U.S. patent application number 10/463190 was filed with the patent office on 2004-01-15 for compositions and methods for increasing bone mineralization.
This patent application is currently assigned to Celltech R&D, Inc.. Invention is credited to Brunkow, Mary E., Galas, David J., Kovacevich, Brian, Mulligan, John T., Paeper, Bryan W., Van Ness, Jeffrey, Winkler, David G..
Application Number | 20040009535 10/463190 |
Document ID | / |
Family ID | 33563705 |
Filed Date | 2004-01-15 |
United States Patent
Application |
20040009535 |
Kind Code |
A1 |
Brunkow, Mary E. ; et
al. |
January 15, 2004 |
Compositions and methods for increasing bone mineralization
Abstract
A novel class or family of TGF-.beta. binding proteins is
disclosed. Also disclosed are assays for selecting molecules for
increasing bone mineralization and methods for utilizing such
molecules. In particular, compositions and methods relating to
antibodies that specifically bind to TGF-beta binding proteins are
provided. These methods and compositions relate to altering bone
mineral density by interfering with the interaction between a
TGF-beta binding protein sclerostin and a TGF-beta superfamily
member, particularly a bone morphogenic protein. Increasing bone
mineral density has uses in diseases and conditions in which low
bone mineral density typifies the condition, such as osteopenia,
osteoporosis, and bone fractures.
Inventors: |
Brunkow, Mary E.; (Seattle,
WA) ; Galas, David J.; (Claremont, CA) ;
Kovacevich, Brian; (Renton, WA) ; Mulligan, John
T.; (Seattle, WA) ; Paeper, Bryan W.;
(Seattle, WA) ; Van Ness, Jeffrey; (Claremont,
CA) ; Winkler, David G.; (Seattle, WA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
Celltech R&D, Inc.
1631 220th Street SE
Bothell
WA
98021
|
Family ID: |
33563705 |
Appl. No.: |
10/463190 |
Filed: |
June 16, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10463190 |
Jun 16, 2003 |
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10095248 |
Mar 7, 2002 |
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10095248 |
Mar 7, 2002 |
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09449218 |
Nov 24, 1999 |
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6395511 |
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60110283 |
Nov 27, 1998 |
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Current U.S.
Class: |
435/7.1 ;
530/388.26 |
Current CPC
Class: |
G01N 2333/495 20130101;
G01N 2500/04 20130101; A61P 19/08 20180101; C07K 2319/00 20130101;
C07K 16/22 20130101; A01K 2217/075 20130101; A61P 19/00 20180101;
C07K 2317/23 20130101; A61P 19/02 20180101; G01N 2800/108 20130101;
A61P 1/02 20180101; A61P 19/10 20180101; C07K 16/18 20130101; C07K
2317/11 20130101; C12N 2799/021 20130101; G01N 33/6872 20130101;
A61K 48/00 20130101; C07K 14/51 20130101; A01K 2217/05
20130101 |
Class at
Publication: |
435/7.1 ;
530/388.26 |
International
Class: |
G01N 033/53; C07K
016/40 |
Claims
We claim the following:
1. An antibody, or an antigen-binding fragment thereof, that binds
specifically to a sclerostin polypeptide, said sclerostin
polypeptide comprising an amino acid sequence set forth in SEQ ID
NOS: 2, 6, 8, 14, 46, or 65, wherein the antibody competitively
inhibits binding of the sclerostin polypeptide to at least one of
(i) a bone morphogenic protein (BMP) Type I Receptor binding site
and (ii) a BMP Type II Receptor binding site, wherein the BMP Type
I Receptor binding site is capable of binding to a BMP Type I
Receptor polypeptide comprising an amino acid sequence set forth in
a sequence selected from the group consisting of GenBank Acc. Nos.
NM.sub.--004329 (SEQ ID NO: 102); D89675 (SEQ ID NO: 103);
NM.sub.--001203 (SEQ ID NO: 104); S75359 (SEQ ID NO: 105);
NM.sub.--030849 (SEQ ID NO: 106); D38082 (SEQ ID NO: 107);
NP.sub.--001194 (SEQ ID NO: 108); BAA19765 (SEQ ID NO: 109); and
AAB33865 (SEQ ID NO: 110) and wherein the BMP Type II Receptor
binding site is capable of binding to a BMP Type II Receptor
polypeptide comprising the amino acid sequence set forth in a
sequence selected from the group consisting of GenBank Ace. NOs.
U25 110 (SEQ ID NO: 111); NM.sub.--033346 (SEQ ID NO: 112); Z48923
(SEQ ID NO: 114); CAA88759 (SEQ ID NO: 115); and NM.sub.--001204
(SEQ ID NO: 113).
2. An antibody, or an antigen-binding fragment thereof, that binds
specifically to a sclerostin polypeptide and that impairs formation
of a sclerostin homodimer, wherein the sclerostin polypeptide
comprises an amino acid sequence set forth in SEQ ID NOS: 2, 6, 8,
14, 46, or 65.
3. The antibody of either claim 1 or claim 2, wherein the antibody
is a polyclonal antibody.
4. The antibody of either claim 1 or claim 2, wherein the antibody
is a monoclonal antibody.
5. The antibody of claim 4 wherein the monoclonal antibody is
selected from the group consisting of a mouse monoclonal antibody,
a human monoclonal antibody, a rat monoclonal antibody, and a
hamster monoclonal antibody.
6. A hybridoma cell producing the antibody of claim 4.
7. A host cell that is capable of expressing the antibody of claim
4.
8. The antibody of either claim 1 or claim 2, wherein the antibody
is a humanized antibody or a chimeric antibody.
9. A host cell that is capable of expressing the antibody of claim
8.
10. The antibody of either claim 1 or claim 2, wherein the
antigen-binding fragment is selected from the group consisting of
F(ab').sub.2, Fab', Fab, Fd, and Fv.
11. The antibody of either claim 1 or claim 2 that comprises a
single chain antibody.
12. A host cell that is capable of expressing the antibody of claim
11.
13. A composition comprising an antibody, or antigen-binding
fragment thereof, according to either claim 1 or claim 2 and a
physiologically acceptable carrier.
14. An immunogen comprising a peptide comprising at least 21
consecutive amino acids and no more than 50 consecutive amino acids
of a SOST polypeptide, said SOST polypeptide comprising an amino
acid sequence set forth in SEQ ID NOS: 2, 6, 8, 14, 46, or 65,
wherein the peptide is capable of eliciting in a non-human animal
an antibody that binds specifically to the SOST polypeptide and
that competitively inhibits binding of the SOST polypeptide to at
least one of (i) a bone morphogenic protein (BMP) Type I Receptor
binding site and (ii) a BMP Type II Receptor binding site, wherein
the BMP Type I Receptor binding site is capable of binding to a BMP
Type I Receptor polypeptide comprising an amino acid sequence set
forth in a sequence selected from the group consisting of GenBank
Acc. Nos. NM.sub.--004329 (SEQ ID NO: 102); D89675 (SEQ ID NO:
103); NM 001203 (SEQ ID NO: 104); S75359 (SEQ ID NO: 105);
NM.sub.--030849 (SEQ ID NO: 106); D38082 (SEQ ID NO: 107);
NP.sub.--001194 (SEQ ID NO: 108); BAA19765 (SEQ ID NO: 109); and
AAB33865 (SEQ ID NO: 110) and wherein the BMP Type II Receptor
binding site is capable of binding to a BMP Type II Receptor
polypeptide comprising the amino acid sequence set forth in a
sequence selected from the group consisting of GenBank Acc. NOs.
U25110 (SEQ ID NO: 111); NM.sub.--033346 (SEQ ID NO: 112); Z48923
(SEQ ID NO: 114); CAA88759 (SEQ ID NO: 115); and NM.sub.--001204
(SEQ ID NO: 113).
15. An immunogen comprising a peptide that comprises at least 21
consecutive amino acids and no more than 50 consecutive amino acids
of a SOST polypeptide, said SOST polypeptide comprising an amino
acid sequence set forth in SEQ ID NOS: 2, 6, 8, 14, 46, or 65,
wherein the peptide is capable of eliciting in a non-human animal
an antibody that binds specifically to the SOST polypeptide and
that impairs formation of a SOST homodimer.
16. The immunogen of either claim 14 or claim 15 wherein the
peptide is associated with a carrier molecule.
17. The immunogen of claim 16 wherein the carrier molecule is
carrier polypeptide.
18. The immunogen of claim 17 wherein the carrier polypeptide is
keyhole limpet hemocyanin.
19. A method for producing an antibody that specifically binds to a
SOST polypeptide, comprising immunizing a non-human animal with an
immunogen according to claim 14, wherein (a) the SOST polypeptide
comprises an amino acid sequence set forth in SEQ ID NOS: 2, 6, 8,
14, 46, or 65; (b) the antibody competitively inhibits binding of
the SOST polypeptide to at least one of (i) a bone morphogenic
protein (BMP) Type I Receptor binding site and (ii) a BMP Type II
Receptor binding site; (c) the BMP Type I Receptor binding site is
capable of binding to a BMP Type I Receptor polypeptide comprising
the amino acid sequence set forth in a sequence selected from the
group consisting of GenBank Acc. Nos. NM.sub.--004329 (SEQ ID NO:
102); D89675 (SEQ ID NO: 103); NM.sub.--001203 (SEQ ID NO: 104);
S75359 (SEQ ID NO: 105); NM.sub.--030849 (SEQ ID NO: 106); D38082
(SEQ ID NO: 107); NP.sub.--001194 (SEQ ID NO: 108); BAA19765 (SEQ
ID NO: 109); and AAB33865 (SEQ ID NO: 110); and (d) the BMP Type II
Receptor binding site is capable of binding to a BMP Type II
Receptor polypeptide comprising the amino acid sequence set forth
in a sequence selected from the group consisting of GenBank Acc.
NOs. U25110 (SEQ ID NO: 111); NM.sub.--033346 (SEQ ID NO: 112);
Z48923 (SEQ ID NO: 114); CAA88759 (SEQ ID NO: 115); and
NM.sub.--001204 (SEQ ID NO: 113).
20. A method for producing an antibody that specifically binds to a
SOST polypeptide, said SOST polypeptide comprising an amino acid
sequence set forth in SEQ ID NOS: 2, 6, 8, 14, 46, or 65,
comprising immunizing a non-human animal with an immunogen
according to claim 15, wherein the antibody impairs formation of a
SOST homodimer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 10/095,248 (filed Mar. 7, 2002), which is a
continuation of U.S. application Ser. No. 09/449,218 (filed Nov.
24, 1999), now issued as U.S. Pat. No. 6,395,511, which claims
priority from U.S. Provisional Application No. 60/110,283 filed
Nov. 27, 1998. The contents of all the above applications are
incorporated herein by reference in their entirety.
TECHNICAL FIELD
[0002] The present invention relates generally to pharmaceutical
products and methods and, more specifically, to methods and
compositions suitable for increasing the mineral content of bone.
Such compositions and methods may be utilized to treat a wide
variety of conditions, including for example, osteopenia,
osteoporosis, fractures and other disorders in which low bone
mineral density are a hallmark of the disease.
BACKGROUND OF THE INVENTION
[0003] Two or three distinct phases of changes to bone mass occur
over the life of an individual (see Riggs, West J Med. 154:63-77,
1991). The first phase occurs in both men and women, and proceeds
to attainment of a peak bone mass. This first phase is achieved
through linear growth of the endochondral growth plates, and radial
growth due to a rate of periosteal apposition. The second phase
begins around age 30 for trabecular bone (flat bones such as the
vertebrae and pelvis) and about age 40 for cortical bone (e.g.,
long bones found in the limbs) and continues to old age. This phase
is characterized by slow bone loss, and occurs in both men and
women. In women, a third phase of bone loss also occurs, most
likely due to postmenopausal estrogen deficiencies. During this
phase alone, women may lose an additional 10% of bone mass from the
cortical bone and 25% from the trabecular compartment (see Riggs,
supra).
[0004] Loss of bone mineral content can be caused by a wide variety
of conditions, and may result in significant medical problems. For
example, osteoporosis is a debilitating disease in humans
characterized by marked decreases in skeletal bone mass and mineral
density, structural deterioration of bone including degradation of
bone microarchitecture and corresponding increases in bone
fragility and susceptibility to fracture in afflicted individuals.
Osteoporosis in humans is preceded by clinical osteopenia (bone
mineral density that is greater than one standard deviation but
less than 2.5 standard deviations below the mean value for young
adult bone), a condition found in approximately 25 million people
in the United States. Another 7-8 million patients in the United
States have been diagnosed with clinical osteoporosis (defined as
bone mineral content greater than 2.5 standard deviations below
that of mature young adult bone). Osteoporosis is one of the most
expensive diseases for the health care system, costing tens of
billions of dollars annually in the United States. In addition to
health care-related costs, long-term residential care and lost
working days add to the financial and social costs of this disease.
Worldwide approximately 75 million people are at risk for
osteoporosis.
[0005] The frequency of osteoporosis in the human population
increases with age, and among Caucasians is predominant in women
(who comprise 80% of the osteoporosis patient pool in the United
States). The increased fragility and susceptibility to fracture of
skeletal bone in the aged is aggravated by the greater risk of
accidental falls in this population. More than 1.5 million
osteoporosis-related bone fractures are reported in the United
States each year. Fractured hips, wrists, and vertebrae are among
the most common injuries associated with osteoporosis. Hip
fractures in particular are extremely uncomfortable and expensive
for the patient, and for women correlate with high rates of
mortality and morbidity.
[0006] Although osteoporosis has been defined as an increase in the
risk of fracture due to decreased bone mass, none of the presently
available treatments for skeletal disorders can substantially
increase the bone density of adults. There is a strong perception
among all physicians that drugs are needed which could increase
bone density in adults, particularly in the bones of the wrist,
spinal column and hip that are at risk in osteopenia and
osteoporosis.
[0007] Current strategies for the prevention of osteoporosis may
offer some benefit to individuals but cannot ensure resolution of
the disease. These strategies include moderating physical activity
(particularly in weight-bearing activities) with the onset of
advanced age, including adequate calcium in the diet, and avoiding
consumption of products containing alcohol or tobacco. For patients
presenting with clinical osteopenia or osteoporosis, all current
therapeutic drugs and strategies are directed to reducing further
loss of bone mass by inhibiting the process of bone absorption, a
natural component of the bone remodeling process that occurs
constitutively.
[0008] For example, estrogen is now being prescribed to retard bone
loss. There is, however, some controversy over whether there is any
long term benefit to patients and whether there is any effect at
all on patients over 75 years old. Moreover, use of estrogen is
believed to increase the risk of breast and endometrial cancer.
[0009] High doses of dietary calcium, with or without vitamin D has
also been suggested for postmenopausal women. However, high doses
of calcium can often have unpleasant gastrointestinal side effects,
and serum and urinary calcium levels must be continuously monitored
(see Khosla and Rigss, Mayo Clin. Proc. 70:978-982, 1995).
[0010] Other therapeutics which have been suggested include
calcitonin, bisphosphonates, anabolic steroids and sodium fluoride.
Such therapeutics however, have undesirable side effects (e.g.,
calcitonin and steroids may cause nausea and provoke an immune
reaction, bisphosphonates and sodium fluoride may inhibit repair of
fractures, even though bone density increases modestly) that may
prevent their usage (see Khosla and Rigss, supra).
[0011] No currently practiced therapeutic strategy involves a drug
that stimulates or enhances the growth of new bone mass. The
present invention provides compositions and methods which can be
utilized to increase bone mineralization, and thus may be utilized
to treat a wide variety of conditions where it is desired to
increase bone mass. Further, the present invention provides other,
related advantages.
SUMMARY OF THE INVENTION
[0012] As noted above, the present invention provides a novel class
or family of TGF-beta binding-proteins, as well as assays for
selecting compounds which increase bone mineral content and bone
mineral density, compounds which increase bone mineral content and
bone mineral density and methods for utilizing such compounds in
the treatment or prevention of a wide variety of conditions.
[0013] Within one aspect of the present invention, isolated nucleic
acid molecules are provided, wherein said nucleic acid molecules
are selected from the group consisting of: (a) an isolated nucleic
acid molecule comprising sequence ID Nos. 1, 5, 7, 9, 11, 13, or,
15, or complementary sequence thereof; (b) an isolated nucleic acid
molecule that specifically hybridizes to the nucleic acid molecule
of (a) under conditions of high stringency; and (c) an isolated
nucleic acid that encodes a TGF-beta binding-protein according to
(a) or (b). Within related aspects of the present invention,
isolated nucleic acid molecules are provided based upon
hybridization to only a portion of one of the above-identified
sequences (e.g., for (a) hybridization may be to a probe of at
least 20, 25, 50, or 100 nucleotides selected from nucleotides 156
to 539 or 555 to 687 of Sequence ID No. 1). As should be readily
evident, the necessary stringency to be utilized for hybridization
may vary based upon the size of the probe. For example, for a
25-mer probe high stringency conditions could include: 60 mM Tris
pH 8.0, 2 mM EDTA, 5.times. Denhardt's, 6.times.SSC, 0.1% (w/v)
N-laurylsarcosine, 0.5% (w/v) NP-40 (nonidet P-40) overnight at 45
degrees C., followed by two washes with 0.2.times.SSC/0.1% SDS at
45-50 degrees. For a 100-mer probe under low stringency conditions,
suitable conditions might include the following: 5.times. SSPE,
5.times. Denhardt's, and 0.5% SDS overnight at 42-50 degrees,
followed by two washes with 2.times. SSPE (or 2.times.SSC)/0.1% SDS
at 42-50 degrees.
[0014] Within related aspects of the present invention, isolated
nucleic acid molecules are provided which have homology to Sequence
ID Nos. 1, 5, 7, 9, 11, 13, or 15, at a 50%, 60%, 75%, 80%, 90%,
95%, or 98% level of homology utilizing a Wilbur-Lipman algorithm.
Representative examples of such isolated molecules include, for
example, nucleic acid molecules which encode a protein comprising
Sequence ID NOs. 2, 6, 10, 12, 14, or 16, or have homology to these
sequences at a level of 50%, 60%, 75%, 80%, 90%, 95%, or 98% level
of homology utilizing a Lipman-Pearson algorithm.
[0015] Isolated nucleic acid molecules are typically less than 100
kb in size, and, within certain embodiments, less than 50 kb, 25
kb, 10 kb, or even 5 kb in size. Further, isolated nucleic acid
molecules, within other embodiments, do not exist in a "library" of
other unrelated nucleic acid molecules (e.g., a subclone BAC such
as described in GenBank Accession No. AC003098 and EMB No.
AQ171546). However, isolated nucleic acid molecules can be found in
libraries of related molecules (e.g., for shuffling, such as is
described in U.S. Pat. Nos. 5,837,458; 5,830,721; and 5,811,238).
Finally, isolated nucleic acid molecules as described herein do not
include nucleic acid molecules which encode Dan, Cerberus, Gremlin,
or SCGF (U.S. Pat. No. 5,780,263).
[0016] Also provided by the present invention are cloning vectors
which contain the above-noted nucleic acid molecules, and
expression vectors which comprise a promoter (e.g., a regulatory
sequence) operably linked to one of the above-noted nucleic acid
molecules. Representative examples of suitable promoters include
tissue-specific promoters, and viral-based promoters (e.g.,
CMV-based promoters such as CMV I-E, SV40 early promoter, and MuLV
LTR). Expression vectors may also be based upon, or derived from
viruses (e.g., a "viral vector"). Representative examples of viral
vectors include herpes simplex viral vectors, adenoviral vectors,
adenovirus-associated viral vectors and retroviral vectors. Also
provided are host cells containing or comprising any of above-noted
vectors (including for example, host cells of human, monkey, dog,
rat, or mouse origin).
[0017] Within other aspects of the present invention, methods of
producing TGF-beta binding-proteins are provided, comprising the
step of culturing the aforementioned host cell containing vector
under conditions and for a time sufficient to produce the TGF-beta
binding protein. Within further embodiments, the protein produced
by this method may be further purified (e.g., by column
chromatography, affinity purification, and the like). Hence,
isolated proteins which are encoded by the above-noted nucleic acid
molecules (e.g., Sequence ID NOs. 2, 4, 6, 8, 10, 12, 14, or 16)
may be readily produced given the disclosure of the subject
application.
[0018] It should also be noted that the aforementioned proteins, or
fragments thereof, may be produced as fusion proteins. For example,
within one aspect fusion proteins are provided comprising a first
polypeptide segment comprising a TGF-beta binding-protein encoded
by a nucleic acid molecule as described above, or a portion thereof
of at least 10, 20, 30, 50, or 100 amino acids in length, and a
second polypeptide segment comprising a non-TGF-beta
binding-protein. Within certain embodiments, the second polypeptide
may be a tag suitable for purification or recognition (e.g., a
polypeptide comprising multiple anionic amino acid residues--see
U.S. Pat. No. 4,851,341), a marker (e.g., green fluorescent
protein, or alkaline phosphatase), or a toxic molecule (e.g.,
ricin).
[0019] Within another aspect of the present invention, antibodies
are provided which are capable of specifically binding the
above-described class of TGF-beta binding proteins (e.g., human
BEER). Within various embodiments, the antibody may be a polyclonal
antibody, or a monoclonal antibody (e.g., of human or murine
origin). Within further embodiments, the antibody is a fragment of
an antibody which retains the binding characteristics of a whole
antibody (e.g., an F(ab').sub.2, F(ab).sub.2, Fab', Fab, or Fv
fragment, or even a CDR). Also provided are hybridomas and other
cells which are capable of producing or expressing the
aforementioned antibodies.
[0020] Within related aspects of the invention, methods are
provided detecting a TGF-beta binding protein, comprising the steps
of incubating an antibody as described above under conditions and
for a time sufficient to permit said antibody to bind to a TGF-beta
binding protein, and detecting the binding. Within various
embodiments the antibody may be bound to a solid support to
facilitate washing or separation, and/or labeled. (e.g., with a
marker selected from the group consisting of enzymes, fluorescent
proteins, and radioisotopes).
[0021] Within other aspects of the present invention, isolated
oligonucleotides are provided which hybridize to a nucleic acid
molecule according to Sequence ID NOs. 1, 3, 5, 7, 9, 11, 13, 15,
17, or 18 or the complement thereto, under conditions of high
stringency. Within further embodiments, the oligonucleotide may be
found in the sequence which encodes Sequence ID Nos. 2, 4, 6, 8,
10, 12, 14, or 16. Within certain embodiments, the oligonucleotide
is at least 15, 20, 30, 50, or 100 nucleotides in length. Within
further embodiments, the oligonucleotide is labeled with another
molecule (e.g., an enzyme, fluorescent molecule, or radioisotope).
Also provided are primers which are capable of specifically
amplifying all or a portion of the above-mentioned nucleic acid
molecules which encode TGF-beta binding-proteins. As utilized
herein, the term "specifically amplifying" should be understood to
refer to primers which amplify the aforementioned TGF-beta
binding-proteins, and not other TGF-beta binding proteins such as
Dan, Cerberus, Gremlin, or SCGF (U.S. Pat. No. 5,780,263).
[0022] Within related aspects of the present invention, methods are
provided for detecting a nucleic acid molecule which encodes a
TGF-beta binding protein, comprising the steps of incubating an
oligonucleotide as described above under conditions of high
stringency, and detecting hybridization of said oligonucleotide.
Within certain embodiments, the oligonucleotide may be labeled
and/or bound to a solid support.
[0023] Within other aspects of the present invention, ribozymes are
provided which are capable of cleaving RNA which encodes one of the
above-mentioned TGF-beta binding-proteins (e.g., Sequence ID NOs.
2, 6, 8, 10, 12, 14, or 16). Such ribozymes may be composed of DNA,
RNA (including 2'-O-methyl ribonucleic acids), nucleic acid analogs
(e.g., nucleic acids having phosphorothioate linkages) or mixtures
thereof. Also provided are nucleic acid molecules (e.g., DNA or
cDNA) which encode these ribozymes, and vectors which are capable
of expressing or producing the ribozymes. Representative examples
of vectors include plasmids, retrotransposons, cosmids, and
viral-based vectors (e.g., viral vectors generated at least in part
from a retrovirus, adenovirus, or, adeno-associated virus). Also
provided are host cells (e.g., human, dog, rat, or mouse cells)
which contain these vectors. In certain embodiments, the host cell
may be stably transformed with the vector.
[0024] Within further aspects of the invention, methods are
provided for producing ribozymes either synthetically, or by in
vitro or in vivo transcription. Within further embodiments, the
ribozymes so produced may be further purified and/or formulated
into pharmaceutical compositions (e.g., the ribozyme or nucleic
acid molecule encoding the ribozyme along with a pharmaceutically
acceptable carrier or diluent). Similarly, the antisense
oligonucleotides and antibodies or other selected molecules
described herein may be formulated into pharmaceutical
compositions.
[0025] Within other aspects of the present invention, antisense
oligonucleotides are provided comprising a nucleic acid molecule
which hybridizes to a nucleic acid molecule according to Sequence
ID NOs. 1, 3, 5, 7, 9, 11, 13, or 15, or the complement thereto,
and wherein said oligonucleotide inhibits the expression of
TGF-beta binding protein as described herein (e.g., human BEER).
Within various embodiments, the oligonucleotide is 15, 20, 25, 30,
35, 40, or 50 nucleotides in length. Preferably, the
oligonucleotide is less than 100, 75, or 60 nucleotides in length.
As should be readily evident, the oligonucleotide may be comprised
of one or more nucleic acid analogs, ribonucleic acids, or
deoxyribonucleic acids. Further, the oligonucleotide may be
modified by one or more linkages, including for example, covalent
linkage such as a phosphorothioate linkage, a phosphotriester
linkage, a methyl phosphonate linkage, a methylene(methylimino)
linkage, a morpholino linkage, an amide linkage, a polyamide
linkage, a short chain alkyl intersugar linkage, a cycloalkyl
intersugar linkage, a short chain heteroatomic intersugar linkage
and a heterocyclic intersugar linkage. One representative example
of a chimeric oligonucleotide is provied in U.S. Pat. No.
5,989,912.
[0026] Within yet another aspect of the present invention, methods
are provided for increasing bone mineralization, comprising
introducing into a warm-blooded animal an effective amount of the
ribozyme as described above. Within related aspects, such methods
comprise the step of introducing into a patient an effective amount
of the nucleic acid molecule or vector as described herein which is
capable of producing the desired ribozyme, under conditions
favoring transcription of the nucleic acid molecule to produce the
ribozyme.
[0027] Within other aspects of the invention transgenic, non-human
animals are provided. Within one embodiment a transgenic animal is
provided whose germ cells and somatic cells contain a nucleic acid
molecule encoding a TGF-beta binding-protein as described above
which is operably linked to a promoter effective for the expression
of the gene, the gene being introduced into the animal, or an
ancestor of the animal, at an embryonic stage, with the proviso
that said animal is not a human. Within other embodiments,
transgenic knockout animals are provided, comprising an animal
whose germ cells and somatic cells comprise a disruption of at
least one allele of an endogenous nucleic acid molecule which
hybridizes to a nucleic acid molecule which encodes a TGF-binding
protein as described herein, wherein the disruption prevents
transcription of messenger RNA from said allele as compared to an
animal without the disruption, with the proviso that the animal is
not a human. Within various embodiments, the disruption is a
nucleic acid deletion, substitution, or, insertion. Within other
embodiments the transgenic animal is a mouse, rat, sheep, pig, or
dog.
[0028] Within further aspects of the invention, kits are provided
for the detection of TGF-beta binding-protein gene expression,
comprising a container that comprises a nucleic acid molecule,
wherein the nucleic acid molecule is selected from the group
consisting of (a) a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 1 1, 13, 15, 100, or 101;
(b) a nucleic acid molecule comprising the complement of the
nucleotide sequence of (a); (c) a nucleic acid molecule that is a
fragment of (a) or (b) of at least 15, 20 30, 50, 75, or, 100
nucleotides in length. Also provided are kits for the detection of
a TGF-beta binding-protein which comprise a container that comprise
one of the TGF-beta binding protein antibodies described
herein.
[0029] For example, within one aspect of the present invention
methods are provided for determining whether a selected molecule is
capable of increasing bone mineral content, comprising the steps of
(a) mixing one or more candidate molecules with
TGF-beta-binding-protein encoded by the nucleic acid molecule
according to claim 1 and a selected member of the TGF-beta family
of proteins (e.g., BMP 5 or 6), (b) determining whether the
candidate molecule alters the signaling of the TGF-beta family
member, or alters the binding of the TGF-beta binding-protein to
the TGF-beta family member. Within certain embodiments, the
molecule alters the ability of TGF-beta to function as a positive
regulator of mesenchymal cell differentiation. Within this aspect
of the present invention, the candidate molecule(s) may alter
signaling or binding by, for example, either decreasing (e.g.,
inhibiting), or increasing (e.g., enhancing) signaling or
binding.
[0030] Within yet another aspect, methods are provided for
determining whether a selected molecule is capable of increasing
bone mineral content, comprising the step of determining whether a
selected molecule inhibits the binding of TGF-beta binding-protein
to bone, or an analogue thereof. Representative examples of bone or
analogues thereof include hydroxyapatite and primary human bone
samples obtained via biopsy.
[0031] Within certain embodiments of the above-recited methods, the
selected molecule is contained within a mixture of molecules and
the methods may further comprise the step of isolating one or more
molecules which are functional within the assay. Within yet other
embodiments, TGF-beta family of proteins is bound to a solid
support and the binding of TGF-beta binding-protein is measured or
TGF-beta binding-protein are bound to a solid support and the
binding of TGF-beta proteins are measured.
[0032] Utilizing methods such as those described above, a wide
variety of molecules may be assayed for their ability to increase
bone mineral content by inhibiting the binding of the TGF-beta
binding-protein to the TGF-beta family of proteins. Representative
examples of such molecules include proteins or peptides, organic
molecules, and nucleic acid molecules.
[0033] Within other related aspects of the invention, methods are
provided for increasing bone mineral content in a warm-blooded
animal, comprising the step of administering to a warm-blooded
animal a therapeutically effective amount of a molecule identified
from the assays recited herein. Within another aspect, methods are
provided for increasing bone mineral content in a warm-blooded
animal, comprising the step of administering to a warm-blooded
animal a therapeutically effective amount of a molecule which
inhibits the binding of the TGF-beta binding-protein to the
TGF-beta super-family of proteins, including bone morphogenic
proteins (BMPs). Representative examples of suitable molecules
include antisense molecules, ribozymes, ribozyme genes, and
antibodies (e.g., a humanized antibody) which specifically
recognize and alter the activity of the TGF-beta
binding-protein.
[0034] Within another aspect of the present invention, methods are
provided for increasing bone mineral content in a warm-blooded
animal, comprising the steps of (a) introducing into cells which
home to the bone a vector which directs the expression of a
molecule which inhibits the binding of the TGF-beta binding-protein
to the TGF-beta family of proteins and bone morphogenic proteins
(BMPs), and (b) administering the vector-containing cells to a
warm-blooded animal. As utilized herein, it should be understood
that cells "home to bone" if they localize within the bone matrix
after peripheral administration. Within one embodiment, such
methods further comprise, prior to the step of introducing,
isolating cells from the marrow of bone which home to the bone.
Within a further embodiment, the cells which home to bone are
selected from the group consisting of CD34+ cells and
osteoblasts.
[0035] Within other aspects of the present invention, molecules are
provided (preferably isolated) which inhibit the binding of the
TGF-beta binding-protein to the TGF-beta super-family of
proteins.
[0036] Within further embodiments, the molecules may be provided as
a composition, and can further comprise an inhibitor of bone
resorption. Representative examples of such inhibitors include
calcitonin, estrogen, a bisphosphonate, a growth factor having
anti-resorptive activity and tamoxifen.
[0037] Representative examples of molecules which may be utilized
in the aforementioned therapeutic contexts include, e.g.,
ribozymes, ribozyme genes, antisense molecules, and/or antibodies
(e.g., humanized antibodies). Such molecules may depending upon
their selection, used to alter, antagonize, or agonize the
signalling or binding of a TGF-beta binding-protein family member
as described herein.
[0038] Within various embodiments of the invention, the
above-described molecules and methods of treatment or prevention
may be utilized on conditions such as osteoporosis, osteomalasia,
periodontal disease, scurvy, Cushing's Disease, bone fracture and
conditions due to limb immobilization and steroid usage.
[0039] The present invention also provides antibodies that
specifically bind to a TGF-beta binding protein, sclerostin (SOST),
and provides immunogens comprising sclerostin peptides derived from
regions of sclerostin that interact with a member of the TGF-beta
superfamily such as a bone morphogenic protein. In one embodiment,
the invention provides an antibody, or an antigen-binding fragment
thereof, that binds specifically to a sclerostin polypeptide, said
sclerostin polypeptide comprising an amino acid sequence set forth
in SEQ ID NOS: 2, 6, 8, 14, 46, or 65, wherein the antibody
competitively inhibits binding of the SOST polypeptide to at least
one of (i) a bone morphogenic protein (BMP) Type I Receptor binding
site and (ii) a BMP Type II Receptor binding site, wherein the BMP
Type I Receptor binding site is capable of binding to a BMP Type I
Receptor polypeptide comprising an amino acid sequence set forth in
GenBank Ace. Nos. NM.sub.--004329 (SEQ ID NO: 102); D89675 (SEQ ID
NO: 103); NM.sub.--001203 (SEQ ID NO: 104); S75359 (SEQ ID NO:
105); NM.sub.--030849 (SEQ ID NO: 106); D38082 (SEQ ID NO: 107);
NP.sub.--001194 (SEQ ID NO: 108); BAA19765 (SEQ ID NO: 109); or
AAB33865 (SEQ ID NO: 110) and wherein the BMP Type II Receptor
binding site is capable of binding to a BMP Type II Receptor
polypeptide comprising the amino acid sequence set forth in GenBank
Ace. NOS. U25110 (SEQ ID NO: 111); NM.sub.--033346 (SEQ ID NO:
112); Z48923 (SEQ ID NO: 114); CAA88759 (SEQ ID NO: 115); or
NM.sub.--001204 (SEQ ID NO: 113). In another embodiment, the
invention provides an antibody, or an antigen-binding fragment
thereof, that binds specifically to a sclerostin polypeptide and
that impairs formation of a sclerostin homodimer, wherein the
sclerostin polypeptide comprises an amino acid sequence set forth
in SEQ ID NOS: 2, 6, 8, 14, 46, or 65.
[0040] In certain particular embodiments of the invention, the
antibody is a polyclonal antibody. In other embodiments, the
antibody is a monoclonal antibody, which is a mouse, human, rat, or
hamster monoclonal antibody. The invention also provides a
hybridoma cell or a host cell that is capable of producing the
monoclonal antibody. In other embodiments of the invention, the
antibody is a humanized antibody or a chimeric antibody. The
invention further provides a host cell that produces the humanized
or chimeric antibody. In certain embodiments the antigen-binding
fragment of the antibody is a F(ab').sub.2, Fab', Fab, Fd, or Fv
fragment. The invention also provides an antibody that is a single
chain antibody and provides a host cell that is capable of
expressing the single chain antibody. In another embodiment, the
invention provides a composition comprising such antibodies and a
physiologically acceptable carrier.
[0041] In another embodiment, the invention provides an immunogen
comprising a peptide comprising at least 21 consecutive amino acids
and no more than 50 consecutive amino acids of a SOST polypeptide,
said SOST polypeptide comprising an amino acid sequence set forth
in SEQ ID NOS: 2, 6, 8, 14, 46, or 65, wherein the peptide is
capable of eliciting in a non-human animal an antibody that binds
specifically to the SOST polypeptide and that competitively
inhibits binding of the SOST polypeptide to at least one of (i) a
bone morphogenic protein (BMP) Type I Receptor binding site and
(ii) a BMP Type II Receptor binding site, wherein the BMP Type I
Receptor binding site is capable of binding to a BMP Type I
Receptor polypeptide comprising an amino acid sequence set forth in
GenBank Ace. Nos. NM.sub.--004329 (SEQ ID NO: 102); D89675 (SEQ ID
NO: 103); NM.sub.--001203 (SEQ ID NO: 104); S75359 (SEQ ID NO:
105); NM.sub.--030849 (SEQ ID NO: 106); D38082 (SEQ ID NO: 107);
NP.sub.--001194 (SEQ ID NO: 108); BAA19765 (SEQ ID NO: 109); or
AAB33865 (SEQ ID NO: 110) and wherein the BMP Type II Receptor
binding site is capable of binding to a BMP Type II Receptor
polypeptide comprising the amino acid sequence set forth in GenBank
Ace. NOs. U25110 (SEQ ID NO: 111); NM.sub.--033346 (SEQ ID NO:
112); Z48923 (SEQ ID NO: 114); CAA88759 (SEQ ID NO: 115); or
NM.sub.--001204 (SEQ ID NO: 113). The invention also provides an
immunogen comprising a peptide that comprises at least 21
consecutive amino acids and no more than 50 consecutive amino acids
of a SOST polypeptide, said SOST polypeptide comprising an amino
acid sequence set forth in SEQ ID NOS: 2, 6, 8, 14, 46, or 65,
wherein the peptide is capable of eliciting in a non-human animal
an antibody that binds specifically to the SOST polypeptide and
that impairs formation of a SOST homodimer.
[0042] In certain particular embodiments, the subject invention
immunogens are associated with a carrier molecule. In certain
embodiments, the carrier molecule is a carrier polypeptide, and in
particular embodiments, the carrier polypeptide is keyhole limpet
hemocyanin.
[0043] The invention also provides a method for producing an
anti-body that specifically binds to a SOST polypeptide, comprising
immunizing a non-human animal with an immunogen comprising a
peptide comprising at least 21 consecutive amino acids and no more
than 50 consecutive amino acids of a SOST polypeptide, wherein (a)
the SOST polypeptide comprises an amino acid sequence set forth in
SEQ ID NOS: 2, 6, 8, 14, 46, or 65; (b) the antibody competitively
inhibits binding of the SOST polypeptide to at least one of (i) a
bone morphogenic protein (BMP) Type I Receptor binding site and
(ii) a BMP Type II Receptor binding site; (c) the BMP Type I
Receptor binding site is capable of binding to a BMP Type I
Receptor polypeptide comprising the amino acid sequence set forth
in GenBank Ace. Nos. NM.sub.--004329 (SEQ ID NO: 102); D89675 (SEQ
ID NO: 103); NM.sub.--001203 (SEQ ID NO: 104); S75359 (SEQ ID NO:
105); NM.sub.--030849 (SEQ ID NO: 106); D38082 (SEQ ID NO: 107);
NP.sub.--001194 (SEQ ID NO: 108); BAA19765 (SEQ ID NO: 109); or
AAB33865 (SEQ ID NO: 110); and (d) the BMP Type II Receptor binding
site is capable of binding to a BMP Type II Receptor polypeptide
comprising the amino acid sequence set forth in GenBank Ace. NOs.
U25110 (SEQ ID NO: 111); NM.sub.--033346 (SEQ ID NO: 112); Z48923
(SEQ ID NO: 114); CAA88759 (SEQ ID NO: 115); or NM.sub.--001204
(SEQ ID NO: 113).
[0044] In another embodiment, the invention provides a method for
producing an antibody that specifically binds to a SOST
polypeptide, said SOST polypeptide comprising an amino acid
sequence set forth in SEQ ID NOS: 2, 6, 8, 14, 46, or 65,
comprising immunizing a non-human animal with an immunogen
comprising a peptide that comprises at least 21 consecutive amino
acids and no more than 50 consecutive amino acids of a SOST
polypeptide, said SOST polypeptide comprising an amino acid
sequence set forth in SEQ ID NOS: 2, 6, 8, 14, 46, or 65, wherein
the antibody impairs formation of a SOST homodimer.
[0045] These and other aspects of the present invention will become
evident upon reference to the following detailed description and
attached drawings. In addition, documents including various
references set forth herein that describe in more detail certain
procedures or compositions (e.g., plasmids, etc.), are incorporated
by reference in their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 is a schematic illustration comparing the amino acid
sequence of Human Dan; Human Gremlin; Human Cerberus and Human
Beer. Arrows indicate the Cysteine backbone.
[0047] FIG. 2 summarizes the results obtained from surveying a
variety of human tissues for the expression of a TGF-beta
binding-protein gene, specifically, the Human Beer gene. A
semi-quantitative Reverse Transcription-Polymerase Chain Reaction
(RT-PCR) procedure was used to amplify a portion of the gene from
first-strand cDNA synthesized from total RNA (described in more
detail in EXAMPLE 2A).
[0048] FIGS. 3A-3D summarize the results obtained from RNA in situ
hybridization of mouse embryo sections, using a cRNA probe that is
complementary to the mouse Beer transcript (described in more
detail in EXAMPLE 2B). Panel 3A is a transverse section of 10.5 dpc
embryo. Panel 3B is a sagittal section of 12.5 dpc embryo and
panels 3C and 3D are sagittal sections of 15.5 dpc embryos.
[0049] FIGS. 4A-4C illustrate, by western blot analysis, the
specificity of three different polyclonal antibodies for their
respective antigens (described in more detail in EXAMPLE 4). FIG.
4A shows specific reactivity of an anti-H. Beer antibody for H.
Beer antigen, but not H. Dan or H. Gremlin. FIG. 4B shows
reactivity of an anti-H. Gremlin antibody for H. Gremlin antigen,
but not H. Beer or H. Dan. FIG. 4C shows reactivity of an anti-H.
Dan antibody for H. Dan, but not H. Beer or H. Gremlin.
[0050] FIG. 5 illustrates, by western blot analysis, the
selectivity of the TGF-beta binding-protein, Beer, for BMP-5 and
BMP-6, but not BMP-4 (described in more detail in EXAMPLE 5).
[0051] FIG. 6 demonstrates that the ionic interaction between the
TGF-beta binding-protein, Beer, and BMP-5 has a dissociation
constant in the 15-30 nM range.
[0052] FIG. 7 presents an alignment of the region containing the
characteristic cystine-knot of a SOST (sclerostin) polypeptide and
its closest homologues. Three disulphide bonds that form the
cystine-knot are illustrated as solid lines. An extra disulphide
bond, shown by a dotted line, is unique to this family, which
connects two .beta.-hairpin tips in the 3D structure. The
polypeptides depicted are SOST: sclerostin (SEQ ID NO: 126); CGHB:
Human Chorionic Gonadotropin .beta. (SEQ ID NO: 127); FSHB:
follicle-stimulating hormone beta subunit (SEQ ID NO: 128); TSHB:
thyrotropin beta chain precursor (SEQ ID NO: 129); VWF: Von
Willebrand factor (SEQ ID NO: 130); MUC2: human mucin 2 precursor
(SEQ ID NO: 131); CER1: Cerberus 1 (Xenopus laevis homolog) (SEQ ID
NO: 132); DRM: gremlin (SEQ ID NO: 133); DAN: (SEQ ID NO: 134);
CTGF: connective tissue growth factor precursor (SEQ ID NO: 135);
NOV: NovH (nephroblastoma overexpressed gene protein homolog) (SEQ
ID NO: 136); CYR6: (SEQ ID NO: 137).
[0053] FIG. 8 illustrates a 3D model of the core region of SOST
(SOST_Core).
[0054] FIG. 9 presents a 3D model of the core region of SOST
homodimer.
[0055] FIGS. 10A and 10B provide an amino acid sequence alignment
of Noggin from five different animals: human (NOGG_HUMAN (SEQ ID
NO: 138); chicken (NOGG_CHICK, SEQ ID NO: 139); African clawed frog
(NOGG_XENLA, SEQ ID NO: 140); NOGG_FUGRU, SEQ ID NO: 141); and
zebrafish (NOGG_ZEBRA, SEQ ID NO: 142); and SOST from human
(SOST_HUMAN, SEQ ID NO: 46), rat (SOST_RAT, SEQ ID NO: 65), and
mouse (SOST Mouse, SEQ ID NO: 143).
[0056] FIG. 11 illustrates the Noggin/BMP-7 complex structure. The
BMP homodimer is shown on the bottom portion of the figure in
surface mode. The Noggin homodimer is shown on top of the BMP dimer
in cartoon mode. The circles outline the N-terminal binding region,
the core region, and the linker between the N-terminal and core
regions.
[0057] FIG. 12 depicts a 3D model of the potential BMP-binding
fragment located at the SOST N-terminal region. A BMP dimer is
shown in surface mode, and the potential BMP-binding fragment is
shown in stick mode. A phenylalanine residue fitting into a
hydrophobic pocket on the BMP surface is noted.
DETAILED DESCRIPTION OF THE INVENTION
[0058] Definitions
[0059] Prior to setting forth the invention in detail, it may be
helpful to an understanding thereof to set forth definitions of
certain terms and to list and to define the abbreviations that will
be used hereinafter.
[0060] "Molecule" should be understood to include proteins or
peptides (e.g., antibodies, recombinant binding partners, peptides
with a desired binding affinity), nucleic acids (e.g., DNA, RNA,
chimeric nucleic acid molecules, and nucleic acid analogues such as
PNA); and organic or inorganic compounds.
[0061] "TGF-beta" should be understood to include any known or
novel member of the TGF-beta super-family, which also includes bone
morphogenic proteins (BMPs).
[0062] "TGF-beta receptor" should be understood to refer to the
receptor specific for a particular member of the TGF-beta
super-family (including bone morphogenic proteins (BMPs)).
[0063] "TGF-beta binding-protein" should be understood to refer to
a protein with specific binding affinity for a particular member or
subset of members of the TGF-beta super-family (including bone
morphogenic proteins (BMPs)). Specific examples of TGF-beta
binding-proteins include proteins encoded by Sequence ID Nos. 1, 5,
7, 9, 11, 13, 15, 100, and 101.
[0064] Inhibiting the "binding of the TGF-beta binding-protein to
the TGF-beta family of proteins and bone morphogenic proteins
(BMPs)" should be understood to refer to molecules which allow the
activation of TGF-beta or bone morphogenic proteins (BMPs), or
allow the binding of TGF-beta family members including bone
morphogenic proteins (BMPs) to their respective receptors, by
removing or preventing TGF-beta from binding to
TGF-binding-protein. Such inhibition may be accomplished, for
example, by molecules which inhibit the binding of the TGF-beta
binding-protein to specific members of the TGF-beta
super-family.
[0065] "Vector" refers to an assembly that is capable of directing
the expression of desired protein. The vector must include
transcriptional promoter elements that are operably linked to the
gene(s) of interest. The vector may be composed of deoxyribonucleic
acids ("DNA"), ribonucleic acids ("RNA"), or a combination of the
two (e.g., a DNA-RNA chimeric). Optionally, the vector may include
a polyadenylation sequence, one or more restriction sites, as well
as one or more selectable markers such as neomycin
phosphotransferase or hygromycin phosphotransferase. Additionally,
depending on the host cell chosen and the vector employed, other
genetic elements such as an origin of replication, additional
nucleic acid restriction sites, enhancers, sequences conferring
inducibility of transcription, and selectable markers, may also be
incorporated into the vectors described herein.
[0066] An "isolated nucleic acid molecule" is a nucleic acid
molecule that is not integrated in the genomic DNA of an organism.
For example, a DNA molecule that encodes a TGF-binding protein that
has been separated from the genomic DNA of a eukaryotic cell is an
isolated DNA molecule. Another example of an isolated nucleic acid
molecule is a chemically-synthesized nucleic acid molecule that is
not integrated in the genome of an organism. The isolated nucleic
acid molecule may be genomic DNA, cDNA, RNA, or composed at least
in part of nucleic acid analogs.
[0067] An "isolated polypeptide" is a polypeptide that is
essentially free from contaminating cellular components, such as
carbohydrate, lipid, or other proteinaceous impurities associated
with the polypeptide in nature. Preferably, such isolated
polypeptides are at least about 90% pure, more preferably at least
about 95% pure, and most preferably at least about 99% pure. Within
certain embodiments, a particular protein preparation contains an
isolated polypeptide if it appears nominally as a single band on
SDS-PAGE gel with Coomassie Blue staining. The term "isolated" when
referring to organic molecules (e.g., organic small molecules)
means that the compounds are greater than 90% pure utilizing
methods which are well known in the art (e.g., NMR, melting
point).
[0068] "Sclerosteosis" is a term that was applied by Hansen (1967)
(Hansen, H. G., Sklerosteose. in: Opitz, H.; Schmid, F., Handbuch
der Kinderheilkunde. Berlin: Springer (pub.) 6 1967. Pp. 351-355)
to a disorder similar to van Buchem hyperostosis corticalis
generalisata but possibly differing in radiologic appearance of the
bone changes and in the presence of asymmetric cutaneous syndactyly
of the index and middle fingers in many cases. The jaw has an
unusually square appearance in this condition.
[0069] "Humanized antibodies" are recombinant proteins in which
murine or other non-human animal complementary determining regions
of monoclonal antibodies have been transferred from heavy and light
variable chains of the murine or other non-human animal
immunoglobulin into a human variable domain.
[0070] As used herein, an "antibody fragment" is a portion of an
antibody such as F(ab').sub.2, F(ab).sub.2, Fab', Fab, and the
like. Regardless of structure, an antibody fragment binds with the
same antigen that is recognized by the intact antibody. For
example, an anti-TGF-beta binding-protein monoclonal antibody
fragment binds to an epitope of TGF-beta binding-protein.
