ctt ggt ccc 1510Gly Leu Ser Met Ser Ser Pro Pro His Gly
Ser Pro Gly Leu Gly Pro455 460 465aac cag cag aac atc atg att tcc
cct cgg aat cgt ggc agc cca aag 1558Asn Gln Gln Asn Ile Met Ile Ser
Pro Arg Asn Arg Gly Ser Pro Lys470 475 480atg gcc tcc cac cag ttc
tct cct gct gca ggt gca cac tca ccc atg 1606Met Ala Ser His Gln Phe
Ser Pro Ala Ala Gly Ala His Ser Pro Met485 490 495gga cct tct ggc
aac aca ggg agc cac agc ttt tct agc agc tcc ctc 1654Gly Pro Ser Gly
Asn Thr Gly Ser His Ser Phe Ser Ser Ser Ser Leu500 505 510 515agt
gcc ttg caa gcc atc agt gaa ggc gtg ggg acc tct ctt tta tct 1702Ser
Ala Leu Gln Ala Ile Ser Glu Gly Val Gly Thr Ser Leu Leu Ser520 525
530act ctg tcc tca cca ggc ccc aaa ctg gat aat tct ccc aat atg aat
1750Thr Leu Ser Ser Pro Gly Pro Lys Leu Asp Asn Ser Pro Asn Met
Asn535 540 545ata agc cag cca agt aaa gtg agt ggt cag gac tct aag
agc ccc cta 1798Ile Ser Gln Pro Ser Lys Val Ser Gly Gln Asp Ser Lys
Ser Pro Leu550 555 560ggc tta tac tgt gaa cag aat cca gtg gag agt
tca gtg tgt cag tca 1846Gly Leu Tyr Cys Glu Gln Asn Pro Val Glu Ser
Ser Val Cys Gln Ser565 570 575aac agc aga gat cac cca agt gaa aaa
gaa agc aag gag agc agt ggg 1894Asn Ser Arg Asp His Pro Ser Glu Lys
Glu Ser Lys Glu Ser Ser Gly580 585 590 595gag gtg tca gag acg ccc
agg gga cct ctg gaa agc aaa ggc cac aag 1942Glu Val Ser Glu Thr Pro
Arg Gly Pro Leu Glu Ser Lys Gly His Lys600 605 610aaa ctg ctg cag
tta ctc acg tgc tcc tcc gac gac cga ggc cat tcc 1990Lys Leu Leu Gln
Leu Leu Thr Cys Ser Ser Asp Asp Arg Gly His Ser615 620 625tcc ttg
acc aac tct ccc ctg gat cca aac tgc aaa gac tct tcc gtt 2038Ser Leu
Thr Asn Ser Pro Leu Asp Pro Asn Cys Lys Asp Ser Ser Val630 635
640agt gtc acc agc ccc tct gga gtg tcc tcc tca aca tca ggg aca gtg
2086Ser Val Thr Ser Pro Ser Gly Val Ser Ser Ser Thr Ser Gly Thr
Val645 650 655tct tcc acc tcc aat gtg cat ggg tct ctg ttg caa gag
aaa cac cgg 2134Ser Ser Thr Ser Asn Val His Gly Ser Leu Leu Gln Glu
Lys His Arg660 665 670 675att ttg cac aag ttg ctg cag aat ggc aac
tcc cca gcg gag gtc gcc 2182Ile Leu His Lys Leu Leu Gln Asn Gly Asn
Ser Pro Ala Glu Val Ala680 685 690aag atc act gca gag gcc act ggg
aag gac acg agc agc act gct tcc 2230Lys Ile Thr Ala Glu Ala Thr Gly
Lys Asp Thr Ser Ser Thr Ala Ser695 700 705tgt gga gag ggg aca acc
agg cag gag cag ctg agt cct aag aag aag 2278Cys Gly Glu Gly Thr Thr
Arg Gln Glu Gln Leu Ser Pro Lys Lys Lys710 715 720gag aat aat gct
ctg ctt aga tac ctg ctg gac agg gat gac ccc agt 2326Glu Asn Asn Ala
Leu Leu Arg Tyr Leu Leu Asp Arg Asp Asp Pro Ser725 730 735gat gtg
ctt gcc aaa gag ctg cag ccc cag gcc gac agt ggg gac agt 2374Asp Val
Leu Ala Lys Glu Leu Gln Pro Gln Ala Asp Ser Gly Asp Ser740 745 750
755aaa ctg agt cag tgc agc tgc tcc acc aat ccc agc tct ggc caa gag
2422Lys Leu Ser Gln Cys Ser Cys Ser Thr Asn Pro Ser Ser Gly Gln
Glu760 765 770aaa gac ccc aaa att aag acc gag acg aac gag gag gta
tcg gga gac 2470Lys Asp Pro Lys Ile Lys Thr Glu Thr Asn Glu Glu Val
Ser Gly Asp775 780 785ctg gat aat cta gat gcc att ctt gga gat ttg
acc agt tct gac ttc 2518Leu Asp Asn Leu Asp Ala Ile Leu Gly Asp Leu
Thr Ser Ser Asp Phe790 795 800tac aac aat cct aca aat ggc ggt cac
cca ggg gcc aaa cag cag atg 2566Tyr Asn Asn Pro Thr Asn Gly Gly His
Pro Gly Ala Lys Gln Gln Met805 810 815ttt gca gga ccg agt tct ctg
ggt ttg cga agt cca cag cct gtg cag 2614Phe Ala Gly Pro Ser Ser Leu
Gly Leu Arg Ser Pro Gln Pro Val Gln820 825 830 835tct gtt cgt cct
cca tat aac cga gcg gtg tct ctg gat agc cct gtg 2662Ser Val Arg Pro
Pro Tyr Asn Arg Ala Val Ser Leu Asp Ser Pro Val840 845 850tct gtt
ggc tca ggt ccg cca gtg aag aat gtc agt gct ttc cct ggg 2710Ser Val
Gly Ser Gly Pro Pro Val Lys Asn Val Ser Ala Phe Pro Gly855 860
865tta cca aaa cag ccc ata ctg gct ggg aat cca aga atg atg gat agt
2758Leu Pro Lys Gln Pro Ile Leu Ala Gly Asn Pro Arg Met Met Asp
Ser870 875 880cag gag aat tac ggt gcc aac atg ggc cca aac aga aat
gtt cct gtg 2806Gln Glu Asn Tyr Gly Ala Asn Met Gly Pro Asn Arg Asn
Val Pro Val885 890 895aat ccg act tcc tcc ccc gga gac tgg ggc tta
gct aac tca agg gcc 2854Asn Pro Thr Ser Ser Pro Gly Asp Trp Gly Leu
Ala Asn Ser Arg Ala900 905 910 915agc aga atg gag cct ctg gca tca
agt ccc ctg gga aga act gga gcc 2902Ser Arg Met Glu Pro Leu Ala Ser
Ser Pro Leu Gly Arg Thr Gly Ala920 925 930gat tac agt gcc act tta
ccc aga cct gcc atg ggg ggc tct gtg cct 2950Asp Tyr Ser Ala Thr Leu
Pro Arg Pro Ala Met Gly Gly Ser Val Pro935 940 945acc ttg cca ctt
cgt tct aat cga ctg cca ggt gca aga cca tcg ttg 2998Thr Leu Pro Leu
Arg Ser Asn Arg Leu Pro Gly Ala Arg Pro Ser Leu950 955 960cag caa
cag cag cag caa cag cag caa cag caa caa caa cag cag caa 3046Gln Gln
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln965 970
975cag cag cag caa cag cag cag cag caa cag cag cag atg ctt caa atg
3094Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Met Leu Gln
Met980 985 990 995aga act ggt gag att ccc atg gga atg gga gtc aat
ccc tat agc cca 3142Arg Thr Gly Glu Ile Pro Met Gly Met Gly Val Asn
Pro Tyr Ser Pro1000 1005 1010gca gtg ccg tct aac caa cca ggt tcc
tgg cca gag ggc atg ctc tct 3190Ala Val Pro Ser Asn Gln Pro Gly Ser
Trp Pro Glu Gly Met Leu Ser1015 1020 1025atg gaa caa ggt cct cac
ggg tct caa aat agg cct ctt ctt aga aac 3238Met Glu Gln Gly Pro His
Gly Ser Gln Asn Arg Pro Leu Leu Arg Asn1030 1035 1040tct ctg gat