[0071] The term antibody fragment or antigen-binding fragment also
includes any synthetic or genetically engineered protein that acts
like an antibody by binding to a specific antigen to form a
complex. For example, antibody fragments include isolated fragments
consisting of the light chain variable region, "Fv" fragments
consisting of the variable regions of the heavy and light chains,
recombinant single chain polypeptide molecules in which light and
heavy variable regions are connected by a peptide linker ("sFv
proteins"), and minimal recognition units consisting of the amino
acid residues that mimic the hypervariable region.
[0072] A "detectable label" is a molecule or atom that can be
conjugated to a polypeptide moiety such as an antibody moiety or a
nucleic acid moiety to produce a molecule useful for diagnosis.
Examples of detectable labels include chelators, photoactive
agents, radioisotopes, fluorescent agents, paramagnetic ions,
enzymes, and other marker moieties.
[0073] As used herein, an "immunoconjugate" is a molecule
comprising an anti-TGF-beta binding-protein antibody, or an
antibody fragment, and a detectable label or an effector molecule.
Preferably, an immunoconjugate has roughly the same, or only
slightly reduced, ability to bind TGF-beta binding-protein after
conjugation as before conjugation.
[0074] Abbreviations: TGF-beta--"Transforming Growth Factor-beta";
TGF-bBP--"Transforming Growth Factor-beta binding-protein" (one
representative TGF-bBP is designated "H. Beer"); BMP--"bone
morphogenic protein"; PCR--"polymerase chain reaction"; RT-PCR--PCR
process in which RNA is first transcribed into DNA using reverse
transcriptase (RT); cDNA--any DNA made by copying an RNA sequence
into DNA form.
[0075] As noted above, the present invention provides a novel class
of TGF-beta binding-proteins, as well as methods and compositions
for increasing bone mineral content in warn-blooded animals.
Briefly, the present inventions are based upon the unexpected
discovery that a mutation in the gene which encodes a novel member
of the TGF-beta binding-protein family results in a rare condition
(sclerosteosis) characterized by bone mineral contents which are
one- to four-fold higher than in normal individuals. Thus, as
discussed in more detail below this discovery has led to the
development of assays which may be utilized to select molecules
which inhibit the binding of the TGF-beta binding-protein to the
TGF-beta family of proteins and bone morphogenic proteins (BMPs),
and methods of utilizing such molecules for increasing the bone
mineral content of warm-blooded animals (including for example,
humans).
[0076] Discussion of the Disease Known as Sclerosteosis
[0077] Sclerosteosis is a disease related to abnormal bone mineral
density in humans. Sclerosteosis is a term that was applied by
Hansen (1967) (Hansen, H. G., Sklerosteose. In: Opitz, H.; Schmid,
F., Handbuch der Kinderheilkunde. Berlin: Springer (pub.) 6 1967.
Pp. 351-355) to a disorder similar to van Buchem hyperostosis
corticalis generalisata but possibly differing in radiologic
appearance of the bone changes and differing in the presence of
asymmetric cutaneous syndactyly of the index and middle fingers in
many cases.
[0078] Sclerosteosis is now known to be an autosomal semi-dominant
disorder that is characterized by widely disseminated sclerotic
lesions of the bone in the adult. The condition is progressive.
Sclerosteosis also has a developmental aspect that is associated
with syndactyly (two or more fingers are fused together). The
Sclerosteosis Syndrome is associated with large stature and many
affected individuals attain a height of six feet or more. The bone
mineral content of homozygotes can be 1 to 6 fold greater than
observed in normal individuals, and bone mineral density can be 1
to 4 fold above normal values (e.g., from unaffected siblings).
[0079] The Sclerosteosis Syndrome occurs primarily in Afrikaaners
of Dutch descent in South Africa. Approximately {fraction (1/140)}
individuals in the Afrikaaner population are carriers of the
mutated gene (heterozygotes). The mutation shows 100% penetrance.
There are anecdotal reports of increased of bone mineral density in
heterozygotes with no associated pathologies (syndactyly or skull
overgrowth).
[0080] No abnormality of the pituitary-hypothalamus axis has been
observed in patients with sclerosteosis. In particular, there
appears to be no over-production of growth hormone and cortisone.
In addition, sex hormone levels are normal in affected individuals.
However, bone turnover markers (osteoblast specific alkaline
phosphatase, osteocalcin, type 1 procollagen C' propeptide (PICP),
and total alkaline phosphatase; (see Comier, C., Curr. Opin. in
Rheu. 7:243, 1995) indicate that there is hyperosteoblastic
activity associated with the disease but that there is normal to
slightly decreased osteoclast activity as measured by markers of
bone resorption (pyridinoline, deoxypryridinoline, N-telopeptide,
urinary hydroxyproline, plasma tartrate-resistant acid phosphatases
and galactosyl hydroxylysine (see Comier, supra)).
[0081] Sclerosteosis is characterized by the continual deposition
of bone throughout the skeleton during the lifetime of the affected
individuals. In homozygotes the continual deposition of bone
mineral leads to an overgrowth of bone in areas of the skeleton
where there is an absence of mechanoreceptors (skull, jaw,
cranium). In homozygotes with Sclerosteosis, the overgrowth of the
bones of the skull leads to cranial compression and eventually to
death due to excessive hydrostatic pressure on the brain stem. In
all other parts of the skeleton there is a generalized and diffuse
sclerosis. Cortical areas of the long bones are greatly thickened
resulting in a substantial increase in bone strength. Trabecular
connections are increased in thickness which in turn increases the
strength of the trabecular bone. Sclerotic bones appear unusually
opaque to x-rays.
[0082] As described in more detail in Example 1, the rare genetic
mutation that is responsible for the Sclerosteosis syndrome has
been localized to the region of human chromosome 17 that encodes a
novel member of the TGF-beta binding-protein family (one
representative example of which is designated "H. Beer"). As
described in more detail below, based upon this discovery, the
mechanism of bone mineralization is more fully understood, allowing
the development of assays for molecules that increase bone
mineralization, and use of such molecules to increase bone mineral
content, and in the treatment or prevention of a wide number of
diseases.
[0083] TGF-Beta Super-Family
[0084] The Transforming Growth Factor-beta (TGF-beta) super-family
contains a variety of growth factors that share common sequence
elements and structural motifs (at both the secondary and tertiary
levels). This protein family is known to exert a wide spectrum of
biological responses that affect a large variety of cell types.
Many of the TGF-beta family members have important functions during
the embryonal development in pattern formation and tissue
specification; in adults the family members are involved, e.g., in
wound healing and bone repair and bone remodeling, and in the
modulation of the immune system. In addition to the TGF-beta's, the
super-family includes the Bone Morphogenic Proteins (BMPs),
Activins, Inhibins, Growth and Differentiation Factors (GDFs), and
Glial-Derived Neurotrophic Factors (GDNFs). Primary classification
is established through general sequence features that bin a
specific protein into a general sub-family. Additional
stratification within the sub-family is possible due to stricter
sequence conservation between members of the smaller group. In
certain instances, such as with BMP-5, BMP-6 and BMP-7, the amino
acid identity can be as high as 75% among members of the smaller
group. This level of identity enables a single representative
sequence to illustrate the key biochemical elements of the
sub-group that separates it from other members of the larger
family.
[0085] The crystal structure of TGF-beta2 has been determined. The
general fold of the TGF-beta2 monomer contains a stable, compact,
cysteine knotlike structure formed by three disulphide bridges.
Dimerization, stabilized by one disulfide bridge, is
antiparallel.
[0086] TGF-beta signals by inducing the formation of
hetero-oligomeric complexes of type I and type II receptors.
Transduction of TGF-beta signals involves these two distinct type I
and type II subfamilies of transmembrane serine/threonine kinase
receptors. At least seven type I receptors and five type II
receptors have been identified (see Kawabata et al., Cytokine
Growth Factor Rev. 9:49-61 (1998); Miyazono et al., Adv. Immunol.
75:115-57 (2000). TGF-beta family members initiate their cellular
action by binding to receptors with intrinsic serine/threonine
kinase activity. Each member of the TGF-beta family binds to a
characteristic combination of type I and type II receptors, both of
which are needed for signaling. In the current model for TGF-beta
receptor activation, a TGF-beta ligand first binds to the type II
receptor (TbR-II), which occurs in the cell membrane in an
oligomeric form with activated kinase. Thereafter, the type I
receptor (TbR-I), which cannot bind ligand in the absence of
TbR-II, is recruited into the complex to form a ligand/type II/type
I ternary complex. TbR-II then phosphorylates TbR-I predominantly
in a domain rich in glycine and serine residues (GS domain) in the
juxtamembrane region, and thereby activates TbR-I. The activated
type I receptor kinase then phosphorylates particular members of
the Smad family of proteins that translocate to the nucleus where
they modulate transcription of specific genes.
[0087] Bone Morphogenic Proteins (BMPS) are Key Regulatory Proteins
in Determining Bone Mineral Density in Humans
[0088] A major advance in the understanding of bone formation was
the identification of the bone morphogenic proteins (BMPs), also
known as osteogenic proteins (OPs), which regulate cartilage and
bone differentiation in vivo. BMPs/OPs induce endochondral bone
differentiation through a cascade of events that include formation
of cartilage, hypertrophy and calcification of the cartilage,
vascular invasion, differentiation of osteoblasts, and formation of
bone. As described above, the BMPs/OPs (BMP 2-14, and osteogenic
protein 1 and -2, OP-1 and OP-2) see, e.g., GenBank P12643 (BMP-2);
GenBank P12645 (BMP3); GenBank P55107 (BMP-3b,
Growth/differentiation factor 10) (GDF-10)); GenBank P12644 (BMP4);
GenBank P22003 (BMP5); GenBank P22004 (BMP6); GenBank P18075
(BMP7); GenBank P34820 (BMP8); GenBank Q9UK05 (BMP9); GenBank
O95393 (BM10); GenBank O95390 (BMP11, Growth/differentiation factor
11 precursor (GDF-11)); GenBank O95972 (BM15)) are members of the
TGF-beta super-family. The striking evolutionary conservation
between members the BMP/OP sub-family suggests that they are
critical in the normal development and function of animals.
Moreover, the presence of multiple forms of BMPs/OPs raises an
important question about the biological relevance of this apparent
redundancy. In addition to postfetal chondrogenesis and
osteogenesis, the BMPs/OPs play multiple roles in skeletogenesis
(including the development of craniofacial and dental tissues) and
in embryonic development and organogenesis of parenchymatous
organs, including the kidney. It is now understood that nature
relies on common (and few) molecular mechanisms tailored to provide
the emergence of specialized tissues and organs. The BMP/OP
super-family is an elegant example of nature parsimony in
programming multiple specialized functions deploying molecular
isoforms with minor variation in amino acid motifs within highly
conserved carboxy-terminal regions.
[0089] BMPs are synthesized as large precursor proteins. Upon
dimerization, the BMPs are proteolyically cleaved within the cell
to yield carboxy-terminal mature proteins that are then secreted
from the cell. BMPs, like other TGF-beta family members, initiate
signal transduction by binding cooperatively to both type I and
type II serine/threonine kinase receptors. Type I receptors for
which BMPs may act as ligands include BMPR-IA (also known as
ALK-3), BMPR-IB (also known as ALK-6), ALK-1, and ALK-2 (also known
as ActR-1). Of the type II receptors, BMPs bind to BMP type II
receptor (BMPR-II), Activin type II (ActR-II), and Activin type IIB
(ActR-IIB). (See Balemans et al., supra, and references cited
therein). Polynucleotide sequences and the encoded amino acid
sequence of BMP type I receptor polypeptides are provided in the
GenBank database, for example, GenBank NM.sub.--004329 (SEQ ID NO:
102 encoded by SEQ ID NO: 116); D89675 (SEQ ID NO: 103 encoded by
SEQ ID NO: 117); NM.sub.--001203 (SEQ ID NO: 104 encoded by SEQ ID
NO: 118); S75359 (SEQ ID NO: 105 encoded by SEQ ID NO: 119);
NM.sub.--030849 (SEQ ID NO: 106 encoded by SEQ ID NO: 120); and
D38082 (SEQ ID NO: 107 encoded by SEQ ID NO: 121). Other
polypeptide sequences of type I receptors are provided in the
GenBank database, for example, NP.sub.--001194 (SEQ ID NO: 108);
BAA19765 (SEQ ID NO: 109); and AAB33865 (SEQ ID NO: 110).
Polynucleotide sequences and the encoded amino acid sequence of BMP
type II receptor polypeptides are provided in the GenBank database
and include, for example, U25110 (SEQ ID NO: 111 encoded by SEQ ID
NO: 122); NM.sub.--033346 (SEQ ID NO: 112 encoded by SEQ ID NO:
123); NM.sub.--001204 (SEQ ID NO: 113 encoded by SEQ ID NO: 124);
and Z48923 (SEQ ID NO: 114 encoded by SEQ ID NO: 125). Additional
polypeptide sequences of type II receptors are also provided in the
GenBank database, for example, CAA88759 (SEQ ID NO: 115).
[0090] BMPs, similar to other cystine-knot proteins, form a
homodimer structure (Scheufler et al., J. Mol. Biol. 287:103-15
(1999)). According to evolutionary trace analysis performed on the
BMP/TGF-.beta. family, the BMP type I receptor binding site and
type II receptor binding site were mapped to the surface of the BMP
structure (Innis et al., Protein Eng. 13:839-47 (2000)). The
location of the type I receptor binding site on BMP was later
confirmed by the x-ray structure of BMP-2/BMP Receptor IA complex
(Nickel et al., J. Joint Surg. Am. 83A(Suppl 1(Pt 1)):S7-S14
(2001)). The predicted type II receptor binding site is in good
agreement with the x-ray structure of TGF-.beta.3/TGF-.beta. Type
II receptor complex (Hart et al., Nat. Struct. Biol. 9:203-208
(2002)), which is highly similar to the BMP/BMP Receptor IIA
system.
[0091] BMP Antagonism
[0092] The BMP and Activin sub-families are subject to significant
post-translational regulation, such as by TGF-beta binding
proteins. An intricate extracellular control system exists, whereby
a high affinity antagonist is synthesized and exported, and
subsequently complexes selectively with BMPs or activins to disrupt
their biological activity (W. C. Smith (1999) TIG 15(1) 3-6). A
number of these natural antagonists have been identified, and on
the basis of sequence divergence, the antagonists appear to have
evolved independently due to the lack of primary sequence
conservation. Earlier studies of these antagonists highlighted a
distinct preference for interacting and neutralizing BMP-2 and
BMP-4. In vertebrates, antagonists include noggin, chordin,
chordin-like, follistatin, FSRP, the DAN/Cerberus protein family,
and sclerostin (SOST) (see Balemans et al., supra, and references
cited therein). The mechanism of antagonism or inhibition seems to
differ for the different antagonists (Iemura et al. (1998) Proc.
Natl. Acad. Sci. USA 95 9337-9342).
[0093] The type I and type II receptor binding sites on the BMP
antagonist noggin have also been mapped. Noggin binds to BMPs with
high affinity (Zimmerman et al., 1996). A study of the noggin/BMP-7
complex structure revealed the binding interactions between the two
proteins (Groppe et al., Nature 420:636-42 (2002)). Superposition
of the noggin-BMP-7 structure onto a model of the BMP signaling
complex showed that noggin binding effectively masks both pairs of
binding epitopes (i.e., BMP Type I and Type II receptor binding
sites) on BMP-7. The cysteine-rich scaffold sequence of noggin is
preceded by an N-terminal segment of about 20 amino acid residues
that are referred to as the "clip" (residues 28-48). The type I
receptor-binding site is occluded by the N-terminal portion of the
clip domain of Noggin, and the type II receptor binding site is
occluded by the carboxy terminal portion of the clip domain. Two
.beta.-strands in the core region near the C-terminus of noggin
also contact BMP-7 at the type II receptor binding site. This
binding mode enables a noggin dimer to efficiently block all the
receptor binding sites (two type I and two type II receptor binding
sites) on a BMP dimer.
[0094] Novel TGF-Beta Binding-Proteins
[0095] As noted above, the present invention provides a novel class
of TGF-beta binding-proteins that possess a nearly identical
cysteine (disulfide) scaffold when compared to Human DAN, Human
Gremlin, and Human Cerberus, and SCGF (U.S. Pat. No. 5,780,263) but
almost no homology at the nucleotide level. (for background
information, see generally Hsu, D. R., Economides, A. N., Wang, X.,
Eimon, P. M., Harland, R. M., "The Xenopus Dorsalizing Factor
Gremlin Identifies a Novel Family of Secreted Proteins that
Antagonize BMP Activities," Molecular Cell 1:673-683, 1998).
[0096] Representative example of the novel class of nucleic acid
molecules encoding TGF-beta binding-proteins are disclosed in SEQ
ID NOS: 1, 5, 7, 9, 11, 13, 15, 100, and 101. The polynucleotides
disclosed herein encode a polypeptide called Beer, which is also
referred to herein as sclerostin or SOST. Representative members of
this class of binding proteins should also be understood to include
variants of the TGF-beta binding-protein (e.g., SEQ ID NOS: 5 and
7). As utilized herein, a "TGF-beta binding-protein variant gene"
(e.g., an isolated nucleic acid molecule that encodes a TGF-beta
binding protein variant) refers to nucleic acid molecules that
encode a polypeptide having an amino acid sequence that is a
modification of SEQ ID NOS: 2, 10, 12, 14, 16, 46, or 65. Such
variants include naturally-occurring polymorphisms or allelic
variants of TGF-beta binding-protein genes, as well as synthetic
genes that contain conservative amino acid substitutions of these
amino acid sequences. A variety of criteria known to those skilled
in the art indicate whether amino acids at a particular position in
a peptide or polypeptide are similar. For example, a similar amino
acid or a conservative amino acid substitution is one in which an
amino acid residue is replaced with an amino acid residue having a
similar side chain, which include amino acids with basic side
chains (e.g., lysine, arginine, histidine); acidic side chains
(e.g., aspartic acid, glutamic acid); uncharged polar side chains
(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine, histidine); nonpolar side chains (e.g., alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan); beta-branched side chains (e.g., threonine, valine,
isoleucine), and aromatic side chains (e.g., tyrosine,
phenylalanine, tryptophan). Proline, which is considered more
difficult to classify, shares properties with amino acids that have
aliphatic side chains (e.g., Leu, Val, Ile, and Ala). In certain
circumstances, substitution of glutamine for glutamic acid or
asparagine for aspartic acid may be considered a similar
substitution in that glutamine and asparagine are amide derivatives
of glutamic acid and aspartic acid, respectively.
[0097] Additional variant forms of a TGF-beta binding-protein gene
are nucleic acid molecules that contain insertions or deletions of
the nucleotide sequences described herein. TGF-beta binding-protein
variant genes can be identified by determining whether the genes
hybridize with a nucleic acid molecule having the nucleotide
sequence of SEQ ID NOS: 1, 5, 7, 9, 11, 13, 15, 100, or 101 under
stringent conditions. In addition, TGF-beta binding-protein variant
genes should encode a protein having a cysteine backbone.
[0098] As an alternative, TGF-beta binding-protein variant genes
can be identified by sequence comparison. As used herein, two amino
acid sequences have "100% amino acid sequence identity" if the
amino acid residues of the two amino acid sequences are the same
when aligned for maximal correspondence. Similarly, two nucleotide
sequences have "100% nucleotide sequence identity" if the
nucleotide residues of the two nucleotide sequences are the same
when aligned for maximal correspondence. Sequence comparisons can
be performed using standard software programs such as those
included in the LASERGENE bioinformatics computing suite, which is
produced by DNASTAR (Madison, Wis.). Other methods for comparing
two nucleotide or amino acid sequences by determining optimal
alignment are well-known to those of skill in the art (see, for
example, Peruski and Peruski, The Internet and the New Biology:
Tools for Genomic and Molecular Research (ASM Press, Inc. 1997), Wu
et al. (eds.), "Information Superhighway and Computer Databases of
Nucleic Acids and Proteins," in Methods in Gene Biotechnology,
pages 123-151 (CRC Press, Inc. 1997), and Bishop (ed.), Guide to
Human Genome Computing, 2nd Edition (Academic Press, Inc.
1998)).
[0099] A variant TGF-beta binding-protein should have at least a
50% amino acid sequence identity to SEQ ID NOS: 2, 6, 10, 12, 14,
16, 46, or 65 and preferably, greater than 60%, 65%, 70%, 75%, 80%,
85%, 90%, or 95% identity. Alternatively, TGF-beta binding-protein
variants can be identified by having at least a 70% nucleotide
sequence identity to SEQ ID NOS: 1, 5, 9, 11, 13, 15, 100, or 101.
Moreover, the present invention contemplates TGF-beta
binding-protein gene variants having greater than 75%, 80%, 85%,
90%, or 95% identity to SEQ ID NO: 1 or SEQ ID NO: 100. Regardless
of the particular method used to identify a TGF-beta
binding-protein variant gene or variant TGF-beta binding-protein, a
variant TGF-beta binding-protein or a polypeptide encoded by a
variant TGF-beta binding-protein gene can be functionally
characterized by, for example, its ability to bind to and/or
inhibit the signaling of a selected member of the TGF-beta family
of proteins, or by its ability to bind specifically to an
anti-TGF-beta binding-protein antibody.
[0100] The present invention includes functional fragments of
TGF-beta binding-protein genes. Within the context of this
invention, a "functional fragment" of a TGF-beta binding-protein
gene refers to a nucleic acid molecule that encodes a portion of a
TGF-beta binding-protein polypeptide which either (1) possesses the
above-noted function activity, or (2) specifically binds with an
anti-TGF-beta binding-protein antibody. For example, a functional
fragment of a TGF-beta binding-protein gene described herein
comprises a portion of the nucleotide sequence of SEQ ID NOS: 1, 5,
9, 11, 13, 15, 100, or 101.
[0101] 2. Isolation of the TGF-beta Binding-Protein Gene
[0102] DNA molecules encoding a TGF-beta binding-protein can be
obtained by screening a human cDNA or genomic library using
polynucleotide probes based upon, for example, SEQ ID NO: 1. For
example, the first step in the preparation of a cDNA library is to
isolate RNA using methods well-known to those of skill in the art.
In general, RNA isolation techniques provide a method for breaking
cells, a means of inhibiting RNase-directed degradation of RNA, and
a method of separating RNA from DNA, protein, and polysaccharide
contaminants. For example, total RNA can be isolated by freezing
tissue in liquid nitrogen, grinding the frozen tissue with a mortar
and pestle to lyse the cells, extracting the ground tissue with a
solution of phenol/chloroform to remove proteins, and separating
RNA from the remaining impurities by selective precipitation with
lithium chloride (see, for example, Ausubel et al. (eds.), Short
Protocols in Molecular Biology, 3rd Edition, pages 4-1 to 4-6 (John
Wiley & Sons 1995) ["Ausubel (1995)"]; Wu et al., Methods in
Gene Biotechnology, pages 33-41 (CRC Press, Inc. 1997) ["Wu
(1997)"]). Alternatively, total RNA can be isolated by extracting
ground tissue with guanidinium isothiocyanate, extracting with
organic solvents, and separating RNA from contaminants using
differential centrifugation (see, for example, Ausubel (1995) at
pages 4-1 to 4-6; Wu (1997) at pages 33-41).
[0103] In order to construct a cDNA library, poly(A).sup.+ RNA is
preferably isolated from a total RNA preparation. Poly(A).sup.+ RNA
can be isolated from total RNA by using the standard technique of
oligo(dT)-cellulose chromatography (see, for example, Ausubel
(1995) at pages 4-11 to 4-12). Double-stranded cDNA molecules may
be synthesized from poly(A).sup.+ RNA using techniques well-known
to those in the art. (see, for example, Wu (1997) at pages 41-46).
Moreover, commercially available kits can be used to synthesize
double-stranded cDNA molecules (for example, Life Technologies,
Inc. (Gaithersburg, Md.); CLONTECH Laboratories, Inc. (Palo Alto,
Calif.); Promega Corporation (Madison, Wis.); and Stratagene
Cloning Systems (La Jolla, Calif.)).
[0104] The basic approach for obtaining TGF-beta binding-protein
cDNA clones can be modified by constructing a subtracted cDNA
library that is enriched in TGF-binding-protein-specific cDNA
molecules. Techniques for constructing subtracted libraries are
well-known to those of skill in the art (see, for example, Sargent,
"Isolation of Differentially Expressed Genes," in Meth. Enzymol.
152:423, 1987; and Wu et al. (eds.), "Construction and Screening of
Subtracted and Complete Expression cDNA Libraries," in Methods in
Gene Biotechnology, pages 29-65 (CRC Press, Inc. 1997)).
[0105] Various cloning vectors are appropriate for the construction
of a cDNA library. For example, a cDNA library can be prepared in a
vector derived from bacteriophage, such as a .lambda.gt10 vector
(see, for example, Huynh et al., "Constructing and Screening cDNA
Libraries in .lambda.gt10 and .lambda.gt11, " in DNA Cloning: A
Practical Approach Vol. I, Glover (ed.), page 49 (IRL Press, 1985);
Wu (1997) at pages 47-52). Alternatively, double-stranded cDNA
molecules can be inserted into a plasmid vector, such as a
pBluescript vector (Stratagene Cloning Systems; La Jolla, Calif.),
a LambdaGEM-4 (Promega Corp.; Madison, Wis.) or other commercially
available vectors. Suitable cloning vectors also can be obtained
from the American Type Culture Collection (Rockville, Md.).
[0106] In order to amplify the cloned cDNA molecules, the cDNA
library is inserted into a prokaryotic host, using standard
techniques. For example, a cDNA library can be introduced into
competent E. coli DH5 cells, which can be obtained from Life
Technologies, Inc. (Gaithersburg, Md.).
[0107] A human genomic DNA library can be prepared by means
well-known in the art (see, for example, Ausubel (1995) at pages
5-1 to 5-6; Wu (1997) at pages 307-327). Genomic DNA can be
isolated by lysing tissue with the detergent Sarkosyl, digesting
the lysate with proteinase K, clearing insoluble debris from the
lysate by centrifugation, precipitating nucleic acid from the
lysate using isopropanol, and purifying resuspended DNA on a cesium
chloride density gradient.
[0108] DNA fragments that are suitable for the production of a
genomic library can be obtained by the random shearing of genomic
DNA or by the partial digestion of genomic DNA with restriction
endonucleases. Genomic DNA fragments can be inserted into a vector,
such as a bacteriophage or cosmid vector, in accordance with
conventional techniques, such as the use of restriction enzyme
digestion to provide appropriate termini, the use of alkaline
phosphatase treatment to avoid undesirable joining of DNA
molecules, and ligation with appropriate ligases. Techniques for
such manipulation are well-known in the art (see, for example,
Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages
307-327).
[0109] Nucleic acid molecules that encode a TGF-beta
binding-protein can also be obtained using the polymerase chain
reaction (PCR) with oligonucleotide primers having nucleotide
sequences that are based upon the nucleotide sequences of the human
TGF-beta binding-protein gene, as described herein. General methods
for screening libraries with PCR are provided by, for example, Yu
et al., "Use of the Polymerase Chain Reaction to Screen Phage
Libraries," in Methods in Molecular Biology, Vol. 15: PCR
Protocols: Current Methods and Applications, White (ed.), pages 211
-215 (Humana Press, Inc. 1993). Moreover, techniques for using PCR
to isolate related genes are described by, for example, Preston,
"Use of Degenerate Oligonucleotide Primers and the Polymerase Chain
Reaction to Clone Gene Family Members," in Methods in Molecular
Biology, Vol. 15: PCR Protocols: Current Methods and Applications,
White (ed.), pages 317-337 (Humana Press, Inc. 1993).
[0110] Alternatively, human genomic libraries can be obtained from
commercial sources such as Research Genetics (Huntsville, Ala.) and
the American Type Culture Collection (Rockville, Md.). A library
containing cDNA or genomic clones can be screened with one or more
polynucleotide probes based upon SEQ ID NO: 1, using standard
methods as described herein and known in the art (see, for example,
Ausubel (1995) at pages 6-1 to 6-11).
[0111] Anti-TGF-beta binding-protein antibodies, produced as
described herein, can also be used to isolate DNA sequences that
encode a TGF-beta binding-protein from cDNA libraries. For example,
the antibodies can be used to screen .lambda.gt11 expression
libraries, or the antibodies can be used for immunoscreening
following hybrid selection and translation (see, for example,
Ausubel (1995) at pages 6-12 to 6-16; Margolis et al., "Screening
.lambda. expression libraries with antibody and protein probes," in
DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.
(eds.), pages 1- 14 (Oxford University Press 1995)).
[0112] The sequence of a TGF-beta binding-protein cDNA or TGF-beta
binding-protein genomic fragment can be determined using standard
methods. Moreover, the identification of genomic fragments
containing a TGF-beta binding-protein promoter or regulatory
element can be achieved using well-established techniques, such as
deletion analysis (see generally Ausubel (1995), supra).
[0113] As an alternative, a TGF-beta binding-protein gene can be
obtained by synthesizing DNA molecules using mutually priming long
oligonucleotides and the nucleotide sequences described herein
(see, for example, Ausubel (1995) at pages 8-8 to 8-9). Established
techniques using the polymerase chain reaction provide the ability
to synthesize DNA molecules at least two kilobases in length (Adang
et al., Plant Molec. Biol. 21:1131, 1993; Bambot et al., PCR
Methods and Applications 2:266, 1993; Dillon et al., "Use of the
Polymerase Chain Reaction for the Rapid Construction of Synthetic
Genes," in Methods in Molecular Biology, Vol. 15: PCR Protocols:
Current Methods and Applications, White (ed.), pages 263-268,
(Humana Press, Inc. 1993); Holowachuk et al., PCR Methods Appl.
4:299, 1995).
[0114] 3. Production of TGF-Beta Binding-Protein Genes
[0115] Nucleic acid molecules encoding variant TGF-beta
binding-protein genes can be obtained by screening various cDNA or
genomic libraries with polynucleotide probes having nucleotide
sequences based upon SEQ ID NO: 1, 5, 9, 11, 13, 15, 100, or 101
using procedures described herein. TGF-beta binding-protein gene
variants can also be constructed synthetically. For example, a
nucleic acid molecule can be devised that encodes a polypeptide
having a conservative amino acid change, compared with the amino
acid sequence of SEQ ID NOS: 2, 6, 8, 10, 12, 14, 16, 46, or 65.
That is, variants can be obtained that contain one or more amino
acid substitutions of SEQ ID NOS: 2, 6, 8, 10, 12, 14, 16, 46, or
65, in which an alkyl amino acid is substituted for an alkyl amino
acid in a TGF-beta binding-protein amino acid sequence, an aromatic
amino acid is substituted for an aromatic amino acid in a TGF-beta
binding-protein amino acid sequence, a sulfur-containing amino acid
is substituted for a sulfur-containing amino acid in a TGF-beta
binding-protein amino acid sequence, a hydroxy-containing amino
acid is substituted for a hydroxy-containing amino acid in a
TGF-beta binding-protein amino acid sequence, an acidic amino acid
is substituted for an acidic amino acid in a TGF-beta
binding-protein amino acid sequence, a basic amino acid is
substituted for a basic amino acid in a TGF-beta binding-protein
amino acid sequence, or a dibasic monocarboxylic amino acid is
substituted for a dibasic monocarboxylic amino acid in a TGF-beta
binding-protein amino acid sequence. Among the common amino acids,
for example, a "conservative amino acid substitution" is
illustrated by a substitution among amino acids within each of the
following groups: (1) glycine, alanine, valine, leucine, and
isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine
and threonine, (4) aspartate and glutamate, (5) glutamine and
asparagine, and (6) lysine, arginine and histidine. In making such
substitutions, it is important, when possible, to maintain the
cysteine backbone outlined in FIG. 1.
[0116] Conservative amino acid changes in a TGF-beta
binding-protein gene can be introduced by substituting nucleotides
for the nucleotides recited in SEQ ID NOS: 1, 5, 9, 11, 13, 15,
100, or 101. Such "conservative amino acid" variants can be
obtained, for example, by oligonucleotide-directed mutagenesis,
linker-scanning mutagenesis, mutagenesis using the polymerase chain
reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22;
McPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL
Press 1991)). The functional ability of such variants can be
determined using a standard method, such as the assay described
herein. Alternatively, a variant TGF-beta binding-protein
polypeptide can be identified by the ability to specifically bind
anti-TGF-beta binding-protein antibodies.
[0117] Routine deletion analyses of nucleic acid molecules can be
performed to obtain "functional fragments" of a nucleic acid
molecule that encodes a TGF-beta binding-protein polypeptide. As an
illustration, DNA molecules having the nucleotide sequence of SEQ
ID NO: 1 can be digested with Bal31 nuclease to obtain a series of
nested deletions. The fragments are then inserted into expression
vectors in proper reading frame, and the expressed polypeptides are
isolated and tested for activity, or for the ability to bind
anti-TGF-beta binding-protein antibodies. One alternative to
exonuclease digestion is to use oligonucleotide-directed
mutagenesis to introduce deletions or stop codons to specify
production of a desired fragment. Alternatively, particular
fragments of a TGF-beta binding-protein gene can be synthesized
using the polymerase chain reaction.
[0118] Standard techniques for functional analysis of proteins are
described by, for example, Treuter et al., Molec. Gen. Genet.
240:113, 1993; Content et al., "Expression and preliminary deletion
analysis of the 42 kDa 2-5A synthetase induced by human
interferon," in Biological Interferon Systems, Proceedings of
ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72
(Nijhoff 1987); Herschman, "The EGF Receptor," in Control of Animal
Cell Proliferation, Vol. 1, Boynton et al., (eds.) pages 169-199
(Academic Press 1985); Coumailleau et al., J. Biol. Chem.
270:29270, 1995; Fukunaga et al., J. Biol. Chem. 270:25291, 1995;
Yamaguchi et al., Biochem. Pharmacol. 50:1295, 1995; Meisel et al.,
Plant Molec. Biol. 30:1, 1996.
[0119] The present invention also contemplates functional fragments
of a TGF-beta binding-protein gene that have conservative amino
acid changes.
[0120] A TGF-beta binding-protein variant gene can be identified on
the basis of structure by determining the level of identity with
nucleotide and amino acid sequences of SEQ ID NOS: 1, 5, 9, 11, 13,
15, 100, or 101 and 2, 6, 10, 12, 14, 16, 46, or 65 as discussed
above. An alternative approach to identifying a variant gene on the
basis of structure is to determine whether a nucleic acid molecule
encoding a potential variant TGF-beta binding-protein gene can
hybridize under stringent conditions to a nucleic acid molecule
having the nucleotide sequence of SEQ ID NOS: 1, 5, 9, 11, 13, 15,
100, or 101, or a portion thereof of at least 15 or 20 nucleotides
in length. As an illustration of stringent hybridization
conditions, a nucleic acid molecule having a variant TGF-beta
binding-protein sequence can bind with a fragment of a nucleic acid
molecule having a sequence from SEQ ID NO: 1 in a buffer
containing, for example, 5.times. SSPE (1.times. SSPE=180 mM sodium
chloride, 10 mM sodium phosphate, 1 mM EDTA (pH 7.7), 5.times.
Denhardt's solution (100.times. Denhardt's=2% (w/v) bovine serum
albumin, 2% (w/v) Ficoll, 2% (w/v) polyvinylpyrrolidone) and 0.5%
SDS incubated overnight at 55-60.degree. C. Post-hybridization
washes at high stringency are typically performed in 0.5.times.SSC
(1.times.SSC=150 mM sodium chloride, 15 mM trisodium citrate) or in
0.5.times. SSPE at 55-60.degree. C.
[0121] Regardless of the particular nucleotide sequence of a
variant TGF-beta binding-protein gene, the gene encodes a
polypeptide that can be characterized by its functional activity,
or by the ability to bind specifically to an anti-TGF-beta
binding-protein antibody. More specifically, variant TGF-beta
binding-protein genes encode polypeptides which exhibit at least
50%, and preferably, greater than 60, 70, 80 or 90%, of the
activity of polypeptides encoded by the human TGF-beta
binding-protein gene described herein.
[0122] 4. Production of TGF-Beta Binding-Protein in Cultured
Cells
[0123] To express a TGF-beta binding-protein gene, a nucleic acid
molecule encoding the polypeptide must be operably linked to
regulatory sequences that control transcriptional expression in an
expression vector and then introduced into a host cell. In addition
to transcriptional regulatory sequences, such as promoters and
enhancers, expression vectors can include translational regulatory
sequences and a marker gene that is suitable for selection of cells
that carry the expression vector. Expression vectors that are
suitable for production of a foreign protein in eukaryotic cells
typically contain (1) prokaryotic DNA elements coding for a
bacterial replication origin and an antibiotic resistance marker to
provide for the growth and selection of the expression vector in a
bacterial host; (2) eukaryotic DNA elements that control initiation
of transcription, such as a promoter; and (3) DNA elements that
control the processing of transcripts, such as a transcription
termination/polyadenylation sequence.
[0124] TGF-beta binding-proteins of the present invention are
preferably expressed in mammalian cells. Examples of mammalian host
cells include African green monkey kidney cells (Vero; ATCC CRL
1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby
hamster kidney cells (BHK-21; ATCC CRL 8544), canine kidney cells
(MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC
CCL61), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC
CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548)
SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650) and
murine embryonic cells (NIH-3T3; ATCC CRL 1658).
[0125] For a mammalian host, the transcriptional and translational
regulatory signals may be derived from viral sources, such as
adenovirus, bovine papilloma virus, simian virus, or the like, in
which the regulatory signals are associated with a particular gene
which has a high level of expression. Suitable transcriptional and
translational regulatory sequences also can be obtained from
mammalian genes, such as actin, collagen, myosin, and
metallothionein genes.
[0126] Transcriptional regulatory sequences include a promoter
region sufficient to direct the initiation of RNA synthesis.
Suitable eukaryotic promoters include the promoter of the mouse
metallothionein I gene [Hamer et al., J. Molec. Appl. Genet. 1:273,
1982], the TK promoter of Herpes virus [McKnight, Cell 31:355,
1982], the SV40 early promoter [Benoist et al., Nature 290:304,
1981], the Rous sarcoma virus promoter [Gorman et al., Proc. Nat'l
Acad. Sci. USA 79:6777, 1982], the cytomegalovirus promoter
[Foecking et al., Gene 45:101, 1980], and the mouse mammary tumor
virus promoter (see, generally, Etcheverry, "Expression of
Engineered Proteins in Mammalian Cell Culture," in Protein
Engineering: Principles and Practice, Cleland et al. (eds.), pages
163-181 (John Wiley & Sons, Inc. 1996)).
[0127] Alternatively, a prokaryotic promoter, such as the
bacteriophage T3 RNA polymerase promoter, can be used to control
TGF-beta binding-protein gene expression in mammalian cells if the
prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et
al., Mol. Cell. Biol. 10:4529, 1990; Kaufman et al., Nucleic Acids
Res. 19:4485, 1991).
[0128] TGF-beta binding-protein genes may also be expressed in
bacterial, yeast, insect, or plant cells. Suitable promoters that
can be used to express TGF-beta binding-protein polypeptides in a
prokaryotic host are well-known to those of skill in the art and
include promoters capable of recognizing the T4, T3, Sp6 and T7
polymerases, the P.sub.R and P.sub.L promoters of bacteriophage
lambda, the trp, recA, heat shock, lacUV5, tac, lpp-lacSpr, phoA,
and lacZ promoters of E. coli, promoters of B. subtilis, the
promoters of the bacteriophages of Bacillus, Streptomyces
promoters, the int promoter of bacteriophage lambda, the bla
promoter of pBR322, and the CAT promoter of the chloramphenicol
acetyl transferase gene. Prokaryotic promoters have been reviewed
by Glick, J. Ind. Microbiol. 1:277, 1987, Watson et al., Molecular
Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), and by
Ausubel et al. (1995).
[0129] Preferred prokaryotic hosts include E. coli and Bacillus
subtilus. Suitable strains of E. coli include BL21(DE3),
BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF',
DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109,
JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for
example, Brown (Ed.), Molecular Biology Labfax (Academic Press
1991)). Suitable strains of Bacillus subtilus include BR151, YB886,
MI119, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning
Methods," in DNA Cloning: A Practical Approach, Glover (Ed.) (IRL
Press 1985)).
[0130] Methods for expressing proteins in prokaryotic hosts are
well-known to those of skill in the art (see, for example, Williams
et al., "Expression of foreign proteins in E. coli using plasmid
vectors and purification of specific polyclonal antibodies," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
page 15 (Oxford University Press 1995); Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, page 137 (Wiley-Liss, Inc.
1995); and Georgiou, "Expression of Proteins in Bacteria," in
Protein Engineering: Principles and Practice, Cleland et al.
(eds.), page 101 (John Wiley & Sons, Inc. 1996)).
[0131] The baculovirus system provides an efficient means to
introduce cloned TGF-beta binding-protein genes into insect cells.
Suitable expression vectors are based upon the Autographa
californica multiple nuclear polyhedrosis virus (AcMNPV), and
contain well-known promoters such as Drosophila heat shock protein
(hsp) 70 promoter, Autographa californica nuclear polyhedrosis
virus immediate-early gene promoter (ie-1) and the delayed early
39K promoter, baculovirus p10 promoter, and the Drosophila
metallothionein promoter. Suitable insect host cells include cell
lines derived from IPLB-Sf-21, a Spodoptera frugiperda pupal
ovarian cell line, such as Sf9 (ATCC CRL 1711), Sf21AE, and Sf21
(Invitrogen Corporation; San Diego, Calif.), as well as Drosophila
Schneider-2 cells. Established techniques for producing recombinant
proteins in baculovirus systems are provided by Bailey et al.,
"Manipulation of Baculovirus Vectors," in Methods in Molecular
Biology, Volume 7: Gene Transfer and Expression Protocols, Murray
(ed.), pages 147-168 (The Humana Press, Inc. 1991), by Patel et
al., "The baculovirus expression system," in DNA Cloning 2:
Expression Systems, 2nd Edition, Glover et al (eds.), pages 205-244
(Oxford University Press 1995), by Ausubel (1995) at pages 16-37 to
16-57, by Richardson (ed.), Baculovirus Expression Protocols (The
Humana Press, Inc. 1995), and by Lucknow, "Insect Cell Expression
Technology," in Protein Engineering: Principles and Practice,
Cleland et al. (eds.), pages 183-218 (John Wiley & Sons, Inc.
1996).
[0132] Promoters for expression in yeast include promoters from
GAL1 (galactose), PGK (phosphoglycerate kinase), ADH (alcohol
dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinol
dehydrogenase), and the like. Many yeast cloning vectors have been
designed and are readily available. These vectors include YIp-based
vectors, such as YIp5, YRp vectors, such as YRp17, YEp vectors such
as YEp13 and YCp vectors, such as YCp19. One skilled in the art
will appreciate that there are a wide variety of suitable vectors
for expression in yeast cells.
[0133] Expression vectors can also be introduced into plant
protoplasts, intact plant tissues, or isolated plant cells. General
methods of culturing plant tissues are provided, for example, by
Miki et al., "Procedures for Introducing Foreign DNA into Plants,"
in Methods in Plant Molecular Biology and Biotechnology, Glick et
al. (eds.), pages 67-88 (CRC Press, 1993).
[0134] An expression vector can be introduced into host cells using
a variety of standard techniques including calcium phosphate
transfection, liposome-mediated transfection,
microprojectile-mediated delivery, electroporation, and the like.
Preferably, the transfected cells are selected and propagated to
provide recombinant host cells that comprise the expression vector
stably integrated in the host cell genome. Techniques for
introducing vectors into eukaryotic cells and techniques for
selecting such stable transformants using a dominant selectable
marker are described, for example, by Ausubel (1995) and- by Murray
(ed.), Gene Transfer and Expression Protocols (Humana Press 1991).
Methods for introducing expression vectors into bacterial, yeast,
insect, and plant cells are also provided by Ausubel (1995).
[0135] General methods for expressing and recovering foreign
protein produced by a mammalian cell system is provided by, for
example, Etcheverry, "Expression of Engineered Proteins in
Mammalian Cell Culture," in Protein Engineering: Principles and
Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc. 1996).
Standard techniques for recovering protein produced by a bacterial
system is provided by, for example, Grisshammer et al.,
"Purification of over-produced proteins from E. coli cells," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
pages 59-92 (Oxford University Press 1995). Established methods for
isolating recombinant proteins from a baculovirus system are
described by Richardson (ed.), Baculovirus Expression Protocols
(The Humana Press, Inc., 1995).
[0136] More generally, TGF-beta binding-protein can be isolated by
standard techniques, such as affinity chromatography, size
exclusion chromatography, ion exchange chromatography, HPLC and the
like. Additional variations in TGF-beta binding-protein isolation
and purification can be devised by those of skill in the art. For
example, anti-TGF-beta binding-protein antibodies, obtained as
described below, can be used to isolate large quantities of protein
by immunoaffinity purification.
[0137] 5. Production of Antibodies to TGF-Beta Binding-Proteins
[0138] The present invention provides antibodies that specifically
bind to sclerostin as described herein in detail. Antibodies to
TGF-beta binding-protein can be obtained, for example, using the
product of an expression vector as an antigen. Antibodies that
specifically bind to sclerostin may also be prepared by using
peptides derived from any one of the sclerostin polypeptide
sequences provided herein (SEQ ID NOS: 2, 6, 8, 10, 12, 14, 16, 46,
and 65). Particularly useful anti-TGF-beta binding-protein
antibodies "bind specifically" with TGF-beta binding-protein of
Sequence ID Nos. 2, 6, 8, 10, 12, 14, 16, 46, or 65 but not to
other TGF-beta binding-proteins such as Dan, Cerberus, SCGF, or
Gremlin. Antibodies of the present invention (including fragments
and derivatives thereof) may be a polyclonal or, especially a
monoclonal antibody. The antibody may belong to any immunoglobulin
class, and may be for example an IgG, (including isotypes of IgG,
which for human antibodies are known in the art as IgG.sub.1,
IgG.sub.2, IgG.sub.3, IgG.sub.4); IgE; IgM; or IgA antibody. An
antibody may be obtained from fowl or mammals, preferably, for
example, from a murine, rat, human or other primate antibody. When
desired the antibody may be an internalising antibody.
[0139] Polyclonal antibodies to recombinant TGF-beta
binding-protein can be prepared using methods well-known to those
of skill in the art (see, for example, Green et al., "Production of
Polyclonal Antisera," in Immunochemical Protocols (Manson, ed.),
pages 1-5 (Humana Press 1992); Williams et al., "Expression of
foreign proteins in E. coli using plasmid vectors and purification
of specific polyclonal antibodies," in DNA Cloning 2: Expression
Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford
University Press 1995)). Although polyclonal antibodies are
typically raised in animals such as rats, mice, rabbits, goats, or
sheep, an anti-TGF-beta binding-protein antibody of the present
invention may also be derived from a subhuman primate antibody.
General techniques for raising diagnostically and therapeutically
useful antibodies in baboons may be found, for example, in
Goldenberg et al., international patent publication No. WO 91/11465
(1991), and in Losman et al., Int. J. Cancer 46:310, 1990.
[0140] The antibody should comprise at least a variable region
domain. The variable region domain may be of any size or amino acid
composition and will generally comprise at least one hypervariable
amino acid sequence responsible for antigen binding embedded in a
framework sequence. In general terms the variable (V) region domain
may be any suitable arrangement of immunoglobulin heavy (V.sub.H)
and/or light (V.sub.L) chain variable domains. Thus for example the
V region domain may be monomeric and be a V.sub.H or V.sub.L domain
where these are capable of independently binding antigen with
acceptable affinity. Alternatively the V region domain may be
dimeric and contain V.sub.H-V.sub.H, V.sub.H-V.sub.L, or
V.sub.L-V.sub.L, dimers in which the V.sub.H and V.sub.L chains are
non-covalently associated (abbreviated hereinafter as F.sub.v).
Where desired, however, the chains may be covalently coupled either
directly, for example via a disulphide bond between the two
variable domains, or through a linker, for example a peptide
linker, to form a single chain domain (abbreviated hereinafter as
scF.sub.v).
[0141] The variable region domain may be any naturally occuring
variable domain or an engineered version thereof. By engineered
version is meant a variable region domain that has been created
using recombinant DNA engineering techniques. Such engineered
versions include those created for example from natural antibody
variable regions by insertions, deletions or changes in or to the
amino acid sequences of the natural antibodies. Particular examples
of this type include those engineered variable region domains
containing at least one CDR and optionally one or more framework
amino acids from one antibody and the remainder of the variable
region domain from a second antibody.
[0142] The variable region domain may be covalently attached at a
C-terminal amino acid to at least one other antibody domain or a
fragment thereof. Thus, for example where a V.sub.H domain is
present in the variable region domain this may be linked to an
immunoglobulin C.sub.H1 domain or a fragment thereof. Similarly a
V.sub.L domain may be linked to a C.sub.K domain or a fragment
thereof. In this way for example the antibody may be a Fab fragment
wherein the antigen binding domain contains associated V.sub.H and
V.sub.L domains covalently linked at their C-termini to a CH1 and
C.sub.K domain respectively. The CH1 domain may be extended with
further amino acids, for example to provide a hinge region domain
as found in a Fab' fragment, or to provide further domains, such as
antibody CH2 and CH3 domains.
[0143] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing cells
(see, for example, Larrick et al., Methods: A Companion to Methods
in Enzymology 2:106, 1991; Courtenay-Luck, "Genetic Manipulation of
Monoclonal Antibodies," in Monoclonal Antibodies: Production,
Engineering and Clinical Application, Ritter et al. (eds.), page
166 (Cambridge University Press 1995); and Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, Birch et al., (eds.), page
137 (Wiley-Liss, Inc. 1995)).