gat ctg ctt ggg cca cct tct aac gca gag ggc cag agt 3286Ser Leu Asp
Asp Leu Leu Gly Pro Pro Ser Asn Ala Glu Gly Gln Ser1045 1050
1055gac gag aga gct ctg ctg gac cag ctg cac aca ctc ctg agc aac aca
3334Asp Glu Arg Ala Leu Leu Asp Gln Leu His Thr Leu Leu Ser Asn
Thr1060 1065 1070 1075gat gcc aca ggt ctg gag gag atc gac agg gcc
ttg gga att cct gag 3382Asp Ala Thr Gly Leu Glu Glu Ile Asp Arg Ala
Leu Gly Ile Pro Glu1080 1085 1090ctc gtg aat cag gga caa gct ttg
gag tcc aaa cag gat gtt ttc caa 3430Leu Val Asn Gln Gly Gln Ala Leu
Glu Ser Lys Gln Asp Val Phe Gln1095 1100 1105ggc caa gaa gca gca
gta atg atg gat cag aag gct gca cta tat gga 3478Gly Gln Glu Ala Ala
Val Met Met Asp Gln Lys Ala Ala Leu Tyr Gly1110 1115 1120cag aca
tac cca gct cag ggt cct ccc ctt caa gga ggc ttt aac ctt 3526Gln Thr
Tyr Pro Ala Gln Gly Pro Pro Leu Gln Gly Gly Phe Asn Leu1125 1130
1135cag gga cag tca cca tcg ttt aac tct atg atg ggt cag att agc cag
3574Gln Gly Gln Ser Pro Ser Phe Asn Ser Met Met Gly Gln Ile Ser
Gln1140 1145 1150 1155caa ggc agc ttt cct ctg caa ggc atg cat cct
aga gcc ggc ctc gtg 3622Gln Gly Ser Phe Pro Leu Gln Gly Met His Pro
Arg Ala Gly Leu Val1160 1165 1170aga cca agg acc aac acc ccg aag
cag ctg aga atg cag ctt cag cag 3670Arg Pro Arg Thr Asn Thr Pro Lys
Gln Leu Arg Met Gln Leu Gln Gln1175 1180 1185agg cta cag ggc cag
cag ttt tta aat cag agc cgg cag gca ctt gaa 3718Arg Leu Gln Gly Gln
Gln Phe Leu Asn Gln Ser Arg Gln Ala Leu Glu1190 1195 1200atg aaa
atg gag aac cct gct ggc act gct gtg atg agg ccc atg atg 3766Met Lys
Met Glu Asn Pro Ala Gly Thr Ala Val Met Arg Pro Met Met1205 1210
1215ccc cag gct ttc ttt aat gcc caa atg gct gcc cag cag aaa cga gag
3814Pro Gln Ala Phe Phe Asn Ala Gln Met Ala Ala Gln Gln Lys Arg
Glu1220 1225 1230 1235ctg atg agc cat cac ctg cag cag cag agg atg
gcg atg atg atg tca 3862Leu Met Ser His His Leu Gln Gln Gln Arg Met
Ala Met Met Met Ser1240 1245 1250caa cca cag cct cag gcc ttc agc
cca cct ccc aac gtc acc gcc tcc 3910Gln Pro Gln Pro Gln Ala Phe Ser
Pro Pro Pro Asn Val Thr Ala Ser1255 1260 1265ccc agc atg gac ggg
gtt ttg gca ggt tca gca atg ccg caa gcc cct 3958Pro Ser Met Asp Gly
Val Leu Ala Gly Ser Ala Met Pro Gln Ala Pro1270 1275 1280cca caa
cag ttt cca tat cca gca aat tac gga atg gga caa cca cca 4006Pro Gln
Gln Phe Pro Tyr Pro Ala Asn Tyr Gly Met Gly Gln Pro Pro1285 1290
1295gag cca gcc ttt ggt cga ggc tcg agt cct ccc agt gca atg atg tca
4054Glu Pro Ala Phe Gly Arg Gly Ser Ser Pro Pro Ser Ala Met Met
Ser1300 1305 1310 1315tca aga atg ggg cct tcc cag aat gcc atg gtg
cag cat cct cag ccc 4102Ser Arg Met Gly Pro Ser Gln Asn Ala Met Val
Gln His Pro Gln Pro1320 1325 1330aca ccc atg tat cag cct tca gat