[0144] Antibodies for use in the invention may in general be
monoclonal (prepared by conventional immunisation and cell fusion
procedures) or in the case of fragments, derived therefrom using
any suitable standard chemical such as reduction or enzymatic
cleavage and/or digestion techniques, for example by treatment with
pepsin. More specifically, monoclonal anti-TGF-beta binding-protein
antibodies can be generated utilizing a variety of techniques.
Rodent monoclonal antibodies to specific antigens may be obtained
by methods known to those skilled in the art (see, for example,
Kohler et al., Nature 256:495, 1975; and Coligan et al. (eds.),
Current Protocols in Immunology, 1:2.5.1-2.6.7 (John Wiley &
Sons 1991) ["Coligan"]; Picksley et al., "Production of monoclonal
antibodies against proteins expressed in E. coli," in DNA Cloning
2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93
(Oxford University Press 1995)).
[0145] Briefly, monoclonal antibodies can be obtained by injecting
mice with a composition comprising a TGF-beta binding-protein gene
product, verifying the presence of antibody production by removing
a serum sample, removing the spleen to obtain B-lymphocytes, fusing
the B-lymphocytes with myeloma cells to produce hybridomas, cloning
the hybridomas, selecting positive clones which produce antibodies
to the antigen, culturing the clones that produce antibodies to the
antigen, and isolating the antibodies from the hybridoma
cultures.
[0146] In addition, an anti-TGF-beta binding-protein antibody of
the present invention may be derived from a human monoclonal
antibody. Human monoclonal antibodies are obtained from transgenic
mice that have been engineered to produce specific human antibodies
in response to antigenic challenge. In this technique, elements of
the human heavy and light chain locus are introduced into strains
of mice derived from embryonic stem cell lines that contain
targeted disruptions of the endogenous heavy chain and light chain
loci. The transgenic mice can synthesize human antibodies specific
for human antigens, and the mice can be used to produce human
antibody-secreting hybridomas. Methods for obtaining human
antibodies from transgenic mice are described, for example, by
Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature
368:856, 1994; and Taylor et al., Int. Immun. 6:579, 1994.
[0147] Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography (see, for example, Coligan at pages
2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., "Purification of
Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol. 10,
pages 79-104 (The Humana Press, Inc. 1992)).
[0148] For particular uses, it may be desirable to prepare
fragments of anti-TGF-beta binding-protein antibodies. Such
antibody fragments can be obtained, for example, by proteolytic
hydrolysis of the antibody. Antibody fragments can be obtained by
pepsin or papain digestion of whole antibodies by conventional
methods. As an illustration, antibody fragments can be produced by
enzymatic cleavage of antibodies with pepsin to provide a 5S
fragment denoted F(ab').sub.2. This fragment can be further cleaved
using a thiol reducing agent to produce 3.5S Fab' monovalent
fragments. Optionally, the cleavage reaction can be performed using
a blocking group for the sulfhydryl groups that result from
cleavage of disulfide linkages. As an alternative, an enzymatic
cleavage using pepsin produces two monovalent Fab fragments and an
Fc fragment directly. These methods are described, for example, by
Goldenberg, U.S. Pat. No. 4,331,647, Nisonoff et al.,Arch Biochem.
Biophys. 89:230, 1960, Porter, Biochem. J. 73:119, 1959, Edelman et
al., in Methods in Enzymology 1:422 (Academic Press 1967), and by
Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
[0149] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0150] Alternatively, the antibody may be a recombinant or
engineered antibody obtained by the use of recombinant DNA
techniques involving the manipulation and re-expression of DNA
encoding antibody variable and/or constant regions. Such DNA is
known and/or is readily available from DNA libraries including for
example phage-antibody libraries (see Chiswell, D J and McCafferty,
J. Tibtech. 10 80-84 (1992)) or where desired can be synthesised.
Standard molecular biology and/or chemistry procedures may be used
to sequence and manipulate the DNA, for example, to introduce
codons to create cysteine residues, to modify, add or delete other
amino acids or domains as desired.
[0151] One or more replicable expression vectors containing the DNA
encoding a variable and/or constant region may be prepared and used
to transform an appropriate cell line, e.g. a non-producing myeloma
cell line, such as a mouse NSO line or a bacterial, such as E.coli,
in which production of the antibody will occur. In order to obtain
efficient transcription and translation, the DNA sequence in each
vector should include appropriate regulatory sequences,
particularly a promoter and leader sequence operably linked to a
variable domain sequence. Particular methods for producing
antibodies in this way are generally well known and routinely used.
For example, basic molecular biology procedures are described by
Maniatis et al (Molecular Cloning, Cold Spring Harbor Laboratory,
New York, 1989); DNA sequencing can be performed as described in
Sanger et al (Proc. Natl. Acad. Sci. USA 74: 5463, (1977)) and the
Amersham International plc sequencing handbook; site directed
mutagenesis can be carried out according to the method of Kramer et
al. (Nucleic Acids Res. 12, 9441, (1984)); the Anglian
Biotechnology Ltd handbook; Kunkel Proc. Natl. Acad. Sci. USA
82:488-92 (1985); Kunkel et al., Methods in Enzymol. 154:367-82
(1987). Additionally, numerous publications detail techniques
suitable for the preparation of antibodies by manipulation of DNA,
creation of expression vectors, and transformation of appropriate
cells, for example as reviewed by Mountain A and Adair, J R in
Biotechnology and Genetic Engineering Reviews (ed. Tombs, M P, 10,
Chapter 1, 1992, Intercept, Andover, UK) and in International
Patent Specification No. WO 91/09967.
[0152] In certain embodiments, the-antibody according to the
invention may have one or more effector or reporter molecules
attached to it and the invention extends to such modified proteins.
A reporter molecule may be a detectable moiety or label such as an
enzyme, cytotoxic agent or other reporter molecule, including a
dye, radionuclide, luminescent group, fluorescent group, or biotin,
or the like. The TGF-beta binding protein-specific immunoglobulin
or fragment thereof may be radiolabeled for diagnostic or
therapeutic applications. Techniques for radiolabeling of
antibodies are known in the art. See, e.g., Adams 1998 In Vivo
12:11-21; Hiltunen 1993 Acta Oncol. 32:831-9. Therapeutic
applications are described in greater detail below and may include
use of the TGF-beta binding protein specific antibody (or fragment
thereof) in conjunction with other therapeutic agents. The effector
or reporter molecules may be attached to the antibody through any
available amino acid side-chain, terminal amino acid or, where
present carbohydrate functional group located in the antibody,
provided that the attachment or the attachment process does not
adversely affect the binding properties and the usefulness of the
molecule. Particular functional groups include, for example any
free amino, imino, thiol, hydroxyl, carboxyl or aldehyde group.
Attachment of the antibody and the effector and/or reporter
molecule(s) may be achieved via such groups and an appropriate
functional group in the effector or reporter molecules. The linkage
may be direct or indirect through spacing or bridging groups.
[0153] Effector molecules include, for example, antineoplastic
agents, toxins (such as enzymatically active toxins of bacterial
(such as P. aeruginosa exotoxin A) or plant origin and fragments
thereof (e.g. ricin and fragments thereof; plant gelonin, bryodin
from Bryonia dioica, or the like. See, e.g., Thrush et al., 1996
Annu. Rev. Immunol. 14:49-71; Frankel et al., 1996 Cancer Res.
56:926-32); biologically active proteins, for example enzymes;
nucleic acids and fragments thereof such as. DNA, RNA and fragments
thereof; naturally occurring and synthetic polymers (e.g.,
polysaccharides and polyalkylene polymers such as poly(ethylene
glycol) and derivatives thereof); radionuclides, particularly
radioiodide; and chelated metals. Suitable reporter groups include
chelated metals, fluorescent compounds, or compounds that may be
detected by NMR or ESR spectroscopy. Particularly useful effector
groups are calichaemicin and derivatives thereof (see, for example,
South African Patent Specifications Nos. 85/8794, 88/8127 and
90/2839).
[0154] Numerous other toxins, including chemotherapeutic agents,
anti-mitotic agents, antibiotics, inducers of apoptosis (or
"apoptogens", see, e.g., Green and Reed, 1998, Science
281:1309-1312), or the like, are known to those familiar with the
art, and the examples provided herein are intended to be
illustrative without limiting the scope and spirit of the
invention. Particular antineoplastic agents include cytotoxic and
cytostatic agents, for example alkylating agents, such as nitrogen
mustards (e.g., chlorambucil, melphalan, mechlorethamine,
cyclophosphamide, or uracil mustard) and derivatives thereof,
triethylenephosphoramide, triethylenethiophosphor-amide, busulphan,
or cisplatin; antimetabolites, such as methotrexate, fluorouracil,
floxuridine, cytarabine, mercaptopurine, thioguanine, fluoroacetic
acid or fluorocitric acid, antibiotics, such as bleomycins (e.g.,
bleomycin sulphate), doxorubicin, daunorubicin, mitomycins (e.g.,
mitomycin C), actinomycins (e.g., dactinomycin) plicamycin,
calichaemicin and derivatives thereof, or esperamicin and
derivatives thereof; mitotic inhibitors, such as etoposide,
vincristine or vinblastine and derivatives thereof, alkaloids, such
as ellipticine; polyols such as taxicin-I or taxicin-II; hormones,
such as androgens (e.g., dromostanolone or testolactone),
progestins (e.g., megestrol acetate or medroxyprogesterone
acetate), estrogens (e.g., dimethylstilbestrol diphosphate,
polyestradiol phosphate or estramustine phosphate) or antiestrogens
(e.g., tamoxifen); anthraquinones, such as mitoxantrone, ureas,
such as hydroxyurea; hydrazines, such as procarbazine; or
imidazoles, such as dacarbazine.
[0155] Chelated metals include chelates of di-or tripositive metals
having a coordination number from 2 to 8 inclusive. Particular
examples of such metals include technetium (Tc), rhenium (Re),
cobalt (Co), copper (Cu), gold (Au), silver (Ag), lead (Pb),
bismuth (Bi), indium (In), gallium (Ga), yttrium (Y), terbium (Tb),
gadolinium (Gd), and scandium (Sc). In general the metal is
preferably a radionuclide. Particular radionuclides include
.sup.99mTc, .sup.186Re, .sup.188Re, .sup.58Co, .sup.60Co,
.sup.67Cu, .sup.195Au, .sup.199Au, .sup.110Ag, .sup.203Pb,
.sup.206Bi, .sup.207Bi, .sup.111In, .sup.67Ga, .sup.68Ga, .sup.88Y,
.sup.90Y, .sup.160Tb, .sup.153Gd, and .sup.47Sc.
[0156] The chelated metal may be for example one of the above types
of metal chelated with any suitable polydentate chelating agent,
for example acyclic or cyclic polyamines, polyethers, (e.g., crown
ethers and derivatives thereof); polyamides; porphyrins; and
carbocyclic derivatives. In general, the type of chelating agent
will depend on the metal in use. One particularly useful group of
chelating agents in conjugates according to the invention, however,
comprises acyclic and cyclic polyamines, especially.
polyaminocarboxylic acids, for example
diethylenetriaminepentaacetic acid and derivatives thereof, and
macrocyclic amines, such as cyclic tri-aza and tetra-aza
derivatives (for example, as described in International Patent
Specification No. WO 92/22583), and polyamides, especially
desferrioxamine and derivatives thereof.
[0157] When a thiol group in the antibody is used as the point of
attachment this may be achieved through reaction with a thiol
reactive group present in the effector or reporter molecule.
Examples of such groups include an -halocarboxylic acid or ester,
such as iodoacetamide, an imide, such as maleimide, a vinyl
sulphone, or a disulphide. These and other suitable linking
procedures are generally and more particularly described in
International Patent Specifications Nos. WO 93/06231, WO 92/22583,
WO 90/091195, and WO 89/01476.
[0158] Assays for Selecting Molecules that Increase Bone
Density
[0159] As discussed above, the present invention provides methods
for selecting and/or isolating compounds that are capable of
increasing bone density. For example, within one aspect of the
present invention methods are provided for determining whether a
selected molecule (e.g., a candidate agent) is capable of
increasing bone mineral content, comprising the steps of (a) mixing
(or contacting) a selected molecule with TGF-beta binding protein
and a selected member of the TGF-beta family of proteins, (b)
determining whether the selected molecule stimulates signaling by
the TGF-beta family of proteins, or inhibits the binding of the
TGF-beta binding protein to at least one member of the TGF-beta
family of proteins. Within certain embodiments, the molecule
enhances the ability of TGF-beta to function as a positive
regulator of mesenchymal cell differentiation.
[0160] Within other aspects of the invention, methods are provided
for determining whether a selected molecule (candidate agent) is
capable of increasing bone mineral content, comprising the steps of
(a) exposing (contacting, mixing, combining) a selected molecule to
cells which express TGF-beta binding-protein and (b) determining
whether the expression (or activity) of TGF-beta binding-protein in
the exposed cells decreases, or whether an activity of the TGF-beta
binding protein decreases, and therefrom determining whether the
compound is capable of increasing bone mineral content. Within one
embodiment, the cells are selected from the group consisting of the
spontaneously transformed or untransformed normal human bone from
bone biopsies and rat parietal bone osteoblasts. Methods for
detecting the level of expression of a TGF-beta binding protein may
be accomplished in a wide variety of assay formats known in the art
and described herein. Immunoassays may be used for detecting and
quantifying the expression of a TGF-beta binding protein and
include, for example, Countercurrent Immuno-Electrophoresis (CIEP),
radioimmunoassays, radioimmunoprecipitations, Enzyme-Linked
Immuno-Sorbent Assays (ELISA), immunoblot assays such as dot blot
assays and Western blots, inhibition or competition assays, and
sandwich assays (see U.S. Pat. Nos. 4,376,110 and 4,486,530; see
also Antibodies: A Laboratory Manual, supra). Such immunoassays may
use an antibody that is specific for a TGF-beta binding protein
such as the anti-sclerostin antibodies described herein, or may use
an antibody that is specific for a reporter molecule that is
attached to the TGF-beta binding protein. The level of polypeptide
expression may also be determined by quantifying the amount of
TGF-beta binding protein that binds to a TGF-beta binding protein
ligand. By way of example, binding of sclerostin in a sample to a
BMP may be detected by surface plasmon resonance (SPR).
Alternatively, the level of expression of mRNA encoding the
specific TGF-beta binding protein may be quantified.
[0161] Representative embodiments of such assays are provided below
in Examples 5 and 6. Briefly, a family member of the TGF-beta
super-family or a TGF-beta binding protein is first bound to a
solid phase, followed by addition of a candidate molecule. A
labeled family member of the TGF-beta super-family or a TGF-beta
binding protein is then added to the assay (i.e., the labeled
polypeptide is the ligand for whichever polypeptide was bound to
the solid phase), the solid phase washed, and the quantity of bound
or labeled TGF-beta super-family member or TGF-beta binding protein
on the solid support determined. Molecules which are suitable for
use in increasing bone mineral content as described herein are
those molecules which decrease the binding of TGF-beta binding
protein to a member or members of the TGF-beta super-family in a
statistically significant manner. Obviously, assays suitable for
use within the present invention should not be limited to the
embodiments described within Examples 2 and 3. In particular,
numerous parameters may be altered, such as by binding TGF-beta to
a solid phase, or by elimination of a solid phase entirely.
[0162] Within other aspects of the invention, methods are provided
for determining whether a selected molecule is capable of
increasing bone mineral content, comprising the steps of (a)
exposing (contacting, mixing, combining) a selected molecule
(candidate agent) to cells which express TGF-beta and (b)
determining whether the activity of TGF-beta from said exposed
cells is altered, and therefrom determining whether the compound is
capable of increasing bone mineral content. Similar to the methods
described herein, a wide variety of methods may be utilized to
assess the changes of TGF-beta binding-protein expression due to a
selected test compound. In one embodiment of the invention, the
candidate agent is an antibody that binds to the TGF-beta binding
protein sclerostin disclosed herein.
[0163] In a preferred embodiment of the invention, a method is
provided for identifying an antibody that modulates a TGF-beta
signaling pathway comprising contacting an antibody that
specifically binds to a SOST polypeptide with a SOST peptide,
including but not limited to the peptides disclosed herein, under
conditions and for a time sufficient to permit formation of an
antibody plus (+) SOST (antibody/SOST) complex and then detecting
the level (e.g., quantifying the amount) of the SOST/antibody
complex to determine the presence of an antibody that modulates a
TGF-beta signaling pathway. The method may be performed using SPR
or any number of different immunoassays known in the art and
disclosed herein, including an ELISA, immunoblot, or the like. A
TGF-beta signaling pathway includes a signaling pathway by which a
BMP binds to a type I and a type II receptor on a cell to stimulate
or induce the pathway that modulates bone mineral content. In
certain preferred embodiments of the invention, an antibody that
specifically binds to SOST stimulates or enhances the pathway for
increasing bone mineral content. Such an antibody may be identified
using the methods disclosed herein to detect binding of an antibody
to SOST specific peptides.
[0164] The subject invention methods may also be used for
identifying antibodies that impair, inhibit (including
competitively inhibit), or prevent binding of a BMP to a SOST
polypeptide by detecting whether an antibody binds to SOST peptides
that are located in regions or portions of regions on SOST to which
a BMP binds, such as peptides at the amino terminal end of SOST and
peptides that include amino terminal amino acid residues and a
portion of the core region (docking core) of SOST (e.g., SEQ ID
NOS: 47-64, 66-73, and 92-95). The methods of the present invention
may also be used to identify an antibody that impairs, prevents, or
inhibits, formation of SOST homodimers. Such an antibody that binds
specifically to SOST may be identified by detecting binding of the
antibody to peptides that are derived from the core or the carboxy
terminal region of SOST (e.g., SEQ ID NOS: 74-91 and 96-99).
[0165] Within another embodiment of the present invention, methods
are provided for determining whether a selected molecule is capable
of increasing bone mineral content, comprising the steps of (a)
mixing or contacting a selected molecule (candidate agent) with a
TGF-beta-binding-protein and a selected member of the TGF-beta
family of proteins, (b) determining whether the selected molecule
up-regulates the signaling of the TGF-beta family of proteins, or
inhibits the binding of the TGF-beta binding-protein to the
TGF-beta family of proteins. Within certain embodiments, the
molecule enhances the ability of TGF-beta to function as a positive
regulator of mesenchymal cell differentiation.
[0166] Similar to the above described methods, a wide variety of
methods may be utilized to assess stimulation of TGF-beta due to a
selected test compound. One such representative method is provided
below in Example 6 (see also Durham et al., Endo. 136:
1374-1380.
[0167] Within yet other aspects of the present invention, methods
are provided for determining whether a selected molecule (candidate
agent) is capable of increasing bone mineral content, comprising
the step of determining whether a selected molecule inhibits the
binding of TGF-beta binding-protein to bone, or an analogue
thereof. As utilized herein, it should be understood that bone or
analogues thereof refers to hydroxyapatite, or a surface composed
of a powdered form of bone, crushed bone or intact bone. Similar to
the above described methods, a wide variety of methods may be
utilized to assess the inhibition of TGF-beta binding-protein
localization to bone matrix. One such representative method is
provided below in Example 7 (see also Nicolas et al., Calcif.
Tissue Int. 47:206-12 (1995)).
[0168] In one embodiment of the invention, an antibody or
antigen-binding fragment thereof that specifically binds to a
sclerostin polypeptide is capable of competitively inhibiting
binding of a TGF-beta family member to the sclerostin polypeptide.
The capability of the antibody or antibody fragment to impair or
blocking binding of a TGF-beta family member, such as a BMP, to
sclerostin may be determined according to any of the methods
described herein. The antibody or fragment thereof that
specifically binds to sclerostin may impair, block, or prevent
binding of a TGF-beta family member to sclerostin by impairing
sclerostin homodimer formation. An antibody that specifically binds
to sclerostin may also be used to identify an activity of
sclerostin by inhibiting or impairing sclerostin from binding to a
BMP. Alternatively, the antibody or fragment thereof may be
incorporated in a cell-based assay or in an animal model in which
sclerostin has a defined activity to determine whether the antibody
alters (increases or decreases in a statistically significant
manner) that activity. An antibody or fragment thereof that
specifically binds to sclerostin may be used to examine the effect
of such an antibody in a signal transduction pathway and thereby
modulate (stimulate or inhibit) the signaling pathway. Preferably,
binding of an antibody to SOST results in a stimulation or
induction of a signaling pathway.
[0169] While the methods recited herein may refer to the analysis
of an individual test molecule, that the present invention should
not be so limited. In particular, the selected molecule may be
contained within a mixture of compounds. Hence, the recited methods
may further comprise the step of isolating a molecule that inhibits
the binding of TGF-beta binding-protein to a TGF-beta family
member.
[0170] Candidate Molecules
[0171] A wide variety of molecules may be assayed for their ability
to inhibit the binding of TGF-beta binding-protein to a TGF-beta
family member. Representative examples discussed in more detail
below include organic molecules (e.g., organic small molecules),
proteins or peptides, and nucleic acid molecules. Although it
should be evident from the discussion below that the candidate
molecules described herein may be utilized in the assays described
herein, it should also be readily apparent that such molecules can
also be utilized in a variety of diagnostic and therapeutic
settins.
[0172] 1. Organic Molecules
[0173] Numerous organic small molecules may be assayed for their
ability to inhibit the binding of TGF-beta binding-protein to a
TGF-beta family member. For example, within one embodiment of the
invention suitable organic molecules may be selected from either a
chemical library, wherein chemicals are assayed individually, or
from combinatorial chemical libraries where multiple compounds are
assayed at once, then deconvoluted to determine and isolate the
most active compounds.
[0174] Representative examples of such combinatorial chemical
libraries include those described by Agrafiotis et al., "System and
method of automatically generating chemical compounds with desired
properties," U.S. Pat. No. 5,463,564; Armstrong, R. W., "Synthesis
of combinatorial arrays of organic compounds through the use of
multiple component combinatorial array syntheses," WO 95/02566;
Baldwin, J. J. et al., "Sulfonamide derivatives and their use," WO
95/24186; Baldwin, J. J. et al., "Combinatorial dihydrobenzopyran
library," WO 95/30642; Brenner, S., "New kit for preparing
combinatorial libraries," WO 95/16918; Chenera, B. et al.,
"Preparation of library of resin-bound aromatic carbocyclic
compounds," WO 95/16712; Ellman, J. A., "Solid phase and
combinatorial synthesis of benzodiazepine compounds on a solid
support," U.S. Pat. No. 5,288,514; Felder, E. et al., "Novel
combinatorial compound libraries," WO 95/16209; Lerner, R. et al.,
"Encoded combinatorial chemical libraries," WO 93/20242; Pavia, M.
R. et al., "A method for preparing and selecting pharmaceutically
useful non-peptide compounds from a structurally diverse universal
library," WO 95/04277; Summerton, J. E. and D. D. Weller,
"Morpholino-subunit combinatorial library and method," U.S. Pat.
No. 5,506,337; Holmes, C., "Methods for the Solid Phase Synthesis
of Thiazolidinones, Metathiazanones, and Derivatives thereof," WO
96/00148; Phillips, G. B. and G. P. Wei, "Solid-phase Synthesis of
Benzimidazoles," Tet. Letters 37:4887-90, 1996; Ruhland, B. et al.,
"Solid-supported Combinatorial Synthesis of Structurally Diverse
.beta.-Lactams," J. Amer. Chem. Soc. 111:253-4, 1996; Look, G. C.
et al., "The Indentification of Cyclooxygenase-1 Inhibitors from
4-Thiazolidinone Combinatorial Libraries," Bioorg and Med. Chem.
Letters 6:707-12, 1996.
[0175] 2. Proteins and Peptides
[0176] A wide range of proteins and peptides may likewise be
utilized as candidate molecules for inhibitors of the binding of
TGF-beta binding-protein to a TGF-beta family member.
[0177] a. Combinatorial Peptide Libraries
[0178] Peptide molecules which are putative inhibitors of the
binding of TGF-beta binding-protein to a TGF-beta family member may
be obtained through the screening of combinatorial peptide
libraries. Such libraries may either be prepared by one of skill in
the art (see e.g., U.S. Pat. Nos. 4,528,266 and 4,359,535, and
Patent Cooperation Treaty Publication Nos. WO 92/15679, WO
92/15677, WO 90/07862, WO 90/02809, or purchased from commercially
available sources (e.g., New England Biolabs Ph.D..TM. Phage
Display Peptide Library Kit).
[0179] b. Antibodies
[0180] The present invention provides antibodies that specifically
bind to a sclerostin polypeptide methods for using such antibodies.
The present invention also provides sclerostin polypeptide
immunogens that may be used for generation and analysis of these
antibodies. The antibodies may be useful to block or impair binding
of a sclerostin polypeptide, which is a TGF-beta binding protein,
to a ligand, particularly a bone morphogenic protein, and may also
block or impair binding of the sclerostin polypeptide to one or
more other ligands.
[0181] A molecule such as an antibody that inhibits the binding of
the TGF-beta binding protein to one or more members of the TGF-beta
family of proteins, including one or more bone morphogenic proteins
(BMPs), should be understood to refer to, for example, a molecule
that allows the activation of a TGF-beta family member or BMP, or
allows binding of TGF-beta family members including one or more
BMPs to their respective receptors by removing or preventing the
TGF-beta member from binding to the TGF-binding-protein.
[0182] The present invention also provides peptide and polypeptide
immunogens that may be used to generate and/or identify antibodies
or fragments thereof that are capable of inhibiting, preventing, or
impairing binding of the TGF-beta binding protein sclerostin to one
or more BMPs. The present invention also provides peptide and
polypeptide immunogens that may be used to generate and/or identify
antibodies or fragments thereof that are capable of inhibiting,
preventing, or impairing (e.g., decreasing in a statistically
significant manner) the formation of sclerostin homodimers. The
antibodies of the present invention are useful for increasing the
mineral content and mineral density of bone, thereby ameliorating
numerous conditions that result in the loss of bone mineral
content, including for example, disease, genetic predisposition,
accidents that result in the lack of use of bone (e.g., due to
fracture), therapeutics that effect bone resorption or that kill
bone forming cells, and normal aging.
[0183] Polypeptides or peptides useful for immunization and/or
analysis of sclerostin-specific antibodies may also be selected by
analyzing the primary, secondary, and tertiary structure of a
TGF-beta binding protein according to methods known to those
skilled in the art and described herein, in order to determine
amino acid sequences more likely to generate an antigenic response
in a host animal. See, e.g., Novotny, Mol. Immunol. 28:201-207
(1991); Berzofsky, Science 229:932-40 (1985)). Modeling and x-ray
crystallography data may also be used to predict and/or identify
which portions or regions of a TGF-beta binding protein interact
with which portions of a TGF-beta binding protein ligand, such as a
BMP. TGF-beta binding protein peptide immunogens may be designed
and prepared that include amino acid sequences within or
surrounding the portions or regions of interaction. These
antibodies may be useful to block or impair binding of the TGF-beta
binding protein to the same ligand and may also block or impair
binding of the TGF-beta binding protein to one or more other
ligands.
[0184] Antibodies or antigen binding fragments thereof contemplated
by the present invention include antibodies that are capable of
specifically binding to sclerostin and competitively inhibiting
binding of a TGF-beta polypeptide, such as a BMP, to sclerostin.
For example, the antibodies contemplated by the present invention
competitively inhibit binding of the sclerostin polypeptide to the
BMP Type I receptor site on a BMP, or to the BMP Type II receptor
binding site, or may competitively inhibit binding of sclerostin to
both the Type I and Type II receptor binding sites on a BMP.
Without wishing to be bound by theory, when an anti-sclerostin
antibody competitively inhibits binding of the Type I and/or Type
II binding sites of the BMP polypeptide to sclerostin, thus
blocking the antagonistic activity of sclerostin, the receptor
binding sites on BMP are available to bind to the Type I and Type
II receptors, thereby increasing bone mineralization. The binding
interaction between a TGF-beta binding protein such as sclerostin
and a TGF-beta polypeptide such as a BMP generally occurs when each
of the ligand pairs forms a homodimer. Therefore instead of or in
addition to using an antibody specific for sclerostin to block,
impair, or prevent binding of sclerostin to a BMP by competitively
inhibiting binding of sclerostin to BMP, a sclerostin specific
antibody may be used to block or impair sclerostin homodimer
formation.
[0185] By way of example, one dimer of human Noggin, which is a BMP
antagonist that has the ability to bind a BMP with high affinity
(Zimmerman et al., supra), was isolated in complex with one dimer
of human BMP-7 and analyzed by multiwavelength anomalous
diffraction (MAD) (Groppe et al., Nature 420:636-42 (2002)). As
discussed herein, this study revealed that Noggin dimer may
efficiently block all the receptor binding sites (two type I and
two type II receptor binding sites) on a BMP dimer. The location of
the amino acids of Noggin that contact BMP-7 may be useful in
modeling the interaction between other TGF-beta binding proteins,
such as sclerostin (SOST), and BMPs, and thus aiding the design of
peptides that may be used as immunogens to generate antibodies that
block or impair such an interaction.
[0186] In one embodiment of the present invention, an antibody, or
an antigen-binding fragment thereof, that binds specifically to a
SOST polypeptide competitively inhibits binding of the SOST
polypeptide to at least one or both of a bone morphogenic protein
(BMP) Type I Receptor binding site and a BMP Type II Receptor
binding site that are located on a BMP. The epitopes on SOST to
which these antibodies bind may include or be included within
contiguous amino acid sequences that are located at the N-terminus
of the SOST polypeptide (amino acids at about positions 1-56 of SEQ
ID NO: 46). The polypeptides may also include a short linker
peptide sequence that connects the N-terminal region to the core
region, for example, polypeptides as provided in SEQ ID NO: 92
(human) and SEQ ID NO: 93 (rat). Shorter representative N-terminus
peptide sequences of human SOST (e.g., SEQ ID NO: 46) include SEQ
ID NOS: 47-51, and representative rat SOST (e.g., SEQ ID NO: 65)
peptide sequences include SEQ ID NOS: 57-60.
[0187] Antibodies that specifically bind to a SOST polypeptide and
block or competitively inhibit binding of the SOST polypeptide to a
BMP, for example, by blocking or inhibiting binding to amino acids
of a BMP corresponding to one or more of the Type I and Type II
receptor binding sites may also specifically bind to peptides that
comprise an amino acid sequence corresponding to the core region of
SOST (amino acids at about positions 57-146 of SEQ ID NO: 46).
Polypeptides that include the core region may also include
additional amino acids extending at either or both the N-terminus
and C-terminus, for example, to include cysteine residues that may
be useful for conjugating the polypeptide to a carrier molecule.
Representative core polypeptides of human and rat SOST, for
example, comprise the amino acid sequences set forth in SEQ ID NO:
94 and SEQ ID NO: 95, respectively. Such antibodies may also bind
shorter polypeptide sequences. Representative human SOST core
peptide sequences are provided in SEQ ID NOS: 66-69 and
representative rat SOST core sequences are provided in SEQ ID NOS:
70-73.
[0188] In another embodiment, antibodies that specifically bind to
a SOST polypeptide impair (inhibit, prevent, or block, e.g.,
decrease in a statistically significant manner) formation of a SOST
homodimer. Because the interaction between SOST and a BMP may
involve a homodimer of SOST and a homodimer of the BMP, an antibody
that prevents or impairs homodimer formation of SOST may thereby
alter bone mineral density, preferably increasing bone mineral
density. In one embodiment, antibodies that bind to the core region
of SOST prevent homodimer formation. Such antibodies may also bind
to peptides that comprise contiguous amino acid sequences
corresponding the core region, for example, SEQ ID NOS: 74, 75, and
98 (human SOST) and SEQ ID NOS: 76 and 99 (rat SOST). Antibodies
that bind to an epitope located on the C-terminal region of a SOST
polypeptide (at about amino acid positions 147-190 of either SEQ ID
NOS: 46 or 65) may also impair homodimer formation. Representative
C-terminal polypeptides of human and rat SOST, for example,
comprise the amino acid sequences set forth in SEQ ID NO: 96 and
SEQ ID NO: 97, respectively. Such antibodies may also bind shorter
polypeptide sequences. Representative human SOST C-terminal peptide
sequences are provided in SEQ ID NOS: 78-81 and representative rat
SOST C-terminal sequences are provided in SEQ ID NOS: 86-88.
[0189] The SOST polypeptides and peptides disclosed herein to which
antibodies may specifically bind are useful as immunogens. These
immunogens of the present invention may be used for immunizing an
animal to generate a humoral immune response that results in
production of antibodies that specifically bind to a Type I or Type
II receptor binding site or both located on a BMP include peptides
derived from the N-terminal region of SOST or that may prevent SOST
homodimer formation.
[0190] Such SOST polypeptides and peptides that are useful as
immunogens may also be used in methods for screening samples
containing antibodies, for example, samples of purified antibodies,
antisera, or cell culture supernatants or any other biological
sample that may contain one or more antibodies specific for SOST.
These peptides may also be used in methods for identifying and
selecting from a biological sample one or more B cells that are
producing an antibody that specifically binds to SOST (e.g., plaque
forming assays and the like). The B cells may then be used as
source of a SOST specific antibody-encoding polynucleotide that can
be cloned and/or modified by recombinant molecular biology
techniques known in the art and described herein.
[0191] A "biological sample" as used herein refers in certain
embodiments to a sample containing at least one antibody specific
for a SOST polypeptide, and a biological sample may be provided by
obtaining a blood sample, biopsy specimen, tissue explant, organ
culture, or any other tissue or cell preparation from a subject or
a biological source. A sample may further refer to a tissue or cell
preparation in which the morphological integrity or physical state
has been disrupted, for example, by dissection, dissociation,
solubilization, fractionation, homogenization, biochemical or
chemical extraction, pulverization, lyophilization, sonication, or
any other means for processing a sample derived from a subject or
biological source. The subject or biological source may be a human
or non-human animal, a primary cell culture (e.g., B cells
immunized in vitro), or culture adapted cell line including but not
limited to genetically engineered cell lines that may contain
chromosomally integrated or episomal recombinant nucleic acid
sequences, immortalized or immortalizable cell lines, somatic cell
hybrid cell lines, differentiated or differentiatable cell lines,
transformed cell lines, and the like.
[0192] SOST peptide immunogens may also be prepared by synthesizing
a series of peptides that, in total, represent the entire
polypeptide sequence of a SOST polypeptide and that each have a
portion of the SOST amino acid sequence in common with another
peptide in the series. This overlapping portion would preferably be
at least four amino acids, and more preferably 5, 6, 7, 8, 9, or 10
amino acids. Each peptide may be used to immunize an animal, the
sera collected from the animal, and tested in an assay to identify
which animal is producing antibodies that impair or block binding
of SOST to a TGF-beta protein. Antibodies are then prepared from
such identified immunized animals according to methods known in the
art and described herein.
[0193] Antibodies which inhibit the binding of TGF-beta
binding-protein to a TGF-beta family member may readily be prepared
given the disclosure provided herein. Particularly useful are
anti-TGF-beta binding-protein antibodies that "specifically bind"
TGF-beta binding-protein of SEQ ID NOS: 2, 6, 8, 10, 12, 14, 16,
46, or 65, but not to other TGF-beta binding-proteins such as Dan,
Cerberus, SCGF, or Gremlin. Within the context of the present
invention, antibodies are understood to include monoclonal
antibodies, polyclonal antibodies, single chain, chimeric,
CDR-grafted immunoglobulings, anti-idiotypic antibodies, and
antibody fragments thereof (e.g., Fab, Fd, Fab', and F(ab').sub.2,
F.sub.V variable regions, or complementarity determining regions).
As discussed above, antibodies are understood to be specific
against TGF-beta binding-protein, or against a specific TGF-beta
family member, if they bind with a K.sub.a of greater than or equal
to 10.sup.7 M.sup.-1, preferably greater than or equal to 10.sup.8
M.sup.-1, and do not bind to other TGF-beta binding-proteins, or
bind with a K.sub.a of less than or equal to 10.sup.6 M.sup.-1.
Affinity of an antibody for its cognate antigen is also commonly
expressed as a dissociation constant K.sub.D, and an anti-SOST
antibody specifically binds to a TGF-beta family member if it binds
with a K.sub.D of less than or equal to about 10.sup.-5 M, more
preferably less than or equal to about 10.sup.-6 M, still more
preferably less than or equal to 10.sup.-7 M, and still more
preferably less than or equal to 10.sup.-8 M. Furthermore,
antibodies of the present invention preferably block, impair, or
inhibit (e.g., decrease with statistical significance) the binding
of TGF-beta binding-protein to a TGF-beta family member. The
affinity of a monoclonal antibody or binding partner, as well as
inhibition of binding can be readily determined by one of ordinary
skill in the art (see Scatchard, Ann. N.Y. Acad. Sci. 51:660-672,
1949). Affinity may also be determined by surface plasmon resonance
(SPR; BIAcore, Biosensor, Piscataway, N.J.). For surface plasmon
resonance, target molecules are immobilized on a solid phase and
exposed to ligands in a mobile phase running along a flow cell. If
ligand binding to the immobilized target occurs, the local
refractive index changes, leading to a change in SPR angle, which
can be monitored in real time by detecting changes in the intensity
of the reflected light. The rates of change of the SPR signal can
be analyzed to yield apparent rate constants for the association
and dissociation phases of the binding reaction. The ratio of these
values gives the apparent equilibrium constant (affinity) (see,
e.g., Wolff et al., Cancer Res. 53:2560-65 (1993)).
[0194] An antibody according to the present invention may belong to
any immunoglobulin class, for example IgG, IgE, IgM, IgD, or IgA,
and may be any one of the different isotypes that may comprise a
class (such as IgG1, IgG2, IgG3, and IgG4 of the human IgG class).
It may be obtained from or derived from an animal, for example,
fowl (e.g., chicken) and mammals, which includes but is not limited
to a mouse, rat, hamster, rabbit, or other rodent, a cow, horse,
sheep, goat, camel, human, or other primate. The antibody may be an
internalising antibody.
[0195] Methods well known in the art may be used to generate
antibodies, polyclonal antisera, or monoclonal antibodies that are
specific for a TGF-beta binding protein such as SOST. Antibodies
also may be produced as genetically engineered immunoglobulins (Ig)
or Ig fragments designed to have desirable properties. For example,
by way of illustration and not limitation, antibodies may include a
recombinant IgG that is a chimeric fusion protein having at least
one variable (V) region domain from a first mammalian species and
at least one constant region domain from a second, distinct
mammalian species. Most commonly, a chimeric antibody has murine
variable region sequences and human constant region sequences. Such
a murine/human chimeric immunoglobulin may be "humanized" by
grafting the complementarity determining regions (CDRs) derived
from a murine antibody, which confer binding specificity for an
antigen, into human-derived V region framework regions and
human-derived constant regions. Fragments of these molecules may be
generated by proteolytic digestion, or optionally, by proteolytic
digestion followed by mild reduction of disulfide bonds and
alkylation. Alternatively, such fragments may also be generated by
recombinant genetic engineering techniques.
[0196] Certain preferred antibodies are those antibodies that
inhibit or block a TGF-beta binding protein activity within an in
vitro assay, as described herein. Binding properties of an antibody
to a TGF-beta binding protein may generally be assessed using
immunodetection methods including, for example, an enzyme-linked
immunosorbent assay (ELISA), immunoprecipitation, immunoblotting,
countercurrent immunoelectrophoresis, radioimmunoassays, dot blot
assays, inhibition or competition assays, and the like, which may
be readily performed by those having ordinary skill in the art
(see, e.g., U.S. Pat. Nos. 4,376,110 and 4,486,530; Harlow et al.,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
(1988)).
[0197] An immunogen may be comprised of cells expressing a TGF-beta
binding protein, purified or partially purified TGF-beta binding
polypeptides, or variants or fragments (i.e., peptides) thereof, or
peptides derived from a TGF-beta binding protein. Such peptides may
be generated by proteolytic cleavage of a larger polypeptide, by
recombinant molecular methodologies, or may be chemically
synthesized. For instance, nucleic acid sequences encoding TGF-beta
binding proteins are provided herein, such that those skilled in
the art may routinely prepare TGF-beta binding proteins for use as
immunogens. Peptides may be chemically synthesized by methods as
described herein and known in the art. Alternatively, peptides may
be generated by proteolytic cleavage of a TGF-beta binding protein,
and individual peptides isolated by methods known in the art such
as polyacrylamide gel electrophoresis or any number of liquid
chromatography or other separation methods. Peptides useful as
immunogens typically may have an amino acid sequence of at least 4
or 5 consecutive amino acids from a TGF-beta binding protein amino
acid sequence such as those described herein, and preferably have
at least 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 19 or 20
consecutive amino acids of a TGF-beta binding protein. Certain
other preferred peptide immunogens comprise at least 6 but no more
than 12 or more consecutive amino acids of a TGF-beta binding
protein sequence, and other preferred peptide immunogens comprise
at least 21 but no more than 50 consecutive amino acids of a SOST
polypeptide. Other preferred peptide immunogens comprise 21-25,
26-30, 31-35, 36-40, 41-50, or any whole integer number of amino
acids between and including 21 and 100 consecutive amino acids, and
between 100 and 190 consecutive amino acids of a TGF-beta binding
protein sequence.
[0198] As disclosed herein, polyclonal antibodies may be readily
generated by one of ordinary skill in the art from a variety of
warm-blooded animals such as horses, cows, various fowl, rabbits,
mice, sheep, goats, baboons, or rats. Typically, the TGF-beta
binding-protein or unique peptide thereof of 13-20 amino acids or
as described herein (preferably conjugated to keyhole limpet
hemocyanin by cross-linking with glutaraldehyde) is used to
immunize the animal through intraperitoneal, intramuscular,
intraocular, intradermal, or subcutaneous injections, along with an
adjuvant such as Freund's complete or incomplete adjuvant, or the
Ribi Adjuvant System (Corixa Corporation, Seattle, Was.). See also,
e.g., Harlow et al., supra. In general, after the first injection,
animals receive one or more booster immunizations according to a
preferred schedule that may vary according to, inter alia, the
antigen, the adjuvant (if any), and/or the particular animal
species. The immune response may be monitored by periodically
bleeding the animal and preparing and analyzing sera in an
immunoassay, such as an ELISA or Ouchterlony diffusion assay, or
the like, to determine the specific antibody titer. Particularly
preferred polyclonal antisera will give a detectable signal on one
of these assays, such as an ELISA, that is preferably at least
three times greater than background. Once the titer of the animal
has reached a plateau in terms of its reactivity to the protein,
larger quantities of antisera may be readily obtained either by
weekly bleedings, or by exsanguinating the animal.
[0199] Polyclonal antibodies that bind specifically to the TGF-beta
binding protein or peptide may then be purified from such antisera,
for example, by affinity chromatography using protein A.
Alternatively, affinity chromatography may be performed wherein the
TGF-beta binding protein or peptide or an antibody specific for an
Ig constant region of the particular immunized animal species is
immobilized on a suitable solid support.
[0200] Antibodies for use in the invention include monoclonal
antibodies that are prepared by conventional immunization and cell
fusion procedures as described herein an known in the art.
Monoclonal antibodies may be readily generated using conventional
techniques (see, e.g., Kohler et al., Nature 256:495, 1975; Coligan
et al. (eds.), Current Protocols in Immunology, 1:2.5.1-2.6.7 (John
Wiley & Sons 1991) ["Coligan"]; U.S. Pat. Nos. RE 32,011,
4,902,614, 4,543,439, and 4,411,993 which are incorporated herein
by reference; see also Monoclonal Antibodies, Hybridomas: A New
Dimension in Biological Analyses, Plenum Press, Kennett, McKearn,
and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual,
Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988,
which are also incorporated herein by reference; Picksley et al.,
"Production of monoclonal antibodies against proteins expressed in
E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover
et al. (eds.), page 93 (Oxford University Press 1995)). Antibody
fragments may be derived therefrom using any suitable standard
technique such as proteolytic digestion, or optionally, by
proteolytic digestion (for example, using papain or pepsin)
followed by mild reduction of disulfide bonds and alkylation.
Alternatively, such fragments may also be generated by recombinant
genetic engineering techniques.
[0201] Briefly, within one embodiment a subject animal such as a
rat or mouse or hamster is immunized with TGF-beta binding-protein
or a portion of a region thereof, including peptides within a
region, as described herein. The protein may be admixed with an
adjuvant such as Freund's complete or incomplete adjuvant or Ribi
adjuvant in order to increase the resultant immune response.
Between one and three weeks after the initial immunization the
animal may be reimmunized with another booster immunization, and
tested for reactivity to the protein using assays described herein.
Once the animal has reached a plateau in its reactivity to the
injected protein, it is sacrificed, and organs which contain large
numbers of B cells such as the spleen and lymph nodes are
harvested. The harvested spleen and/or lymph node cell suspensions
are fused with a suitable myeloma cell that is drug-sensitized in
order to create a "hybridoma" which secretes monoclonal antibody.
Suitable myeloma lines include, for example, NS-0, SP20, NS-1 (ATCC
No. TIB 18), and P3X63-Ag 8.653 (ATCC No. CRL 1580).
[0202] The lymphoid (e.g., spleen) cells and the myeloma cells may
be combined for a few minutes with a membrane fusion-promoting
agent, such as polyethylene glycol or a nonionic detergent, and
then plated at low density on a selective medium that supports the
growth of hybridoma cells but not unfused myeloma cells. Following
the fusion, the cells may be placed into culture plates containing
a suitable medium, such as RPMI 1640, or DMEM (Dulbecco's Modified
Eagles Medium) (JRH Biosciences, Lenexa, Kans.), as well as
additional ingredients, such as fetal bovine serum (FBS, i.e., from
Hyclone, Logan, Utah, or JRH Biosciences). Additionally, the medium
should contain a reagent which selectively allows for the growth of
fused spleen and myeloma cells such as HAT (hypoxanthine,
aminopterin, and thymidine) (Sigma Chemical Co., St. Louis, Mo.).
After about seven days, the resulting fused cells or hybridomas may
be screened in order to determine the presence of antibodies which
are reactive with TGF-beta binding-protein (depending on the
antigen used), and which block, impair, or inhibit the binding of
TGF-beta binding-protein to a TGF-beta family member. Hybridomas
that produce monoclonal antibodies that specifically bind to
sclerostin or a variant thereof are preferred.
[0203] A wide variety of assays may be utilized to determine the
presence of antibodies which are reactive against the proteins of
the present invention, including for example countercurrent
immuno-electrophoresis, radioimmunoassays,
radioimmunoprecipitations, enzyme-linked immuno-sorbent assays
(ELISA), dot blot assays, western blots, immunoprecipitation,
inhibition or competition assays, and sandwich assays (see U.S.
Pat. Nos. 4,376,110 and 4,486,530; see also Antibodies: A
Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor
Laboratory Press, 1988). The hybridomas are cloned, for example, by
limited dilution cloning or by soft agar plaque isolation, and
reassayed. Thus, a hybridoma producing antibodies reactive against
the desired protein may be isolated.
[0204] The monoclonal antibodies from the hybridoma cultures may be
isolated from the supernatants of hybridoma cultures. An
alternative method for production of a murine monoclonal antibody
is to inject the hybridoma cells into the peritoneal cavity of a
syngeneic mouse, for example, a mouse that has been treated (e.g.,
pristane-primed) to promote formation of ascites fluid containing
the monoclonal antibody. Monoclonal antibodies can be isolated and
purified by a variety of well-established techniques. Such
isolation techniques include affinity chromatography with Protein-A
Sepharose, size-exclusion chromatography, and ion-exchange
chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and
pages 2.9.1-2.9.3; Baines et al., "Purification of Immunoglobulin G
(IgG)," in Methods in Molecular Biology, Vol. 10, pages 79-104 (The
Humana Press, Inc. 1992)). Monoclonal antibodies may be purified by
affinity chromatography using an appropriate ligand selected based
on particular properties of the antibody (e.g., heavy or light
chain isotype, binding specificity, etc.). Examples of a suitable
ligand, immobilized on a solid support, include Protein A, Protein
G, an anti-constant region (light chain or heavy chain) antibody,
an anti-idiotype antibody, and a TGF-beta binding protein, or
fragment or variant thereof.
[0205] In addition, an anti-TGF-beta binding-protein antibody of
the present invention may be a human monoclonal antibody. Human
monoclonal antibodies may be generated by any number of techniques
with which those having ordinary skill in the art will be familiar.
Such methods include, but are not limited to, Epstein Barr Virus
(EBV) transformation of human peripheral blood cells (e.g.,
containing B lymphocytes), in vitro immunization of human B cells,
fusion of spleen cells from immunized transgenic mice carrying
inserted human immunoglobulin genes, isolation from human
immunoglobulin V region phage libraries, or other procedures as
known in the art and based on the disclosure herein. For example,
human monoclonal antibodies may be obtained from transgenic mice
that have been engineered to produce specific human antibodies in
response to antigenic challenge. Methods for obtaining human
antibodies from transgenic mice are described, for example, by
Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature
368:856, 1994; Taylor et al., Int. Immun. 6:579, 1994; U.S. Pat.
No. 5,877,397; Bruggemann et al., 1997 Curr. Opin. Biotechnol.
8:455-58; Jakobovits et al., 1995 Ann. N. Y. Acad. Sci. 764:525-35.
In this technique, elements of the human heavy and light chain
locus are introduced into strains of mice derived from embryonic
stem cell lines that contain targeted disruptions of the endogenous
heavy chain and light chain loci. (See also Bruggemann et al.,
Curr. Opin. Biotechnol. 8:455-58 (1997)). For example, human
immunoglobulin transgenes may be mini-gene constructs, or transloci
on yeast artificial chromosomes, which undergo B cell-specific DNA
rearrangement and hypermutation in the mouse lymphoid tissue. Human
monoclonal antibodies may be obtained by immunizing the transgenic
mice, which may then produce human antibodies specific for the
antigen. Lymphoid cells of the immunized transgenic mice can be
used to produce human antibody-secreting hybridomas according to
the methods described herein. Polyclonal sera containing human
antibodies may also be obtained from the blood of the immunized
animals.
[0206] Another method for generating human TGF-beta binding protein
specific monoclonal antibodies includes immortalizing human
peripheral blood cells by EBV transformation. See, e.g., U.S. Pat.
No. 4,464,456. Such an immortalized B cell line (or lymphoblastoid
cell line) producing a monoclonal antibody that specifically binds
to a TGF-beta binding protein (or a variant or fragment thereof)
can be identified by immunodetection methods as provided herein,
for example, an ELISA, and then isolated by standard cloning
techniques. The stability of the lymphoblastoid cell line producing
an anti-TGF-beta binding protein antibody may be improved by fusing
the transformed cell line with a murine myeloma to produce a
mouse-human hybrid cell line according to methods known in the art
(see, e.g., Glasky et al., Hybridoma 8:377-89 (1989)). Still
another method to generate human monoclonal antibodies is in vitro
immunization, which includes priming human splenic B cells with
antigen, followed by fusion of primed B cells with a heterohybrid
fusion partner. See, e.g., Boerner et al., 1991 J Immunol.
147:86-95.
[0207] In certain embodiments, a B cell that is producing an
anti-SOST antibody is selected and the light chain and heavy chain
variable regions are cloned from the B cell according to molecular
biology techniques known in the art (WO 92/02551; U.S. Pat. No.
5,627,052; Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48
(1996)) and described herein. Preferably B cells from an immunized
animal are isolated from the spleen, lymph node, or peripheral
blood sample by selecting a cell that is producing an antibody that
specifically binds to SOST. B cells may also be isolated from
humans, for example, from a peripheral blood sample. Methods for
detecting single B cells that are producing an antibody with the
desired specificity are well known in the art, for example, by
plaque formation, fluorescence-activated cell sorting, in vitro
stimulation followed by detection of specific antibody, and the
like. Methods for selection of specific antibody producing B cells
include, for example, preparing a single cell suspension of B cells
in soft agar that contains SOST or a peptide fragment thereof.
Binding of the specific antibody produced by the B cell to the
antigen results in the formation of a complex, which may be visible
as an immunoprecipitate. After the B cells producing the specific
antibody are selected, the specific antibody genes may be cloned by
isolating and amplifying DNA or mRNA according to methods known in
the art and described herein.
[0208] For particular uses, fragments of anti-TGF-beta binding
protein antibodies may be desired. Antibody fragments,
F(ab').sub.2, Fab, Fab', Fv, Fe, Fd, retain the antigen binding
site of the whole antibody and therefore bind to the same epitope.
These antigen-binding fragments derived from an antibody can be
obtained, for example, by proteolytic hydrolysis of the antibody,
for example, pepsin or papain digestion of whole antibodies
according to conventional methods. As an illustration, antibody
fragments can be produced by enzymatic cleavage of antibodies with
pepsin to provide a 5S fragment denoted F(ab').sub.2. This fragment
can be further cleaved using a thiol reducing agent to produce 3.5S
Fab' monovalent fragments. Optionally, the cleavage reaction can be
performed using a blocking group for the sulfhydryl groups that
result from cleavage of disulfide linkages. As an alternative, an
enzymatic cleavage using papain produces two monovalent Fab
fragments and an Fc fragment directly. These methods are described,
for example, by Goldenberg, U.S. Pat. No. 4,331,647, Nisonoff et
al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J.
73:119, 1959; Edelman et al., in Methods in Enzymology 1:422
(Academic Press 1967); and by Coligan at pages 2.8.1-2.8.10 and
2.10.-2.10.4. Other methods for cleaving antibodies, such as
separating heavy chains to form monovalent light-heavy chain
fragments (Fd), further cleaving of fragments, or other enzymatic,
chemical, or genetic techniques may also be used, so long as the
fragments bind to the antigen that is recognized by the intact
antibody.
[0209] An antibody fragment may also be any synthetic or
genetically engineered protein that acts like an antibody in that
it binds to a specific antigen to form a complex. For example,
antibody fragments include isolated fragments consisting of the
light chain variable region, "Fv" fragments consisting of the
variable regions of the heavy and light chains, recombinant single
chain polypeptide molecules in which light and heavy variable
regions are connected by a peptide linker (scFv proteins), and
minimal recognition units consisting of the amino acid residues
that mimic the hypervariable region. The antibody of the present
invention preferably comprises at least one variable region domain.
The variable region domain may be of any size or amino acid
composition and will generally comprise at least one hypervariable
amino acid sequence responsible for antigen binding and which is
adjacent to or in frame with one or more framework sequences. In
general terms, the variable (V) region domain may be any suitable
arrangement of immunoglobulin heavy (V.sub.H) and/or light
(V.sub.L) chain variable domains. Thus, for example, the V region
domain may be monomeric and be a V.sub.H or V.sub.L domain, which
is capable of independently binding antigen with acceptable
affinity. Alternatively, the V region domain may be dimeric and
contain V.sub.H-V.sub.H, V.sub.H-V.sub.L, or V.sub.L-V.sub.L,
dimers. Preferably, the V region dimer comprises at least one
V.sub.H and at least one V.sub.L chain that are non-covalently
associated (hereinafter referred to as F.sub.v). If desired, the
chains may be covalently coupled either directly, for example via a
disulphide bond between the two variable domains, or through a
linker, for example a peptide linker, to form a single chain Fv
(scF.sub.v).
[0210] The variable region domain may be any naturally occurring
variable domain or an engineered version thereof. By engineered
version is meant a variable region domain that has been created
using recombinant DNA engineering techniques. Such engineered
versions include those created, for example, from a specific
antibody variable region by insertions, deletions, or changes in or
to the amino acid sequences of the specific antibody. Particular
examples include engineered variable region domains containing at
least one CDR and optionally one or more framework amino acids from
a first antibody and the remainder of the variable region domain
from a second antibody.
[0211] The variable region domain may be covalently attached at a
C-terminal amino acid to at least one other antibody domain or a
fragment thereof. Thus, for example, a V.sub.H, domain that is
present in the variable region domain may be linked to an
immunoglobulin C.sub.H1 domain, or a fragment thereof. Similarly a
V.sub.L domain may be linked to a C.sub.K domain or a fragment
thereof. In this way, for example, the antibody may be a Fab
fragment wherein the antigen binding domain contains associated
V.sub.H and V.sub.L domains covalently linked at their C-termini to
a CH1 and C.sub.K domain, respectively. The CH1 domain may be
extended with further amino acids, for example to provide a hinge
region or a portion of a hinge region domain as found in a Fab'
fragment, or to provide further domains, such as antibody CH2 and
CH3 domains.
[0212] Another form of an antibody fragment is a peptide comprising
for a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing
polynucleotides that encode the CDR of an antibody of interest.
Such polynucleotides are prepared, for example, by using the
polymerase chain reaction to synthesize the variable region using
mRNA of antibody-producing cells as a template (see, for example,
Larrick et al., Methods: A Companion to Methods in Enzymology
2:106, 1991; Courtenay-Luck, "Genetic Manipulation of Monoclonal
Antibodies," in Monoclonal Antibodies: Production, Engineering and
Clinical Application, Ritter et al. (eds.), page 166 (Cambridge
University Press 1995); and Ward et al., "Genetic Manipulation and
Expression of Antibodies," in Monoclonal Antibodies: Principles and
Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc.
1995)).
[0213] Alternatively, the antibody may be a recombinant or
engineered antibody obtained by the use of recombinant DNA
techniques involving the manipulation and re-expression of DNA
encoding antibody variable and/or constant regions. Such DNA is
known and/or is readily available from DNA libraries including for
example phage-antibody libraries (see Chiswell and McCafferty,
Tibtech. 10:80-84 (1992)) or if desired can be synthesized.
Standard molecular biology and/or chemistry procedures may be used
to sequence and manipulate the DNA, for example, to introduce
codons to create cysteine residues, or to modify, add or delete
other amino acids or domains as desired.
[0214] Chimeric antibodies, specific for a TGF-beta binding
protein, and which include humanized antibodies, may also be
generated according to the present invention. A chimeric antibody
has at least one constant region domain derived from a first
mammalian species and at least one variable region domain derived
from a second, distinct mammalian species (see, e.g., Morrison et
al., Proc. Natl. Acad. Sci. USA, 81:6851-55 (1984)). In preferred
embodiments, a chimeric antibody may be constructed by cloning the
polynucleotide sequence that encodes at least one variable region
domain derived from a non-human monoclonal antibody, such as the
variable region derived from a murine, rat, or hamster monoclonal
antibody, into a vector containing a nucleotide sequence that
encodes at least one human constant region (see, e.g., Shin et al.,
Methods Enzymol. 178:459-76 (1989); Walls et al., Nucleic Acids
Res. 21:2921-29 (1993)). By way of example, the polynucleotide
sequence encoding the light chain variable region of a murine
monoclonal -antibody may be inserted into a vector containing a
nucleotide sequence encoding the human kappa light chain constant
region sequence. In a separate vector, the polynucleotide sequence
encoding the heavy chain variable region of the monoclonal antibody
may be cloned in frame with sequences encoding a human IgG constant
region, for example, the human IgG1 constant region. The particular
human constant region selected may depend upon the effector
functions desired for the particular antibody (e.g. complement
fixing, binding to a particular Fc receptor, etc.). Preferably, the
constructed vectors will be transfected into eukaryotic cells for
stable expression of the chimeric antibody. Another method known in
the art for generating chimeric antibodies is homologous
recombination (e.g., U.S. Pat. No. 5,482,856).
[0215] A non-human/human chimeric antibody may be further
genetically engineered to create a "humanized" antibody. Such a
humanized antibody may comprise a plurality of CDRs derived from an
immunoglobulin of a non-human mammalian species, at least one human
variable framework region, and at least one human immunoglobulin
constant region. Useful strategies for designing humanized
antibodies may include, for example by way of illustration and not
limitation, identification of human variable framework regions that
are most homologous to the non-human framework regions of the
chimeric antibody. Without wishing to be bound by theory, such a
strategy may increase the likelihood that the humanized antibody
will retain specific binding affinity for a TGF-beta binding
protein, which in some preferred embodiments may be substantially
the same affinity for a TGF-beta binding protein or variant or
fragment thereof, and in certain other preferred embodiments may be
a greater affinity for TGF-beta binding protein. See, e.g., Jones
et al., 1986 Nature 321:522-25; Riechmann et al., 1988 Nature
332:323-27. Designing such a humanized antibody may therefore
include determining CDR loop conformations and structural
determinants of the non-human variable regions, for example, by
computer modeling, and then comparing the CDR loops and
determinants to known human CDR loop structures and determinants.
See, e.g., Padlan et al., 1995 FASEB 9:133-39; Chothia et al., 1989
Nature, 342:377-383. Computer modeling may also be used to compare
human structural templates selected by sequence homology with the
non-human variable regions. See, e.g., Bajorath et al., 1995 Ther.
Immunol. 2:95-103; EP-0578515-A3. If humanization of the non-human
CDRs results in a decrease in binding affinity, computer modeling
may aid in identifying specific amino acid residues that could be
changed by site-directed or other mutagenesis techniques to
partially, completely or supra-optimally (i.e., increase to a level
greater than that of the non-humanized antibody) restore affinity.
Those having ordinary skill in the art are familiar with these
techniques, and will readily appreciate numerous variations and
modifications to such design strategies.
[0216] One such method for preparing a humanized antibody is called
veneering. As used herein, the terms "veneered FRs" and
"recombinantly veneered FRs" refer to the selective replacement of
FR residues from, e.g., a rodent heavy or light chain V region,
with human FR residues in order to provide a xenogeneic molecule
comprising an antigen-binding site that retains substantially all
of the native FR polypeptide folding structure. Veneering
techniques are based on the understanding that the ligand binding
characteristics of an antigen-binding site are determined primarily
by the structure and relative disposition of the heavy and light
chain CDR sets within the antigen-binding surface. Davies et al.,
Ann. Rev. Biochem. 59:439-73, 1990. Thus, antigen binding
specificity can be preserved in a humanized antibody only wherein
the CDR structures, their interaction with each other, and their
interaction with the rest of the V region domains are carefully
maintained. By using veneering techniques, exterior (e.g.,
solvent-accessible) FR residues that are readily encountered by the
immune system are selectively replaced with human residues to
provide a hybrid molecule that comprises either a weakly
immunogenic, or substantially non-immunogenic veneered surface.
[0217] The process of veneering makes use of the available sequence
data for human antibody variable domains compiled by Kabat et al.,
in Sequences of Proteins of Immunological Interest, 4th ed., (U.S.
Dept. of Health and Human Services, U.S. Government Printing
Office, 1987), updates to the Kabat database, and other accessible
U.S. and foreign databases (both nucleic acid and protein). Solvent
accessibilities of V region amino acids can be deduced from the
known three-dimensional structure for human and murine antibody
fragments. Initially, the FRs of the variable domains of an
antibody molecule of interest are compared with corresponding FR
sequences of human variable domains obtained from the
above-identified sources. The most homologous human V regions are
then compared residue by residue to corresponding murine amino
acids. The residues in the murine FR that differ from the human
counterpart are replaced by the residues present in the human
moiety using recombinant techniques well known in the art. Residue
switching is only carried out with moieties which are at least
partially exposed (solvent accessible), and care is exercised in
the replacement of amino acid residues that may have a significant
effect on the tertiary structure of V region domains, such as
proline, glycine, and charged amino acids.
[0218] In this manner, the resultant "veneered" antigen-binding
sites are thus designed to retain the rodent CDR residues, the
residues substantially adjacent to the CDRs, the residues
identified as buried or mostly buried (solvent inaccessible), the
residues believed to participate in non-covalent (e.g.,
electrostatic and hydrophobic) contacts between heavy and light
chain domains, and the residues from conserved structural regions
of the FRs which are believed to influence the "canonical" tertiary
structures of the CDR loops. These design criteria are then used to
prepare recombinant nucleotide sequences that combine the CDRs of
both the heavy and light chain of a antigen-binding site into
human-appearing FRs that can be used to transfect mammalian cells
for the expression of recombinant human antibodies that exhibit the
antigen specificity of the rodent antibody molecule.
[0219] An additional method for selecting antibodies that
specifically bind to a TGF-beta binding protein or variant or
fragment thereof is by phage display. See, e.g., Winter et al.,
1994 Annu. Rev. Immunol. 12:433-55; Burton et al., 1994 Adv.
Immunol. 57:191-280. Human or murine immunoglobulin variable region
gene combinatorial libraries may be created in phage vectors that
can be screened to select Ig fragments (Fab, Fv, sFv, or multimers
thereof) that bind specifically to TGF-beta binding protein or
variant or fragment thereof. See, e.g., U.S. Pat. No. 5,223,409;
William D. Huse et al., "Generation of a Large Combinational
Library of the Immunoglobulin Repertoire in Phage Lambda," Science
246:1275-1281, December 1989; see also L. Sastry et al., "Cloning
of the Immunological Repertoire in Escherichia coli for Generation
of Monoclonal Catalytic Antibodies: Construction of a Heavy Chain
Variable Region-Specific cDNA Library," Proc. Natl. Acad. Sci. USA
86:5728-5732, August 1989; see also Michelle Alting-Mees et al.,
"Monoclonal Antibody Expression Libraries: A Rapid Alternative to
Hybridomas," Strategies in Molecular Biology 3:1-9, January 1990;
Kang et al., 1991 Proc. Natl. Acad. Sci. USA 88:4363-66; Hoogenboom
et al., 1992 J. Molec. Biol. 227:381-388; Schlebusch et al., 1997
Hybridoma 16:47-52 and references cited therein). A commercial
system is available from Stratagene (La Jolla, Calif.) which
enables the production of antibodies through recombinant
techniques. Briefly, mRNA is isolated from a B cell population, and
utilized to create heavy and light chain immunoglobulin cDNA
expression libraries in the .lambda. ImmunoZap(H) and
.lambda.ImmunoZap(L) vectors. Positive plaques may subsequently be
converted to a non-lytic plasmid which allows high level expression
of monoclonal antibody fragments from E. coli. Alternatively, a
library containing a plurality of polynucleotide sequences encoding
Ig variable region fragments may be inserted into the genome of a
filamentous bacteriophage, such as M13 or a variant thereof, in
frame with the sequence encoding a phage coat protein. A fusion
protein may be a fusion of the coat protein with the light chain
variable region domain and/or with the heavy chain variable region
domain. According to certain embodiments, immunoglobulin Fab
fragments may also be displayed on a phage particle (see, e.g.,
U.S. Pat. No. 5,698,426). These vectors may be screened
individually or co-expressed to form Fab fragments or antibodies
(see Huse et al., supra; see also Sastry et al., supra).
[0220] Similarly, portions or fragments, such as Fab and Fv
fragments, of antibodies may also be constructed utilizing
conventional enzymatic digestion or recombinant DNA techniques to
incorporate the variable regions of a gene which encodes a
specifically binding antibody. Within one embodiment, the genes
which encode the variable region from a hybridoma producing a
monoclonal antibody of interest are amplified using nucleotide
primers for the variable region. These primers may be synthesized
by one of ordinary skill in the art, or may be purchased from
commercially available sources. Stratagene (La Jolla, Calif.) sells
primers for mouse and human variable regions including, among
others, primers for V.sub.Ha, V.sub.Hb, V.sub.Hc, V.sub.Hd,
C.sub.H1, V.sub.L and C.sub.L regions. These primers may be
utilized to amplify heavy or light chain variable regions, which
may then be inserted into vectors such as ImmunoZAP.TM. H or
ImmunoZAP.TM. L (Stratagene), respectively. These vectors may then
be introduced into E. coli, yeast, or mammalian-based systems for
expression. Utilizing these techniques, large amounts of a
single-chain protein containing a fusion of the V.sub.H and V.sub.L
domains may be produced (see Bird et al., Science 242:423-426,
1988). In addition, such techniques may be utilized to change a
"murine" antibody to a "human" antibody, without altering the
binding specificity of the antibody.
[0221] In certain particular embodiments of the invention,
combinatorial phage libraries may also be used for humanization of
non-human variable regions. See, e.g., Rosok et al., 1996 J. Biol.
Chem. 271:22611-18; Rader et al., 1998 Proc. Natl. Acad. Sci. USA
95:8910-15. A phage library may be screened to select an Ig
variable region fragment of interest by immunodetection methods
known in the art and described herein, and the DNA sequence of the
inserted immunoglobulin gene in the phage so selected may be
determined by standard techniques. See, Sambrook et al., 2001
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press.
The selected Ig-encoding sequence may then be cloned into another
suitable vector for expression of the Ig fragment or, optionally,
may be cloned into a vector containing Ig constant regions, for
expression of whole immunoglobulin chains.
[0222] In certain other embodiments, the invention contemplates
SOST-specific antibodies that are multimeric antibody fragments.
Useful methodologies are described generally, for example in Hayden
et al. 1997, Curr Opin. Immunol. 9:201-12; Coloma et al., 1997 Nat.
Biotechnol. 15:159-63). For example, multimeric antibody fragments
may be created by phage techniques to form miniantibodies (U.S.
Pat. No. 5,910,573) or diabodies (Holliger et al., 1997, Cancer
Immunol. Immunother. 45:128-130).
[0223] In certain embodiments of the invention, an antibody
specific for SOST may be an antibody that is expressed as an
intracellular protein. Such intracellular antibodies are also
referred to as intrabodies and may comprise an Fab fragment, or
preferably comprise a scFv fragment (see, e.g., Lecerf et al.,
Proc. Natl. Acad. Sci. USA 98:4764-49 (2001). The framework regions
flanking the CDR regions can be modified to improve expression
levels and solubility of an intrabody in an intracellular reducing
environment (see, e.g., Worn et al., J. Biol. Chem. 275:2795-803
(2000). An intrabody may be directed to a particular cellular
location or organelle, for example by constructing a vector that
comprises a polynucleotide sequence encoding the variable regions
of an intrabody that may be operatively fused to a polynucleotide
sequence that encodes a particular target antigen within the cell
(see, e.g., Graus-Porta et al., Mol. Cell Biol. 15:1182-91 (1995);
Lener et al., Eur. J. Biochem. 267:1196-205 (2000)). An intrabody
may be introduced into a cell by a variety of techniques available
to the skilled artisan including via a gene therapy vector, or a
lipid mixture (e.g., Provectin.TM. manufactured by Imgenex
Corporation, San Diego, Calif.), or according to photochemical
internalization methods.
[0224] Introducing amino acid mutations into an immunoglobulin
molecule specific for a TGF-beta binding protein may be useful to
increase the specificity or affinity for TGF-beta binding protein
or to alter an effector function. Immunoglobulins with higher
affinity for TGF-beta binding protein may be generated by
site-directed mutagenesis of particular residues. Computer assisted
three-dimensional molecular modeling may be employed to identify
the amino acid residues to be changed, in order to improve affinity
for the TGF-beta binding protein. See, e.g., Mountain et al., 1992,
Biotechnol. Genet Eng. Rev. 10: 1-142. Alternatively, combinatorial
libraries of CDRs may be generated in M13 phage and screened for
immunoglobulin fragments with improved affinity. See, e.g., Glaser
et al., 1992, J. Immunol. 149:3903-3913; Barbas et al., 1994 Proc.
Natl. Acad. Sci. USA 91:3809-13; U.S. Pat. No. 5,792,456.
[0225] Effector functions may also be altered by site-directed
mutagenesis. See, e.g., Duncan et al., 1988 Nature 332:563-64;
Morgan et al., 1995 Immunology 86:319-24; Eghtedarzedeh-Kondri et
al., 1997 Biotechniques 23:830-34. For example, mutation of the
glycosylation site on the Fc portion of the immunoglobulin may
alter the ability of the immunoglobulin to fix complement. See,
e.g., Wright et al., 1997 Trends Biotechnol. 15:26-32. Other
mutations in the constant region domains may alter the ability of
the immunoglobulin to fix complement, or to effect
antibody-dependent cellular cytotoxicity. See, e.g., Duncan et al.,
1988 Nature 332:563-64; Morgan et al., 1995 Immunology 86:319-24;
Sensel et al., 1997 Mol. Immunol. 34:1019-29.
[0226] According to certain embodiments, non-human, human, or
humanized heavy chain and light chain variable regions of any of
the Ig molecules described herein may be constructed as single
chain Fv (scFv) polypeptide fragments (single chain antibodies).
See, e.g., Bird et al., 1988 Science 242:423-426; Huston et al.,
1988 Proc. Natl. Acad. Sci. USA 85:5879-5883. Multi-functional scFv
fusion proteins may be generated by linking a polynucleotide
sequence encoding an scFv polypeptide in-frame with at least one
polynucleotide sequence encoding any of a variety of known effector
proteins. These methods are known in the art, and are disclosed,
for example, in EP-B1-0318554, U.S. Pat. No. 5,132,405, U.S. Pat.
No. 5,091,513, and U.S. Pat. No. 5,476,786. By way of example,
effector proteins may include immunoglobulin constant region
sequences. See, e.g., Hollenbaugh et al., 1995 J. Immunol. Methods
188:1-7. Other examples of effector proteins are enzymes. As a
non-limiting example, such an enzyme may provide a biological
activity for therapeutic purposes (see, e.g., Siemers et al., 1997
Bioconjug. Chem. 8:510-19), or may provide a detectable activity,
such as horseradish peroxidase-catalyzed conversion of any of a
number of well-known substrates into a detectable product, for
diagnostic uses. Still other examples of scFv fusion proteins
include Ig-toxin fusions, or immunotoxins, wherein the scFv
polypeptide is linked to a toxin.
[0227] The scFv or any antibody fragment described herein may, in
certain embodiments, be fused to peptide or polypeptide domains
that permits detection of specific binding between the fusion
protein and antigen (e.g., a TGF-beta binding protein). For
example, the fusion polypeptide domain may be an affinity tag
polypeptide for detecting binding of the scFv fusion protein to a
TGF-beta binding protein by any of a variety of techniques with
which those skilled in the art will be familiar. Examples of a
peptide tag, include avidin, streptavidin or His (e.g.,
polyhistidine). Detection techniques may also include, for example,
binding of an avidin or streptavidin fusion protein to biotin or to
a biotin mimetic sequence (see, e.g., Luo et al., 1998 J.
Biotechnol. 65:225 and references cited therein), direct covalent
modification of a fusion protein with a detectable moiety (e.g., a
labeling moiety), non-covalent binding of the fusion protein to a
specific labeled reporter molecule, enzymatic modification of a
detectable substrate by a fusion protein that includes a portion
having enzyme activity, or immobilization (covalent or
non-covalent) of the fusion protein on a solid-phase support. Other
useful affinity polypeptides for construction of scFv fusion
proteins may include streptavidin fusion proteins, as disclosed,
for example, in WO 89/03422, U.S. Pat. No. 5,489,528, U.S. Pat. No.
5,672,691, WO 93/24631, U.S. Pat. No. 5,168,049, U.S. Pat. No.
5,272,254; avidin fusion proteins (see, e.g., EP 511,747); an
enzyme such as glutathione-S-transferase; and Staphylococcus aureus
protein A polypeptide.
[0228] The polynucleotides encoding an antibody or fragment thereof
that specifically bind a TGF-beta binding protein, as described
herein, may be propagated and expressed according to any of a
variety of well-known procedures for nucleic acid excision,
ligation, transformation, and transfection using any number of
known expression vectors. Thus, in certain embodiments expression
of an antibody fragment may be preferred in a prokaryotic host,
such as Escherichia coli (see, e.g., Pluckthun et al., 1989 Methods
Enzymol. 178:497-515). In certain other embodiments, expression of
the antibody or a fragment thereof may be preferred in a eukaryotic
host cell, including yeast (e.g., Saccharomyces cerevisiae,
Schizosaccharomyces pombe, and Pichia pastoris), animal cells
(including mammalian cells) or plant cells. Examples of suitable
animal cells include, but are not limited to, myeloma (such as a
mouse NSO line), COS, CHO, or hybridoma cells. Examples of plant
cells include tobacco, corn, soybean, and rice cells.
[0229] Once suitable antibodies have been obtained, they may be
isolated or purified by many techniques well known to those of
ordinary skill in the art (see Antibodies: A Laboratory Manual,
Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988).
Suitable techniques include peptide or protein affinity columns
(including use of anti-constant region antibodies attached to the
column matrix), HPLC or RP-HPLC, purification on protein A or
protein G columns, or any combination of these techniques.
[0230] c. Mutant TGF-Beta Binding-Proteins
[0231] As described herein and below in the Examples (e.g.,
Examples 8 and 9), altered versions of TGF-beta binding-protein
which compete with native TGF-beta binding-protein's ability to
block the activity of a particular TGF-beta family member should
lead to increased bone density. Thus, mutants of TGF-beta
binding-protein which bind to the TGF-beta family member but do not
inhibit the function of the TGF-beta family member would meet the
criteria. The mutant versions must effectively compete with the
endogenous inhibitory functions of TGF-beta binding-protein.
[0232] d. Production of Proteins
[0233] Polypeptides described herein include the TGF binding
protein sclerostin and variants thereof and antibodies or fragments
thereof that specifically bind to sclerostin. The polynucleotides
that encode these polypeptides include derivatives of the genes
that are substantially similar to the genes and isolated nucleic
acid molecules, and, when appropriate, the proteins (including
peptides and polypeptides) that are encoded by the genes and their
derivatives. As used herein, a nucleotide sequence is deemed to be
"substantially similar" if (a) the nucleotide sequence is derived
from the coding region of the above-described genes and nucleic
acid molecules and includes, for example, portions of the sequence
or allelic variations of the sequences discussed above, or
alternatively, encodes a molecule which inhibits the binding of
TGF-beta binding-protein to a member of the TGF-beta family; (b)
the nucleotide sequence is capable of hybridization to nucleotide
sequences of the present invention under moderate, high or very
high stringency (see Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press,
NY, 1989); and/or (c) the DNA sequences are degenerate as a result
of the genetic code to the DNA sequences defined in (a) or (b).
Further, the nucleic acid molecule disclosed herein includes both
complementary and non-complementary sequences, provided the
sequences otherwise meet the criteria set forth herein. Within the
context of the present invention, high stringency means standard
hybridization conditions (e.g., 5.times. SSPE, 0.5% SDS at
65.degree. C., or the equivalent).
[0234] The structure of the proteins encoded by the nucleic acid
molecules described herein may be predicted from the primary
translation products using the hydrophobicity plot function of, for
example, P/C Gene or Intelligenetics Suite (Intelligenetics,
Mountain View, Calif.), or according to the methods described by
Kyte and Doolittle (J. Mol. Biol. 157:105-132, 1982).
[0235] Proteins of the present invention may be prepared in the
form of acidic or basic salts, or in neutral form. In addition,
individual amino acid residues may be modified by oxidation or
reduction. Furthermore, various substitutions, deletions, or
additions may be made to the amino acid or nucleic acid sequences,
the net effect of which is to retain or further enhance or decrease
the biological activity of the mutant or wild-type protein.
Moreover, due to degeneracy in the genetic code, for example, there
may be considerable variation in nucleotide sequences encoding the
same amino acid sequence.
[0236] Other derivatives of the proteins disclosed herein include
conjugates of the proteins along with other proteins or
polypeptides. This may be accomplished, for example, by the
synthesis of N-terminal or C-terminal fusion proteins which may be
added to facilitate purification or identification of proteins (see
U.S. Pat. No. 4,851,341, see also, Hopp et al., Bio/Technology
6:1204, 1988.) Alternatively, fusion proteins such as
Flag.RTM./TGF-beta binding-protein be constructed in order to
assist in the identification, expression, and analysis of the
protein.
[0237] Proteins of the present invention may be constructed using a
wide variety of techniques described herein. Further, mutations may
be introduced at particular loci by synthesizing oligonucleotides
containing a mutant sequence, flanked by restriction sites enabling
ligation to fragments of the native sequence. Following ligation,
the resulting reconstructed sequence encodes a derivative having
the desired amino acid insertion, substitution, or deletion.
[0238] Alternatively, oligonucleotide-directed site-specific (or
segment specific) mutagenesis procedures may be employed to provide
an altered gene or nucleic acid molecule having particular codons
altered according to the substitution, deletion, or insertion
required. Exemplary methods of making the alterations set forth
above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et
al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19);
Smith et al. (Genetic Engineering: Principles and Methods, Plenum
Press, 1981); and Sambrook et al. (supra). Deletion or truncation
derivatives of proteins (e.g., a soluble extracellular portion) may
also be constructed by utilizing convenient restriction
endonuclease sites adjacent to the desired deletion. Subsequent to
restriction, overhangs may be filled in and the DNA religated.
Exemplary methods of making the alterations set forth above are
disclosed by Sambrook et al. (Molecular Cloning: A Laboratory
Manual, 2d Ed., Cold Spring Harbor Laboratory Press, 1989).
[0239] Mutations which are made in the nucleic acid molecules of
the present invention preferably preserve the reading frame of the
coding sequences. Furthermore, the mutations will preferably not
create complementary regions that when transcribed could hybridize
to produce secondary mRNA structures, such as loops or hairpins,
that would adversely affect translation of the mRNA. Although a
mutation site may be predetermined, it is not necessary that the
nature of the mutation per se be predetermined. For example, in
order to select for optimum characteristics of mutants at a given
site, random mutagenesis may be conducted at the target codon and
the expressed mutants screened for gain or loss or retention of
biological activity. Alternatively, mutations may be introduced at
particular loci by synthesizing oligonucleotides containing a
mutant sequence, flanked by restriction sites enabling ligation to
fragments of the native sequence. Following ligation, the resulting
reconstructed sequence encodes a derivative having the desired
amino acid insertion, substitution, or deletion.
[0240] Nucleic acid molecules which encode proteins of the present
invention may also be constructed utilizing techniques such as PCR
mutagenesis, chemical mutagenesis (Drinkwater and Klinedinst, PNAS
83:3402-3406, 1986), by forced nucleotide misincorporation (e.g.,
Liao and Wise Gene 88:107-111, 1990), or by use of randomly
mutagenized oligonucleotides (Horwitz et al., Genome 3:112-117,
1989).
[0241] The present invention also provides for the manipulation and
expression of the above described genes and nucleic acid molecules
by culturing host cells containing a vector capable of expressing
the above-described genes. Such vectors or vector constructs
include either synthetic or cDNA-derived nucleic acid molecules
encoding the desired protein, which are operably linked to suitable
transcriptional or translational regulatory elements. Suitable
regulatory elements may be derived from a variety of sources,
including bacterial, fungal, viral, mammalian, insect, or plant
genes. Selection of appropriate regulatory elements is dependent on
the host cell chosen, and may be readily accomplished by one of
ordinary skill in the art. Examples of regulatory elements include
a transcriptional promoter and enhancer or RNA polymerase binding
sequence, a transcriptional terminator, and a ribosomal binding
sequence, including a translation initiation signal.
[0242] Nucleic acid molecules that encode any of the proteins
described above may be readily expressed by a wide variety of
prokaryotic and eukaryotic host cells, including bacterial,
mammalian, yeast or other fungi, viral, insect, or plant cells.
Methods for transforming or transfecting such cells to express
foreign DNA are well known in the art (see, e.g., Itakura et al.,
U.S. Pat. No. 4,704,362; Hinnen et al., Proc. Natl. Acad. Sci. USA
75:1929-1933, 1978; Murray et al., U.S. Pat. No. 4,801,542; Upshall
et al., U.S. Pat. No. 4,935,349; Hagen et al., U.S. Pat. No.
4,784,950; Axel et al., U.S. Pat. No. 4,399,216; Goeddel et al.,
U.S. Pat. No. 4,766,075; and Sambrook et al. Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press,
1989; for plant cells see Czako and Marton, Plant Physiol.
104:1067-1071, 1994; and Paszkowski et al., Biotech. 24:387-392,
1992).
[0243] Bacterial host cells suitable for carrying out the present
invention include E. coli, B. subtilis, Salmonella typhimurium, and
various species within the genera Pseudomonas, Streptomyces, and
Staphylococcus, as well as many other bacterial species well known
to one of ordinary skill in the art and described herein. A
representative example of a bacterial host cell includes E. coli
DH5.alpha. (Stratagene, La Jolla, Calif.).
[0244] Bacterial expression vectors preferably comprise a promoter
which functions in the host cell, one or more selectable phenotypic
markers, and a bacterial origin of replication. Representative
promoters include the .beta.-lactamase (penicillinase) and lactose
promoter system (see Chang et al., Nature 275:615, 1978), the T7
RNA polymerase promoter (Studier et al., Meth. Enzymol. 185:60-89,
1990), the lambda promoter (Elvin et al., Gene 87:123-126, 1990),
the trp promoter (Nichols and Yanofsky, Meth. in Enzymology
101:155, 1983), and the tac promoter (Russell et al., Gene 20:231,
1982). Representative selectable markers include various antibiotic
resistance markers such as the kanamycin or ampicillin resistance
genes. Many plasmids suitable for transforming host cells are well
known in the art, including among others, pBR322 (see Bolivar et
al., Gene 2:95, 1977), the pUC plasmids pUC18, pUC19, pUC118,
pUC119 (see Messing, Meth. in Enzymology 101:20-77, 1983 and Vieira
and Messing, Gene 19:259-268, 1982), and pNH8A, pNH16a, pNH18a, and
Bluescript M13 (Stratagene, La Jolla, Calif.).
[0245] Yeast and fungi host cells suitable for carrying out the
present invention include, among others, Saccharomyces pombe,
Saccharomyces cerevisiae, the genera Pichia or Kluyveromyces and
various species of the genus Aspergillus (McKnight et al., U.S.
Pat. No. 4,935,349). Suitable expression vectors for yeast and
fungi include, among others, YCp50 (ATCC No. 37419) for yeast, and
the amdS cloning vector pV3 (Turnbull, Bio/Technology 7:169, 1989),
YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76:1035-1039,
1978), YEp13 (Broach et al., Gene 8:121-133, 1979), pJDB249 and
pJDB219 (Beggs, Nature 275:104-108, 1978) and derivatives
thereof.
[0246] Preferred promoters for use in yeast include promoters from
yeast glycolytic genes (Hitzeman et al., J. Biol. Chem.
255:12073-12080, 1980; Alber and Kawasaki, J. Mol. Appl. Genet.
1:419-434, 1982) or alcohol dehydrogenase genes (Young et al., in
Genetic Engineering of Microorganisms for Chemicals, Hollaender et
al. (eds.), p. 355, Plenum, New York, 1982; Ammerer, Meth. Enzymol.
101:192-201, 1983). Examples of useful promoters for fungi vectors
include those derived from Aspergillus nidulans glycolytic genes,
such as the adh3 promoter (McKnight et al., EMBO J 4:2093-2099,
1985). The expression units may also include a transcriptional
terminator. An example of a suitable terminator is the adh3
terminator (McKnight et al., supra, 1985).
[0247] As with bacterial vectors, the yeast vectors will generally
include a selectable marker, which may be one of any number of
genes that exhibit a dominant phenotype for which a phenotypic
assay exists to enable transformants to be selected. Preferred
selectable markers are those that complement host cell auxotrophy,
provide antibiotic resistance, or enable a cell to utilize specific
carbon sources, and include leu2 (Broach et al., ibid.), ura3
(Botstein et al., Gene 8:17, 1979), or his3 (Struhl et al., ibid.).
Another suitable selectable marker is the cat gene, which confers
chloramphenicol resistance on yeast cells.
[0248] Techniques for transforming fungi are well known in the
literature and have been described, for instance, by Beggs (ibid.),
Hinnen et al. (Proc. Natl. Acad. Sci. USA 75:1929-1933, 1978),
Yelton et al. (Proc. Natl Acad. Sci. USA 81:1740-1747, 1984), and
Russell (Nature 301:167-169, 1983). The genotype of the host cell
may contain a genetic defect that is complemented by the selectable
marker present on the expression vector. Choice of a particular
host and selectable marker is well within the level of ordinary
skill in the art.
[0249] Protocols for the transformation of yeast are also well
known to those of ordinary skill in the art. For example,
transformation may be readily accomplished either by preparation of
spheroplasts of yeast with DNA (see Hinnen et al., PNAS USA
75:1929, 1978) or by treatment with alkaline salts such as LiCl
(see Itoh et al., J. Bacteriology 153:163, 1983). Transformation of
fungi may also be carried out using polyethylene glycol as
described by Cullen et al. (Bio/Technology 5:369, 1987).
[0250] Viral vectors include those that comprise a promoter that
directs the expression of an isolated nucleic acid molecule that
encodes a desired protein as described above. A wide variety of
promoters may be utilized within the context of the present
invention, including for example, promoters such as MoMLV LTR, RSV
LTR, Friend MuLV LTR, adenoviral promoter (Ohno et al., Science
265:781-784, 1994), neomycin phosphotransferase promoter/enhancer,
late parvovirus promoter (Koering et al., Hum. Gene Therap.
5:457-463, 1994), Herpes TK promoter, SV40 promoter,
metallothionein IIa gene enhancer/promoter, cytomegalovirus
immediate early promoter, and the cytomegalovirus immediate late
promoter. Within particularly preferred embodiments of the
invention, the promoter is a tissue-specific promoter (see e.g., WO
91/02805; EP 0,415,731; and WO 90/07936). Representative examples
of suitable tissue specific promoters include neural specific
enolase promoter, platelet derived growth factor beta promoter,
bone morphogenic protein promoter, human alpha1-chimaerin promoter,
synapsin I promoter and synapsin II promoter. In addition to the
above-noted promoters, other viral-specific promoters (e.g.,
retroviral promoters (including those noted above, as well as
others such as HIV promoters), hepatitis, herpes (e.g., EBV), and
bacterial, fungal or parasitic (e.g., malarial)-specific promoters
may be utilized in order to target a specific cell or tissue which
is infected with a virus, bacteria, fungus, or parasite.
[0251] Mammalian cells suitable for carrying out the present
invention include, among others COS, CHO, SaOS, osteosarcomas,
KS483, MG-63, primary osteoblasts, and human or mammalian bone
marrow stroma. Mammalian expression vectors for use in carrying out
the present invention will include a promoter capable of directing
the transcription of a cloned gene, nucleic acid molecule, or cDNA.
Preferred promoters include viral promoters and cellular promoters.
Bone specific promoters include the promoter for bone sialo-protein
and the promoter for osteocalcin. Viral promoters include the
cytomegalovirus immediate early promoter (Boshart et al., Cell
41:521-530, 1985), cytomegalovirus immediate late promoter, SV40
promoter (Subramani et al., Mol. Cell. Biol. 1:854-864, 1981), MMTV
LTR, RSV LTR, metallothionein-1, adenovirus E1a. Cellular promoters
include the mouse metallothionein-1 promoter (Palmiter et al., U.S.
Pat. No. 4,579,821), a mouse V.sub..kappa. promoter (Bergman et
al., Proc. Natl Acad. Sci. USA 81:7041-7045, 1983; Grant et al.,
Nucleic Acids Res. 15:5496, 1987) and a mouse V.sub.H promoter (Loh
et al., Cell 33:85-93, 1983). The choice of promoter will depend,
at least in part, upon the level of expression desired or the
recipient cell line to be transfected.
[0252] Such expression vectors may also contain a set of RNA splice
sites located downstream from the promoter and upstream from the
DNA sequence encoding the peptide or protein of interest. Preferred
RNA splice sites may be obtained from adenovirus and/or
immunoglobulin genes. Also contained in the expression vectors is a
polyadenylation signal located downstream of the coding sequence of
interest. Suitable polyadenylation signals include the early or
late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.),
the polyadenylation signal from the Adenovirus 5 E1B region and the
human growth hormone gene terminator (DeNoto et al., Nucleic Acids
Res. 9:3719-3730, 1981). The expression vectors may include a
noncoding viral leader sequence, such as the Adenovirus 2
tripartite leader, located between the promoter and the RNA splice
sites. Preferred vectors may also include enhancer sequences, such
as the SV40 enhancer. Expression vectors may also include sequences
encoding the adenovirus VA RNAs. Suitable expression vectors can be
obtained from commercial sources (e.g., Stratagene, La Jolla,
Calif.).
[0253] Vector constructs comprising cloned DNA sequences can be
introduced into cultured mammalian cells by, for example, calcium
phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978;
Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and
Van der Eb, Virology 52:456, 1973), electroporation (Neumann et
al., EMBO J. 1:841-845, 1982), or DEAE-dextran mediated
transfection (Ausubel et al. (eds.), Current Protocols in Molecular
Biology, John Wiley and Sons, Inc., NY, 1987). To identify cells
that have stably transfected with the vector or have integrated the
cloned DNA, a selectable marker is generally introduced into the
cells along with the gene or cDNA of interest. Preferred selectable
markers for use in cultured mammalian cells include genes that
confer resistance to drugs, such as neomycin, hygromycin, and
methotrexate. The selectable marker may be an amplifiable
selectable marker. Preferred amplifiable selectable markers are the
DHFR gene and the neomycin resistance gene. Selectable markers are
reviewed by Thilly (Mammalian Cell Technology, Butterworth
Publishers, Stoneham, Mass., which is incorporated herein by
reference).
[0254] Mammalian cells containing a suitable vector are allowed to
grow for a period of time, typically 1-2 days, to begin expressing
the DNA sequence(s) of interest. Drug selection is then applied to
select for growth of cells that are expressing the selectable
marker in a stable fashion. For cells that have been transfected
with an amplifiable, selectable marker, the drug concentration may
be increased in a stepwise manner to select for increased copy
number of the cloned sequences, thereby increasing expression
levels. Cells expressing the introduced sequences are selected and
screened for production of the protein of interest in the desired
form or at the desired level. Cells that satisfy these criteria can
then be cloned and scaled up for production.
[0255] Protocols for the transfection of mammalian cells are well
known to those of ordinary skill in the art. Representative methods
include calcium phosphate mediated transfection, electroporation,
lipofection, retroviral, adenoviral and protoplast fusion-mediated
transfection (see Sambrook et al., supra). Naked vector constructs
can also be taken up by muscular cells or other suitable cells
subsequent to injection into the muscle of a mammal (or other
animals).
[0256] Methods for using insect host cells and plant host cells for
production of polypeptides are known in the art and described
herein. Numerous insect host cells known in the art can also be
useful within the present invention. For example, the use of
baculoviruses as vectors for expressing heterologous DNA sequences
in insect cells has been reviewed by Atkinson et al. (Pestic. Sci.
28:215-224,1990). Numerous vectors and plant host cells known in
the art can also be useful within the present invention, for
example, the use of Agrobacterium rhizogenes as vectors for
expressing genes and nucleic acid molecules in plant cells (see
review by Sinkar et al., J. Biosci. (Bangalore 11:47-58, 1987).
[0257] Within related aspects of the present invention, proteins of
the present invention may be expressed in a transgenic animal whose
germ cells and somatic cells contain a gene which encodes the
desired protein and which is operably linked to a promoter
effective for the expression of the gene. Alternatively, in a
similar manner transgenic animals may be prepared that lack the
desired gene (e.g., "knock-out" mice). Such transgenics may be
prepared in a variety of non-human animals, including mice, rats,
rabbits, sheep, dogs, goats, and pigs (see Hammer et al., Nature
315:680-683, 1985, Palmiter et al., Science 222:809-814, 1983,
Brinster et al., Proc. Natl Acad. Sci. USA 82:4438-4442, 1985,
Palmiter and Brinster, Cell 41:343-345, 1985, and U.S. Pat. Nos.
5,175,383, 5,087,571, 4,736,866, 5,387,742, 5,347,075, 5,221,778,
and 5,175,384). Briefly, an expression vector, including a nucleic
acid molecule to be expressed together with appropriately
positioned expression control sequences, is introduced into
pronuclei of fertilized eggs, for example, by microinjection.
Integration of the injected DNA is detected by blot analysis of DNA
from tissue samples. It is preferred that the introduced DNA be
incorporated into the germ line of the animal so that it is passed
on to the animal's progeny. Tissue-specific expression may be
achieved through the use of a tissue-specific promoter, or through
the use of an inducible promoter, such as the metallothionein gene
promoter (Palmiter et al., 1983, supra), which allows regulated
expression of the transgene.
[0258] Proteins can be isolated by, among other methods, culturing
suitable host and vector systems to produce the recombinant
translation products as described herein. Supernatants from such
cell lines, or protein inclusions, or whole cells from which the
protein is not excreted into the supernatant, can then be treated
by a variety of purification procedures in order to isolate the
desired proteins. For example, the supernatant may be first
concentrated using commercially available protein concentration
filters, such as an Amicon or Millipore Pellicon ultrafiltration
unit. Following concentration, the concentrate may be applied to a
suitable purification matrix such as, for example, an anti-protein
antibody (e.g., an antibody that specifically binds to the
polypeptide to be isolated) bound to a suitable support.
Alternatively, anion or cation exchange resins may be employed in
order to purify the protein. As a further alternative, one or more
reverse-phase high performance liquid chromatography (RP-HPLC)
steps may be employed to further purify the protein. Other methods
of isolating the proteins of the present invention are well known
in the art.
[0259] The purity of an isolated polypeptide may be determined by
techniques known in the art and described herein, such as gel
electrophoresis and chromatography methods. Preferably, such
isolated polypeptides are at least about 90% pure, more preferably
at least about 95% pure, and most preferably at least about 99%
pure. Within certain specific embodiments, a protein is deemed to
be "isolated" within the context of the present invention if no
other undesired protein is detected pursuant to SDS-PAGE analysis
followed by Coomassie blue staining. Within other embodiments, the
desired protein can be isolated such that no other undesired
protein is detected pursuant to SDS-PAGE analysis followed by
silver staining.
[0260] 3. Nucleic Acid Molecules
[0261] Within other aspects of the invention, nucleic acid
molecules are provided which are capable of inhibiting TGF-beta
binding-protein binding to a member of the TGF-beta family. For
example, within one embodiment antisense oligonucleotide molecules
are provided which specifically inhibit expression of TGF-beta
binding-protein nucleic acid sequences (see generally, Hirashima et
al. in Molecular Biology of RNA: New Perspectives (M. Inouye and B.
S. Dudock, eds., 1987 Academic Press, San Diego, p. 401);
Oligonucleotides: Antisense Inhibitors of Gene Expression (J. S.
Cohen, ed., 1989 MacMillan Press, London); Stein and Cheng, Science
261:1004-1012, 1993; WO 95/10607; U.S. Pat. No. 5,359,051; WO
92/06693; and EP-A2-612844). Briefly, such molecules are
constructed such that they are complementary to, and able to form
Watson-Crick base pairs with, a region of transcribed TGF-beta
binding-protein mRNA sequence. The resultant double-stranded
nucleic acid interferes with subsequent processing of the mRNA,
thereby preventing protein synthesis (see Example 10).