atg aag ggg tgg ccg tca ggg aac 4150Thr Pro Met Tyr Gln Pro Ser Asp
Met Lys Gly Trp Pro Ser Gly Asn1335 1340 1345ctg gcc agg aat ggc
tcc ttc ccc cag cag cag ttt gct ccc cag ggg 4198Leu Ala Arg Asn Gly
Ser Phe Pro Gln Gln Gln Phe Ala Pro Gln Gly1350 1355 1360aac cct
gca gcc tac aac atg gtg cat atg aac agc agc ggt ggg cac 4246Asn Pro
Ala Ala Tyr Asn Met Val His Met Asn Ser Ser Gly Gly His1365 1370
1375ttg gga cag atg gcc atg acc ccc atg ccc atg tct ggc atg ccc atg
4294Leu Gly Gln Met Ala Met Thr Pro Met Pro Met Ser Gly Met Pro
Met1380 1385 1390 1395ggc ccc gat cag aaa tac tgc tga catctcccta
gtgggactga ctgtacagat 4348Gly Pro Asp Gln Lys Tyr Cys1400gacactgcac
aggatcatca ggacgtggcg gcgagtcatt gtctaagcat ccagcttgga
4408aacaaggcca gcgtgaccag cagcggggtc tgtgctgtca tttgagcaga
gctgggtctc 4468gctgaagcgc actgtctacc tgatgccctg cctctgtgtg
gcaaggtgtt ctgcctcatg 4528aggatgtgat tctggagatg gggtgttcgt
aagcaccgct ctcttacgtc actcccttct 4588gcctcgccag ccaaagtctt
cacgtagatc tag 46211022DNAArtificial SequenceDescription of
Artificial Sequence forward primer A1B1/mESTF1 to screen mouse
BAC
10tccttttccc agcagcagtt tg 221120DNAArtificial SequenceDescription
of Artificial Sequence reverse primer A1B1/mESTR1 used to screen
mouse BAC 11atgccagaca tgggcatggg 20121402PRTMus musculus 12Met Ser
Gly Leu Gly Glu Ser Ser Leu Asp Pro Leu Ala Ala Glu Ser1 5 10 15Arg
Lys Arg Lys Leu Pro Cys Asp Ala Pro Gly Gln Gly Leu Val Tyr20 25
30Ser Gly Glu Lys Trp Arg Arg Glu Gln Glu Ser Lys Tyr Ile Glu Glu35
40 45Leu Ala Glu Leu Ile Ser Ala Asn Leu Ser Asp Ile Asp Asn Phe
Asn50 55 60Val Lys Pro Asp Lys Cys Ala Ile Leu Lys Glu Thr Val Arg
Gln Ile65 70 75 80Arg Gln Ile Lys Glu Gln Gly Lys Thr Ile Ser Ser
Asp Asp Asp Val85 90 95Gln Lys Ala Asp Val Ser Ser Thr Gly Gln Gly
Val Ile Asp Lys Asp100 105 110Ser Leu Gly Pro Leu Leu Leu Gln Ala
Leu Asp Gly Phe Leu Phe Val115 120 125Val Asn Arg Asp Gly Asn Ile
Val Phe Val Ser Glu Asn Val Thr Gln130 135 140Tyr Leu Gln Tyr Lys
Gln Glu Asp Leu Val Asn Thr Ser Val Tyr Ser145 150 155 160Ile Leu
His Glu Gln Asp Arg Lys Asp Phe Leu Lys His Leu Pro Lys165 170
175Ser Thr Val Asn Gly Val Ser Trp Thr Asn Glu Asn Gln Arg Gln
Lys180 185 190Ser His Thr Phe Asn Cys Arg Met Leu Met Lys Thr His
Asp Ile Leu195 200 205Glu Asp Val Asn Ala Ser Pro Glu Thr Arg Gln
Arg Tyr Glu Thr Met210 215 220Gln Cys Phe Ala Leu Ser Gln Pro Arg
Ala Met Leu Glu Glu Gly Glu225 230 235 240Asp Leu Gln Cys Cys Met
Ile Cys Val Ala Arg Arg Val Thr Ala Pro245 250 255Phe Pro Ser Ser
Pro Glu Ser Phe Ile Thr Arg His Asp Leu Ser Gly260 265 270Lys Val
Val Asn Ile Asp Thr Asn Ser Leu Arg Ser Ser Met Arg Pro275 280
285Gly Phe Glu Asp Ile Ile Arg Arg Cys Ile Gln Arg Phe Phe Ser
Leu290 295 300Asn Asp Gly Gln Ser Trp Ser Gln Lys Arg His Tyr Gln
Glu Ala Tyr305 310 315 320Val His Gly His Ala Glu Thr Pro Val Tyr
Arg Phe Ser Leu Ala Asp325 330 335Gly Thr Ile Val Ser Ala Gln Thr
Lys Ser Lys Leu Phe Arg Asn Pro340 345 350Val Thr Asn Asp Arg His
Gly Phe Ile Ser Thr His Phe Leu Gln Arg355 360 365Glu Gln Asn Gly
Tyr Arg Pro Asn Pro Asn Pro Ala Gly Gln Gly Ile370 375 380Arg Pro
Pro Ala Ala Gly Cys Gly Val Ser Met Ser Pro Asn Gln Asn385 390 395
400Val Gln Met Met Gly Ser Arg Thr Tyr Gly Val Pro Asp Pro Ser
Asn405 410 415Thr Gly Gln Met Gly Gly Ala Arg Tyr Gly Ala Ser Ser
Ser Val Ala420 425 430Ser Leu Thr Pro Gly Gln Ser Leu Gln Ser Pro
Ser Ser Tyr Gln Asn435 440 445Ser Ser Tyr Gly Leu Ser Met Ser Ser
Pro Pro His Gly Ser Pro Gly450 455 460Leu Gly Pro Asn Gln Gln Asn
Ile Met Ile Ser Pro Arg Asn Arg Gly465 470 475 480Ser Pro Lys Met
Ala Ser His Gln Phe Ser Pro Ala Ala Gly Ala His485 490 495Ser Pro
Met Gly Pro Ser Gly Asn Thr Gly Ser His Ser Phe Ser Ser500 505
510Ser Ser Leu Ser Ala Leu Gln Ala Ile Ser Glu Gly Val Gly Thr
Ser515 520 525Leu Leu Ser Thr Leu Ser Ser Pro Gly Pro Lys Leu Asp
Asn Ser Pro530 535 540Asn Met Asn Ile Ser Gln Pro Ser Lys Val Ser
Gly Gln Asp Ser Lys545 550 555 560Ser Pro Leu Gly Leu Tyr Cys Glu
Gln Asn Pro Val Glu Ser Ser Val565 570 575Cys Gln Ser Asn Ser Arg
Asp His Pro Ser Glu Lys Glu Ser Lys Glu580 585 590Ser Ser Gly Glu
Val Ser Glu Thr Pro Arg Gly Pro Leu Glu Ser Lys595 600 605Gly His
Lys Lys Leu Leu Gln Leu Leu Thr Cys Ser Ser Asp Asp Arg610 615
620Gly His Ser Ser Leu Thr Asn Ser Pro Leu Asp Pro Asn Cys Lys
Asp625 630 635 640Ser Ser Val Ser Val Thr Ser Pro Ser Gly Val Ser
Ser Ser Thr Ser645 650 655Gly Thr Val Ser Ser Thr Ser Asn Val His
Gly Ser Leu Leu Gln Glu660 665 670Lys His Arg Ile Leu His Lys Leu
Leu Gln Asn Gly Asn Ser Pro Ala675 680 685Glu Val Ala Lys Ile Thr
Ala Glu Ala Thr Gly Lys Asp Thr Ser Ser690 695 700Thr Ala Ser Cys
Gly Glu Gly Thr Thr Arg Gln Glu Gln Leu Ser Pro705 710 715 720Lys
Lys Lys Glu Asn Asn Ala Leu Leu Arg Tyr Leu Leu Asp Arg Asp725 730
735Asp Pro Ser Asp Val Leu Ala Lys Glu Leu Gln Pro Gln Ala Asp
Ser740 745 750Gly Asp Ser Lys Leu Ser Gln Cys Ser Cys Ser Thr Asn
Pro Ser Ser755 760 765Gly Gln Glu Lys Asp Pro Lys Ile Lys Thr Glu
Thr Asn Glu Glu Val770 775 780Ser Gly Asp Leu Asp Asn Leu Asp Ala
Ile Leu Gly Asp Leu Thr Ser785 790 795 800Ser Asp Phe Tyr Asn Asn
Pro Thr Asn Gly Gly His Pro Gly Ala Lys805 810 815Gln Gln Met Phe
Ala Gly Pro Ser Ser Leu Gly Leu Arg Ser Pro Gln820 825 830Pro Val
Gln Ser Val Arg Pro Pro Tyr Asn Arg Ala Val Ser Leu Asp835 840
845Ser Pro Val Ser Val Gly Ser Gly Pro Pro Val Lys Asn Val Ser