[0262] Within other aspects of the invention, ribozymes are
provided which are capable of inhibiting the TGF-beta
binding-protein binding to a member of the TGF-beta family. As used
herein, "ribozymes" are intended to include RNA molecules that
contain anti-sense sequences -for specific recognition, and an
RNA-cleaving enzymatic activity. The catalytic strand cleaves a
specific site in a target RNA at greater than stoichiometric
concentration. A wide variety of ribozymes may be utilized within
the context of the present invention, including for example, the
hammerhead ribozyme (for example, as described by Forster and
Symons, Cell 48:211-220, 1987; Haseloff and Gerlach, Nature
328:596-600, 1988; Walbot and Bruening, Nature 334:196, 1988;
Haseloff and Gerlach, Nature 334:585, 1988); the hairpin ribozyme
(for example, as described by Haseloff et al., U.S. Pat. No.
5,254,678, issued Oct. 19, 1993 and Hempel et al., European Patent
Publication No. 0 360 257, published Mar. 26, 1990); and
Tetrahymena ribosomal RNA-based ribozymes (see Cech et al., U.S.
Pat. No. 4,987,071). Ribozymes of the present invention typically
consist of RNA, but may also be composed of DNA, nucleic acid
analogs (e.g., phosphorothioates), or chimerics thereof (e.g.,
DNA/RNA/RNA).
[0263] 4. Labels
[0264] The gene product or any of the candidate molecules described
above and below, may be labeled with a variety of compounds,
including for example, fluorescent molecules, toxins, and
radionuclides. Representative examples of fluorescent molecules
include fluorescein, Phycobili proteins, such as phycoerythrin,
rhodamine, Texas red and luciferase. Representative examples of
toxins include ricin, abrin diphtheria toxin, cholera toxin,
gelonin, pokeweed antiviral protein, tritin, Shigella toxin, and
Pseudomonas exotoxin A. Representative examples of radionuclides
include Cu-64, Ga-67, Ga-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109,
In-111, I-123, I-125, I-131, Re-186, Re-188, Au-198, Au-199,
Pb-203, At-211, Pb-212 and Bi-212. In addition, the antibodies
described above may also be labeled or conjugated to one partner of
a ligand binding pair. Representative examples include
avidin-biotin, streptavidin-biotin, and riboflavin-riboflavin
binding protein.
[0265] Methods for conjugating or labeling the molecules described
herein with the representative labels set forth above may be
readily accomplished by one of ordinary skill in the art (see
Trichothecene Antibody Conjugate, U.S. Pat. No. 4,744,981; Antibody
Conjugate, U.S. Pat. No. 5,106,951; Fluorogenic Materials and
Labeling Techniques, U.S. Pat. No. 4,018,884; Metal Radionuclide
Labeled Proteins for Diagnosis and Therapy, U.S. Pat. No.
4,897,255; and Metal Radionuclide Chelating Compounds for Improved
Chelation Kinetics, U.S. Pat. No. 4,988,496; see also Inman,
Methods In Enzymology, Vol. 34, Affinity Techniques, Enzyme
Purification: Part B, Jakoby and Wilchek (eds.), Academic Press,
New York, p. 30, 1974; see also Wilchek and Bayer, "The
Avidin-Biotin Complex in Bioanalytical Applications," Anal.
Biochem. 171:1-32, 1988).
[0266] Pharmaceutical Compositions
[0267] As noted above, the present invention also provides a
variety of pharmaceutical compositions, comprising one of the
above-described molecules which inhibits the TGF-beta
binding-protein binding to a member of the TGF-beta family along
with a pharmaceutically or physiologically acceptable carrier,
excipients or diluents. Generally, such carriers should be nontoxic
to recipients at the dosages and concentrations employed.
Ordinarily, the preparation of such compositions entails combining
the therapeutic agent with buffers, antioxidants such as ascorbic
acid, low molecular weight (less than about 10 residues)
polypeptides, proteins, amino acids, carbohydrates including
glucose, maltose, sucrose or dextrins, chelating agents such as
EDTA, glutathione and other stabilizers and excipients. Neutral
buffered saline or saline mixed with nonspecific serum albumin are
exemplary appropriate diluents.
[0268] The pharmaceutical compositions of the present invention may
be prepared for administration by a variety of different routes. In
general, the type of carrier is selected based on the mode of
administration. Pharmaceutical compositions may be formulated for
any appropriate manner of administration, including, for example,
topical, oral, nasal, intrathecal, rectal, vaginal, sublingual or
parenteral administration, including subcutaneous, intravenous,
intramuscular, intrasternal, intracavernous, intrameatal, or
intraurethral injection or infusion. A pharmaceutical composition
(e.g., for oral administration or delivery by injection) may be in
the form of a liquid (e.g., an elixir, syrup, solution, emulsion or
suspension). A liquid pharmaceutical composition may include, for
example, one or more of the following: sterile diluents such as
water for injection, saline solution, preferably physiological
saline, Ringer's solution, isotonic sodium chloride, fixed oils
that may serve as the solvent or suspending medium, polyethylene
glycols, glycerin, propylene glycol or other solvents;
antibacterial agents; antioxidants; chelating agents; buffers such
as acetates, citrates or phosphates and agents for the adjustment
of tonicity such as sodium chloride or dextrose. A parenteral
preparation can be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass or plastic. The use of
physiological saline is preferred, and an injectable pharmaceutical
composition is preferably sterile.
[0269] The compositions described herein may be formulated for
sustained release (i.e., a formulation such as a capsule or sponge
that effects a slow release of compound following administration).
Such compositions may generally be prepared using well known
technology and administered by, for example, oral, rectal or
subcutaneous implantation, or by implantation at the desired target
site. Sustained-release formulations may contain an agent dispersed
in a carrier matrix and/or contained within a reservoir surrounded
by a rate controlling membrane. Carriers for use within such
formulations are biocompatible, and may also be biodegradable;
preferably the formulation provides a relatively constant level of
active component release. The amount of active compound contained
within a sustained release formulation depends upon the site of
implantation, the rate and expected duration of release and the
nature of the condition to be treated or prevented. Illustrative
carriers useful in this regard include microparticles of
poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose,
dextran and the like. Other illustrative delayed-release carriers
include supramolecular biovectors, which comprise a non-liquid
hydrophilic core (e.g., a cross-linked polysaccharide or
oligosaccharide) and, optionally, an external layer comprising an
amphiphilic compound, such as a phospholipid (see e.g., U.S. Pat.
No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO
96/06638).
[0270] In another illustrative embodiment, biodegradable
microspheres (e.g., polylactate polyglycolate) are employed as
carriers for the compositions of this invention. Suitable
biodegradable microspheres are disclosed, for example, in U.S. Pat.
Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883;
5,853,763; 5,814,344, 5,407,609 and 5,942,252. Modified hepatitis B
core protein carrier systems, such as described in WO/99 40934, and
references cited therein, will also be useful for many
applications. Another illustrative carrier/delivery system employs
a carrier comprising particulate-protein complexes, such as those
described in U.S. Pat. No. 5,928,647, which are capable of inducing
a class I-restricted cytotoxic T lymphocyte responses in a
host.
[0271] In another illustrative embodiment, calcium phosphate core
particles are employed as carriers or as controlled release
matrices for the compositions of this invention. Exemplary calcium
phosphate particles are disclosed, for example, in published patent
application No. WO/0046147.
[0272] For pharmaceutical compositions comprising a polynucleotide
encoding an anti-SOST antibody and/or modulating agent (such that
the polypeptide and/or modulating agent is generated in situ), the
polynucleotide may be present within any of a variety of delivery
systems known to those of ordinary skill in the art, including
nucleic acid, and bacterial, viral and mammalian expression
systems. Techniques for incorporating DNA into such expression
systems are well known to those of ordinary skill in the art. The
DNA may also be "naked," as described, for example, in Ulmer et
al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science
259:1691-1692, 1993. The uptake of naked DNA may be increased by
coating the DNA onto biodegradable beads, which are efficiently
transported into the cells.
[0273] The development of suitable dosing and treatment regimens
for using the particular compositions described herein in a variety
of treatment regimens, including e.g., oral, parenteral,
intravenous, intranasal, and intramuscular administration and
formulation, is well known in the art, some of which are briefly
discussed below for general purposes of illustration.
[0274] In certain applications, the pharmaceutical compositions
disclosed herein may be delivered via oral administration to an
animal. As such, these compositions may be formulated with an inert
diluent or with an assimilable edible carrier, or they may be
enclosed in hard- or soft-shell gelatin capsule, or they may be
compressed into tablets, or they may be incorporated directly with
the food of the diet.
[0275] In certain circumstances it will be desirable to deliver the
pharmaceutical compositions disclosed herein parenterally,
intravenously, intramuscularly, or even intraperitoneally. Such
approaches are well known to the skilled artisan, some of which are
further described, for example, in U.S. Pat. No. 5,543,158; U.S.
Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain
embodiments, solutions of the active compounds as free base or
pharmacologically acceptable salts may be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions may also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations generally will
contain a preservative to prevent the growth of microorganisms.
[0276] Illustrative pharmaceutical forms suitable for injectable
use include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions (for example, see U.S. Pat. No.
5,466,468). In all cases the form must be sterile and must be fluid
to the extent that easy syringability exists. It must be stable
under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g.,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and/or vegetable oils. Proper
fluidity may be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and/or by the use of surfactants. The
prevention of the action of microorganisms can be facilitated by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0277] In one embodiment, for parenteral administration in an
aqueous solution, the solution should be suitably buffered if
necessary and the liquid diluent first rendered isotonic with
sufficient saline or glucose. These particular aqueous solutions
are especially suitable for intravenous, intramuscular,
subcutaneous and intraperitoneal administration. In this
connection, a sterile aqueous medium that can be employed will be
known to those of skill in the art in light of the present
disclosure. For example, one dosage may be dissolved in 1 ml of
isotonic NaCl solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, Remington's Pharmaceutical Sciences, 15th ed.,
pp. 1035-1038 and 1570-1580). Some variation in dosage will
necessarily occur depending on the condition of the subject being
treated. Moreover, for human administration, preparations will of
course preferably meet sterility, pyrogenicity, and the general
safety and purity standards as required by FDA Office of Biologics
standards.
[0278] In another embodiment of the invention, the compositions
disclosed herein may be formulated in a neutral or salt form.
Illustrative pharmaceutically-acceptable salts include the acid
addition salts (formed with the free amino groups of the protein)
and which are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic, tartaric, mandelic, and the like. Salts formed with the
free carboxyl groups can also be derived from inorganic bases such
as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. Upon formulation,
solutions will be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically
effective.
[0279] The carriers can further comprise any and all solvents,
dispersion media, vehicles, coatings, diluents, antibacterial and
antifungal agents, isotonic and absorption delaying agents,
buffers, carrier solutions, suspensions, colloids, and the like.
The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions. The phrase
"pharmaceutically-acceptable" refers to molecular entities and
compositions that do not produce an allergic or similar untoward
reaction when administered to a human.
[0280] In certain embodiments, liposomes, nanocapsules,
microparticles, lipid particles, vesicles, and the like, are used
for the introduction of the compositions of the present invention
into suitable host cells/organisms. In particular, the compositions
of the present invention may be formulated for delivery either
encapsulated in a lipid particle, a liposome, a vesicle, a
nanosphere, or a nanoparticle or the like. Alternatively,
compositions of the present invention can be bound, either
covalently or non-covalently, to the surface of such carrier
vehicles.
[0281] The formation and use of liposome and liposome-like
preparations as potential drug carriers is generally known to those
of skill in the art (see for example, Lasic, Trends Biotechnol.
16(7):307-21, 1998; Takakura, Nippon Rinsho 56(3):691-95, 1998;
Chandran et al., Indian J. Exp. Biol. 35(8):801-09, 1997; Margalit,
Crit. Rev. Ther. Drug Carrier Syst. 12(2-3):233-61, 1995; U.S. Pat.
No. 5,567,434; U.S. Pat. No. 5,552,157; U.S. Pat. No. 5,565,213;
U.S. Pat. No. 5,738,868 and U.S. Pat. No. 5,795,587, each
specifically incorporated herein by reference in its entirety).
[0282] Liposomes have been used successfully with a number of cell
types that are normally difficult to transfect by other procedures,
including T cell suspensions, primary hepatocyte cultures and PC 12
cells (Renneisen et al., J. Biol. Chem. 265(27):16337-42, 1990;
Muller et al., DNA Cell Biol. 9(3):221-29, 1990). In addition,
liposomes are free of the DNA length constraints that are typical
of viral-based delivery systems. Liposomes have been used
effectively to introduce genes, various drugs, radiotherapeutic
agents, enzymes, viruses, transcription factors, allosteric
effectors and the like, into a variety of cultured cell lines and
animals. Furthermore, he use of liposomes does not appear to be
associated with autoimmune responses or unacceptable toxicity after
systemic delivery.
[0283] In certain embodiments, liposomes are formed from
phospholipids that are dispersed in an aqueous medium and
spontaneously form multilamellar concentric bilayer vesicles (also
termed multilamellar vesicles (MLVs).
[0284] Alternatively, in other embodiments, the invention provides
for pharmaceutically-acceptable nanocapsule formulations of the
compositions of the present invention. Nanocapsules can generally
entrap compounds in a stable and reproducible way (see, for
example, Quintanar-Guerrero et al., Drug Dev. Ind. Pharm.
24(12):1113-28, 1998). To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
.mu.m) may be designed using polymers able to be degraded in vivo.
Such particles can be made as described, for example, by Couvreur
et al., Crit. Rev. Ther. Drug Carrier Syst. 5(1):1-20, 1988; zur
Muhlen et al., Eur. J. Pharm. Biopharm. 45(2):149-55, 1998; Zambaux
et al., J. Controlled Release 50(1-3):31-40, 1998; and U.S. Pat.
No. 5,145,684.
[0285] In addition, pharmaceutical compositions of the present
invention may be placed within containers, along with packaging
material that provides instructions regarding the use of such
pharmaceutical compositions. Generally, such instructions will
include a tangible expression describing the reagent concentration,
as well as within certain embodiments, relative amounts of
excipient ingredients or diluents (e.g., water, saline or PBS)
which may be necessary to reconstitute the pharmaceutical
composition.
[0286] Methods of Treatment
[0287] The present invention also provides methods for increasing
the mineral content and mineral density of bone. Briefly, numerous
conditions result in the loss of bone mineral content, including
for example, disease, genetic predisposition, accidents which
result in the lack of use of bone (e.g., due to fracture),
therapeutics which effect bone resorption, or which kill bone
forming cells and normal aging. Through use of the molecules
described herein which inhibit the TGF-beta binding-protein binding
to a TGF-beta family member such conditions may be treated or
prevented. As utilized herein, it should be understood that bone
mineral content has been increased if bone mineral content has been
increased in a statistically significant manner (e.g., greater than
one-half standard deviation), at a selected site.
[0288] A wide variety of conditions that result in loss of bone
mineral content may be treated with the molecules described herein.
Patients with such conditions may be identified through clinical
diagnosis utilizing well known techniques (see, e.g., Harrison's
Principles of Internal Medicine, McGraw-Hill, Inc.). Representative
examples of diseases that may be treated included dysplasias,
wherein there is abnormal growth or development of bone.
Representative examples of such conditions include achondroplasia,
cleidocranial dysostosis, enchondromatosis, fibrous dysplasia,
Gaucher's Disease, hypophosphatemic rickets, Marfan's Syndrome,
multiple hereditary exotoses, neurofibromatosis, osteogenesis
imperfecta, osteopetrosis, osteopoikilosis, sclerotic lesions,
fractures, periodontal disease, pseudoarthrosis, and pyogenic
osteomyelitis.
[0289] Other conditions which may be treated or prevented include a
wide variety of causes of osteopenia (i.e., a condition that causes
greater than one standard deviation of bone mineral content or
density below peak skeletal mineral content at youth).
Representative examples of such conditions include anemic states,
conditions caused by steroids, conditions caused by heparin, bone
marrow disorders, scurvy, malnutrition, calcium deficiency,
idiopathic osteoporosis, congenital osteopenia or osteoporosis,
alcoholism, chronic liver disease, senility, postmenopausal state,
oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus,
hyperthyroidism, Cushing's disease, acromegaly, hypogonadism,
immobilization or disuse, reflex sympathetic dystrophy syndrome,
transient regional osteoporosis, and osteomalacia.
[0290] Within one aspect of the present invention, bone mineral
content or density may be increased by administering to a
warm-blooded animal a therapeutically effective amount of a
molecule that inhibits binding of the TGF-beta binding-protein to a
TGF-beta family member. Examples of warm-blooded animals that may
be treated include both vertebrates and mammals, including for
example humans, horses, cows, pigs, sheep, dogs, cats, rats and
mice. Representative examples of therapeutic molecules include
ribozymes, ribozyme genes, antisense oligonucleotides, and
antibodies (e.g., humanized antibodies or any other antibody
described herein).
[0291] Within other aspects of the present invention, methods are
provided for increasing bone density, comprising the steps of
introducing into cells which home to bone, a vector that directs
the expression of a molecule which inhibits binding of the TGF-beta
binding-protein to a member of the TGF-beta family, and
administering the vector-containing cells to a warm-blooded animal.
Briefly, cells that home to bone may be obtained directly from the
bone of patients (e.g., cells obtained from the bone marrow such as
CD34+, osteoblasts, osteocytes, and the like), from peripheral
blood, or from cultures.
[0292] A vector that directs the expression of a molecule that
inhibits the binding of TGF-beta binding-protein to a member of the
TGF-beta family may be introduced into cells. Representative
examples of suitable vectors include viral vectors such as herpes
viral vectors (e.g., U.S. Pat. No. 5,288,641), adenoviral vectors
(e.g., WO 94/26914, WO 93/9191; Kolls et al., PNAS 91(1):215-219,
1994; Kass-Eisler et al., PNAS 90(24):11498-502, 1993; Guzman et
al., Circulation 88(6):2838-48, 1993; Guzman et al., Cir. Res.
73(6):1202-1207, 1993; Zabner et al., Cell 75(2):207-216, 1993; Li
et al., Hum Gene Ther. 4(4):403-409, 1993; Caillaud et al., Eur. J.
Neurosci. 5(10:1287-1291, 1993; Vincent et al., Nat. Genet.
5(2):130-134, 1993; Jaffe et al., Nat. Genet. 1(5):372-378, 1992;
and Levrero et al., Gene 101(2):195-202, 1991), adeno-associated
viral vectors (WO 95/13365; Flotte et al., PNAS 90(22):10613-10617,
1993), baculovirus vectors, parvovirus vectors (Koering et al.,
Hum. Gene Therap. 5:457-463, 1994), pox virus vectors (Panicali and
Paoletti, PNAS 79:4927-4931, 1982; and Ozaki et al., Biochem.
Biophys. Res. Comm. 193(2):653-660, 1993), and retroviruses (e.g.,
EP 0,415,731; WO 90/07936; WO 91/0285, WO 94/03622; WO 93/25698; WO
93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218). Viral
vectors may likewise be constructed which contain a mixture of
different elements (e.g., promoters, envelope sequences, and the
like) from different viruses, or non-viral sources. Within various
embodiments, either the viral vector itself, or a viral particle
which contains the viral vector may be utilized in the methods and
compositions described below. Within other embodiments of the
invention, nucleic acid molecules which encode a molecule which
inhibits binding of the TGF-beta binding-protein to a member of the
TGF-beta family may be administered by a variety of techniques,
including, for example, administration of asialoosomucoid (ASOR)
conjugated with poly-L-lysine DNA complexes (Cristano et al., PNAS
92122-92126, 1993), DNA linked to killed adenovirus (Curiel et al.,
Hum. Gene Ther. 3(2):147-154, 1992), cytofectin-mediated
introduction (DMRIE-DOPE, Vical, Calif.), direct DNA injection
(Acsadi et al., Nature 352:815-818, 1991); DNA ligand (Wu et al.,
J. of Biol. Chem. 264:16985-16987, 1989); lipofection (Felgner et
al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1989); liposomes
(Pickering et al., Circ. 89(l):13-21, 1994; and Wang et al., PNAS
84:7851-7855, 1987); microprojectile bombardment (Williams et al.,
PNAS 88:2726-2730, 1991); and direct delivery of nucleic acids
which encode the protein itself either alone (Vile and Hart, Cancer
Res. 53: 3860-3864, 1993), or utilizing PEG-nucleic acid complexes.
Representative examples of molecules that may be expressed by the
vectors of present invention include ribozymes and antisense
molecules, each of which are discussed in more detail above.
[0293] Determination of increased bone mineral content may be
determined directly through the use of X-rays (e.g., Dual Energy
X-ray Absorptometry or "DEXA"), or by inference through bone
turnover markers (such as osteoblast specific alkaline phosphatase,
osteocalcin, type 1 procollagen C' propeptide (PICP), and total
alkaline phosphatase; see Comier, C., Curr. Opin. in Rheu. 7:243,
1995), or by markers of bone resorption (pyridinoline,
deoxypryridinoline, N-telopeptide, urinary hydroxyproline, plasma
tartrate-resistant acid phosphatases and galactosyl hydroxylysine;
see Comier, supra). The amount of bone mass may also be calculated
from body weights or by other methods known in the art (see
Guinness-Hey, Metab. Bone Dis. and Relat. Res. 5:177-181,
1984).
[0294] As will be evident to one of skill in the art, the amount
and frequency of administration will depend, of course, on such
factors as the nature and severity of the indication being treated,
the desired response, the condition of the patient, and so forth.
Typically, the compositions may be administered by a variety of
techniques, as noted above.
[0295] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
[0296] Sclerosteosis Maps to the Long Arm of Human Chromosome
17
[0297] Genetic mapping of the defect responsible for sclerosteosis
in humans localized the gene responsible for this disorder to the
region of human chromosome 17 that encodes a novel TGF-beta
binding-protein family member. In sclerosteosis, skeletal bone
displays a substantial increase in mineral density relative to that
of unafflicted individuals. Bone in the head displays overgrowth as
well. Sclerosteosis patients are generally healthy although they
may exhibit variable degrees of syndactyly at birth and variable
degrees of cranial compression and nerve compression in the
skull.
[0298] Linkage analysis of the gene defect associated with
sclerosteosis was conducted by applying the homozygosity mapping
method to DNA samples collected from 24 South African Afrikaaner
families in which the disease occurred. (Sheffield et al., 1994,
Human Molecular Genetics 3:1331-1335. "Identification of a
Bardet-Biedl syndrome locus on chromosome 3 and evaluation of an
efficient approach to homozygosity mapping"). The Afrikaaner
population of South Africa is genetically homogeneous; the
population is descended from a small number of founders who
colonized the area several centuries ago, and it has been isolated
by geographic and social barriers since the founding. Sclerosteosis
is rare everywhere in the world outside the Afrikaaner community,
which suggests that a mutation in the gene was present in the
founding population and has since increased in numbers along with
the increase in the population. The use of homozygosity mapping is
based on the assumption that DNA mapping markers adjacent to a
recessive mutation are likely to be homozygous in affected
individuals from consanguineous families and isolated
populations.
[0299] A set of 371 microsatellite markers (Research Genetics, Set
6) from the autosomal chromosomes was selected to type pools of DNA
from sclerosteosis patient samples. The DNA samples for this
analysis came from 29 sclerosteosis patients in 24 families, 59
unaffected family members and a set of unrelated control
individuals from the same population. The pools consisted of 4-6
individuals, either affected individuals, affected individuals from
consanguineous families, parents and unaffected siblings, or
unrelated controls. In the pools of unrelated individuals and in
most of the pools with affected individuals or family members
analysis of the markers showed several allele sizes for each
marker. One marker, D17S1299, showed an indication of homozygosity:
one band in several of the pools of affected individuals.
[0300] All 24 sclerosteosis families were typed with a total of 19
markers in the region of D17S1299 (at 17q12-q21). Affected
individuals from every family were shown to be homozygous in this
region, and 25 of the 29 individuals were homozygous for a core
haplotype; they each had the same alleles between D17S1787 and
D17S930. The other four individuals had one chromosome which
matched this haplotype and a second which did not. In sum, the data
compellingly suggested that this 3 megabase region contained the
sclerosteosis mutation. Sequence analysis of most of the exons in
this 3 megabase region identified a nonsense mutation in the novel
TGF-beta binding-protein coding sequence (C>T mutation at
position 117 of Sequence ID No. 1 results in a stop codon). This
mutation was shown to be unique to sclerosteosis patients and
carriers of Afrikaaner descent. The identity of the gene was
further confirmed by identifying a mutation in its intron (A>T
mutation at position +3 of the intron) which results in improper
mRNA processing in a single, unrelated patient with diagnosed
sclerosteosis.
Example 2
[0301] Tissue-Specificity of TGF-Beta Binding-Protein
Expression
[0302] A. Human Beer Gene Expression by RT-PCR:
[0303] First-strand cDNA was prepared from the following total RNA
samples using a commercially available kit ("Superscript
Preamplification System for First-Strand cDNA Synthesis", Life
Technologies, Rockville, Md.): human brain, human liver, human
spleen, human thymus, human placenta, human skeletal muscle, human
thyroid, human pituitary, human osteoblast (NHOst from Clonetics
Corp., San Diego, Calif.), human osteosarcoma cell line (Saos-2,
ATCC# HTB-85), human bone, human bone marrow, human cartilage,
vervet monkey bone, saccharomyces cerevisiae, and human peripheral
blood monocytes. All RNA samples were purchased from a commercial
source (Clontech, Palo Alto, Calif.), except the following which
were prepared in-house: human osteoblast, human osteosarcoma cell
line, human bone, human cartilage and vervet monkey bone. These
in-house RNA samples were prepared using a commercially available
kit ("TRI Reagent", Molecular Research Center, Inc., Cincinnati,
Ohio).
[0304] PCR was performed on these samples, and additionally on a
human genomic sample as a control. The sense Beer oligonucleotide
primer had the sequence 5'-CCGGAGCTGGAGAACAACAAG-3' (SEQ ID NO:
19). The antisense Beer oligonucleotide primer had the sequence
5'-GCACTGGCCGGAGCACACC-3' (SEQ ID NO: 20). In addition, PCR was
performed using primers for the human beta-actin gene, as a
control. The sense beta-actin oligonucleotide primer had the
sequence 5'-AGGCCAACCGCGAGAAGATGA CC-3' (SEQ ID NO: 21). The
antisense beta-actin oligonucleotide primer had the sequence
5'-GAAGT CCAGGGCGACGTAGCA-3' (SEQ ID NO: 22). PCR was performed
using standard conditions in 25 ul reactions, with an annealing
temperature of 61 degrees Celsius. Thirty-two cycles of PCR were
performed with the Beer primers and twenty-four cycles were
performed with the beta-actin primers.
[0305] Following amplification, 12 ul from each reaction were
analyzed by agarose gel electrophoresis and ethidium bromide
staining. See FIG. 2A.
[0306] B. RNA In-situ Hybridization of Mouse Embryo Sections:
[0307] The full length mouse Beer cDNA (Sequence ID No. 11) was
cloned into the pCR2.1 vector (Invitrogen, Carlsbad, Calif.) in the
antisense and sense direction using the manufacturer's protocol.
.sup.35S-alpha-GTP-labeled cRNA sense and antisense transcripts
were synthesized using in-vitro transcription reagents supplied by
Ambion, Inc (Austin, Tex.). In-situ hybridization was performed
according to the protocols of Lyons et al. (J. Cell Biol.
111:2427-2436, 1990).
[0308] The mouse Beer cRNA probe detected a specific message
expressed in the neural tube, limb buds, blood vessels and
ossifying cartilages of developing mouse embryos. Panel A in FIG. 3
shows expression in the apical ectodermal ridge (aer) of the limb
(l) bud, blood vessels (bv) and the neural tube (nt). Panel B shows
expression in the 4.sup.th ventricle of the brain (4). Panel C
shows expression in the mandible (ma) cervical vertebrae (cv),
occipital bone (oc), palate (pa) and a blood vessel (bv). Panel D
shows expression in the ribs (r) and a heart valve (va). Panel A is
a transverse section of 10.5 dpc embryo. Panel B is a sagittal
section of 12.5 dpc embryo and panels C and D are sagittal sections
of 15.5 dpc embryos.
[0309] ba=branchial arch, h=heart, te=telencephalon (forebrain),
b=brain, f=frontonasal mass, g=gut, h=heart, j=jaw, li=liver,
lu=lung, ot=otic vesicle, ao=, sc=spinal cord, skm=skeletal muscle,
ns=nasal sinus, th=thymus, to=tongue, fl=forelimb, di=diaphragm
Example 3
[0310] Expression and Purification of Recombinant Beer Protein
[0311] A. Expression in COS-1 Cells:
[0312] The DNA sequence encoding the full length human Beer protein
was amplified using the following PCR oligonucleotide primers: The
5' oligonucleotide primer had the sequence
5'-AAGCTTGGTACCATGCAGCTCCCAC-3' (SEQ ID NO: 23) and contained a
HindIII restriction enzyme site (in bold) followed by 19
nucleotides of the Beer gene starting 6 base pairs prior to the
presumed amino terminal start codon (ATG). The 3' oligonucleotide
primer had the sequence 5'-AAGCTTCTACTTGTCATCGTCGTCCT
TGTAGTCGTAGGCGTTCTCCAGCT-3' (SEQ ID NO: 24) and contained a HindIII
restriction enzyme site (in bold) followed by a reverse complement
stop codon (CTA) followed by the reverse complement of the FLAG
epitope (underlined, Sigma-Aldrich Co., St. Louis, Mo.) flanked by
the reverse complement of nucleotides coding for the carboxy
terminal 5 amino acids of the Beer. The PCR product was TA cloned
("Original TA Cloning Kit", Invitrogen, Carlsbad, Calif.) and
individual clones were screened by DNA sequencing. A
sequence-verified clone was then digested by HindIII and purified
on a 1.5% agarose gel using a commercially available reagents
("QIAquick Gel Extraction Kit", Qiagen Inc., Valencia, Calif.).
This fragment was then ligated to HindIII digested,
phosphatase-treated pcDNA3.1 (Invitrogen, Carlsbad, Calif.) plasmid
with T4 DNA ligase. DH10B E. coli were transformed and plated on
LB, 100 .mu.g/ml ampicillin plates. Colonies bearing the desired
recombinant in the proper orientation were identified by a
PCR-based screen, using a 5' primer corresponding to the T7
promoter/priming site in pcDNA3.1 and a 3' primer with the sequence
5'-GCACTGGCCGGAGCACACC-3' (SEQ ID NO: 25) that corresponds to the
reverse complement of internal BEER sequence. The sequence of the
cloned fragment was confirmed by DNA sequencing.
[0313] COS-1 cells (ATCC# CRL-1650) were used for transfection. 50
.mu.g of the expression plasmid pcDNA-Beer-Flag was transfected
using a commercially available kit following protocols supplied by
the manufacturer ("DEAE-Dextran Transfection Kit", Sigma Chemical
Co., St. Louis, Mo.). The final media following transfection was
DMEM (Life Technologies, Rockville, Md.) containing 0.1% Fetal
Bovine Serum. After 4 days in culture, the media was removed.
Expression of recombinant BEER was analyzed by SDS-PAGE and Western
Blot using anti-FLAG.RTM. M2 monoclonal antibody (Sigma-Aldrich
Co., St. Louis, Mo.). Purification of recombinant BEER protein was
performed using an anti-FLAG M2 affinity column ("Mammalian
Transient Expression System", Sigma-Aldrich Co., St. Louis, Mo.).
The column profile was analyzed via SDS-PAGE and Western Blot using
anti-FLAG M2 monoclonal antibody.
[0314] B. Expression in SF9 Insect Cells:
[0315] The human Beer gene sequence was amplified using PCR with
standard conditions and the following primers:
1 Sense primer: 5'-GTCGTCGGATCCATGGGGTGGCAGGCGTTCAAGAATGAT-3' (SEQ
ID NO:26) Antisense primer: 5'-GTCGTCAAGCTTCTACTTGTCATCGTCCTTGTAG-
TCGTAGGCGTTCTCCAGCTCGGC-3' (SEQ ID NO:27)
[0316] The resulting cDNA contained the coding region of Beer with
two modifications. The N-terminal secretion signal was removed and
a FLAG epitope tag (Sigma) was fused in frame to the C-terminal end
of the insert. BamH1 and HindIII cloning sites were added and the
gene was subcloned into pMelBac vector (Invitrogen) for transfer
into a baculoviral expression vector using standard methods.
[0317] Recombinant baculoviruses expressing Beer protein were made
using the Bac-N-Blue transfection kit (Invitrogen) and purified
according to the manufacturers instructions.
[0318] SF9 cells (Invitrogen) were maintained in TNM_FH media
(Invitrogen) containing 10% fetal calf serum. For protein
expression, SF9 cultures in spinner flasks were infected at an MOI
of greater than 10. Samples of the media and cells were taken daily
for five days, and Beer expression monitored by western blot using
an anti-FLAG M2 monoclonal antibody (Sigma) or an anti-Beer rabbit
polyclonal antiserum.
[0319] After five days the baculovirus-infected SF9 cells were
harvested by centrifugation and cell associated protein was
extracted from the cell pellet using a high salt extraction buffer
(1.5 M NaCl, 50 mM Tris pH 7.5). The extract (20 ml per 300 ml
culture) was clarified by centrifugation, dialyzed three times
against four liters of Tris buffered saline (150 mM NaCl, 50 mM
Tris pH 7.5), and clarified by centrifugation again. This high salt
fraction was applied to Hitrap Heparin (Pharmacia; 5 ml bed
volume), washed extensively with HEPES buffered saline (25 mM HEPES
7.5, 150 mM Nacl) and bound proteins were eluted with a gradient
from 150 mM NaCl to 1200 mM NaCl. Beer elution was observed at
aproximately 800 mM NaCl. Beer containing fractions were
supplemented to 10% glycerol and 1 mM DTT and frozen at -80 degrees
C.
Example 4
[0320] Preparation and Testing of Polyclonal Antibodies to Beer,
Gremlin, and Dan
[0321] A. Preparation of Antigen:
[0322] The DNA sequences of Human Beer, Human Gremlin, and Human
Dan were amplified using standard PCR methods with the following
oligonucleotide primers:
2 H. Beer Sense: (SEQ ID NO:28) 5'-GACTTGGATCCCAGGGGTGGCAGGCGTTC-3'
Antisense (SEQ ID NO:29) 5'-AGCATAAGCTTCTAGTAGGCGTTCTCCAG-3' H.
Gremlin Sense: (SEQ ID NO:30) 5'-GACTTGGATCCGAAGGGAAAAA- GAAAGGG-3'
Antisense: (SEQ ID NO:31) 5'-AGCATAAGCTTTTAATCCAAATCGATGGA-3' H.
Dan Sense: (SEQ ID NO:32) 5'-ACTACGAGCTCGGCCCCACCACCCATCAACAAG-3'
Antisense: (SEQ ID NO:33)
5'-ACTTAGAAGCTTTCAGTCCTCAGCCCCCTCTTCC-3'
[0323] In each case the listed primers amplified the entire coding
region minus the secretion signal sequence. These include
restriction sites for subcloning into the bacterial expression
vector pQE-30 (Qiagen Inc., Valencia, Calif.) at sites
BamHI/HindIII for Beer and Gremlin, and sites SacI/HindIII for Dan.
pQE30 contains a coding sequence for a 6.times. His tag at the 5'
end of the cloning region. The completed constructs were
transformed into E. coli strain M-15/pRep (Qiagen Inc) and
individual clones verified by sequencing. Protein expression in
M-15/pRep and purification (6.times. His affinity tag binding to
Ni-NTA coupled to Sepharose) were performed as described by the
manufacturer (Qiagen, The QIAexpressionist).
[0324] The E. coli-derived Beer protein was recovered in
significant quantity using solubilization in 6M guanidine and
dialyzed to 2-4M to prevent precipitation during storage. Gremlin
and Dan protein were recovered in higher quantity with
solubilization in 6M guanidine and a post purification guanidine
concentration of 0.5M.
[0325] B. Production and Testing of Polyclonal Antibodies:
[0326] Polyclonal antibodies to each of the three antigens were
produced in rabbit and in chicken hosts using standard protocols (R
& R Antibody, Stanwood, Wash.; standard protocol for rabbit
immunization and antisera recovery; Short Protocols in Molecular
Biology. 2nd edition. 1992. 11.37-11.41. Contributors Helen M.
Cooper and Yvonne Paterson; chicken antisera was generated with
Strategic Biosolutions, Ramona, Calif.).
[0327] Rabbit antisera and chicken egg Igy fraction were screened
for activity via Western blot. Each of the three antigens was
separated by PAGE and transferred to 0.45 um nitrocellulose (Novex,
San Diego, Calif.). The membrane was cut into strips with each
strip containing approximately 75 ng of antigen. The strips were
blocked in 3% Blotting Grade Block (Bio-Rad Laboratories, Hercules,
Calif.) and washed 3 times in 1.times. Tris buffer saline
(TBS)/0.02% TWEEN buffer. The primary antibody (preimmunization
bleeds, rabbit antisera or chicken egg IgY in dilutions ranging
from 1:100 to 1:10,000 in blocking buffer) was incubated with the
strips for one hour with gentle rocking. A second series of three
washes 1.times. TBS/0.02% TWEEN was followed by an one hour
incubation with the secondary antibody (peroxidase conjugated
donkey anti-rabbit, Amersham Life Science, Piscataway, N.J.; or
peroxidase conjugated donkey anti-chicken, Jackson ImmunoResearch,
West Grove, Pa.). A final cycle of 3.times. washes of 1.times.
TBS/0.02% TWEEN was performed and the strips were developed with
Lumi-Light Western Blotting Substrate (Roche Molecular
Biochemicals, Mannheim, Germany).
[0328] C. Antibody Cross-Reactivity Test:
[0329] Following the protocol described in the previous section,
nitrocellulose strips of Beer, Gremlin or Dan were incubated with
dilutions (1:5000 and 1:10,000) of their respective rabbit antisera
or chicken egg IgY as well as to antisera or chicken egg Igy
(dilutions 1:1000 and 1:5000) made to the remaining two antigens.
The increased levels of nonmatching antibodies was performed to
detect low affinity binding by those antibodies that may be seen
only at increased concentration. The protocol and duration of
development is the same for all three binding events using the
protocol described above. There was no antigen cross-reactivity
observed for any of the antigens tested.
Example 5
[0330] Interaction of Beer with TGF-Beta Super-Family Proteins
[0331] The interaction of Beer with proteins from different
phylogenetic arms of the TGF-.beta. superfamily were studied using
immunoprecipitation methods. Purified TGF.beta.-1, TGF.beta.-2,
TGF.beta.-3, BMP-4, BMP-5, BMP-6 and GDNF were obtained from
commerical sources (R&D systems; Minneapolis, Minn.). A
representative protocol is as follows. Partially purified Beer was
dialyzed into HEPES buffered saline (25 mM HEPES 7.5, 150 mM NaCl).
Immunoprecipitations were done in 300 ul of IP buffer (150 mM NaCl,
25 mM Tris pH 7.5, 1 mM EDTA, 1.4 mM .beta.-mercaptoethanol, 0.5 %
triton .times.100, and 10% glycerol). 30 ng recombinant human BMP-5
protein (R&D systems) was applied to 15 ul of FLAG affinity
matrix (Sigma; St Louis Mo.)) in the presence and absence of 500 ng
FLAG epitope-tagged Beer. The proteins were incubated for 4 hours@
4.degree. C. and then the affinity matrix-associated proteins were
washed 5 times in IP buffer (1 ml per wash). The bound proteins
were eluted from the affinity matrix in 60 microliters of 1.times.
SDS PAGE sample buffer. The proteins were resolved by SDS PAGE and
Beer associated BMP-5 was detected by western blot using anti-BMP-5
antiserum (Research Diagnostics, Inc) (see FIG. 5).
[0332] BEER Ligand Binding Assay:
[0333] FLAG-Beer protein (20 ng) is added to 100 ul PBS/0.2% BSA
and adsorbed into each well of 96 well microtiter plate previously
coated with anti-FLAG monoclonal antibody (Sigma; St Louis Mo.) and
blocked with 10% BSA in PBS. This is conducted at room temperature
for 60 minutes. This protein solution is removed and the wells are
washed to remove unbound protein. BMP-5 is added to each well in
concentrations ranging from 10 pM to 500 nM in PBS/0.2% BSA and
incubated for 2 hours at room temperature. The binding solution is
removed and the plate washed with three times with 200 ul volumes
of PBS/0.2% BSA. BMP-5 levels are then detected using BMP-5
anti-serum via ELISA (F. M. Ausubel et al (1998) Current Protocols
in Mol Biol. Vol 2 11.2.1-11.2.22). Specific binding is calculated
by subtracting non-specific binding from total binding and analyzed
by the LIGAND program (Munson and Podbard, Anal. Biochem., 107,
p220-239, (1980).
[0334] In a variation of this method, Beer is engineered and
expressed as a human Fc fusion protein. Likewise the ligand BMP is
engineered and expressed as mouse Fc fusion. These proteins are
incubated together and the assay conducted as described by Mellor
et al using homogeneous time resolved fluorescence detection (G. W.
Mellor et al., J of Biomol Screening, 3(2) 91-99, 1998).
Example 6
[0335] Screening Assay for Inhibition of TGF-Beta Binding-Protein
Binding to TGF-Beta Family Members
[0336] The assay described above is replicated with two exceptions.
First, BMP concentration is held fixed at the Kd determined
previously. Second, a collection of antagonist candidates is added
at a fixed concentration (20 uM in the case of the small organic
molecule collections and 1 uM in antibody studies). These candidate
molecules (antagonists) of TGF-beta binding-protein binding include
organic compounds derived from commercial or internal collections
representing diverse chemical structures. These compounds are
prepared as stock solutions in DMSO and are added to assay wells at
.ltoreq.1% of final volume under the standard assay conditions.
These are incubated for 2 hours at room temperature with the BMP
and Beer, the solution removed and the bound BMP is quantitated as
described. Agents that inhibit 40% of the BMP binding observed in
the absence of compound or antibody are considered antagonists of
this interaction. These are further evaluated as potential
inhibitors based on titration studies to determine their inhibition
constants and their influence on TGF-beta binding-protein binding
affinity. Comparable specificity control assays may also be
conducted to establish the selectivity profile for the identified
antagonist through studies using assays dependent on the BMP ligand
action (e.g. BMP/BMP receptor competition study).
Example 7
[0337] Inhibition of TGF-Beta Binding-Protein Localization to Bone
Matrix
[0338] Evaluation of inhibition of localization to bone matrix
(hydroxyapatite) is conducted using modifications to the method of
Nicolas (Nicolas, V. Calcif Tissue Int 57:206, 1995). Briefly,
.sup.125I-labelled TGF-beta binding-protein is prepared as
described by Nicolas (supra). Hydroxyapatite is added to each well
of a 96 well microtiter plate equipped with a polypropylene
filtration membrane (Polyfiltroninc, Weymouth Mass.). TGF-beta
binding-protein is added to 0.2% albumin in PBS buffer. The wells
containing matrix are washed 3 times with this buffer. Adsorbed
TGF-beta binding-protein is eluted using 0.3M NaOH and
quantitated.
[0339] Inhibitor identification is conducted via incubation of
TGF-beta binding-protein with test molecules and applying the
mixture to the matrix as described above. The matrix is washed 3
times with 0.2% albumin in PBS buffer. Adsorbed TGF-beta
binding-protein is eluted using 0.3 M NaOH and quantitated. Agents
that inhibit 40% of the TGF-beta binding-protein binding observed
in the absence of compound or antibody are considered bone
localization inhibitors. These inhibitors are further characterized
through dose response studies to determine their inhibition
constants and their influence on TGF-beta binding-protein binding
affinity.
Example 8
[0340] Construction of TGF-Beta Binding-Protein Mutant
[0341] A. Mutagenesis:
[0342] A full-length TGF-beta binding-protein cDNA in pBluescript
SK serves as a template for mutagenesis. Briefly, appropriate
primers (see the discussion provided above) are utilized to
generate the DNA fragment by polymerase chain reaction using Vent
DNA polymerase (New England Biolabs, Beverly, Mass.). The
polymerase chain reaction is run for 23 cycles in buffers provided
by the manufacturer using a 57.degree. C. annealing temperature.
The product is then exposed to two restriction enzymes and after
isolation using agarose gel electrophoresis, ligated back into
pRBP4-503 from which the matching sequence has been removed by
enzymatic digestion. Integrity of the mutant is verified by DNA
sequencing.
[0343] B. Mammalian Cell Expression and Isolation of Mutant
TGF-Beta Binding-Protein:
[0344] The mutant TGF-beta binding-protein cDNAs are transferred
into the pcDNA3.1 mammalian expression vector described in EXAMPLE
3. After verifying the sequence, the resultant constructs are
transfected into COS-1 cells, and secreted protein is purified as
described in EXAMPLE 3.
Example 9
[0345] Animal Models-I Generation of Transgenic Mice Overexpressing
the Beer Gene
[0346] The .about.200 kilobase (kb) BAC clone 15G5, isolated from
the CITB mouse genomic DNA library (distributed by Research
Genetics, Huntsville, Ala.) was used to determine the complete
sequence of the mouse Beer gene and its 5' and 3' flanking regions.
A 41 kb SalI fragment, containing the entire gene body, plus
.about.17 kb of 5' flanking and .about.20 kb of 3' flanking
sequence was sub-cloned into the BamHI site of the SuperCosI cosmid
vector (Stratagene, La Jolla, Calif.) and propagated in the E. coli
strain DH10B. From this cosmid construct, a 35 kb MluI--AviII
restriction fragment (Sequence No. 6), including the entire mouse
Beer gene, as well as 17 kb and 14 kb of 5' and 3' flanking
sequence, respectively, was then gel purified, using conventional
means, and used for microinjection of mouse zygotes (DNX
Transgenics; U.S. Pat. No. 4,873,191). Founder animals in which the
cloned DNA fragment was integrated randomly into the genome were
obtained at a frequency of 5-30% of live-born pups. The presence of
the transgene was ascertained by performing Southern blot analysis
of genomic DNA extracted from a small amount of mouse tissue, such
as the tip of a tail. DNA was extracted using the following
protocol: tissue was digested overnight at 55.degree. C. in a lysis
buffer containing 200 mM NaCl, 100 mM Tris pH8.5, 5 mM EDTA, 0.2%
SDS and 0.5 mg/ml Proteinase K. The following day, the DNA was
extracted once with phenol/chloroform (50:50), once with
chloroform/isoamylalcohol (24:1) and precipitated with ethanol.
Upon resuspension in TE (10 mM Tris pH7.5, 1 mM EDTA) 8-10 ug of
each DNA sample were digested with a restriction endonuclease, such
as EcoRI, subjected to gel electrophoresis and transferred to a
charged nylon membrane, such as HyBondN+ (Amersham, Arlington
Heights, Ill.). The resulting filter was then hybridized with a
radioactively labelled fragment of DNA deriving from the mouse Beer
gene locus, and able to recognize both a fragment from the
endogenous gene locus and a fragment of a different size deriving
from the transgene. Founder animals were bred to normal
non-transgenic mice to generate sufficient numbers of transgenic
and non-transgenic progeny in which to determine the effects of
Beer gene overexpression. For these studies, animals at various
ages (for example, 1 day, 3 weeks, 6 weeks, 4 months) are subjected
to a number of different assays designed to ascertain gross
skeletal formation, bone mineral density, bone mineral content,
osteoclast and osteoblast activity, extent of endochondral
ossification, cartilage formation, etc. The transcriptional
activity from the transgene may be determined by extracting RNA
from various tissues, and using an RT-PCR assay which takes
advantage of single nucleotide polymorphisms between the mouse
strain from which the transgene is derived (129 Sv/J) and the
strain of mice used for DNA microinjection
[(C57BL5/J.times.SJL/J)F2].
[0347] Animal Models--II
[0348] Disruption of the Mouse Beer Gene By Homologous
Recombination
[0349] Homologous recombination in embryonic stem (ES) cells can be
used to inactivate the endogenous mouse Beer gene and subsequently
generate animals carrying the loss-of-function mutation. A reporter
gene, such as the E. coli .beta.-galactosidase gene, was engineered
into the targeting vector so that its expression is controlled by
the endogenous Beer gene's promoter and translational initiation
signal. In this way, the spatial and temporal patterns of Beer gene
expression can be determined in animals carrying a targeted
allele.
[0350] The targeting vector was constructed by first cloning the
drug-selectable phosphoglycerate kinase (PGK) promoter driven
neomycin-resistance gene (neo) cassette from pGT-N29 (New England
Biolabs, Beverly, Mass.) into the cloning vector pSP72 (Promega,
Madson, Wis.). PCR was used to flank the PGKneo cassette with
bacteriophage P1 loxP sites, which are recognition sites for the P1
Cre recombinase (Hoess et al., PNAS USA, 79:3398, 1982). This
allows subsequent removal of the neo-resistance marker in targeted
ES cells or ES cell-derived animals (U.S. Pat. No. 4,959,317). The
PCR primers were comprised of the 34 nucleotide (ntd) loxP
sequence, 15-25 ntd complementary to the 5' and 3' ends of the
PGKneo cassette, as well as restriction enzyme recognition sites
(BamHI in the sense primer and EcoRI in the anti-sense primer) for
cloning into pSP72. The sequence of the sense primer was
5'-AATCTGGATCCATAACTTCGTATAGCATACATTATACGAAGTTATCTGCAG
GATTCGAGGGCCCCT-3' (SEQ ID NO: 34); sequence of the anti-sense
primer was 5'-AATCTGAATTCCACCGGTGTTAATTAAATAACTTCGT
ATAATGTATGCTATACGAAGTTATAGATCTAG- AG TCAGCTTCTGA-3' (SEQ ID NO:
35).
[0351] The next step was to clone a 3.6 kb XhoI-HindIII fragment,
containing the E. coli .beta.-galactosidase gene and SV40
polyadenylation signal from pSV.beta. (Clontech, Palo Alto, Calif.)
into the pSP72-PGKneo plasmid. The "short arm" of homology from the
mouse Beer gene locus was generated by amplifying a 2.4 kb fragment
from the BAC clone 15G5. The 3' end of the fragment coincided with
the translational initiation site of the Beer gene, and the
anti-sense primer used in the PCR also included 30 ntd
complementary to the 5' end of the .beta.-galactosidase gene so
that its coding region could be fused to the Beer initiation site
in-frame. The approach taken for introducing the "short arm" into
the pSP72-.beta.gal-PGKneo plasmid was to linearize the plasmid at
a site upstream of the .beta.-gal gene and then to co-transform
this fragment with the "short arm" PCR product and to select for
plasmids in which the PCR product was integrated by homologous
recombination. The sense primer for the "short arm" amplification
included 30 ntd complementary to the pSP72 vector to allow for this
recombination event. The sequence of the sense primer was
5'-ATTTAGGTGACACT ATAGAACTCGAGCAGCTGAAGCTTAACCACATGGTGGC-
TCACAACCAT-3' (SEQ ID NO: 36) and the sequence of the anti-sense
primer was 5'-AACGACGGCCAGTGAATCCGTA
ATCATGGTCATGCTGCCAGGTGGAGGAGGGCA-3' (SEQ ID NO: 37).
[0352] The "long arm" from the Beer gene locus was generated by
amplifying a 6.1 kb fragment from BAC clone 15G5 with primers which
also introduce the rare-cutting restriction enzyme sites SgrAI,
FseI, AscI and PacI. Specifically, the sequence of the sense primer
was 5'-ATTACCACCGGTGACACCCGCTTCCTGACAG-3' (SEQ ID NO: 38); the
sequence of the anti-sense primer was
5'-ATTACTTAATTAAACATGGCGCGCCAT
ATGGCCGGCCCCTAATTGCGGCGCATCGTTAATT-3' (SEQ ID NO: 39). The
resulting PCR product was cloned into the TA vector (Invitrogen,
Carlsbad, Calif.) as an intermediate step.
[0353] The mouse Beer gene targeting construct also included a
second selectable marker, the herpes simplex virus I thymidine
kinase gene (HSVTK) under the control of rous sarcoma virus long
terminal repeat element (RSV LTR). Expression of this gene renders
mammalian cells sensitive (and inviable) to gancyclovir; it is
therefore a convenient way to select against neomycin-resistant
cells in which the construct has integrated by a non-homologous
event (U.S. Pat. No. 5,464,764). The RSVLTR-HSVTK cassette was
amplified from pPS1337 using primers that allow subsequent cloning
into the FseI and AscI sites of the "long arm"-TA vector plasmid.
For this PCR, the sequence of the sense primer was
5'-ATTACGGCCGGCCGCAAAGGAATTCAAGA TCTGA-3' (SEQ ID NO: 40); the
sequence of the anti-sense primer was 5'-ATTACGGCGCGCCCCTC
ACAGGCCGCACCCAGCT-3' (SEQ ID NO: 41).
[0354] The final step in the construction of the targeting vector
involved cloning the 8.8 kb SgrAI-AscI fragment containing the
"long arm" and RSVLTR-HSVTK gene into the SgrAI and AscI sites of
the pSP72-"short arm"-.beta.gal-PGKneo plasmid. This targeting
vector was linearized by digestion with either AscI or PacI before
electroporation into ES cells.
Example 10
[0355] Antisense-Mediated Beer Inactivation
[0356] 17-nucleotide antisense oligonucleotides are prepared in an
overlapping format, in such a way that the 5' end of the first
oligonucleotide overlaps the translation initiating AUG of the Beer
transcript, and the 5' ends of successive oligonucleotides occur in
5 nucleotide increments moving in the 5' direction (up to 50
nucleotides away), relative to the Beer AUG. Corresponding control
oligonucleotides are designed and prepared using equivalent base
composition but redistributed in sequence to inhibit any
significant hybridization to the coding mRNA. Reagent delivery to
the test cellular system is conducted through cationic lipid
delivery (P. L. Felgner, Proc. Natl. Acad. Sci. USA 84:7413, 1987).
2 ug of antisense oligonucleotide is added to 100 ul of reduced
serum media (Opti-MEM I reduced serum media; Life Technologies,
Gaithersburg Md.) and this is mixed with Lipofectin reagent (6 ul)
(Life Technologies, Gaithersburg Md.) in the 100 ul of reduced
serum media. These are mixed, allowed to complex for 30 minutes at
room temperature and the mixture is added to previously seeded
MC3T3E21 or KS483 cells. These cells are cultured and the mRNA
recovered. Beer mRNA is monitored using RT-PCR in conjunction with
Beer specific primers. In addition, separate experimental wells are
collected and protein levels characterized through western blot
methods described in Example 4. The cells are harvested,
resuspended in lysis buffer (50 mM Tris pH 7.5, 20 mM NaCl, 1 mM
EDTA, 1% SDS) and the soluble protein collected. This material is
applied to 10-20 % gradient denaturing SDS PAGE. The separated
proteins are transferred to nitrocellulose and the western blot
conducted as above using the antibody reagents described. In
parallel, the control oligonucleotides are added to identical
cultures and experimental operations are repeated. Decrease in Beer
mRNA or protein levels are considered significant if the treatment
with the antisense oligonucleotide results in a 50% change in
either instance compared to the control scrambled oligonucleotide.
This methodology enables selective gene inactivation and subsequent
phenotype characterization of the mineralized nodules in the tissue
culture model.
Example 11
[0357] Modeling of Sclerostin Core Region
[0358] Homology recognition techniques (e.g., PSI-BLAST (Altschul
et al., Nucleic Acids Res. 25:3389-402 (1997)), FUGUE (Shi et al.,
J. Mol. Biol. 310:243-57 (2001)) suggested that the core region of
SOST (SOST_Core) adopts a cystine-knot fold. FUGUE is a sensitive
method for detecting homology between sequences and structures.
Human Chorionic Gonadotropin .beta. (hCG-.beta.), for which an
experimentally determined 3D structure is known, was identified by
FUGUE (Shi et al., supra) as the closest homologue of SOST_Core.
Therefore, hCG-.beta. was used as the structural template to build
3D models for SOST_Core.
[0359] An alignment of SOST_Core and its close homologues is shown
in FIG. 7. Among the homologues shown in the alignment, only
hCG-.beta. (CGHB) had known 3D structure. The sequence identity
between SOST_Core and hCG-.beta. was approximately 25%. Eight CYS
residues were conserved throughout the family, emphasizing the
overall structural similarity between SOST_Core and hCG-.beta..
Three pairs of cystines (1-5, 3-7, 4-8) formed disulfide bonds
(shown with solid lines in FIG. 7) in a "knot" configuration, which
was characteristic to the cystine-knot fold. An extra disulfide
bond (2-6), shown as a dotted line in FIG. 7, was unique to this
family and distinguished the family of proteins from other
cystine-knot families (e.g., TGF-.beta., BMP).
[0360] SOST_Core was modeled using PDB (Berman et al., Acta
Crystallogr. D. Biol. Crystallogr. 58(Pt 6 Pt1):899-907 (2002))
entry 1HCN, the 3D structure of hCG-.beta. (Wu et al., Structure
2:545-58 (1994)), as the structural template. Models were
calculated with MODELER (Sali & Blundell, J. Mol. Biol.
234:779-815 (1993)). A snapshot of the best model is shown in FIG.
8.
[0361] Most of the cystine-knot proteins form dimers because of the
lack of hydrophobic core in a monomer (Scheufler et al., supra;
Schlunegger and Grutter, J. Mol. Biol. 231:445-58 (1993)); Wu et
al., supra). SOST likely follows the same rule and forms a
homodimer to increase its stability. Constructing a model for the
dimerized SOST_Core region presented several challenges because (1)
the sequence similarity between SOST_Core and hCG-.beta. was low
(25%); (2) instead of a homodimer, hCG-.beta. formed a heterodimer
with hCG-.alpha.; and (3) a number of different relative
conformations of monomers have been observed in dimerized
cystine-knot proteins from different families (e.g., PDGF,
TGF-.beta., Neurotrophin, IL-17F, Gonadotropin), which suggested
that the dimer conformation of SOST could deviate significantly
from the hCG-.alpha./.beta. heterodimer conformation. In
constructing the model, hCG-.alpha. was replaced with hCG-.beta.
from the heterodimer structure (1HCN) using structure
superimposition techniques combined with manual adjustment, and
then a SOST_Core homodimer model was built according to the pseudo
hCG-.beta. homodimer structure. The final model is shown in FIG.
9.
Example 12
[0362] Modeling SOST-BMP Interaction
[0363] This example describes protein modeling of type I and type
II receptor binding sites on BMP that are involved with interaction
between BMP and SOST.
[0364] Competition studies demonstrated that SOST competed with
both type I and type II receptors for binding to BMP. In an
ELISA-based competition assay, BMP-6 selectively interacted with
the sclerostin-coated surface (300 ng/well) with high affinity
(K.sub.D=3.4 nM). Increasing amounts of BMP receptor IA (FC fusion
construct) competed with sclerostin for binding to BMP-6 (11 nM)
(IC.sub.50=114 nM). A 10-fold molar excess of the BMP receptor was
sufficient to reduce binding of sclerostin to BMP-6 by
approximately 50%. This competition was also observed with a BMP
receptor II-FC fusion protein (IC.sub.50=36 nM) and DAN
(IC.sub.50=43 nM). Specificity of the assay was shown by lack of
competition for binding to BMP-6 between sclerostin and a rActivin
R1B-FC fusion protein, a TGF-.beta. receptor family member that did
not bind BMP.
[0365] The type I and type II receptor binding sites on a BMP
polypeptide have been mapped and were spatially separated
(Scheufler et al., supra; Innis et al., supra; Nickel et al.,
supra; Hart et al. supra). Noggin, another BMP antagonist that
binds to BMP with high affinity, contacts BMP at both type I and
type II receptor binding sites via the N-terminal portion of Noggin
(Groppe et al., supra). The two .beta.-strands in the core region
near the C-terminal also contact BMP at the type II receptor
binding site.
[0366] A manually tuned alignment of Noggin and SOST indicated that
the two polypeptides shared sequence similarity between the
N-terminal portions of the proteins and between the core regions.
An amino acid sequence alignment is presented in FIG. 10. The
cysteine residues that form the characteristic cys-knot were
conserved between Noggin and SOST. The overall sequence identity
was 24%, and the sequence identity within the N-terminal binding
region (alignment positions 1-45) was 33%. Two residues in the
Noggin N-terminal binding region, namely Leu (L) at alignment
position 21 and Glu (E) at position 23, were reported to play
important roles in BMP binding (Groppe et al., supra). Both
residues were conserved in SOST as well. The sequence similarity
within the core region (alignment positions 131-228) was about 20%,
but the cys-knot scaffold was maintained and a sufficient number of
key residues was conserved, supporting homology between Noggin and
SOST.
[0367] The Noggin structure was compared to SOST also to understand
how two SOST monomers dimerize. As shown in FIG. 11, the Noggin
structure suggested that the linker between the N-terminal region
and the core region not only played a role in connecting the two
regions, but also formed part of the dimerization interface between
two Noggin monomers. One major difference between Noggin and SOST
was that the linker between the N-terminal region and the core
region was much shorter in SOST.
[0368] The C-terminal region of SOST may play a role in SOST
dimerization. The sequence of Noggin ended with the core region,
while SOST had an extra C-terminal region. In the Noggin structure
a disulfide bond connected the C-termini of two Noggin monomers.
Thus, the C-terminal region of SOST started close to the interface
of two monomers and could contribute to dimerization. In addition,
secondary structure prediction showed that some portions of the
C-terminal region of SOST had a tendency to form helices. This
region in SOST may be responsible for the dimerization activity,
possibly through helix-helix packing, which mimicked the function
of the longer linker in Noggin. Another difference between the
structure of Noggin and SOST was the amino acid insertion in the
SOST core region at alignment positions 169-185 (see FIG. 10). This
insertion extended a .beta.-hairpin, which pointed towards the
dimerization interface in the Noggin structure (shown in FIG. 11 as
a loop region in the middle of the monomers and above the
C-terminal Cys residue). This elongated .beta.-hairpin could also
contribute to SOST dimerization.
Example 13
[0369] Design and Preparation of SOST Peptide Immunogens
[0370] This Example describes the design of SOST peptide immunogens
that are used for immunizing animals and generating antibodies that
block interactions between BMP and SOST and prevent dimer formation
of SOST monomers.
[0371] BMP Binding Fragments
[0372] The overall similarity between SOST and Noggin and the
similarity between the N-terminal regions of the two polypeptides
suggest that SOST may interact with BMP in a similar manner to
Noggin. That is, the N-terminal region of SOST may interact with
both the type I and type II receptor binding sites on BMP, and a
portion of the core region (amino acid alignment positions 190-220
in FIG. 10) may interact with the type II receptor binding site
such that antibodies specific for these SOST regions may block or
impair binding of BMP to SOST.
[0373] The amino acid sequences of these SOST polypeptide fragments
for rat and human SOST are provided as follows.
[0374] SOST_N_Linker: The N-terminal region (includes the short
linker that connects to the core region)
3 Human: QGWQAFKNDATEIIPELGEYPEPPPELENNKTMNRAENGGRPPHHPFETKDVSEYS
(SEQ ID NO:92) Rat: QGWQAFKNDATEIIPGLREYPEPPQELENNQTMNRA-
ENGGRPPHHPYDTKDVSEYS (SEQ ID NO:93)
[0375] SOST_Core_Bind: Portion of the core region that is likely to
contact BMP at its type II receptor binding site (extended slightly
at both termini to include the CYS residue anchors):
4 Human: CIPDRYRAQRVQLLCPGGEAPRARKVRLVASC (SEQ ID NO:94) Rat:
CIPDRYRAQRVQLLCPGGAAPRSRKVRLVASC (SEQ ID NO:95)
[0376] SOST Dimerization Fragments
[0377] The C-terminal region of SOST is likely to be involved in
the formation of SOST homodimers (see Example 12). The elongated
.beta.-hairpin may also play a role in homodimer formation.
Antibodies that specifically bind to such regions may prevent or
impair dimerization of SOST monomers, which may in turn interfere
with interaction between SOST and BMP. Polypeptide fragments in rat
and human SOST corresponding to these regions are as follows.
[0378] SOST_C: the C-terminal region
5 (SEQ ID NO:96) Human: LTRFHNQSELKDFGTEAARPQKGRKPRPRARSAKA-
NQAELENAY (SEQ ID NO:97) Rat:
LTRFHNQSELKDFGPETARPQKGRKPRPRARGAKANQAELENAY
[0379] SOST_Core_Dimer: Portion of the core region that is likely
involved in SOST dimerization (extended slightly at both termini to
include the Cys residue anchors):
6 Human: CGPARLLPNAIGRGKWWRPSGPDFRC (SEQ ID NO:98) Rat:
CGPARLLPNAIGRVKWWRPNGPDFRC (SEQ ID NO:99)
[0380] BMP Binding Fragment at SOST N-Terminus
[0381] The key N-terminal binding region of SOST (alignment
positions 1-35 in FIG. 10) was modeled on the basis of the
Noggin/BMP-7 complex structure (Protein Data Bank Entry No: 1M4U)
and the amino acid sequence alignment (see FIG. 10) to identify
amino acid residues of the SOST N-terminus that likely interact
with BMP. The model of SOST is presented in FIG. 12. In the
comparative model, phenylalanine (Phe, F) at alignment position 8
(see arrow and accompanying text) in the SOST sequence projects
into a hydrophobic pocket on the surface of the BMP dimer. The same
"knob-into-hole" feature has been observed in the BMP and type I
receptor complex structure (Nickel et al., supra), where Phe85 of
the receptor fits into the same pocket, which is a key feature in
ligand-type I receptor recognition for TGF-.beta. superfamily
members (including, for example, TGF-.beta. family, BMP family, and
the like). According to the model, a proline (Pro) directed turn is
also conserved, which allows the N-terminal binding fragment to
thread along the BMP dimer surface, traveling from type I receptor
binding site to type II receptor binding site on the other side of
the complex. Also conserved is another Pro-directed turn near the
carboxy end of the binding fragment, which then connects to the
linker region. Extensive contacts between SOST and BMP are evident
in FIG. 12.
[0382] Peptide Immunogens
[0383] Peptides were designed to encompass the SOST N-terminal
region predicted to make contact with BMP proteins. The peptide
sequences are presented below. For immunizing animals, the peptide
sequences were designed to overlap, and an additional cysteine was
added to the C-terminal end to facilitate crosslinking to KLH. The
peptides were then used for immunization. The peptide sequences of
the immunogens are as follows.
7 Human SOST: QGWQAFKNDATEIIPELGEY (SEQ ID NO:47)
TEIIPELGEYPEPPPELENN (SEQ ID NO:48) PEPPPELENNKTMNRAENGG (SEQ ID
NO:49) KTMNRAENGGRPPHHPFETK (SEQ ID NO:50) RPPHHPFETKDVSEYS (SEQ ID
NO:51) Human SOST Peptides with Additional Cys:
QGWQAFKNDATEIIPELGEY-C (SEQ ID NO:52) TEIIPELGEYPEPPPELENN-C (SEQ
ID NO:53) PEPPPELENNKTMNRAENGG-C (SEQ ID NO:54)
KTMNRAENGGRPPHHPFETK-C (SEQ ID NO:55) RPPHHPFETKDVSEYS-C (SEQ ID
NO:56) Rat SOST: QGWQAFKNDATEIIPGLREYPEPP (SEQ ID NO:57)
PEPPQELENNQTMNRAENGG (SEQ ID NO:58) ENGGRPPHHPYDTKDVSEYS (SEQ ID
NO:59) TEIIPGLREYPEPPQELENN (SEQ ID NO:60) Rat SOST Peptides with
Additional Cys: QGWQAFKNDATEIIPGLREYPEPP-C (SEQ ID NO:61)
PEPPQELENNQTMNRAENGG-C (SEQ ID NO:62) ENGGRPPHHPYDTKDVSEYS-C (SEQ
ID NO:63) TEIIPGLREYPEPPQELENN-C (SEQ ID NO:64)
[0384] The following peptides were designed to contain the amino
acid portion of core region that was predicted to make contact with
BMP proteins. Cysteine was added at the C-terminal end of each
peptide for conjugation to KLH, and the conjugated peptides were
used for immunization. In the Docking Core N-terminal Peptide an
internal cysteine was changed to a serine to avoid double
conjugation to KLH.
8 For Human SOST: Amino acid sequence without Cys residues added:
Docking_Core_N-terminal Peptide: IPDRYRAQRVQLLCPGGEAP (SEQ ID
NO:66) Docking_Core_Cterm_Peptide: QLLCPGGEAPRARKVRLVAS (SEQ ID
NO:67) Docking_Core_N-terminal_Peptide: IPDRYRAQRVQLLCPGGEAP-C (SEQ
ID NO:68) Docking_Core_Cterm_Petide: QLLCPGGEAPRARKVRLVAS-C (SEQ ID
NO:69) For Rat SOST: Amino acid sequence without Cys residues added
or substituted: Docking_Core_N-terminal- _Peptide:
IPDRYRAQRVQLLSPGG (SEQ ID NO:70) Docking_Core_Cterm_Pept- ide:
PGGAAPRSRKVRLVAS (SEQ ID NO:71) Peptide immunogens with Cys added
and substituted: Docking_Core_N-terminal_Peptide:
IPDRYRAQRVQLLSPGG-C (SEQ ID NO:72) Docking_Core_Cterm_Peptide:
PGGAAPRSRKVRLVAS-C (SEQ ID NO:73)
[0385] Two regions within SOST that potentially interact to form
SOST homodimers include the amino acids with the SOST core region
that are not present in Noggin. Human SOST peptides designed to
contain this sequence had a C-terminal or N-terminal Cys that was
conjugated to KLH. For the rat SOST peptide, a cysteine was added
to the carboxy terminus of the sequence (SEQ ID NO: 76). The KLH
conjugated peptides were used for immunization.
9 For Human SOST: CGPARLLPNAIGRGKWWRPS (SEQ ID NO:74)
IGRGKWWRPSGPDFRC (SEQ ID NO:75) For Rat SOST: PNAIGRVKWWRPNGPDFR
(SEQ ID NO:76) Rat SOST peptide with cysteine added
PNAIGRVKWWRPNGPDFR-C (SEQ ID NO:77)
[0386] The second region within SOST that potentially interacts to
form SOST homodimers includes the C-terminal region. Peptide
immunogens were designed to include amino acid sequences within
this region (see below). For conjugation to KLH, a cysteine residue
was added to the C-terminal end, and the conjugated peptides were
used for immunization.
10 For Human SOST: (SEQ ID NO:78) KRLTRFHNQS ELKDFGTEAA (SEQ ID
NO:79) ELKDFGTEAA RPQKGRKPRP (SEQ ID NO:80) RPQKGRKPRP RARSAKANQA
(SEQ ID NO:81) RARSAKANQA ELENAY Peptide immunogens with Cys added
at C-terminus: (SEQ ID NO:82) KRLTRFHNQS ELKDFGTEAA-C (SEQ ID
NO:83) ELKDFGTEAA RPQKGRKPRP-C (SEQ ID NO:84) RPQKGRKPRP
RARSAKANQA-C (SEQ ID NO:85) RARSAKANQA ELENAY-C For Rat SOST: (SEQ
ID NO:86) KRLTRFHNQSELKDFGPETARPQ (SEQ ID NO:87)
KGRKPRPRARGAKANQAELENAY (SEQ ID NO:88) SELKDFGPETARPQKGRKPRPRAR
Peptide immunogens with Cys added at C-terminus: (SEQ ID NO:89)
KRLTRFHNQSELKDFGPETARPQ-C (SEQ ID NO:90) KGRKPRPRARGAKANQAELENAY-C
(SEQ ID NO:91) SELKDFGPETARPQKGRKPRPRAR-C
Example 14
[0387] Assay for Detecting Binding of Antibodies to a TGF-Beta
Binding-Protein
[0388] This example describes an assay for detecting binding of a
ligand, for example, an antibody or antibody fragment thereof, to
sclerostin.
[0389] A FLAG.RTM.-sclerostin fusion protein was prepared according
to protocols provided by the manufacturer (Sigma Aldrich, St.
Louis, Mo.) and as described in U.S. Pat. No. 6,395,511. Each well
of a 96 well microtiter plate is coated with anti-FLAG.RTM.
monoclonal antibody (Sigma Aldrich) and then blocked with 10% BSA
in PBS. The fusion protein (20 ng) is added to 100 .mu.l PBS/0.2%
BSA and adsorbed onto the 96-well plate for 60 minutes at room
temperature. This protein solution is removed and the wells are
washed to remove unbound fusion protein. A BMP, for example, BMP-4,
BMP-5, BMP-6, or BMP-7, is diluted in PBS/0.2% BSA and added to
each well at concentrations ranging from 10 pM to 500 nM. After an
incubation for 2 hours at room temperature, the binding solution is
removed and the plate is washed three times with 200 .mu.l volumes
of PBS/0.2% BSA. Binding of the BMP to sclerostin is detected using
polyclonal antiserum or monoclonal antibody specific for the BMP
and an appropriate enzyme-conjugated second step reagent according
to standard ELISA techniques (see, e.g., Ausubel et al., Current
Protocols in Mol Biol. Vol 2 11.2.1-11.2.22 (1998)). Specific
binding is calculated by subtracting non-specific binding from
total binding and analyzed using the LIGAND program (Munson and
Podbard, Anal. Biochem. 107:220-39 (1980)).
[0390] Binding of sclerostin to a BMP is also detected by
homogeneous time resolved fluorescence detection (Mellor et al., J
Biomol. Screening, 3:91-99 (1998)). A polynucleotide sequence
encoding sclerostin is operatively linked to a human immunoglobulin
constant region in a recombinant nucleic acid construct and
expressed as a human Fc-sclerostin fusion protein according to
methods known in the art and described herein. Similarly, a BMP
ligand is engineered and expressed as a BMP-mouse Fe fusion
protein. These two fusion proteins are incubated together and the
assay conducted as described by Mellor et al.
Example 15
[0391] Screening Assay for Antibodies that Inhibit Binding of
TGF-Beta Family Members to TGF-Beta Binding Protein
[0392] This example describes a method for detecting an antibody
that inhibits binding of a TGF-beta family member to sclerostin. An
ELISA is performed essentially as described in Example 14 except
that the BMP concentration is held fixed at its Kd (determined, for
example, by BIAcore analysis). In addition, an antibody or a
library or collection of antibodies is added to the wells to a
concentration of 1 .mu.M. Antibodies are incubated for 2 hours at
room temperature with the BMP and sclerostin, the solution removed,
and the bound BMP is quantified as described (see Example 14).
Antibodies that inhibit 40% of the BMP binding observed in the
absence of antibody are considered antagonists of this interaction.
These antibodies are further evaluated as potential inhibitors by
performing titration studies to determine their inhibition
constants and their effect on TGF-beta binding-protein binding
affinity. Comparable specificity control assays may also be
conducted to establish the selectivity profile for the identified
antagonist using assays dependent on the BMP ligand action (e.g., a
BMP/BMP receptor competition study).
Example 16
[0393] Inhibition of TGF-Beta Binding-Protein Localization to Bone
Matrix
[0394] Evaluation of inhibition of localization to bone matrix
(hydroxyapatite) is conducted using modifications to the method of
Nicolas (Calcif. Tissue Int. 57:206-12 (1995)). Briefly,
.sup.125I-labelled TGF-beta binding-protein is prepared as
described by Nicolas (supra). Hydroxyapatite is added to each well
of a 96-well microtiter plate equipped with a polypropylene
filtration membrane (Polyfiltroninc, Weymouth Mass.). TGF-beta
binding-protein diluted in 0.2% albumin in PBS buffer is then added
to the wells. The wells containing matrix are washed 3 times with
0.2% albumin in PBS buffer. Adsorbed TGF-beta binding-protein is
eluted using 0.3 M NaOH and then quantified.
[0395] An antibody that inhibits or impairs binding of the
sclerostin TGF-beta binding protein to the hydroxyapatite is
identified by incubating the TGF-beta binding protein with the
antibody and applying the mixture to the matrix as described above.
The matrix is washed 3 times with 0.2% albumin in PBS buffer.
Adsorbed sclerostin is eluted with 0.3 M NaOH and then quantified.
An antibody that inhibits the level of binding of sclerostin to the
hydroxyapatite by at least 40% compared to the level of binding
observed in the absence of antibody is considered a bone
localization inhibitor. Such an antibody is further characterized
in dose response studies to determine its inhibition constant and
its effect on TGF-beta binding-protein binding affinity.
[0396] From the foregoing, although specific embodiments of the
invention have been described herein for purposes of illustration,
various modifications may be made without deviating from the spirit
and scope of the invention. Accordingly, the invention is not
limited except as by the appended claims.
Sequence CWU 1
1
143 1 2301 DNA Homo sapien 1 agagcctgtg ctactggaag gtggcgtgcc
ctcctctggc tggtaccatg cagctcccac 60 tggccctgtg tctcgtctgc
ctgctggtac acacagcctt ccgtgtagtg gagggccagg 120 ggtggcaggc
gttcaagaat gatgccacgg aaatcatccc cgagctcgga gagtaccccg 180
agcctccacc ggagctggag aacaacaaga ccatgaaccg ggcggagaac ggagggcggc
240 ctccccacca cccctttgag accaaagacg tgtccgagta cagctgccgc
gagctgcact 300 tcacccgcta cgtgaccgat gggccgtgcc gcagcgccaa
gccggtcacc gagctggtgt 360 gctccggcca gtgcggcccg gcgcgcctgc
tgcccaacgc catcggccgc ggcaagtggt 420 ggcgacctag tgggcccgac
ttccgctgca tccccgaccg ctaccgcgcg cagcgcgtgc 480 agctgctgtg
tcccggtggt gaggcgccgc gcgcgcgcaa ggtgcgcctg gtggcctcgt 540
gcaagtgcaa gcgcctcacc cgcttccaca accagtcgga gctcaaggac ttcgggaccg
600 aggccgctcg gccgcagaag ggccggaagc cgcggccccg cgcccggagc
gccaaagcca 660 accaggccga gctggagaac gcctactaga gcccgcccgc
gcccctcccc accggcgggc 720 gccccggccc tgaacccgcg ccccacattt
ctgtcctctg cgcgtggttt gattgtttat 780 atttcattgt aaatgcctgc
aacccagggc agggggctga gaccttccag gccctgagga 840 atcccgggcg
ccggcaaggc ccccctcagc ccgccagctg aggggtccca cggggcaggg 900
gagggaattg agagtcacag acactgagcc acgcagcccc gcctctgggg ccgcctacct
960 ttgctggtcc cacttcagag gaggcagaaa tggaagcatt ttcaccgccc
tggggtttta 1020 agggagcggt gtgggagtgg gaaagtccag ggactggtta
agaaagttgg ataagattcc 1080 cccttgcacc tcgctgccca tcagaaagcc
tgaggcgtgc ccagagcaca agactggggg 1140 caactgtaga tgtggtttct
agtcctggct ctgccactaa cttgctgtgt aaccttgaac 1200 tacacaattc
tccttcggga cctcaatttc cactttgtaa aatgagggtg gaggtgggaa 1260
taggatctcg aggagactat tggcatatga ttccaaggac tccagtgcct tttgaatggg
1320 cagaggtgag agagagagag agaaagagag agaatgaatg cagttgcatt
gattcagtgc 1380 caaggtcact tccagaattc agagttgtga tgctctcttc
tgacagccaa agatgaaaaa 1440 caaacagaaa aaaaaaagta aagagtctat
ttatggctga catatttacg gctgacaaac 1500 tcctggaaga agctatgctg
cttcccagcc tggcttcccc ggatgtttgg ctacctccac 1560 ccctccatct
caaagaaata acatcatcca ttggggtaga aaaggagagg gtccgagggt 1620
ggtgggaggg atagaaatca catccgcccc aacttcccaa agagcagcat ccctcccccg
1680 acccatagcc atgttttaaa gtcaccttcc gaagagaagt gaaaggttca
aggacactgg 1740 ccttgcaggc ccgagggagc agccatcaca aactcacaga
ccagcacatc ccttttgaga 1800 caccgccttc tgcccaccac tcacggacac
atttctgcct agaaaacagc ttcttactgc 1860 tcttacatgt gatggcatat
cttacactaa aagaatatta ttgggggaaa aactacaagt 1920 gctgtacata
tgctgagaaa ctgcagagca taatagctgc cacccaaaaa tctttttgaa 1980
aatcatttcc agacaacctc ttactttctg tgtagttttt aattgttaaa aaaaaaaagt
2040 tttaaacaga agcacatgac atatgaaagc ctgcaggact ggtcgttttt
ttggcaattc 2100 ttccacgtgg gacttgtcca caagaatgaa agtagtggtt
tttaaagagt taagttacat 2160 atttattttc tcacttaagt tatttatgca
aaagtttttc ttgtagagaa tgacaatgtt 2220 aatattgctt tatgaattaa
cagtctgttc ttccagagtc cagagacatt gttaataaag 2280 acaatgaatc
atgaccgaaa g 2301 2 213 PRT Homo sapien 2 Met Gln Leu Pro Leu Ala
Leu Cys Leu Val Cys Leu Leu Val His Thr 1 5 10 15 Ala Phe Arg Val
Val Glu Gly Gln Gly Trp Gln Ala Phe Lys Asn Asp 20 25 30 Ala Thr
Glu Ile Ile Pro Glu Leu Gly Glu Tyr Pro Glu Pro Pro Pro 35 40 45
Glu Leu Glu Asn Asn Lys Thr Met Asn Arg Ala Glu Asn Gly Gly Arg 50
55 60 Pro Pro His His Pro Phe Glu Thr Lys Asp Val Ser Glu Tyr Ser
Cys 65 70 75 80 Arg Glu Leu His Phe Thr Arg Tyr Val Thr Asp Gly Pro
Cys Arg Ser 85 90 95 Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly
Gln Cys Gly Pro Ala 100 105 110 Arg Leu Leu Pro Asn Ala Ile Gly Arg
Gly Lys Trp Trp Arg Pro Ser 115 120 125 Gly Pro Asp Phe Arg Cys Ile
Pro Asp Arg Tyr Arg Ala Gln Arg Val 130 135 140 Gln Leu Leu Cys Pro
Gly Gly Glu Ala Pro Arg Ala Arg Lys Val Arg 145 150 155 160 Leu Val
Ala Ser Cys Lys Cys Lys Arg Leu Thr Arg Phe His Asn Gln 165 170 175
Ser Glu Leu Lys Asp Phe Gly Thr Glu Ala Ala Arg Pro Gln Lys Gly 180
185 190 Arg Lys Pro Arg Pro Arg Ala Arg Ser Ala Lys Ala Asn Gln Ala
Glu 195 200 205 Leu Glu Asn Ala Tyr 210 3 2301 DNA Homo sapien 3
agagcctgtg ctactggaag gtggcgtgcc ctcctctggc tggtaccatg cagctcccac
60 tggccctgtg tctcgtctgc ctgctggtac acacagcctt ccgtgtagtg
gagggctagg 120 ggtggcaggc gttcaagaat gatgccacgg aaatcatccc
cgagctcgga gagtaccccg 180 agcctccacc ggagctggag aacaacaaga
ccatgaaccg ggcggagaac ggagggcggc 240 ctccccacca cccctttgag
accaaagacg tgtccgagta cagctgccgc gagctgcact 300 tcacccgcta
cgtgaccgat gggccgtgcc gcagcgccaa gccggtcacc gagctggtgt 360
gctccggcca gtgcggcccg gcgcgcctgc tgcccaacgc catcggccgc ggcaagtggt
420 ggcgacctag tgggcccgac ttccgctgca tccccgaccg ctaccgcgcg
cagcgcgtgc 480 agctgctgtg tcccggtggt gaggcgccgc gcgcgcgcaa
ggtgcgcctg gtggcctcgt 540 gcaagtgcaa gcgcctcacc cgcttccaca
accagtcgga gctcaaggac ttcgggaccg 600 aggccgctcg gccgcagaag
ggccggaagc cgcggccccg cgcccggagc gccaaagcca 660 accaggccga
gctggagaac gcctactaga gcccgcccgc gcccctcccc accggcgggc 720
gccccggccc tgaacccgcg ccccacattt ctgtcctctg cgcgtggttt gattgtttat
780 atttcattgt aaatgcctgc aacccagggc agggggctga gaccttccag
gccctgagga 840 atcccgggcg ccggcaaggc ccccctcagc ccgccagctg
aggggtccca cggggcaggg 900 gagggaattg agagtcacag acactgagcc
acgcagcccc gcctctgggg ccgcctacct 960 ttgctggtcc cacttcagag
gaggcagaaa tggaagcatt ttcaccgccc tggggtttta 1020 agggagcggt
gtgggagtgg gaaagtccag ggactggtta agaaagttgg ataagattcc 1080
cccttgcacc tcgctgccca tcagaaagcc tgaggcgtgc ccagagcaca agactggggg
1140 caactgtaga tgtggtttct agtcctggct ctgccactaa cttgctgtgt
aaccttgaac 1200 tacacaattc tccttcggga cctcaatttc cactttgtaa
aatgagggtg gaggtgggaa 1260 taggatctcg aggagactat tggcatatga
ttccaaggac tccagtgcct tttgaatggg 1320 cagaggtgag agagagagag
agaaagagag agaatgaatg cagttgcatt gattcagtgc 1380 caaggtcact
tccagaattc agagttgtga tgctctcttc tgacagccaa agatgaaaaa 1440
caaacagaaa aaaaaaagta aagagtctat ttatggctga catatttacg gctgacaaac
1500 tcctggaaga agctatgctg cttcccagcc tggcttcccc ggatgtttgg
ctacctccac 1560 ccctccatct caaagaaata acatcatcca ttggggtaga
aaaggagagg gtccgagggt 1620 ggtgggaggg atagaaatca catccgcccc
aacttcccaa agagcagcat ccctcccccg 1680 acccatagcc atgttttaaa
gtcaccttcc gaagagaagt gaaaggttca aggacactgg 1740 ccttgcaggc
ccgagggagc agccatcaca aactcacaga ccagcacatc ccttttgaga 1800
caccgccttc tgcccaccac tcacggacac atttctgcct agaaaacagc ttcttactgc
1860 tcttacatgt gatggcatat cttacactaa aagaatatta ttgggggaaa
aactacaagt 1920 gctgtacata tgctgagaaa ctgcagagca taatagctgc
cacccaaaaa tctttttgaa 1980 aatcatttcc agacaacctc ttactttctg
tgtagttttt aattgttaaa aaaaaaaagt 2040 tttaaacaga agcacatgac
atatgaaagc ctgcaggact ggtcgttttt ttggcaattc 2100 ttccacgtgg
gacttgtcca caagaatgaa agtagtggtt tttaaagagt taagttacat 2160
atttattttc tcacttaagt tatttatgca aaagtttttc ttgtagagaa tgacaatgtt
2220 aatattgctt tatgaattaa cagtctgttc ttccagagtc cagagacatt
gttaataaag 2280 acaatgaatc atgaccgaaa g 2301 4 23 PRT Homo sapien 4
Met Gln Leu Pro Leu Ala Leu Cys Leu Val Cys Leu Leu Val His Thr 1 5
10 15 Ala Phe Arg Val Val Glu Gly 20 5 2301 DNA Homo sapien 5
agagcctgtg ctactggaag gtggcgtgcc ctcctctggc tggtaccatg cagctcccac
60 tggccctgtg tctcatctgc ctgctggtac acacagcctt ccgtgtagtg
gagggccagg 120 ggtggcaggc gttcaagaat gatgccacgg aaatcatccg
cgagctcgga gagtaccccg 180 agcctccacc ggagctggag aacaacaaga
ccatgaaccg ggcggagaac ggagggcggc 240 ctccccacca cccctttgag
accaaagacg tgtccgagta cagctgccgc gagctgcact 300 tcacccgcta
cgtgaccgat gggccgtgcc gcagcgccaa gccggtcacc gagctggtgt 360
gctccggcca gtgcggcccg gcgcgcctgc tgcccaacgc catcggccgc ggcaagtggt
420 ggcgacctag tgggcccgac ttccgctgca tccccgaccg ctaccgcgcg
cagcgcgtgc 480 agctgctgtg tcccggtggt gaggcgccgc gcgcgcgcaa
ggtgcgcctg gtggcctcgt 540 gcaagtgcaa gcgcctcacc cgcttccaca
accagtcgga gctcaaggac ttcgggaccg 600 aggccgctcg gccgcagaag
ggccggaagc cgcggccccg cgcccggagc gccaaagcca 660 accaggccga
gctggagaac gcctactaga gcccgcccgc gcccctcccc accggcgggc 720
gccccggccc tgaacccgcg ccccacattt ctgtcctctg cgcgtggttt gattgtttat
780 atttcattgt aaatgcctgc aacccagggc agggggctga gaccttccag
gccctgagga 840 atcccgggcg ccggcaaggc ccccctcagc ccgccagctg
aggggtccca cggggcaggg 900 gagggaattg agagtcacag acactgagcc
acgcagcccc gcctctgggg ccgcctacct 960 ttgctggtcc cacttcagag
gaggcagaaa tggaagcatt ttcaccgccc tggggtttta 1020 agggagcggt
gtgggagtgg gaaagtccag ggactggtta agaaagttgg ataagattcc 1080
cccttgcacc tcgctgccca tcagaaagcc tgaggcgtgc ccagagcaca agactggggg
1140 caactgtaga tgtggtttct agtcctggct ctgccactaa cttgctgtgt
aaccttgaac 1200 tacacaattc tccttcggga cctcaatttc cactttgtaa
aatgagggtg gaggtgggaa 1260 taggatctcg aggagactat tggcatatga
ttccaaggac tccagtgcct tttgaatggg 1320 cagaggtgag agagagagag
agaaagagag agaatgaatg cagttgcatt gattcagtgc 1380 caaggtcact
tccagaattc agagttgtga tgctctcttc tgacagccaa agatgaaaaa 1440
caaacagaaa aaaaaaagta aagagtctat ttatggctga catatttacg gctgacaaac
1500 tcctggaaga agctatgctg cttcccagcc tggcttcccc ggatgtttgg
ctacctccac 1560 ccctccatct caaagaaata acatcatcca ttggggtaga
aaaggagagg gtccgagggt 1620 ggtgggaggg atagaaatca catccgcccc
aacttcccaa agagcagcat ccctcccccg 1680 acccatagcc atgttttaaa
gtcaccttcc gaagagaagt gaaaggttca aggacactgg 1740 ccttgcaggc
ccgagggagc agccatcaca aactcacaga ccagcacatc ccttttgaga 1800
caccgccttc tgcccaccac tcacggacac atttctgcct agaaaacagc ttcttactgc
1860 tcttacatgt gatggcatat cttacactaa aagaatatta ttgggggaaa
aactacaagt 1920 gctgtacata tgctgagaaa ctgcagagca taatagctgc
cacccaaaaa tctttttgaa 1980 aatcatttcc agacaacctc ttactttctg
tgtagttttt aattgttaaa aaaaaaaagt 2040 tttaaacaga agcacatgac