Ala850 855 860Phe Pro Gly Leu Pro Lys Gln Pro Ile Leu Ala Gly Asn
Pro Arg Met865 870 875 880Met Asp Ser Gln Glu Asn Tyr Gly Ala Asn
Met Gly Pro Asn Arg Asn885 890 895Val Pro Val Asn Pro Thr Ser Ser
Pro Gly Asp Trp Gly Leu Ala Asn900 905 910Ser Arg Ala Ser Arg Met
Glu Pro Leu Ala Ser Ser Pro Leu Gly Arg915 920 925Thr Gly Ala Asp
Tyr Ser Ala Thr Leu Pro Arg Pro Ala Met Gly Gly930 935 940Ser Val
Pro Thr Leu Pro Leu Arg Ser Asn Arg Leu Pro Gly Ala Arg945 950 955
960Pro Ser Leu Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln965 970 975Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln Gln Met980 985 990Leu Gln Met Arg Thr Gly Glu Ile Pro Met Gly
Met Gly Val Asn Pro995 1000 1005Tyr Ser Pro Ala Val Pro Ser Asn Gln
Pro Gly Ser Trp Pro Glu Gly1010 1015 1020Met Leu Ser Met Glu Gln
Gly Pro His Gly Ser Gln Asn Arg Pro Leu1025 1030 1035 1040Leu Arg
Asn Ser Leu Asp Asp Leu Leu Gly Pro Pro Ser Asn Ala Glu1045 1050
1055Gly Gln Ser Asp Glu Arg Ala Leu Leu Asp Gln Leu His Thr Leu
Leu1060 1065 1070Ser Asn Thr Asp Ala Thr Gly Leu Glu Glu Ile Asp
Arg Ala Leu Gly1075 1080 1085Ile Pro Glu Leu Val Asn Gln Gly Gln
Ala Leu Glu Ser Lys Gln Asp1090 1095 1100Val Phe Gln Gly Gln Glu
Ala Ala Val Met Met Asp Gln Lys Ala Ala1105 1110 1115 1120Leu Tyr
Gly Gln Thr Tyr Pro Ala Gln Gly Pro Pro Leu Gln Gly Gly1125 1130
1135Phe Asn Leu Gln Gly Gln Ser Pro Ser Phe Asn Ser Met Met Gly
Gln1140 1145 1150Ile Ser Gln Gln Gly Ser Phe Pro Leu Gln Gly Met
His Pro Arg Ala1155 1160 1165Gly Leu Val Arg Pro Arg Thr Asn Thr
Pro Lys Gln Leu Arg Met Gln1170 1175 1180Leu Gln Gln Arg Leu Gln
Gly Gln Gln Phe Leu Asn Gln Ser Arg Gln1185 1190 1195 1200Ala Leu
Glu Met Lys Met Glu Asn Pro Ala Gly Thr Ala Val Met Arg1205 1210
1215Pro Met Met Pro Gln Ala Phe Phe Asn Ala Gln Met Ala Ala Gln
Gln1220 1225 1230Lys Arg Glu Leu Met Ser His His Leu Gln Gln Gln
Arg Met Ala Met1235 1240 1245Met Met Ser Gln Pro Gln Pro Gln Ala
Phe Ser Pro Pro Pro Asn Val1250 1255 1260Thr Ala Ser Pro Ser Met
Asp Gly Val Leu Ala Gly Ser Ala Met Pro1265 1270 1275 1280Gln Ala
Pro Pro Gln Gln Phe Pro Tyr Pro Ala Asn Tyr Gly Met Gly1285 1290
1295Gln Pro Pro Glu Pro Ala Phe Gly Arg Gly Ser Ser Pro Pro Ser
Ala1300 1305 1310Met Met Ser Ser Arg Met Gly Pro Ser Gln Asn Ala
Met Val Gln His1315 1320 1325Pro Gln Pro Thr Pro Met Tyr Gln Pro
Ser Asp Met Lys Gly Trp Pro1330 1335 1340Ser Gly Asn Leu Ala Arg
Asn Gly Ser Phe Pro Gln Gln Gln Phe Ala1345 1350 1355 1360Pro Gln
Gly Asn Pro Ala Ala Tyr Asn Met Val His Met Asn Ser Ser1365 1370
1375Gly Gly His Leu Gly Gln Met Ala Met Thr Pro Met Pro Met Ser
Gly1380 1385 1390Met Pro Met Gly Pro Asp Gln Lys Tyr Cys1395
1400
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