atatgaaagc ctgcaggact ggtcgttttt ttggcaattc 2100 ttccacgtgg
gacttgtcca caagaatgaa agtagtggtt tttaaagagt taagttacat 2160
atttattttc tcacttaagt tatttatgca aaagtttttc ttgtagagaa tgacaatgtt
2220 aatattgctt tatgaattaa cagtctgttc ttccagagtc cagagacatt
gttaataaag 2280 acaatgaatc atgaccgaaa g 2301 6 213 PRT Homo sapien
6 Met Gln Leu Pro Leu Ala Leu Cys Leu Ile Cys Leu Leu Val His Thr 1
5 10 15 Ala Phe Arg Val Val Glu Gly Gln Gly Trp Gln Ala Phe Lys Asn
Asp 20 25 30 Ala Thr Glu Ile Ile Arg Glu Leu Gly Glu Tyr Pro Glu
Pro Pro Pro 35 40 45 Glu Leu Glu Asn Asn Lys Thr Met Asn Arg Ala
Glu Asn Gly Gly Arg 50 55 60 Pro Pro His His Pro Phe Glu Thr Lys
Asp Val Ser Glu Tyr Ser Cys 65 70 75 80 Arg Glu Leu His Phe Thr Arg
Tyr Val Thr Asp Gly Pro Cys Arg Ser 85 90 95 Ala Lys Pro Val Thr
Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala 100 105 110 Arg Leu Leu
Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro Ser 115 120 125 Gly
Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gln Arg Val 130 135
140 Gln Leu Leu Cys Pro Gly Gly Glu Ala Pro Arg Ala Arg Lys Val Arg
145 150 155 160 Leu Val Ala Ser Cys Lys Cys Lys Arg Leu Thr Arg Phe
His Asn Gln 165 170 175 Ser Glu Leu Lys Asp Phe Gly Thr Glu Ala Ala
Arg Pro Gln Lys Gly 180 185 190 Arg Lys Pro Arg Pro Arg Ala Arg Ser
Ala Lys Ala Asn Gln Ala Glu 195 200 205 Leu Glu Asn Ala Tyr 210 7
2301 DNA Homo sapien 7 agagcctgtg ctactggaag gtggcgtgcc ctcctctggc
tggtaccatg cagctcccac 60 tggccctgtg tctcgtctgc ctgctggtac
acacagcctt ccgtgtagtg gagggccagg 120 ggtggcaggc gttcaagaat
gatgccacgg aaatcatccg cgagctcgga gagtaccccg 180 agcctccacc
ggagctggag aacaacaaga ccatgaaccg ggcggagaac ggagggcggc 240
ctccccacca cccctttgag accaaagacg tgtccgagta cagctgccgc gagctgcact
300 tcacccgcta cgtgaccgat gggccgtgcc gcagcgccaa gccggtcacc
gagctggtgt 360 gctccggcca gtgcggcccg gcgcgcctgc tgcccaacgc
catcggccgc ggcaagtggt 420 ggcgacctag tgggcccgac ttccgctgca
tccccgaccg ctaccgcgcg cagcgcgtgc 480 agctgctgtg tcccggtggt
gaggcgccgc gcgcgcgcaa ggtgcgcctg gtggcctcgt 540 gcaagtgcaa
gcgcctcacc cgcttccaca accagtcgga gctcaaggac ttcgggaccg 600
aggccgctcg gccgcagaag ggccggaagc cgcggccccg cgcccggagc gccaaagcca
660 accaggccga gctggagaac gcctactaga gcccgcccgc gcccctcccc
accggcgggc 720 gccccggccc tgaacccgcg ccccacattt ctgtcctctg
cgcgtggttt gattgtttat 780 atttcattgt aaatgcctgc aacccagggc
agggggctga gaccttccag gccctgagga 840 atcccgggcg ccggcaaggc
ccccctcagc ccgccagctg aggggtccca cggggcaggg 900 gagggaattg
agagtcacag acactgagcc acgcagcccc gcctctgggg ccgcctacct 960
ttgctggtcc cacttcagag gaggcagaaa tggaagcatt ttcaccgccc tggggtttta
1020 agggagcggt gtgggagtgg gaaagtccag ggactggtta agaaagttgg
ataagattcc 1080 cccttgcacc tcgctgccca tcagaaagcc tgaggcgtgc
ccagagcaca agactggggg 1140 caactgtaga tgtggtttct agtcctggct
ctgccactaa cttgctgtgt aaccttgaac 1200 tacacaattc tccttcggga
cctcaatttc cactttgtaa aatgagggtg gaggtgggaa 1260 taggatctcg
aggagactat tggcatatga ttccaaggac tccagtgcct tttgaatggg 1320
cagaggtgag agagagagag agaaagagag agaatgaatg cagttgcatt gattcagtgc
1380 caaggtcact tccagaattc agagttgtga tgctctcttc tgacagccaa
agatgaaaaa 1440 caaacagaaa aaaaaaagta aagagtctat ttatggctga
catatttacg gctgacaaac 1500 tcctggaaga agctatgctg cttcccagcc
tggcttcccc ggatgtttgg ctacctccac 1560 ccctccatct caaagaaata
acatcatcca ttggggtaga aaaggagagg gtccgagggt 1620 ggtgggaggg
atagaaatca catccgcccc aacttcccaa agagcagcat ccctcccccg 1680
acccatagcc atgttttaaa gtcaccttcc gaagagaagt gaaaggttca aggacactgg
1740 ccttgcaggc ccgagggagc agccatcaca aactcacaga ccagcacatc
ccttttgaga 1800 caccgccttc tgcccaccac tcacggacac atttctgcct
agaaaacagc ttcttactgc 1860 tcttacatgt gatggcatat cttacactaa
aagaatatta ttgggggaaa aactacaagt 1920 gctgtacata tgctgagaaa
ctgcagagca taatagctgc cacccaaaaa tctttttgaa 1980 aatcatttcc
agacaacctc ttactttctg tgtagttttt aattgttaaa aaaaaaaagt 2040
tttaaacaga agcacatgac atatgaaagc ctgcaggact ggtcgttttt ttggcaattc
2100 ttccacgtgg gacttgtcca caagaatgaa agtagtggtt tttaaagagt
taagttacat 2160 atttattttc tcacttaagt tatttatgca aaagtttttc
ttgtagagaa tgacaatgtt 2220 aatattgctt tatgaattaa cagtctgttc
ttccagagtc cagagacatt gttaataaag 2280 acaatgaatc atgaccgaaa g 2301
8 213 PRT Homo sapien 8 Met Gln Leu Pro Leu Ala Leu Cys Leu Val Cys
Leu Leu Val His Thr 1 5 10 15 Ala Phe Arg Val Val Glu Gly Gln Gly
Trp Gln Ala Phe Lys Asn Asp 20 25 30 Ala Thr Glu Ile Ile Arg Glu
Leu Gly Glu Tyr Pro Glu Pro Pro Pro 35 40 45 Glu Leu Glu Asn Asn
Lys Thr Met Asn Arg Ala Glu Asn Gly Gly Arg 50 55 60 Pro Pro His
His Pro Phe Glu Thr Lys Asp Val Ser Glu Tyr Ser Cys 65 70 75 80 Arg
Glu Leu His Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg Ser 85 90
95 Ala Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala
100 105 110 Arg Leu Leu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg
Pro Ser 115 120 125 Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg
Ala Gln Arg Val 130 135 140 Gln Leu Leu Cys Pro Gly Gly Glu Ala Pro
Arg Ala Arg Lys Val Arg 145 150 155 160 Leu Val Ala Ser Cys Lys Cys
Lys Arg Leu Thr Arg Phe His Asn Gln 165 170 175 Ser Glu Leu Lys Asp
Phe Gly Thr Glu Ala Ala Arg Pro Gln Lys Gly 180 185 190 Arg Lys Pro
Arg Pro Arg Ala Arg Ser Ala Lys Ala Asn Gln Ala Glu 195 200 205 Leu
Glu Asn Ala Tyr 210 9 642 DNA Cercopithecus pygerythrus 9
atgcagctcc cactggccct gtgtcttgtc tgcctgctgg tacacgcagc cttccgtgta
60 gtggagggcc aggggtggca ggccttcaag aatgatgcca cggaaatcat
ccccgagctc 120 ggagagtacc ccgagcctcc accggagctg gagaacaaca
agaccatgaa ccgggcggag 180 aatggagggc ggcctcccca ccaccccttt
gagaccaaag acgtgtccga gtacagctgc 240 cgagagctgc acttcacccg
ctacgtgacc gatgggccgt gccgcagcgc caagccagtc 300 accgagttgg
tgtgctccgg ccagtgcggc ccggcacgcc tgctgcccaa cgccatcggc 360
cgcggcaagt ggtggcgccc gagtgggccc gacttccgct gcatccccga ccgctaccgc
420 gcgcagcgtg tgcagctgct gtgtcccggt ggtgccgcgc cgcgcgcgcg
caaggtgcgc 480 ctggtggcct cgtgcaagtg caagcgcctc acccgcttcc
acaaccagtc ggagctcaag 540 gacttcggtc ccgaggccgc tcggccgcag
aagggccgga agccgcggcc ccgcgcccgg 600 ggggccaaag ccaatcaggc
cgagctggag aacgcctact ag 642 10 213 PRT Cercopithecus pygerythrus
10 Met Gln Leu Pro Leu Ala Leu Cys Leu Val Cys Leu Leu Val His Ala
1 5 10 15 Ala Phe Arg Val Val Glu Gly Gln Gly Trp Gln Ala Phe Lys
Asn Asp 20 25 30 Ala Thr Glu Ile Ile Pro Glu Leu Gly Glu Tyr Pro
Glu Pro Pro Pro 35 40 45 Glu Leu Glu Asn Asn Lys Thr Met Asn Arg
Ala Glu Asn Gly Gly Arg 50 55 60 Pro Pro His His Pro Phe Glu Thr
Lys Asp Val Ser Glu Tyr Ser Cys 65 70 75 80 Arg Glu Leu His Phe Thr
Arg Tyr Val Thr Asp Gly Pro Cys Arg Ser 85 90 95 Ala Lys Pro Val
Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala 100 105 110 Arg Leu
Leu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro Ser
115 120 125 Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gln
Arg Val 130 135 140 Gln Leu Leu Cys Pro Gly Gly Ala Ala Pro Arg Ala
Arg Lys Val Arg 145 150 155 160 Leu Val Ala Ser Cys Lys Cys Lys Arg
Leu Thr Arg Phe His Asn Gln 165 170 175 Ser Glu Leu Lys Asp Phe Gly
Pro Glu Ala Ala Arg Pro Gln Lys Gly 180 185 190 Arg Lys Pro Arg Pro
Arg Ala Arg Gly Ala Lys Ala Asn Gln Ala Glu 195 200 205 Leu Glu Asn
Ala Tyr 210 11 638 DNA Mus musculus 11 atgcagccct cactagcccc
gtgcctcatc tgcctacttg tgcacgctgc cttctgtgct 60 gtggagggcc
aggggtggca agccttcagg aatgatgcca cagaggtcat cccagggctt 120
ggagagtacc ccgagcctcc tcctgagaac aaccagacca tgaaccgggc ggagaatgga
180 ggcagacctc cccaccatcc ctatgacgcc aaaggtgtgt ccgagtacag
ctgccgcgag 240 ctgcactaca cccgcttcct gacagacggc ccatgccgca
gcgccaagcc ggtcaccgag 300 ttggtgtgct ccggccagtg cggccccgcg
cggctgctgc ccaacgccat cgggcgcgtg 360 aagtggtggc gcccgaacgg
accggatttc cgctgcatcc cggatcgcta ccgcgcgcag 420 cgggtgcagc
tgctgtgccc cgggggcgcg gcgccgcgct cgcgcaaggt gcgtctggtg 480
gcctcgtgca agtgcaagcg cctcacccgc ttccacaacc agtcggagct caaggacttc
540 gggccggaga ccgcgcggcc gcagaagggt cgcaagccgc ggcccggcgc
ccggggagcc 600 aaagccaacc aggcggagct ggagaacgcc tactagag 638 12 211
PRT Mus musculus 12 Met Gln Pro Ser Leu Ala Pro Cys Leu Ile Cys Leu
Leu Val His Ala 1 5 10 15 Ala Phe Cys Ala Val Glu Gly Gln Gly Trp
Gln Ala Phe Arg Asn Asp 20 25 30 Ala Thr Glu Val Ile Pro Gly Leu
Gly Glu Tyr Pro Glu Pro Pro Pro 35 40 45 Glu Asn Asn Gln Thr Met
Asn Arg Ala Glu Asn Gly Gly Arg Pro Pro 50 55 60 His His Pro Tyr
Asp Ala Lys Asp Val Ser Glu Tyr Ser Cys Arg Glu 65 70 75 80 Leu His
Tyr Thr Arg Phe Leu Thr Asp Gly Pro Cys Arg Ser Ala Lys 85 90 95
Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala Arg Leu 100
105 110 Leu Pro Asn Ala Ile Gly Arg Val Lys Trp Trp Arg Pro Asn Gly
Pro 115 120 125 Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gln Arg
Val Gln Leu 130 135 140 Leu Cys Pro Gly Gly Ala Ala Pro Arg Ser Arg
Lys Val Arg Leu Val 145 150 155 160 Ala Ser Cys Lys Cys Lys Arg Leu
Thr Arg Phe His Asn Gln Ser Glu 165 170 175 Leu Lys Asp Phe Gly Pro
Glu Thr Ala Arg Pro Gln Lys Gly Arg Lys 180 185 190 Pro Arg Pro Gly
Ala Arg Gly Ala Lys Ala Asn Gln Ala Glu Leu Glu 195 200 205 Asn Ala
Tyr 210 13 674 DNA Rattus norvegicus 13 gaggaccgag tgcccttcct
ccttctggca ccatgcagct ctcactagcc ccttgccttg 60 cctgcctgct
tgtacatgca gccttcgttg ctgtggagag ccaggggtgg caagccttca 120
agaatgatgc cacagaaatc atcccgggac tcagagagta cccagagcct cctcaggaac
180 tagagaacaa ccagaccatg aaccgggccg agaacggagg cagacccccc
caccatcctt 240 atgacaccaa agacgtgtcc gagtacagct gccgcgagct
gcactacacc cgcttcgtga 300 ccgacggccc gtgccgcagt gccaagccgg
tcaccgagtt ggtgtgctcg ggccagtgcg 360 gccccgcgcg gctgctgccc
aacgccatcg ggcgcgtgaa gtggtggcgc ccgaacggac 420 ccgacttccg
ctgcatcccg gatcgctacc gcgcgcagcg ggtgcagctg ctgtgccccg 480
gcggcgcggc gccgcgctcg cgcaaggtgc gtctggtggc ctcgtgcaag tgcaagcgcc
540 tcacccgctt ccacaaccag tcggagctca aggacttcgg acctgagacc
gcgcggccgc 600 agaagggtcg caagccgcgg ccccgcgccc ggggagccaa
agccaaccag gcggagctgg 660 agaacgccta ctag 674 14 213 PRT Rattus
norvegicus 14 Met Gln Leu Ser Leu Ala Pro Cys Leu Ala Cys Leu Leu
Val His Ala 1 5 10 15 Ala Phe Val Ala Val Glu Ser Gln Gly Trp Gln
Ala Phe Lys Asn Asp 20 25 30 Ala Thr Glu Ile Ile Pro Gly Leu Arg
Glu Tyr Pro Glu Pro Pro Gln 35 40 45 Glu Leu Glu Asn Asn Gln Thr
Met Asn Arg Ala Glu Asn Gly Gly Arg 50 55 60 Pro Pro His His Pro
Tyr Asp Thr Lys Asp Val Ser Glu Tyr Ser Cys 65 70 75 80 Arg Glu Leu
His Tyr Thr Arg Phe Val Thr Asp Gly Pro Cys Arg Ser 85 90 95 Ala
Lys Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro Ala 100 105
110 Arg Leu Leu Pro Asn Ala Ile Gly Arg Val Lys Trp Trp Arg Pro Asn
115 120 125 Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gln
Arg Val 130 135 140 Gln Leu Leu Cys Pro Gly Gly Ala Ala Pro Arg Ser
Arg Lys Val Arg 145 150 155 160 Leu Val Ala Ser Cys Lys Cys Lys Arg
Leu Thr Arg Phe His Asn Gln 165 170 175 Ser Glu Leu Lys Asp Phe Gly
Pro Glu Thr Ala Arg Pro Gln Lys Gly 180 185 190 Arg Lys Pro Arg Pro
Arg Ala Arg Gly Ala Lys Ala Asn Gln Ala Glu 195 200 205 Leu Glu Asn
Ala Tyr 210 15 532 DNA Bos torus 15 agaatgatgc cacagaaatc
atccccgagc tgggcgagta ccccgagcct ctgccagagc 60 tgaacaacaa
gaccatgaac cgggcggaga acggagggag acctccccac cacccctttg 120
agaccaaaga cgcctccgag tacagctgcc gggagctgca cttcacccgc tacgtgaccg
180 atgggccgtg ccgcagcgcc aagccggtca ccgagctggt gtgctcgggc
cagtgcggcc 240 cggcgcgcct gctgcccaac gccatcggcc gcggcaagtg
gtggcgccca agcgggcccg 300 acttccgctg catccccgac cgctaccgcg
cgcagcgggt gcagctgttg tgtcctggcg 360 gcgcggcgcc gcgcgcgcgc
aaggtgcgcc tggtggcctc gtgcaagtgc aagcgcctca 420 ctcgcttcca
caaccagtcc gagctcaagg acttcgggcc cgaggccgcg cggccgcaaa 480
cgggccggaa gctgcggccc cgcgcccggg gcaccaaagc cagccgggcc ga 532 16
176 PRT Bos torus 16 Asn Asp Ala Thr Glu Ile Ile Pro Glu Leu Gly
Glu Tyr Pro Glu Pro 1 5 10 15 Leu Pro Glu Leu Asn Asn Lys Thr Met
Asn Arg Ala Glu Asn Gly Gly 20 25 30 Arg Pro Pro His His Pro Phe
Glu Thr Lys Asp Ala Ser Glu Tyr Ser 35 40 45 Cys Arg Glu Leu His
Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg 50 55 60 Ser Ala Lys
Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro 65 70 75 80 Ala
Arg Leu Leu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro 85 90
95 Ser Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gln Arg
100 105 110 Val Gln Leu Leu Cys Pro Gly Gly Ala Ala Pro Arg Ala Arg
Lys Val 115 120 125 Arg Leu Val Ala Ser Cys Lys Cys Lys Arg Leu Thr
Arg Phe His Asn 130 135 140 Gln Ser Glu Leu Lys Asp Phe Gly Pro Glu
Ala Ala Arg Pro Gln Thr 145 150 155 160 Gly Arg Lys Leu Arg Pro Arg
Ala Arg Gly Thr Lys Ala Ser Arg Ala 165 170 175 17 35828 DNA Mus
musculus misc_feature (1)..(35828) n = A,T,C or G 17 cgcgttttgg
tgagcagcaa tattgcgctt cgatgagcct tggcgttgag attgatacct 60
ctgctgcaca aaaggcaatc gaccgagctg gaccagcgca ttcgtgacac cgtctccttc
120 gaacttattc gcaatggagt gtcattcatc aaggacngcc tgatcgcaaa
tggtgctatc 180 cacgcagcgg caatcgaaaa ccctcagccg gtgaccaata
tctacaacat cagccttggt 240 atcctgcgtg atgagccagc gcagaacaag
gtaaccgtca gtgccgataa gttcaaagtt 300 aaacctggtg ttgataccaa
cattgaaacg ttgatcgaaa acgcgctgaa aaacgctgct 360 gaatgtgcgg
cgctggatgt cacaaagcaa atggcagcag acaagaaagc gatggatgaa 420
ctggcttcct atgtccgcac ggccatcatg atggaatgtt tccccggtgg tgttatctgg
480 cagcagtgcc gtcgatagta tgcaattgat aattattatc atttgcgggt
cctttccggc 540 gatccgcctt gttacggggc ggcgacctcg cgggttttcg
ctatttatga aaattttccg 600 gtttaaggcg tttccgttct tcttcgtcat
aacttaatgt ttttatttaa aataccctct 660 gaaaagaaag gaaacgacag
gtgctgaaag cgagcttttt ggcctctgtc gtttcctttc 720 tctgtttttg
tccgtggaat gaacaatgga agtcaacaaa aagcagagct tatcgatgat 780
aagcggtcaa acatgagaat tcgcggccgc ataatacgac tcactatagg gatcgacgcc
840 tactccccgc gcatgaagcg gaggagctgg actccgcatg cccagagacg
ccccccaacc 900 cccaaagtgc ctgacctcag cctctaccag ctctggcttg
ggcttgggcg gggtcaaggc 960 taccacgttc tcttaacagg tggctgggct
gtctcttggc cgcgcgtcat gtgacagctg 1020 cctagttctg cagtgaggtc
accgtggaat gtctgccttc gttgccatgg caacgggatg 1080 acgttacaat
ctgggtgtgg agcttttcct gtccgtgtca ggaaatccaa ataccctaaa 1140
ataccctaga agaggaagta gctgagccaa ggctttcctg gcttctccag ataaagtttg
1200 acttagatgg aaaaaaacaa aatgataaag acccgagcca tctgaaaatt
cctcctaatt 1260 gcaccactag gaaatgtgta tattattgag ctcgtatgtg
ttcttatttt aaaaagaaaa 1320 ctttagtcat gttattaata agaatttctc
agcagtggga gagaaccaat attaacacca 1380 agataaaagt tggcatgatc
cacattgcag gaagatccac gttgggtttt catgaatgtg 1440 aagaccccat
ttattaaagt cctaagctct gtttttgcac actaggaagc gatggccggg 1500
atggctgagg ggctgtaagg atctttcaat gtcttacatg tgtgtttcct gtcctgcacc
1560 taggacctgc tgcctagcct gcagcagagc cagaggggtt tcacatgatt
agtctcagac 1620 acttgggggc aggttgcatg tactgcatcg cttatttcca
tacggagcac ctactatgtg 1680 tcaaacacca tatggtgttc actcttcaga
acggtggtgg tcatcatggt gcatttgctg 1740 acggttggat tggtggtaga
gagctgagat atatggacgc actcttcagc attctgtcaa 1800 cgtggctgtg
cattcttgct cctgagcaag tggctaaaca gactcacagg gtcagcctcc 1860
agctcagtcg ctgcatagtc ttagggaacc tctcccagtc ctccctacct caactatcca
1920 agaagccagg gggcttggcg gtctcaggag cctgcttgct gggggacagg
ttgttgagtt 1980 ttatctgcag taggttgcct aggcatagtg tcaggactga
tggctgcctt ggagaacaca 2040 tcctttgccc tctatgcaaa tctgaccttg
acatgggggc gctgctcagc tgggaggatc 2100 aactgcatac ctaaagccaa
gcctaaagct tcttcgtcca cctgaaactc ctggaccaag 2160 gggcttccgg
cacatcctct caggccagtg agggagtctg tgtgagctgc actttccaat 2220
ctcagggcgt gagaggcaga gggaggtggg ggcagagcct tgcagctctt tcctcccatc
2280 tggacagcgc tctggctcag cagcccatat gagcacaggc acatccccac
cccaccccca 2340 cctttcctgt cctgcagaat ttaggctctg ttcacggggg
gggggggggg ggggcagtcc 2400 tatcctctct taggtagaca ggactctgca
ggagacactg ctttgtaaga tactgcagtt 2460 taaatttgga tgttgtgagg
ggaaagcgaa gggcctcttt gaccattcag tcaaggtacc 2520 ttctaactcc
catcgtattg gggggctact ctagtgctag acattgcaga gagcctcaga 2580
actgtagtta ccagtgtggt aggattgatc cttcagggag cctgacatgt gacagttcca
2640 ttcttcaccc agtcaccgaa catttattca gtacctaccc cgtaacaggc
accgtagcag 2700 gtactgaggg acggaccact caaagaactg acagaccgaa
gccttggaat ataaacacca 2760 aagcatcagg ctctgccaac agaacactct
ttaacactca ggccctttaa cactcaggac 2820 ccccaccccc accccaagca
gttggcactg ctatccacat tttacagaga ggaaaaacta 2880 ggcacaggac
gatataagtg gcttgcttaa gcttgtctgc atggtaaatg gcagggctgg 2940
attgagaccc agacattcca actctagggt ctatttttct tttttctcgt tgttcgaatc
3000 tgggtcttac tgggtaaact caggctagcc tcacactcat atccttctcc
catggcttac 3060 gagtgctagg attccaggtg tgtgctacca tgtctgactc
cctgtagctt gtctatacca 3120 tcctcacaac ataggaattg tgatagcagc
acacacaccg gaaggagctg gggaaatccc 3180 acagagggct ccgcaggatg
acaggcgaat gcctacacag aaggtgggga agggaagcag 3240 agggaacagc
atgggcgtgg gaccacaagt ctatttgggg aagctgccgg taaccgtata 3300
tggctggggt gaggggagag gtcatgagat gaggcaggaa gagccacagc aggcagcggg
3360 tacgggctcc ttattgccaa gaggctcgga tcttcctcct cttcctcctt
ccggggctgc 3420 ctgttcattt tccaccactg cctcccatcc aggtctgtgg
ctcaggacat cacccagctg 3480 cagaaactgg gcatcaccca cgtcctgaat
gctgccgagg gcaggtcctt catgcacgtc 3540 aacaccagtg ctagcttcta
cgaggattct ggcatcacct acttgggcat caaggccaat 3600 gatacgcagg
agttcaacct cagtgcttac tttgaaaggg ccacagattt cattgaccag 3660
gcgctggccc ataaaaatgg taaggaacgt acattccggc acccatggag cgtaagccct
3720 ctgggacctg cttcctccaa agaggccccc acttgaaaaa ggttccagaa
agatcccaaa 3780 atatgccacc aactagggat taagtgtcct acatgtgagc
cgatgggggc cactgcatat 3840 agtctgtgcc atagacatga caatggataa
taatatttca gacagagagc aggagttagg 3900 tagctgtgct cctttccctt
taattgagtg tgcccatttt tttattcatg tatgtgtata 3960 catgtgtgtg
cacacatgcc ataggttgat actgaacacc gtcttcaatc gttccccacc 4020
ccaccttatt ttttgaggca gggtctcttc cctgatcctg gggctcattg gtttatctag
4080 gctgctggcc agtgagctct ggagttctgc ttttctctac ctccctagcc
ctgggactgc 4140 aggggcatgt gctgggccag gcttttatgt cgcgttgggg
atctgaactt aggtccctag 4200 gcctgagcac cgtaaagact ctgccacatc
cccagcctgt ttgagcaagt gaaccattcc 4260 ccagaattcc cccagtgggg
ctttcctacc cttttattgg ctaggcattc atgagtggtc 4320 acctcgccag
aggaatgagt ggccacgact ggctcagggt cagcagccta gagatactgg 4380
gttaagtctt cctgccgctc gctccctgca gccgcagaca gaaagtagga ctgaatgaga
4440 gctggctagt ggtcagacag gacagaaggc tgagagggtc acagggcaga
tgtcagcaga 4500 gcagacaggt tctccctctg tgggggaggg gtggcccact
gcaggtgtaa ttggccttct 4560 ttgtgctcca tagaggcttc ctgggtacac
agcagcttcc ctgtcctggt gattcccaaa 4620 gagaactccc taccactgga
cttacagaag ttctattgac tggtgtaacg gttcaacagc 4680 tttggctctt
ggtggacggt gcatactgct gtatcagctc aagagctcat tcacgaatga 4740
acacacacac acacacacac acacacacac acacaagcta attttgatat gccttaacta
4800 gctcagtgac tgggcatttc tgaacatccc tgaagttagc acacatttcc
ctctggtgtt 4860 cctggcttaa caccttctaa atctatattt tatctttgct
gccctgttac cttctgagaa 4920 gcccctaggg ccacttccct tcgcacctac
attgctggat ggtttctctc ctgcagctct 4980 taaatctgat ccctctgcct
ctgagccatg ggaacagccc aataactgag ttagacataa 5040 aaacgtctct
agccaaaact tcagctaaat ttagacaata aatcttactg gttgtggaat 5100
ccttaagatt cttcatgacc tccttcacat ggcacgagta tgaagcttta ttacaattgt
5160 ttattgatca aactaactca taaaaagcca gttgtctttc acctgctcaa
ggaaggaaca 5220 aaattcatcc ttaactgatc tgtgcacctt gcacaatcca
tacgaatatc ttaagagtac 5280 taagattttg gttgtgagag tcacatgtta
cagaatgtac agctttgaca aggtgcatcc 5340 ttgggatgcc gaagtgacct
gctgttccag ccccctacct tctgaggctg ttttggaagc 5400 aatgctctgg
aagcaacttt aggaggtagg atgctggaac agcgggtcac ttcagcatcc 5460
cgatgacgaa tcccgtcaaa gctgtacatt ctgtaacaga ctgggaaagc tgcagacttt
5520 aaggccaggg ccctatggtc cctcttaatc cctgtcacac ccaacccgag
cccttctcct 5580 ccagccgttc tgtgcttctc actctggata gatggagaac
acggccttgc tagttaaagg 5640 agtgaggctt cacccttctc acatggcagt
ggttggtcat cctcattcag ggaactctgg 5700 ggcattctgc ctttacttcc
tctttttgga ctacagggaa tatatgctga cttgttttga 5760 ccttgtgtat
ggggagactg gatctttggt ctggaatgtt tcctgctagt ttttccccat 5820
cctttggcaa accctatcta tatcttacca ctaggcatag tggccctcgt tctggagcct
5880 gccttcaggc tggttctcgg ggaccatgtc cctggtttct ccccagcata
tggtgttcac 5940 agtgttcact gcgggtggtt gctgaacaaa gcggggattg
catcccagag ctccggtgcc 6000 ttgtgggtac actgctaaga taaaatggat
actggcctct ctctgaccac ttgcagagct 6060 ctggtgcctt gtgggtacac
tgctaagata aaatggatac tggcctctct ctatccactt 6120 gcaggactct
agggaacagg aatccattac tgagaaaacc aggggctagg agcagggagg 6180
tagctgggca gctgaagtgc ttggcgacta accaatgaat accagagttt ggatctctag
6240 aatactctta aaatctgggt gggcagagtg gcctgcctgt aatcccagaa
ctcgggaggc 6300 ggagacaggg aatcatcaga gcaaactggc taaccagaat
agcaaaacac tgagctctgg 6360 gctctgtgag agatcctgcc ttaacatata
agagagagaa taaaacattg aagaagacag 6420 tagatgccaa ttttaagccc
ccacatgcac atggacaagt gtgcgtttga acacacatat 6480 gcactcatgt
gaaccaggca tgcacactcg ggcttatcac acacataatt tgaaagagag 6540
agtgagagag gagagtgcac attagagttc acaggaaagt gtgagtgagc acacccatgc
6600 acacagacat gtgtgccagg gagtaggaaa ggagcctggg tttgtgtata
agagggagcc 6660 atcatgtgtt tctaaggagg gcgtgtgaag gaggcgttgt
gtgggctggg actggagcat 6720 ggttgtaact gagcatgctc cctgtgggaa
acaggagggt ggccaccctg cagagggtcc 6780 cactgtccag cgggatcagt
aaaagcccct gctgagaact ttaggtaata gccagagaga 6840 gaaaggtagg
aaagtggggg gactcccatc tctgatgtag gaggatctgg gcaagtagag 6900
gtgcgtttga ggtagaaaga ggggtgcaga ggagatgctc ttaattctgg gtcagcagtt
6960 tctttccaaa taatgcctgt gaggaggtgt aggtggtggc cattcactca
ctcagcagag 7020 ggatgatgat gcccggtgga tgctggaaat ggccgagcat
caaccctggc tctggaagaa 7080 ctccatcttt cagaaggaga gtggatctgt
gtatggccag cggggtcaca ggtgcttggg 7140 gcccctgggg gactcctagc
actgggtgat gtttatcgag tgctcttgtg tgccaggcac 7200 tggcctgggg
ctttgtttct gtctctgttt tgtttcgttt tttgagacag actcttgcta 7260
tgtatccgtg tcaatcttgg aatctcactg catagcccag gctgcggaga gaggggaggg
7320 caataggcct tgtaagcaag ccacacttca gagactagac tccaccctgc
gaatgatgac 7380 aggtcagagc tgagttccgg aagatttttt ttccagctgc
caggtggagt gtggagtggc 7440 agctagcggc aagggtagag ggcgagctcc
ctgtgcagga gaaatgcaag caagagatgg 7500 caagccagtg agttaagcat
tctgtgtggg gagcaggtgg atgaagagag aggctgggct 7560 ttcgcctctg
gggggggggt gaggggtggg gatgaggtga gaggagggca gctccctgca 7620
gtgtgatgag atttttcctg acagtgacct ttggcctctc cctcccccac ttcccttctt
7680 tcctttcttc ccaccattgc tttccttgtc cttgagaaat tctgagtttc
cacttcactg 7740 gtgatgcaga cggaaacaga agccgtgtgt gtgtgtgtgt
gtgtgtgtgt gtgtgtgtgt 7800 gtgtgtgtgt ttgtgtgtat gtgtgtgtgt
gtgtttgtgt gtatgtgtgt cagtgggaat 7860 ggctcatagt ctgcaggaag
gtgggcagga aggaataagc tgtaggctga ggcagtgtgg 7920 gatgcaggga
gagaggagag gagggatacc agagaaggaa attaagggag ctacaagagg 7980
gcattgttgg ggtgtgtgtg tgtgtgtgtt gtttatattt gtattggaaa tacattcttt
8040 taaaaaatac ttatccattt atttattttt atgtgcacgt gtgtgtgcct
gcatgagttc 8100 atgtgtgcca cgtgtgtgcg ggaacccttg gaggccacaa
gggggcatct gatcccctgg 8160 aactggagtt ggaggaggtt gtgagtcccc
tgacatgttt gctgggaact gaaccccggt 8220 cctatgcaag agcaggaagt
gcagttatct gctgagccat ctctccagtc ctgaaatcca 8280 ttctcttaaa
atacacgtgg cagagacatg atgggattta cgtatggatt taatgtggcg 8340
gtcattaagt tccggcacag gcaagcacct gtaaagccat caccacaacc gcaacagtga
8400 atgtgaccat cacccccatg ttcttcatgt cccctgtccc ctccatcctc
cattctcaag 8460 cacctcttgc tctgcctctg tcgctggaga acagtgtgca
tctgcacact cttatgtcag 8520 tgaagtcaca cagcctgcac cccttcctgg
tctgagtatt tgggttctga ctctgctatc 8580 acacactact gtactgcatt
ctctcgctct ctttttttaa acatattttt atttgtttgt 8640 gtgtatgcac
atgtgccaca tgtgtacaga tactatggag gccagaagag
gccatggccg 8700 tccctggagc tggagttaca ggcagcgtgt gagctgcctg
gtgtgggtgc tgggaaccaa 8760 acttgaatct aaagcaagca cttttaactg
ctgaggcagc tctcagtacc cttcttcatt 8820 tctccgcctg ggttccattg
tatggacaca tgtagctaga atatcttgct tatctaatta 8880 tgtacattgt
tttgtgctaa gagagagtaa tgctctatag cctgagctgg cctcaacctt 8940
gccatcctcc tgcctcagcc tcctcctcct gagtgctagg atgacaggcg agtggtaact
9000 tacatggttt catgttttgt tcaagactga aggataacat tcatacagag
aaggtctggg 9060 tcacaaagtg tgcagttcac tgaatggcac aacccgtgat
caagaaacaa aactcagggg 9120 ctggagagat ggcactgact gctcttccag
aggtccggag ttcaattccc agcaaccaca 9180 tggtggctca cagccatcta
taacgagatc tgacgccctc ttctggtgtg tctgaagaca 9240 gctacagtgt
actcacataa aataaataaa tctttaaaac acacacacac acacaattac 9300
caccccagaa agcccactcc atgttccctc ccacgtctct gcctacagta ctcccaggtt
9360 accactgttc aggcttctaa caacctggtt tacttgggcc tcttttctgc
tctgtggagc 9420 cacacatttg tgtgcctcat acacgttctt tctagtaagt
tgcatattac tctgcgtttt 9480 tacatgtatt tatttattgt agttgtgtgt
gcgtgtgggc ccatgcatgg cacagtgtgt 9540 ggggatgtca gagtattgtg
aacaggggac agttcttttc ttcaatcatg tgggttccag 9600 aggttgaact
caggtcatca tgtgtggcag caaatgcctt tacccactga gacatctcca 9660
tattcttttt ttttcccctg aggtgggggc ttgttccata gcccaaactg gctttgcact
9720 tgcagttcaa agtgactccc tgtctccacc tcttagagta ttggaattac
gatgtgtact 9780 accacacctg actggatcat taattctttg atgggggcgg
ggaagcgcac atgctgcagg 9840 tgaagggatg actggactgg acatgagcgt
ggaagccaga gaacagcttc agtctaatgc 9900 tctcccaact gagctatttc
ggtttgccag agaacaactt acagaaagtt ctcagtgcca 9960 tgtggattcg
gggttggagt tcaactcatc agcttgacat tggctcctct acccactgag 10020
ccttctcact actctctacc tagatcatta attctttttt aaaaagactt attagggggc
10080 tggagagatg gctcagccgt taagagcacc gaatgccctt ccagaggtcc
tgagttcaat 10140 tcccagcatg ccattgctgg gcagtagggg gcgcaggtgt
tcaacgtgag tagctgttgc 10200 cagttttccg cggtggagaa cctcttgaca
ccctgctgtc cctggtcatt ctgggtgggt 10260 gcatggtgat atgcttgttg
tatggaagac tttgactgtt acagtgaagt tgggcttcca 10320 cagttaccac
gtctcccctg tttcttgcag gccgggtgct tgtccattgc cgcgagggct 10380
acagccgctc cccaacgcta gttatcgcct acctcatgat gcggcagaag atggacgtca
10440 agtctgctct gagtactgtg aggcagaatc gtgagatcgg ccccaacgat
ggcttcctgg 10500 cccaactctg ccagctcaat gacagactag ccaaggaggg
caaggtgaaa ctctagggtg 10560 cccacagcct cttttgcaga ggtctgactg
ggagggccct ggcagccatg tttaggaaac 10620 acagtatacc cactccctgc
accaccagac acgtgcccac atctgtccca ctctggtcct 10680 cgggggccac
tccaccctta gggagcacat gaagaagctc cctaagaagt tctgctcctt 10740
agccatcctt tcctgtaatt tatgtctctc cctgaggtga ggttcaggtt tatgtccctg
10800 tctgtggcat agatacatct cagtgaccca gggtgggagg gctatcaggg
tgcatggccc 10860 gggacacggg cactcttcat gacccctccc ccacctgggt
tcttcctgtg tggtccagaa 10920 ccacgagcct ggtaaaggaa ctatgcaaac
acaggccctg acctccccat gtctgttcct 10980 ggtcctcaca gcccgacacg
ccctgctgag gcagacgaat gacattaagt tctgaagcag 11040 agtggagata
gattagtgac tagatttcca aaaagaagga aaaaaaaggc tgcattttaa 11100
aattatttcc ttagaattaa agatactaca taggggccct tgggtaagca aatccatttt
11160 tcccagaggc tatcttgatt ctttggaatg tttaaagtgt gccttgccag
agagcttacg 11220 atctatatct gctgcttcag agccttccct gaggatggct
ctgttccttt gcttgttaga 11280 agagcgatgc cttgggcagg gtttccccct
tttcagaata cagggtgtaa agtccagcct 11340 attacaaaca aacaaacaaa
caaacaaaca aaggacctcc atttggagaa ttgcaaggat 11400 tttatcctga
attatagtgt tggtgagttc aagtcatcac gccaagtgct tgccatcctg 11460
gttgctattc taagaataat taggaggagg aacctagcca attgcagctc atgtccgtgg
11520 gtgtgtgcac gggtgcatat gttggaaggg gtgcctgtcc ccttggggac
agaaggaaaa 11580 tgaaaggccc ctctgctcac cctggccatt tacgggaggc
tctgctggtt ccacggtgtc 11640 tgtgcaggat cctgaaactg actcgctgga
cagaaacgag acttggcggc accatgagaa 11700 tggagagaga gagagcaaag
aaagaaacag cctttaaaag aactttctaa gggtggtttt 11760 tgaacctcgc
tggaccttgt atgtgtgcac atttgccaga gattgaacat aatcctcttg 11820
ggacttcacg ttctcattat ttgtatgtct ccggggtcac gcagagccgt cagccaccac
11880 cccagcaccc ggcacatagg cgtctcataa aagcccattt tatgagaacc
agagctgttt 11940 gagtaccccg tgtatagaga gagttgttgt cgtggggcac
ccggatccca gcagcctggt 12000 tgcctgcctg taggatgtct tacaggagtt
tgcagagaaa ccttccttgg agggaaagaa 12060 atatcaggga tttttgttga
atatttcaaa ttcagcttta agtgtaagac tcagcagtgt 12120 tcatggttaa
ggtaaggaac atgccttttc cagagctgct gcaagaggca ggagaagcag 12180
acctgtctta ggatgtcact cccagggtaa agacctctga tcacagcagg agcagagctg
12240 tgcagcctgg atggtcattg tcccctattc tgtgtgacca cagcaaccct
ggtcacatag 12300 ggctggtcat cctttttttt tttttttttt tttttttttg
gcccagaatg aagtgaccat 12360 agccaagttg tgtacctcag tctttagttt
ccaagcggct ctcttgctca atacaatgtg 12420 catttcaaaa taacactgta
gagttgacag aactggttca tgtgttatga gagaggaaaa 12480 gagaggaaag
aacaaaacaa aacaaaacac cacaaaccaa aaacatctgg gctagccagg 12540
catgattgca atgtctacag gcccagttca tgagaggcag agacaggaag accgccgaaa
12600 ggtcaaggat agcatggtct acgtatcgag actccagcca gggctacggt
cccaagatcc 12660 taggttttgg attttgggct ttggtttttg agacagggtt
tctctgtgta gccctggctg 12720 tcctggaact cgctctgtag accaggctgg
cctcaaactt agagatctgc ctgactctgc 12780 ctttgagggc tgggacgaat
gccaccactg cccaactaag attccattaa aaaaaaaaaa 12840 agttcaagat
aattaagagt tgccagctcg ttaaagctaa gtagaagcag tctcaggcct 12900
gctgcttgag gctgttcttg gcttggacct gaaatctgcc cccaacagtg tccaagtgca
12960 catgactttg agccatctcc agagaaggaa gtgaaaattg tggctcccca
gtcgattggg 13020 acacagtctc tctttgtcta ggtaacacat ggtgacacat
agcattgaac tctccactct 13080 gagggtgggt ttccctcccc ctgcctcttc
tgggttggtc accccatagg acagccacag 13140 gacagtcact agcacctact
ggaaacctct ttgtgggaac atgaagaaag agcctttggg 13200 agattcctgg
ctttccatta gggctgaaag tacaacggtt cttggttggc tttgcctcgt 13260
gtttataaaa ctagctacta ttcttcaggt aaaataccga tgttgtggaa aagccaaccc
13320 cgtggctgcc cgtgagtagg gggtggggtt gggaatcctg gatagtgttc
tatccatgga 13380 aagtggtgga ataggaatta agggtgttcc cccccccccc
aacctcttcc tcagacccag 13440 ccactttcta tgacttataa acatccaggt
aaaaattaca aacataaaaa tggtttctct 13500 tctcaatctt ctaaagtctg
cctgcctttt ccaggggtag gtctgtttct ttgctgttct 13560 attgtcttga
gagcacagac taacacttac caaatgaggg aactcttggc ccatactaag 13620
gctcttctgg gctccagcac tcttaagtta ttttaagaat tctcacttgg cctttagcac
13680 acccgccacc cccaagtggg tgtggataat gccatggcca gcagggggca
ctgttgaggc 13740 gggtgccttt ccaccttaag ttgcttatag tatttaagat
gctaaatgtt ttaatcaaga 13800 gaagcactga tcttataata cgaggataag
agattttctc acaggaaatt gtctttttca 13860 taattctttt acaggctttg
tcctgatcgt agcatagaga gaatagctgg atatttaact 13920 tgtattccat
tttcctctgc cagcgttagg ttaactccgt aaaaagtgat tcagtggacc 13980
gaagaggctc agagggcagg ggatggtggg gtgaggcaga gcactgtcac ctgccaggca
14040 tgggaggtcc tgccatccgg gaggaaaagg aaagtttagc ctctagtcta
ccaccagtgt 14100 taacgcactc taaagttgta accaaaataa atgtcttaca
ttacaaagac gtctgttttg 14160 tgtttccttt tgtgtgtttg ggctttttat
gtgtgcttta taactgctgt ggtggtgctg 14220 ttgttagttt tgaggtagga
tctcaggctg gccttgaact tctgatcgcc tgcccctgcc 14280 cctgcccctg
cccctgtccc tgcctccaag tgctaggact aaaagcacat gccaccacac 14340
cagtacagca tttttctaac atttaaaaat aatcacctag gggctggaga gagggttcca
14400 gctaagagtg cacactgctc ttgggtagga cctgagttta gttcccagaa
cctatactgg 14460 gtggctccag gtccagagga tccaggacct ctggcctcca
tgggcatctg ctcttagcac 14520 atacccacat acagatacac acataaaaat
aaaatgaagc ctttaaaaac ctcctaaaac 14580 ctagcccttg gaggtacgac
tctggaaagc tggcatactg tgtaagtcca tctcatggtg 14640 ttctggctaa
cgtaagactt acagagacag aaaagaactc agggtgtgct gggggttggg 14700
atggaggaag agggatgagt agggggagca cggggaactt gggcagtgaa aattctttgc
14760 aggacactag aggaggataa ataccagtca ttgcacccac tactggacaa
ctccagggaa 14820 ttatgctggg tgaaaagaga aggccccagg tattggctgc
attggctgca tttgcgtaac 14880 atttttttaa attgaaaaga aaaagatgta
aatcaaggtt agatgagtgg ttgctgtgag 14940 ctgagagctg gggtgagtga
gacatgtgga caactccatc aaaaagcgac agaaagaacg 15000 ggctgtggtg
acagctacct ctaatctcca cctccgggag gtgatcaagg ttagccctca 15060
gctagcctgt ggtgcatgag accctgtttc aaaaacttta ataaagaaat aatgaaaaaa
15120 gacatcaggg cagatccttg gggccaaagg cggacaggcg agtctcgtgg
taaggtcgtg 15180 tagaagcgga tgcatgagca cgtgccgcag gcatcatgag
agagccctag gtaagtaagg 15240 atggatgtga gtgtgtcggc gtcggcgcac
tgcacgtcct ggctgtggtg ctggactggc 15300 atctttggtg agctgtggag
gggaaatggg tagggagatc ataaaatccc tccgaattat 15360 ttcaagaact
gtctattaca attatctcaa aatattaaaa aaaaagaaga attaaaaaac 15420
aaaaaaccta tccaggtgtg gtggtgtgca cctatagcca cgggcacttg gaaagctgga
15480 gcaagaggat ggcgagtttg aaggtatctg gggctgtaca gcaagaccgt
cgtccccaaa 15540 ccaaaccaaa cagcaaaccc attatgtcac acaagagtgt
ttatagtgag cggcctcgct 15600 gagagcatgg ggtgggggtg ggggtggggg
acagaaatat ctaaactgca gtcaataggg 15660 atccactgag accctggggc
ttgactgcag cttaaccttg ggaaatgata agggttttgt 15720 gttgagtaaa
agcatcgatt actgacttaa cctcaaatga agaaaaagaa aaaaagaaaa 15780
caacaaaagc caaaccaagg ggctggtgag atggctcagt gggtaagagc acccgactgc
15840 tcttccgaag gtccagagtt caaatcccag caaccacatg gtggctcaca
accatctgta 15900 acgagatatg atgccctctt ctggtgtgtc tgaagacagc
tacagtgtac ttacatataa 15960 taaataaatc ttaaaaaaaa aaaaaaaaaa
aaaagccaaa ccgagcaaac caggccccca 16020 aacagaaggc aggcacgacg
gcaggcacca cgagccatcc tgtgaaaagg cagggctacc 16080 catgggccga
ggagggtcca gagagatagg ctggtaagct cagtttctct gtataccctt 16140
tttcttgttg acactacttc aattacagat aaaataacaa ataaacaaaa tctagagcct
16200 ggccactctc tgctcgcttg atttttcctg ttacgtccag caggtggcgg
aagtgttcca 16260 aggacagatc gcatcattaa ggtggccagc ataatctccc
atcagcaggt ggtgctgtga 16320 gaaccattat ggtgctcaca gaatcccggg
cccaggagct gccctctccc aagtctggag 16380 caataggaaa gctttctggc
ccagacaggg ttaacagtcc acattccaga gcaggggaaa 16440 aggagactgg
aggtcacaga caaaagggcc agcttctaac aacttcacag ctctggtagg 16500
agagatagat cacccccaac aatggccaca gctggttttg tctgccccga aggaaactga
16560 cttaggaagc aggtatcaga gtccccttcc tgaggggact tctgtctgcc
ttgtaaagct 16620 gtcagagcag ctgcattgat gtgtgggtga cagaagatga
aaaggaggac ccaggcagat 16680 cgccacagat ggaccggcca cttacaagtc
gaggcaggtg gcagagcctt gcagaagctc 16740 tgcaggtgga cgacactgat
tcattaccca gttagcatac cacagcgggc taggcggacc 16800 acagcctcct
tcccagtctt cctccagggc tggggagtcc tccaaccttc tgtctcagtg 16860
cagcttccgc cagcccctcc tccttttgca cctcaggtgt gaaccctccc tcctctcctt
16920 ctccctgtgg catggccctc ctgctactgc aggctgagca ttggatttct
ttgtgcttag 16980 atagacctga gatggctttc tgatttatat atatatatcc
atcccttgga tcttacatct 17040 aggacccaga gctgtttgtg ataccataag
aggctgggga gatgatatgg taagagtgct 17100 tgctgtacaa gcatgaagac
atgagttcga atccccagca accatgtgga aaaataacct 17160 tctaacctca
gagttgaggg gaaaggcagg tggattctgg gggcttactg gccagctagc 17220
cagcctaacc taaatgtctc agtcagagat cctgtctcag ggaataactt gggagaatga
17280 ctgagaaaga cacctcctca ggtctcccat gcacccacac agacacacgg
ggggggggta 17340 atgtaataag ctaagaaata atgagggaaa tgattttttg
ctaagaaatg aaattctgtg 17400 ttggccgcaa gaagcctggc cagggaagga
actgcctttg gcacaccagc ctataagtca 17460 ccatgagttc cctggctaag
aatcacatgt aatggagccc aggtccctct tgcctggtgg 17520 ttgcctctcc
cactggtttt gaagagaaat tcaagagaga tctccttggt cagaattgta 17580
ggtgctgagc aatgtggagc tggggtcaat gggattcctt taaaggcatc cttcccaggg
17640 ctgggtcata cttcaatagt agggtgcttg cacagcaagc gtgagaccct
aggttagagt 17700 ccccagaatc tgcccccaac cccccaaaaa ggcatccttc
tgcctctggg tgggtggggg 17760 gagcaaacac ctttaactaa gaccattagc
tggcaggggt aacaaatgac cttggctaga 17820 ggaatttggt caagctggat
tccgccttct gtagaagccc cacttgtttc ctttgttaag 17880 ctggcccaca
gtttgttttg agaatgcctg aggggcccag ggagccagac aattaaaagc 17940
caagctcatt ttgatatctg aaaaccacag cctgactgcc ctgcccgtgg gaggtactgg
18000 gagagctggc tgtgtccctg cctcaccaac gccccccccc ccaacacaca
ctcctcgggt 18060 cacctgggag gtgccagcag caatttggaa gtttactgag
cttgagaagt cttgggaggg 18120 ctgacgctaa gcacacccct tctccacccc
cccccacccc acccccgtga ggaggagggt 18180 gaggaaacat gggaccagcc
ctgctccagc ccgtccttat tggctggcat gaggcagagg 18240 gggctttaaa
aaggcaaccg tatctaggct ggacactgga gcctgtgcta ccgagtgccc 18300
tcctccacct ggcagcatgc agccctcact agccccgtgc ctcatctgcc tacttgtgca
18360 cgctgccttc tgtgctgtgg agggccaggg gtggcaagcc ttcaggaatg
atgccacaga 18420 ggtcatccca gggcttggag agtaccccga gcctcctcct
gagaacaacc agaccatgaa 18480 ccgggcggag aatggaggca gacctcccca
ccatccctat gacgccaaag gtacgggatg 18540 aagaagcaca ttagtggggg
ggggggtcct gggaggtgac tggggtggtt ttagcatctt 18600 cttcagaggt
ttgtgtgggt ggctagcctc tgctacatca gggcagggac acatttgcct 18660
ggaagaatac tagcacagca ttagaacctg gagggcagca ttggggggct ggtagagagc
18720 acccaaggca gggtggaggc tgaggtcagc cgaagctggc attaacacgg
gcatgggctt 18780 gtatgatggt ccagagaatc tcctcctaag gatgaggaca
caggtcagat ctagctgctg 18840 accagtgggg aagtgatatg gtgaggctgg
atgccagatg ccatccatgg ctgtactata 18900 tcccacatga ccaccacatg
aggtaaagaa ggccccagct tgaagatgga gaaaccgaga 18960 ggctcctgag
ataaagtcac ctgggagtaa gaagagctga gactggaagc tggtttgatc 19020
cagatgcaag gcaaccctag attgggtttg ggtgggaacc tgaagccagg aggaatccct
19080 ttagttcccc cttgcccagg gtctgctcaa tgagcccaga gggttagcat
taaaagaaca 19140 gggtttgtag gtggcatgtg acatgagggg cagctgagtg
aaatgtcccc tgtatgagca 19200 caggtggcac cacttgccct gagcttgcac
cctgacccca gctttgcctc attcctgagg 19260 acagcagaaa ctgtggaggc
agagccagca cagagagatg cctggggtgg gggtgggggt 19320 atcacgcacg
gaactagcag caatgaatgg ggtggggtgg cagctggagg gacactccag 19380
agaaatgacc ttgctggtca ccatttgtgt gggaggagag ctcattttcc agcttgccac
19440 cacatgctgt ccctcctgtc tcctagccag taagggatgt ggaggaaagg
gccaccccaa 19500 aggagcatgc aatgcagtca cgtttttgca gaggaagtgc
ttgacctaag ggcactattc 19560 ttggaaagcc ccaaaactag tccttccctg
ggcaaacagg cctcccccac ataccacctc 19620 tgcaggggtg agtaaattaa
gccagccaca gaagggtggc aaggcctaca cctcccccct 19680 gttgtgcccc
cccccccccc gtgaaggtgc atcctggcct ctgcccctct ggctttggta 19740
ctgggatttt ttttttcctt ttatgtcata ttgatcctga caccatggaa cttttggagg
19800 tagacaggac ccacacatgg attagttaaa agcctcccat ccatctaagc
tcatggtagg 19860 agatagagca tgtccaagag aggagggcag gcatcagacc
tagaagatat ggctgggcat 19920 ccaacccaat ctccttcccc ggagaacaga
ctctaagtca gatccagcca cccttgagta 19980 accagctcaa ggtacacaga
acaagagagt ctggtataca gcaggtgcta aacaaatgct 20040 tgtggtagca
aaagctatag gttttgggtc agaactccga cccaagtcgc gagtgaagag 20100
cgaaaggccc tctactcgcc accgccccgc ccccacctgg ggtcctataa cagatcactt
20160 tcacccttgc gggagccaga gagccctggc atcctaggta gccccccccg
cccccccccc 20220 gcaagcagcc cagccctgcc tttggggcaa gttcttttct
cagcctggac ctgtgataat 20280 gagggggttg gacgcgccgc ctttggtcgc
tttcaagtct aatgaattct tatccctacc 20340 acctgccctt ctaccccgct
cctccacagc agctgtcctg atttattacc ttcaattaac 20400 ctccactcct
ttctccatct cctgggatac cgcccctgtc ccagtggctg gtaaaggagc 20460
ttaggaagga ccagagccag gtgtggctag aggctaccag gcagggctgg ggatgaggag
20520 ctaaactgga agagtgtttg gttagtaggc acaaagcctt gggtgggatc
cctagtaccg 20580 gagaagtgga gatgggcgct gagaagttca agaccatcca
tccttaacta cacagccagt 20640 ttgaggccag cctgggctac ataaaaaccc
aatctcaaaa gctgccaatt ctgattctgt 20700 gccacgtagt gcccgatgta
atagtggatg aagtcgttga atcctggggc aacctatttt 20760 acagatgtgg
ggaaaagcaa ctttaagtac cctgcccaca gatcacaaag aaagtaagtg 20820
acagagctcc agtgtttcat ccctgggttc caaggacagg gagagagaag ccagggtggg
20880 atctcactgc tccccggtgc ctccttccta taatccatac agattcgaaa
gcgcagggca 20940 ggtttggaaa aagagagaag ggtggaagga gcagaccagt
ctggcctagg ctgcagcccc 21000 tcacgcatcc ctctctccgc agatgtgtcc
gagtacagct gccgcgagct gcactacacc 21060 cgcttcctga cagacggccc
atgccgcagc gccaagccgg tcaccgagtt ggtgtgctcc 21120 ggccagtgcg
gccccgcgcg gctgctgccc aacgccatcg ggcgcgtgaa gtggtggcgc 21180
ccgaacggac cggatttccg ctgcatcccg gatcgctacc gcgcgcagcg ggtgcagctg
21240 ctgtgccccg ggggcgcggc gccgcgctcg cgcaaggtgc gtctggtggc
ctcgtgcaag 21300 tgcaagcgcc tcacccgctt ccacaaccag tcggagctca
aggacttcgg gccggagacc 21360 gcgcggccgc agaagggtcg caagccgcgg
cccggcgccc ggggagccaa agccaaccag 21420 gcggagctgg agaacgccta
ctagagcgag cccgcgccta tgcagccccc gcgcgatccg 21480 attcgttttc
agtgtaaagc ctgcagccca ggccaggggt gccaaacttt ccagaccgtg 21540
tggagttccc agcccagtag agaccgcagg tccttctgcc cgctgcgggg gatggggagg
21600 gggtggggtt cccgcgggcc aggagaggaa gcttgagtcc cagactctgc
ctagccccgg 21660 gtgggatggg ggtctttcta ccctcgccgg acctatacag
gacaaggcag tgtttccacc 21720 ttaaagggaa gggagtgtgg aacgaaagac
ctgggactgg ttatggacgt acagtaagat 21780 ctactccttc cacccaaatg
taaagcctgc gtgggctaga tagggtttct gaccctgacc 21840 tggccactga
gtgtgatgtt gggctacgtg gttctctttt ggtacggtct tctttgtaaa 21900
atagggaccg gaactctgct gagattccaa ggattggggt accccgtgta gactggtgag
21960 agagaggaga acaggggagg ggttagggga gagattgtgg tgggcaaccg
cctagaagaa 22020 gctgtttgtt ggctcccagc ctcgccgcct cagaggtttg
gcttccccca ctccttcctc 22080 tcaaatctgc cttcaaatcc atatctggga
tagggaaggc cagggtccga gagatggtgg 22140 aagggccaga aatcacactc
ctggcccccc gaagagcagt gtcccgcccc caactgcctt 22200 gtcatattgt
aaagggattt tctacacaac agtttaaggt cgttggagga aactgggctt 22260
gccagtcacc tcccatcctt gtcccttgcc aggacaccac ctcctgcctg ccacccacgg
22320 acacatttct gtctagaaac agagcgtcgt cgtgctgtcc tctgagacag
catatcttac 22380 attaaaaaga ataatacggg gggggggggc ggagggcgca
agtgttatac atatgctgag 22440 aagctgtcag gcgccacagc accacccaca
atctttttgt aaatcatttc cagacacctc 22500 ttactttctg tgtagatttt
aattgttaaa aggggaggag agagagcgtt tgtaacagaa 22560 gcacatggag
gggggggtag gggggttggg gctggtgagt ttggcgaact ttccatgtga 22620
gactcatcca caaagactga aagccgcgtt ttttttttta agagttcagt gacatattta
22680 ttttctcatt taagttattt atgccaacat ttttttcttg tagagaaagg
cagtgttaat 22740 atcgctttgt gaagcacaag tgtgtgtggt tttttgtttt
ttgttttttc cccgaccaga 22800 ggcattgtta ataaagacaa tgaatctcga
gcaggaggct gtggtcttgt tttgtcaacc 22860 acacacaatg tctcgccact
gtcatctcac tcccttccct tggtcacaag acccaaacct 22920 tgacaacacc
tccgactgct ctctggtagc ccttgtggca atacgtgttt cctttgaaaa 22980
gtcacattca tcctttcctt tgcaaacctg gctctcattc cccagctggg tcatcgtcat
23040 accctcaccc cagcctccct ttagctgacc actctccaca ctgtcttcca
aaagtgcacg 23100 tttcaccgag ccagttccct ggtccaggtc atcccattgc
tcctccttgc tccagaccct 23160 tctcccacaa agatgttcat ctcccactcc
atcaagcccc agtggccctg cggctatccc 23220 tgtctcttca gttagctgaa
tctacttgct gacaccacat gaattccttc ccctgtctta 23280 aggttcatgg
aactcttgcc tgcccctgaa ccttccagga ctgtcccagc gtctgatgtg 23340
tcctctctct tgtaaagccc caccccacta tttgattccc aattctagat cttcccttgt
23400 tcattccttc acgggatagt gtctcatctg gccaagtcct gcttgatatt
gggataaatg 23460 caaagccaag tacaattgag gaccagttca tcattgggcc
aagctttttc aaaatgtgaa 23520 ttttacacct atagaagtgt aaaagccttc
caaagcagag gcaatgcctg gctcttcctt 23580 caacatcagg gctcctgctt
tatgggtctg gtggggtagt acattcataa acccaacact 23640 aggggtgtga
aagcaagatg attgggagtt cgaggccaat cttggctatg aggccctgtc 23700
tcaacctctc ctccctccct ccagggtttt gttttgtttt gtttttttga
tttgaaactg 23760 caacacttta aatccagtca agtgcatctt tgcgtgaggg
gaactctatc cctaatataa 23820 gcttccatct tgatttgtgt atgtgcacac
tgggggttga acctgggcct ttgtacctgc 23880 cgggcaagct ctctactgct
ctaaacccag ccctcactgg ctttctgttt caactcccaa 23940 tgaattcccc
taaatgaatt atcaatatca tgtctttgaa aaataccatt gagtgctgct 24000
ggtgtccctg tggttccaga ttccaggaag gacttttcag ggaatccagg catcctgaag
24060 aatgtcttag agcaggaggc catggagacc ttggccagcc ccacaaggca
gtgtggtgca 24120 gagggtgagg atggaggcag gcttgcaatt gaagctgaga
cagggtactc aggattaaaa 24180 agcttccccc aaaacaattc caagatcagt
tcctggtact tgcacctgtt cagctatgca 24240 gagcccagtg ggcataggtg
aagacaccgg ttgtactgtc atgtactaac tgtgcttcag 24300 agccggcaga
gacaaataat gttatggtga ccccagggga cagtgattcc agaaggaaca 24360
cagaagagag tgctgctaga ggctgcctga aggagaaggg gtcccagact ctctaagcaa
24420 agactccact cacataaaga cacaggctga gcagagctgg ccgtggatgc
agggagccca 24480 tccaccatcc tttagcatgc ccttgtattc ccatcacatg
ccagggatga ggggcatcag 24540 agagtccaag tgatgcccaa acccaaacac
acctaggact tgctttctgg gacagacaga 24600 tgcaggagag actaggttgg
gctgtgatcc cattaccaca aagagggaaa aaacaaaaaa 24660 caaacaaaca
aacaaaaaaa aacaaaacaa aacaaaaaaa aacccaaggt ccaaattgta 24720
ggtcaggtta gagtttattt atggaaagtt atattctacc tccatggggt ctacaaggct
24780 ggcgcccatc agaaagaaca aacaacaggc tgatctggga ggggtggtac
tctatggcag 24840 ggagcacgtg tgcttggggt acagccagac acggggcttg
tattaatcac agggcttgta 24900 ttaataggct gagagtcaag cagacagaga
gacagaagga aacacacaca cacacacaca 24960 cacacacaca cacacacaca
catgcacaca ccactcactt ctcactcgaa gagcccctac 25020 ttacattcta
agaacaaacc attcctcctc ataaaggaga caaagttgca gaaacccaaa 25080
agagccacag ggtccccact ctctttgaaa tgacttggac ttgttgcagg gaagacagag
25140 gggtctgcag aggcttcctg ggtgacccag agccacagac actgaaatct
ggtgctgaga 25200 cctgtataaa ccctcttcca caggttccct gaaaggagcc
cacattcccc aaccctgtct 25260 cctgaccact gaggatgaga gcacttgggc
cttccccatt cttggagtgc accctggttt 25320 ccccatctga gggcacatga
ggtctcaggt cttgggaaag ttccacaagt attgaaagtg 25380 ttcttgtttt
gtttgtgatt taatttaggt gtatgagtgc ttttgcttga atatatgcct 25440
gtgtagcatt tacaagcctg gtgcctgagg agatcagaag atggcatcag ataccctgga
25500 actggacttg cagacagtta tgagccactg tgtgggtgct aggaacagaa
cctggatcct 25560 ccggaagagc agacagccag cgctcttagc cactaagcca
tcactgaggt tctttctgtg 25620 gctaaagaga caggagacaa aggagagttt
cttttagtca ataggaccat gaatgttcct 25680 cgtaacgtga gactagggca
gggtgatccc ccagtgacac cgatggccct gtgtagttat 25740 tagcagctct
agtcttattc cttaataagt cccagtttgg ggcaggagat atgtattccc 25800
tgctttgaag tggctgaggt ccagttatct acttccaagt acttgtttct ctttctggag
25860 ttggggaagc tccctgcctg cctgtaaatg tgtccattct tcaaccttag
acaagatcac 25920 tttccctgag cagtcaggcc agtccaaagc ccttcaattt
agctttcata aggaacaccc 25980 cttttgttgg gtggaggtag cacttgcctt
gaatcccagc attaagaagg cagagacagt 26040 cggatctctg tgagttcaca
gccagcctgg tctacggagt gagttccaag acagccaggc 26100 ctacacagag
aaaccctgtc tcgaaaaaaa caaaaacaaa agaaataaag aaaaagaaaa 26160
caaaaacgaa caaacagaaa aacaagccag agtgtttgtc cccgtatttt attaatcata
26220 tttttgtccc tttgccattt tagactaaaa gactcgggaa agcaggtctc
tctctgtttc 26280 tcatccggac acacccagaa ccagatgtat ggaagatggc
taatgtgctg cagttgcaca 26340 tctggggctg ggtggattgg ttagatggca
tgggctgggt gtggttacga tgactgcagg 26400 agcaaggagt atgtggtgca
tagcaaacga ggaagtttgc acagaacaac actgtgtgta 26460 ctgatgtgca
ggtatgggca catgcaagca gaagccaagg gacagcctta gggtagtgtt 26520
tccacagacc cctcccccct tttaacatgg gcatctctca ttggcctgga gcttgccaac
26580 tgggctgggc tggctagctt gtaggtccca gggatctgca tatctctgcc
tccctagtgc 26640 tgggattaca gtcatatatg agcacacctg gcttttttat
gtgggttctg ggctttgaac 26700 ccagatctga gtgcttgcaa ggcaatcggt
tgaatgactg cttcatctcc ccagaccctg 26760 ggattctact ttctattaaa
gtatttctat taaatcaatg agcccctgcc cctgcactca 26820 gcagttctta
ggcctgctga gagtcaagtg gggagtgaga gcaagcctcg agaccccatc 26880
agcgaagcag aggacaaaga aatgaaaact tgggattcga ggctcgggat atggagatac
26940 agaaagggtc agggaaggaa atgaaccaga tgaatagagg caggaagggt
agggccctgc 27000 atacatggaa cctggtgtac atgttatctg catggggttt
gcattgcaat ggctcttcag 27060 caggttcacc acactgggaa acagaagcca
aaaagaagag taggtggtgt tggagtcaga 27120 tactgtcagt catgcctgaa
gaaatggaag caattaacga tgcgccgcaa ttaggatatt 27180 agctccctga
agaaaggcaa gaagctgggc tgtgggcact gaagggagct ttgaatgatg 27240
tcacattctc tgtatgccta gcagggcagt attggagact gagacttgac ttgtgtgtcc
27300 atatgattcc tccttttcct acagtcatct ggggctcctg agcttcgtcc
ttgtccaaga 27360 acctggagct ggcagtgggc agctgcagtg atagatgtct
gcaagaaaga tctgaaaaga 27420 gggaggaaga tgaaggaccc agaggaccac
cgacctctgc tgcctgacaa agctgcagga 27480 ccagtctctc ctacagatgg
gagacagagg cgagagatga atggtcaggg gaggagtcag 27540 agaaaggaga
gggtgaggca gagaccaaag gagggaaaca cttgtgctct acagctactg 27600
actgagtacc agctgcgtgg cagacagcca atgccaaggc tcggctgatc atggcacctc
27660 gtgggactcc tagcccagtg ctggcagagg ggagtgctga atggtgcatg
gtttggatat 27720 gatctgaatg tggtccagcc ctagtttcct tccagttgct
gggataaagc accctgacca 27780 aagctacttt tttgtttgtt tgttttggtt
tggttttgtt tggtttttcg aggcagggtt 27840 tctctgtatc accctagctg
tcctggaact cactctgtag accaggctgg cctcgaactc 27900 agaaatcccc
ctgcctctgc ctcctaagtg ctggaattaa aggcctgcgc caccactgcc 27960
ggcccaaagc tactttaaga gagagagagg aatgtataag tattataatt ccaggttata
28020 gttcattgct gtagaattgg agtcttcata ttccaggtaa tctcccacag
acatgccaca 28080 aaacaacctg ttctacgaaa tctctcatgg actcccttcc
ccagtaattc taaactgtgt 28140 caaatctaca agaaatagtg acagtcacag
tctctaacgt tttgggcatg agtctgaagt 28200 ctcattgcta agtactggga
agatgaaaac tttacctagt gtcagcattt ggagcagagc 28260 ctttgggatt
tgagatggtc ttttgcagag ctcctaatgg ctacatggag agagggggcc 28320
tgggagagac ccatacacct tttgctgcct tatgtcacct gacctgctcc ttgggaagct
28380 ctagcaagaa ggccttccct ggatcaccca ccaccttgca cctccagaac
tcagagccaa 28440 attaaacttt cttgttactg tcgtcaaagc acagtcggtc
tgggttgtat cactgtcaat 28500 gggaaacaga cttgcctgga tggataactt
gtacattgca taatgtctag aaatgaaaag 28560 tcctatagag aaaaagaaaa
ttagctggca cacagataga ggccctggag gaggctggct 28620 ttgtcctccc
cgaggaggtg gcgagtaagg tgtaaatgtt catggatgta aatgggccca 28680
tatatgaggg tctggggtaa caagaaggcc tgtgaatata aagcactgaa ggtatgtcta
28740 gtctggagaa ggtcactaca gagagttctc caactcagtg cccatacaca
cacacacaca 28800 cacacacaca cacacacaca cacacacaca ccacaaagaa
aaaaaggaag aaaaatctga 28860 gagcaagtac agtacttaaa attgtgtgat
tgtgtgtgtg actctgatgt cacatgctca 28920 tcttgcccta tgagttgaaa
accaaatggc ccctgagagg cataacaacc acactgttgg 28980 ctgtgtgctc
acgtttttct taaagcgtct gtctggtttg ctgctagcat caggcagact 29040
tgcagcagac tacatatgct cagccctgaa gtccttctag ggtgcatgtc tcttcagaat
29100 ttcagaaagt catctgtggc tccaggaccg cctgcactct ccctctgccg
cgaggctgca 29160 gactctaggc tggggtggaa gcaacgctta cctctgggac
aagtataaca tgttggcttt 29220 tctttccctc tgtggctcca acctggacat
aaaatagatg caagctgtgt aataaatatt 29280 tcctcccgtc cacttagttc
tcaacaataa ctactctgag agcacttatt aataggtggc 29340 ttagacataa
gctttggctc attcccccac tagctcttac ttctttaact ctttcaaacc 29400
attctgtgtc ttccacatgg ttagttacct ctccttccat cctggttcgc ttcttccttc
29460 gagtcgccct cagtgtctct aggtgatgct tgtaagatat tctttctaca
aagctgagag 29520 tggtggcact ctgggagttc aaagccagcc tgatctacac
agcaagctcc aggatatcca 29580 gggcaatgtt gggaaaacct ttctcaaaca
aaaagagggg ttcagttgtc aggaggagac 29640 ccatgggtta agaagtctag
acgagccatg gtgatgcata cctttcatcc aagcacttag 29700 gaggcaaaga
aaggtgaaac tctttgactt tgaggccagc taggttacat agtgataccc 29760
tgcttagtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtaatt taaaagtcta
29820 aaaatgcatt cttttaaaaa tatgtataag tatttgcctg cacatatgta
tgtatgtatg 29880 tataccatgt gtgtgtctgg tgctgaagga ctaggcatag
actccctaga actagagtca 29940 tagacagttg tgacactccc caacccccca
ccatgtgggt gcttgaagct aaactcctgt 30000 cctttgtaaa gcagcaggtg
tctatgaacc ctgaaccatc tctccagtct ccagatgtgc 30060 attctcaaag
aggagtcctt catatttccc taaactgaac atccttatca gtgagcatcc 30120
tcgagtcacc aaagctactg caaaccctct tagggaacat tcactattca cttctacttg
30180 gctcatgaaa cttaagtaca cacacacaaa cacacacaca cacacagagt
catgcactca 30240 caaaagcatg catgtacacc attcttatta gactatgctt
tgctaaaaga ctttcctaga 30300 tactttaaaa catcacttct gccttttggt
gggcaggttc caagattggt actggcgtac 30360 tggaaactga acaaggtaga
gatctagaaa tcacagcagg tcagaagggc cagcctgtac 30420 aagagagagt
tccacacctt ccaggaacac tgagcagggg gctgggacct tgcctctcag 30480
cccaagaaac tagtgcgttt cctgtatgca tgcctctcag agattccata agatctgcct
30540 tctgccataa gatctcctgc atccagacaa gcctagggga agttgagagg
ctgcctgagt 30600 ctctcccaca ggccccttct tgcctggcag tattttttta
tctggaggag aggaatcagg 30660 gtgggaatga tcaaatacaa ttatcaagga
aaaagtaaaa aacatatata tatatatatt 30720 aactgatcta gggagctggc
tcagcagtta agagttctgg ctgcccttgc ttcagatctt 30780 gctttgattc
ccagcaccca catgatggct ttcaactgta tctctgcttc caggggatcc 30840
aacagcctct tctgacctcc atagacaaga cctagtcctc tgcaagagca ccaaatgctc
30900 ttatctgttg atccatctct ctagcctcat gccagatcat ttaaaactac
tggacactgt 30960 cccattttac gaagatgtca ctgcccagtc atttgccatg
agtggatatt tcgattcttt 31020 ctatgttctc acccttgcaa tttataagaa
agatatctgc atttgtctcc tgagagaaca 31080 aagggtggag ggctactgag
atggctctag gggtaaaggt gcttgccaca aaatctgaca 31140 acttaagttt
ggtcttggaa tccacatggt ggagagagag aagagattcc cgtaagttgt 31200
cctcaaactt cccacacatg tgctgtggct tatgtgtaac cccaataagt aaagatagtt
31260 ttaaacacta cataaggtag ggtttcttca tgaccccaag gaatgatgcc
cctgatagag 31320 cttatgctga aaccccatct ccattgtgcc atctggaaag
agacaattgc atcccggaaa 31380 cagaatcttc atgaatggat taatgagcta
ttaagaaagt ggcttggtta ttgcacatgc 31440 tggcggcgta atgacctcca
ccatgatgtt atccagcatg aaggtcctca ccagaagtca 31500 tacaaatctt
cttaggcttc cagagtcgtg agcaaaaaaa gcacacctct aaataaatta 31560
actagcctca ggtagttaac caccgaaaat gaaccaaggc agttctaata caaaaccact
31620 tcccttccct gttcaaacca cagtgcccta ttatctaaaa gataaacttc
aagccaagct 31680 tttaggttgc cagtatttat gtaacaacaa ggcccgttga
cacacatctg taactcctag 31740 tactgggcct caggggcaga gacaggtgga
gccctggagt ttgaattcca ggttctgtga 31800 gaaactctgt ctgaaaagac
aatatggtga gtgacccggg aggatatctg atattgactt 31860 ctggccaaca
cacagccatc tctgcacatc tgtagttgca agccttttgc actaagtttg 31920
gccagagtca gagtttgcaa gtgtttgtgg actgaatgca cgtgttgctg gtgatctaca
31980 aagtcaccct ccttctcaag ctagcagcac tggcttcggc cagctgctca
ttcaagcctc 32040 tttgcagagt catcacgggg atgggggagc agggcccctc
cctagaacac caagcctgtg 32100 gttgtttatt caggacatta ttgagggcca
agatgacaga taactctatc acttggccaa 32160 cagtcgggtg ttgcggtgtt
aggttatttc tgtgtctgca gaaaacagtg caacctggac 32220 aaaagaaata
aatgatatca tttttcattc aggcaactag attccgtggt acaaaaggct 32280
ccctggggaa cgaggccggg acagcgcggc tcctgagtcg ctatttccgt ctgtcaactt
32340 ctctaatctc ttgatttcct ccctctgtct gtttccttcc tcttgctggg
gcccagtgga 32400 gtctgtgtac tcacagggag gagggtggca aagccctggt
cctctacggg ctgggggaag 32460 gggggaagct gtcggcccag tgactttttc
ccctttctct ttttcttaga aaccagtctc 32520 aatttaagat aatgagtctc
ctcattcacg tgtgctcact attcataggg acttatccac 32580 ccccgccctg
tcaatctggc taagtaagac aagtcaaatt taaaagggaa cgtttttcta 32640
aaaatgtggc tggaccgtgt gccggcacga aaccagggat ggcggtctaa gttacatgct
32700 ctctgccagc cccggtgcct tttcctttcg gaaaggagac ccggaggtaa
aacgaagttg 32760 ccaacttttg atgatggtgt gcgccgggtg actctttaaa
atgtcatcca tacctgggat 32820 agggaaggct cttcagggag tcatctagcc
ctcccttcag gaaaagattc cacttccggt 32880 ttagttagct tccacctggt
cccttatccg ctgtctctgc ccactagtcc tcatccatcc 32940 ggtttccgcc
ctcatccacc ttgccctttt agttcctaga aagcagcacc gtagtcttgg 33000
caggtgggcc attggtcact ccgctaccac tgttaccatg gccaccaagg tgtcatttaa
33060 atatgagctc actgagtcct gcgggatggc ttggttggta atatgcttgc
tgcaaaatcg 33120 tgagaactgg agttcaattc ccagcacatg gatgtatttc
cagcacctgg aaggcaggga 33180 gcagagatct taaagctcct ggccagacag
cccagcctaa ttagtaatca gtgagagacc 33240 ctgtctcaag aaacaagatg
gaacatcaaa ggtcaacctc ttgtctccac acacacaaat 33300 acacacatgc
acatacatcc acacacaggc aaacacatgc acacacctga acaccctcca 33360
caaatacata cataaaaaaa taaatacata cacacataca tacatacacc aacattccct
33420 ctccttagtc tcctggctac gctcttgtca cccccactaa ggcttcaact
tcttctattt 33480 cttcatcttg actcctctgt actttgcatg ccttttccag
caaaggcttt tctttaaatc 33540 tccgtcattc ataaactccc tctaaatttc
ttcccctgcc cttttctttc tctctaggga 33600 gataaagaca cacactacaa
agtcaccgtg ggaccagttt attcacccac ccacccctgc 33660 ttctgttcat
ccggccagct aagtagtcca acctctctgg tgctgtaccc tggaccctgg 33720
cttcaccaca gctcctccat gctacccagc cctgcaaacc ttcagcctag cctctggttc
33780 tccaaccagc acaggcccag tctggcttct atgtcctaga aatctccttc
attctctcca 33840 tttccctcct gaatctacca ccttctttct cccttctcct
gacctctaat gtcttggtca 33900 aacgattaca aggaagccaa tgaaattagc
agtttggggt acctcagagt cagcagggga 33960 gctgggatga attcacattt
ccaggccttt gctttgctcc ccggattctg acaggcagtt 34020 ccgaagctga
gtccaggaag ctgaatttaa aatcacactc cagctgggtt ctgaggcagc 34080
cctaccacat cagctggccc tgactgagct gtgtctgggt ggcagtggtg ctggtggtgc
34140 tggtggtgct ggtggtggtg gtggtggtgg tggtggtggt ggtggtggtg
tgtgtgtgtg 34200 ttttctgctt ttacaaaact tttctaattc ttatacaaag
gacaaatctg cctcatatag 34260 gcagaaagat gacttatgcc tatataagat
ataaagatga ctttatgcca cttattagca 34320 atagttactg tcaaaagtaa
ttctatttat acacccttat acatggtatt gcttttgttg 34380 gagactctaa
aatccagatt atgtatttaa aaaaaaattc cccagtcctt aaaaggtgaa 34440
gaatggaccc agatagaagg tcacggcaca agtatggagt cggagtgtgg agtcctgcca
34500 atggtctgga cagaagcatc cagagagggt ccaagacaaa tgcctcgcct
cctaaggaac 34560 actggcagcc ctgatgaggt accagagatt gctaagtgga
ggaatacagg atcagaccca 34620 tggaggggct taaagcgtga ctgtagcagc
cctccgctga ggggctccag gtgggcgccc 34680 aaggtgctgc agtgggagcc
acatgagagg tgatgtcttg gagtcacctc gggtaccatt 34740 gtttagggag
gtggggattt gtggtgtgga gacaggcagc ctcaaggatg cttttcaaca 34800
atggttgatg agttggaact aaaacagggg ccatcacact ggctcccata gctctgggct
34860 tgccagcttc cacatctgcc ccccaccccc tgtctggcac cagctcaagc
tctgtgattc 34920 tacacatcca aaagaggaag agtagcctac tgggcatgcc
acctcttctg gaccatcagg 34980 tgagagtgtg gcaagcccta ggctcctgtc
caggatgcag ggctgccaga taggatgctc 35040 agctatctcc tgagctggaa
ctattttagg aataaggatt atgcccgccc ggggttggcc 35100 agcaccccag
cagcctgtgc ttgcgtaaaa gcaagtgctg ttgatttatc taaaaacaga 35160
gccgtggacc cacccacagg acaagtatgt atgcatctgt ttcatgtatc tgaaaagcga
35220 cacaaccatt tttcacatca tggcatcttc ctaaccccca ttcttttttg
ttttgttttt 35280 ttgagacagg gtttctctgt gtagtcctgg ctgtcctgga
actcactttg tagaccaggc 35340 tggcctcgaa ctcagaaatc ctgggattaa
aggtgtgtgc caccacgccc ggccctaacc 35400 cccattctta atggtgatcc
agtggttgaa atttcgggcc acacacatgt ccattaggga 35460 ttagctgctg
tcttctgagc tacctggtac aatctttatc ccctggggcc tgggctcctg 35520
atccctgact cgggcccgat caagtccagt tcctgggccc gatcaagtcc agttcctggg
35580 cccgaacaag tccagtccct agctcgatta gctcatcctg gctccctggc
ctgttcttac 35640 ttacactctt ccccttgctc tggacttgtt gctttcttta
ctcaagttgt ctgccacagt 35700 ccctaagcca cctctgtaag acaactaaga
taatacttcc ctcaagcacg gaaagtcctg 35760 agtcaccaca ccctctggag
gtgtgtggac acatgttcat gcgtgtggtt gcgcttacgt 35820 acgtgtgc 35828 18
9301 DNA Homo sapien 18 tagaggagaa gtctttgggg agggtttgct ctgagcacac
ccctttccct ccctccgggg 60 ctgagggaaa catgggacca gccctgcccc
agcctgtcct cattggctgg catgaagcag 120 agaggggctt taaaaaggcg
accgtgtctc ggctggagac cagagcctgt gctactggaa 180 ggtggcgtgc
cctcctctgg ctggtaccat gcagctccca ctggccctgt gtctcgtctg 240
cctgctggta cacacagcct tccgtgtagt ggagggccag gggtggcagg cgttcaagaa
300 tgatgccacg gaaatcatcc ccgagctcgg agagtacccc gagcctccac
cggagctgga 360 gaacaacaag accatgaacc gggcggagaa cggagggcgg
cctccccacc acccctttga 420 gaccaaaggt atggggtgga ggagagaatt
cttagtaaaa gatcctgggg aggttttaga 480 aacttctctt tgggaggctt
ggaagactgg ggtagaccca gtgaagattg ctggcctctg 540 ccagcactgg
tcgaggaaca gtcttgcctg gaggtggggg aagaatggct cgctggtgca 600
gccttcaaat tcaggtgcag aggcatgagg caacagacgc tggtgagagc ccagggcagg
660 gaggacgctg gggtggtgag ggtatggcat cagggcatca gaacaggctc
aggggctcag 720 aaaagaaaag gtttcaaaga atctcctcct gggaatatag
gagccacgtc cagctgctgg 780 taccactggg aagggaacaa ggtaagggag
cctcccatcc acagaacagc acctgtgggg 840 caccggacac tctatgctgg
tggtggctgt ccccaccaca cagacccaca tcatggaatc 900 cccaggaggt
gaacccccag ctcgaagggg aagaaacagg ttccaggcac tcagtaactt 960
ggtagtgaga agagctgagg tgtgaacctg gtttgatcca actgcaagat agccctggtg
1020 tgtggggggg tgtgggggac agatctccac aaagcagtgg ggaggaaggc
cagagaggca 1080 cccctgcagt gtgcattgcc catggcctgc ccagggagct
ggcacttgaa ggaatgggag 1140 ttttcggcac agttttagcc cctgacatgg
gtgcagctga gtccaggccc tggaggggag 1200 agcagcatcc tctgtgcagg
agtagggaca tctgtcctca gcagccaccc cagtcccaac 1260 cttgcctcat
tccaggggag ggagaaggaa gaggaaccct gggttcctgg tcaggcctgc 1320
acagagaagc ccaggtgaca gtgtgcatct ggctctataa ttggcaggaa tcctgaggcc
1380 atgggggcgt ctgaaatgac acttcagact aagagcttcc ctgtcctctg
gccattatcc 1440 aggtggcaga gaagtccact gcccaggctc ctggacccca
gccctccccg cctcacaacc 1500 tgttgggact atggggtgct aaaaagggca
actgcatggg aggccagcca ggaccctccg 1560 tcttcaaaat ggaggacaag
ggcgcctccc cccacagctc cccttctagg caaggtcagc 1620 tgggctccag
cgactgcctg aagggctgta aggaacccaa acacaaaatg tccaccttgc 1680
tggactccca cgagaggcca cagcccctga ggaagccaca tgctcaaaac aaagtcatga
1740 tctgcagagg aagtgcctgg cctaggggcg ctattctcga aaagccgcaa
aatgccccct 1800 tccctgggca aatgcccccc tgaccacaca cacattccag
ccctgcagag gtgaggatgc 1860 aaaccagccc acagaccaga aagcagcccc
agacgatggc agtggccaca tctcccctgc 1920 tgtgcttgct cttcagagtg
ggggtggggg gtggccttct ctgtcccctc tctggtttgg 1980 tcttaagact
atttttcatt ctttcttgtc acattggaac tatccccatg aaacctttgg 2040
gggtggactg gtactcacac gacgaccagc tatttaaaaa gctcccaccc atctaagtcc
2100 accataggag acatggtcaa ggtgtgtgca ggggatcagg ccaggcctcg
gagcccaatc 2160 tctgcctgcc cagggagtat caccatgagg cgcccattca
gataacacag aacaagaaat 2220 gtgcccagca gagagccagg tcaatgtttg
tggcagctga acctgtaggt tttgggtcag 2280 agctcagggc ccctatggta
ggaaagtaac gacagtaaaa agcagccctc agctccatcc 2340 cccagcccag
cctcccatgg atgctcgaac gcagagcctc cactcttgcc ggagccaaaa 2400
ggtgctggga ccccagggaa gtggagtccg gagatgcagc ccagcctttt gggcaagttc
2460 ttttctctgg ctgggcctca gtattctcat tgataatgag ggggttggac
acactgcctt 2520 tgattccttt caagtctaat gaattcctgt cctgatcacc
tccccttcag tccctcgcct 2580 ccacagcagc tgccctgatt tattaccttc
aattaacctc tactcctttc tccatcccct 2640 gtccacccct cccaagtggc
tggaaaagga atttgggaga agccagagcc aggcagaagg 2700 tgtgctgagt
acttaccctg cccaggccag ggaccctgcg gcacaagtgt ggcttaaatc 2760
ataagaagac cccagaagag aaatgataat aataatacat aacagccgac gctttcagct
2820 atatgtgcca aatggtattt tctgcattgc gtgtgtaatg gattaactcg
caatgcttgg 2880 ggcggcccat tttgcagaca
ggaagaagag agaggttaag gaacttgccc aagatgacac 2940 ctgcagtgag
cgatggagcc ctggtgtttg aaccccagca gtcatttggc tccgagggga 3000
cagggtgcgc aggagagctt tccaccagct ctagagcatc tgggaccttc ctgcaataga
3060 tgttcagggg caaaagcctc tggagacagg cttggcaaaa gcagggctgg
ggtggagaga 3120 gacgggccgg tccagggcag gggtggccag gcgggcggcc
accctcacgc gcgcctctct 3180 ccacagacgt gtccgagtac agctgccgcg
agctgcactt cacccgctac gtgaccgatg 3240 ggccgtgccg cagcgccaag
ccggtcaccg agctggtgtg ctccggccag tgcggcccgg 3300 cgcgcctgct
gcccaacgcc atcggccgcg gcaagtggtg gcgacctagt gggcccgact 3360
tccgctgcat ccccgaccgc taccgcgcgc agcgcgtgca gctgctgtgt cccggtggtg
3420 aggcgccgcg cgcgcgcaag gtgcgcctgg tggcctcgtg caagtgcaag
cgcctcaccc 3480 gcttccacaa ccagtcggag ctcaaggact tcgggaccga
ggccgctcgg ccgcagaagg 3540 gccggaagcc gcggccccgc gcccggagcg
ccaaagccaa ccaggccgag ctggagaacg 3600 cctactagag cccgcccgcg
cccctcccca ccggcgggcg ccccggccct gaacccgcgc 3660 cccacatttc
tgtcctctgc gcgtggtttg attgtttata tttcattgta aatgcctgca 3720
acccagggca gggggctgag accttccagg ccctgaggaa tcccgggcgc cggcaaggcc
3780 cccctcagcc cgccagctga ggggtcccac ggggcagggg agggaattga
gagtcacaga 3840 cactgagcca cgcagccccg cctctggggc cgcctacctt
tgctggtccc acttcagagg 3900 aggcagaaat ggaagcattt tcaccgccct
ggggttttaa gggagcggtg tgggagtggg 3960 aaagtccagg gactggttaa
gaaagttgga taagattccc ccttgcacct cgctgcccat 4020 cagaaagcct
gaggcgtgcc cagagcacaa gactgggggc aactgtagat gtggtttcta 4080
gtcctggctc tgccactaac ttgctgtgta accttgaact acacaattct ccttcgggac
4140 ctcaatttcc actttgtaaa atgagggtgg aggtgggaat aggatctcga
ggagactatt 4200 ggcatatgat tccaaggact ccagtgcctt ttgaatgggc
agaggtgaga gagagagaga 4260 gaaagagaga gaatgaatgc agttgcattg
attcagtgcc aaggtcactt ccagaattca 4320 gagttgtgat gctctcttct
gacagccaaa gatgaaaaac aaacagaaaa aaaaaagtaa 4380 agagtctatt
tatggctgac atatttacgg ctgacaaact cctggaagaa gctatgctgc 4440
ttcccagcct ggcttccccg gatgtttggc tacctccacc cctccatctc aaagaaataa
4500 catcatccat tggggtagaa aaggagaggg tccgagggtg gtgggaggga
tagaaatcac 4560 atccgcccca acttcccaaa gagcagcatc cctcccccga
cccatagcca tgttttaaag 4620 tcaccttccg aagagaagtg aaaggttcaa
ggacactggc cttgcaggcc cgagggagca 4680 gccatcacaa actcacagac
cagcacatcc cttttgagac accgccttct gcccaccact 4740 cacggacaca
tttctgccta gaaaacagct tcttactgct cttacatgtg atggcatatc 4800
ttacactaaa agaatattat tgggggaaaa actacaagtg ctgtacatat gctgagaaac
4860 tgcagagcat aatagctgcc acccaaaaat ctttttgaaa atcatttcca
gacaacctct 4920 tactttctgt gtagttttta attgttaaaa aaaaaaagtt
ttaaacagaa gcacatgaca 4980 tatgaaagcc tgcaggactg gtcgtttttt
tggcaattct tccacgtggg acttgtccac 5040 aagaatgaaa gtagtggttt
ttaaagagtt aagttacata tttattttct cacttaagtt 5100 atttatgcaa
aagtttttct tgtagagaat gacaatgtta atattgcttt atgaattaac 5160
agtctgttct tccagagtcc agagacattg ttaataaaga caatgaatca tgaccgaaag
5220 gatgtggtct cattttgtca accacacatg acgtcatttc tgtcaaagtt
gacacccttc 5280 tcttggtcac tagagctcca accttggaca cacctttgac
tgctctctgg tggcccttgt 5340 ggcaattatg tcttcctttg aaaagtcatg
tttatccctt cctttccaaa cccagaccgc 5400 atttcttcac ccagggcatg
gtaataacct cagccttgta tccttttagc agcctcccct 5460 ccatgctggc
ttccaaaatg ctgttctcat tgtatcactc ccctgctcaa aagccttcca 5520
tagctccccc ttgcccagga tcaagtgcag tttccctatc tgacatggga ggccttctct
5580 gcttgactcc cacctcccac tccaccaagc ttcctactga ctccaaatgg
tcatgcagat 5640 ccctgcttcc ttagtttgcc atccacactt agcaccccca
ataactaatc ctctttcttt 5700 aggattcaca ttacttgtca tctcttcccc
taaccttcca gagatgttcc aatctcccat 5760 gatccctctc tcctctgagg
ttccagcccc ttttgtctac accactactt tggttcctaa 5820 ttctgttttc
catttgacag tcattcatgg aggaccagcc tggccaagtc ctgcttagta 5880
ctggcataga caacacaaag ccaagtacaa ttcaggacca gctcacagga aacttcatct
5940 tcttcgaagt gtggatttga tgcctcctgg gtagaaatgt aggatcttca
aaagtgggcc 6000 agcctcctgc acttctctca aagtctcgcc tccccaaggt
gtcttaatag tgctggatgc 6060 tagctgagtt agcatcttca gatgaagagt
aaccctaaag ttactcttca gttgccctaa 6120 ggtgggatgg tcaactggaa
agctttaaat taagtccagc ctaccttggg ggaacccacc 6180 cccacaaaga
aagctgaggt ccctcctgat gacttgtcag tttaactacc aataacccac 6240
ttgaattaat catcatcatc aagtctttga taggtgtgag tgggtatcag tggccggtcc
6300 cttcctgggg ctccagcccc cgaggaggcc tcagtgagcc cctgcagaaa
atccatgcat 6360 catgagtgtc tcagggccca gaatatgaga gcaggtagga
aacagagaca tcttccatcc 6420 ctgagaggca gtgcggtcca gtgggtgggg
acacgggctc tgggtcaggt ttgtgttgtt 6480 tgtttgtttg ttttgagaca
gagtctcgct ctattgccca ggctggagtg cagtgtcaca 6540 atctcggctt
actgcaactt ctgccttccc ggattcaagt gattctcctg cctcagcctc 6600
cagagtagct gggattacag gtgcgtgcca ccacgcctgg ctaatttttg tatttttgat
6660 agagacgggg tttcaccatg ttggccaggc tagtctcgaa ctcttgacct
caagtgatct 6720 gcctgcctcg gcctcccaaa gtgctgggat tacaggcgtg
agccaccaca cccagcccca 6780 ggttggtgtt tgaatctgag gagactgaag
caccaagggg ttaaatgttt tgcccacagc 6840 catacttggg ctcagttcct
tgccctaccc ctcacttgag ctgcttagaa cctggtgggc 6900 acatgggcaa
taaccaggtc acactgtttt gtaccaagtg ttatgggaat ccaagatagg 6960
agtaatttgc tctgtggagg ggatgaggga tagtggttag ggaaagcttc acaaagtggg
7020 tgttgcttag agattttcca ggtggagaag ggggcttcta ggcagaaggc
atagcccaag 7080 caaagactgc aagtgcatgg ctgctcatgg gtagaagaga
atccaccatt cctcaacatg 7140 taccgagtcc ttgccatgtg caaggcaaca
tgggggtacc aggaattcca agcaatgtcc 7200 aaacctaggg tctgctttct
gggacctgaa gatacaggat ggatcagccc aggctgcaat 7260 cccattacca
cgagggggaa aaaaacctga aggctaaatt gtaggtcggg ttagaggtta 7320
tttatggaaa gttatattct acctacatgg ggtctataag cctggcgcca atcagaaaag
7380 gaacaaacaa cagacctagc tgggaggggc agcattttgt tgtagggggc
ggggcacatg 7440 ttctgggggt acagccagac tcagggcttg tattaatagt
ctgagagtaa gacagacaga 7500 gggatagaag gaaataggtc cctttctctc
tctctctctc tctctctctc actctctctc 7560 tctctcacac acacacacag
acacacacac acgctctgta ggggtctact tatgctccaa 7620 gtacaaatca
ggccacattt acacaaggag gtaaaggaaa agaacgttgg aggagccaca 7680
ggaccccaaa attccctgtt ttccttgaat caggcaggac ttacgcagct gggagggtgg
7740 agagcctgca gaagccacct gcgagtaagc caagttcaga gtcacagaca
ccaaaagctg 7800 gtgccatgtc ccacacccgc ccacctccca cctgctcctt
gacacagccc tgtgctccac 7860 aacccggctc ccagatcatt gattatagct
ctggggcctg caccgtcctt cctgccacat 7920 ccccacccca ttcttggaac
ctgccctctg tcttctccct tgtccaaggg caggcaaggg 7980 ctcagctatt
gggcagcttt gaccaacagc tgaggctcct tttgtggctg gagatgcagg 8040
aggcagggga atattcctct tagtcaatgc gaccatgtgc ctggtttgcc cagggtggtc
8100 tcgtttacac ctgtaggcca agcgtaatta ttaacagctc ccacttctac
tctaaaaaat 8160 gacccaatct gggcagtaaa ttatatggtg cccatgctat
taagagctgc aacttgctgg 8220 gcgtggtggc tcacacctgt aatcccagta
ctttgggacg tcaaggcggg tggatcacct 8280 gaggtcacga gttagagact
ggcctggcca gcatggcaaa accccatctt tactaaaaat 8340 acaaaaatta
gcaaggcatg gtggcatgca cctgtaatcc caggtactcg ggaggctgag 8400
acaggagaat ggcttgaacc caggaggcag aggttgcagt gagccaagat tgtgccactg
8460 ccctccagcc ctggcaacag agcaagactt catctcaaaa gaaaaaggat
actgtcaatc 8520 actgcaggaa gaacccaggt aatgaatgag gagaagagag
gggctgagtc accatagtgg 8580 cagcaccgac tcctgcagga aaggcgagac
actgggtcat gggtactgaa gggtgccctg 8640 aatgacgttc tgctttagag
accgaacctg agccctgaaa gtgcatgcct gttcatgggt 8700 gagagactaa
attcatcatt ccttggcagg tactgaatcc tttcttacgg ctgccctcca 8760
atgcccaatt tccctacaat tgtctggggt gcctaagctt ctgcccacca agagggccag
8820 agctggcagc gagcagctgc aggtaggaga gataggtacc cataagggag
gtgggaaaga 8880 gagatggaag gagaggggtg cagagcacac acctcccctg
cctgacaact tcctgagggc 8940 tggtcatgcc agcagattta aggcggaggc
aggggagatg gggcgggaga ggaagtgaaa 9000 aaggagaggg tggggatgga
gaggaagaga gggtgatcat tcattcattc cattgctact 9060 gactggatgc
cagctgtgag ccaggcacca ccctagctct gggcatgtgg ttgtaatctt 9120
ggagcctcat ggagctcaca gggagtgctg gcaaggagat ggataatgga cggataacaa
9180 ataaacattt agtacaatgt ccgggaatgg aaagttctcg aaagaaaaat
aaagctggtg 9240 agcatataga cagccctgaa ggcggccagg ccaggcattt
ctgaggaggt ggcatttgag 9300 c 9301 19 21 DNA Artificial Sequence
Primer for PCR 19 ccggagctgg agaacaacaa g 21 20 19 DNA Artificial
Sequence PRimer for PCR 20 gcactggccg gagcacacc 19 21 23 DNA
Artificial Sequence Primer for PCR 21 aggccaaccg cgagaagatg acc 23
22 21 DNA Artificial Sequence Primer for PCR 22 gaagtccagg
gcgacgtagc a 21 23 25 DNA Artificial Sequence Primer for PCR 23
aagcttggta ccatgcagct cccac 25 24 50 DNA Artificial Sequence Primer
for PCR 24 aagcttctac ttgtcatcgt cgtccttgta gtcgtaggcg ttctccagct
50 25 19 DNA Artificial Sequence Primer for PCR 25 gcactggccg
gagcacacc 19 26 39 DNA Artificial Sequence Primer for PCR 26
gtcgtcggat ccatggggtg gcaggcgttc aagaatgat 39 27 57 DNA Artificial
Sequence Primer for PCR 27 gtcgtcaagc ttctacttgt catcgtcctt
gtagtcgtag gcgttctcca gctcggc 57 28 29 DNA Artificial Sequence
Primer for PCR 28 gacttggatc ccaggggtgg caggcgttc 29 29 29 DNA
Artificial Sequence Primer for PCR 29 agcataagct tctagtaggc
gttctccag 29 30 29 DNA Artificial Sequence Primer for PCR 30
gacttggatc cgaagggaaa aagaaaggg 29 31 29 DNA Artificial Sequence
Primer for PCR 31 agcataagct tttaatccaa atcgatgga 29 32 33 DNA
Artificial Sequence Primer for PCR 32 actacgagct cggccccacc
acccatcaac aag 33 33 34 DNA Artificial Sequence Primer for PCR 33
acttagaagc tttcagtcct cagccccctc ttcc 34 34 66 DNA Artificial
Sequence Primer for PCR 34 aatctggatc cataacttcg tatagcatac
attatacgaa gttatctgca ggattcgagg 60 gcccct 66 35 82 DNA Artificial
Sequence Primer for PCR 35 aatctgaatt ccaccggtgt taattaaata
acttcgtata atgtatgcta tacgaagtta 60 tagatctaga gtcagcttct ga 82 36
62 DNA Artificial Sequence Primer for PCR 36 atttaggtga cactatagaa
ctcgagcagc tgaagcttaa ccacatggtg gctcacaacc 60 at 62 37 54 DNA
Artificial Sequence Primer for PCR 37 aacgacggcc agtgaatccg
taatcatggt catgctgcca ggtggaggag ggca 54 38 31 DNA Artificial
Sequence Primer for PCR 38 attaccaccg gtgacacccg cttcctgaca g 31 39
61 DNA Artificial Sequence Primer for PCR 39 attacttaat taaacatggc
gcgccatatg gccggcccct aattgcggcg catcgttaat 60 t 61 40 34 DNA
Artificial Sequence Primer for PCR 40 attacggccg gccgcaaagg
aattcaagat ctga 34 41 34 DNA Artificial Sequence Primer for PCR 41
attacggcgc gcccctcaca ggccgcaccc agct 34 42 184 PRT Homo sapiens 42
Met Ser Arg Thr Ala Tyr Thr Val Gly Ala Leu Leu Leu Leu Leu Gly 1 5
10 15 Thr Leu Leu Pro Ala Ala Glu Gly Lys Lys Lys Gly Ser Gln Gly
Ala 20 25 30 Ile Pro Pro Pro Asp Lys Ala Gln His Asn Asp Ser Glu
Gln Thr Gln 35 40 45 Ser Pro Gln Gln Pro Gly Ser Arg Asn Arg Gly
Arg Gly Gln Gly Arg 50 55 60 Gly Thr Ala Met Pro Gly Glu Glu Val
Leu Glu Ser Ser Gln Glu Ala 65 70 75 80 Leu His Val Thr Glu Arg Lys
Tyr Leu Lys Arg Asp Trp Cys Lys Thr 85 90 95 Gln Pro Leu Lys Gln
Thr Ile His Glu Glu Gly Cys Asn Ser Arg Thr 100 105 110 Ile Ile Asn
Arg Phe Cys Tyr Gly Gln Cys Asn Ser Phe Tyr Ile Pro 115 120 125 Arg
His Ile Arg Lys Glu Glu Gly Ser Phe Gln Ser Cys Ser Phe Cys 130 135
140 Lys Pro Lys Lys Phe Thr Thr Met Met Val Thr Leu Asn Cys Pro Glu
145 150 155 160 Leu Gln Pro Pro Thr Lys Lys Lys Arg Val Thr Arg Val
Lys Gln Cys 165 170 175 Arg Cys Ile Ser Ile Asp Leu Asp 180 43 267
PRT Homo sapiens 43 Met His Leu Leu Leu Phe Gln Leu Leu Val Leu Leu
Pro Leu Gly Lys 1 5 10 15 Thr Thr Arg His Gln Asp Gly Arg Gln Asn
Gln Ser Ser Leu Ser Pro 20 25 30 Val Leu Leu Pro Arg Asn Gln Arg
Glu Leu Pro Thr Gly Asn His Glu 35 40 45 Glu Ala Glu Glu Lys Pro
Asp Leu Phe Val Ala Val Pro His Leu Val 50 55 60 Ala Thr Ser Pro
Ala Gly Glu Gly Gln Arg Gln Arg Glu Lys Met Leu 65 70 75 80 Ser Arg
Phe Gly Arg Phe Trp Lys Lys Pro Glu Arg Glu Met His Pro 85 90 95
Ser Arg Asp Ser Asp Ser Glu Pro Phe Pro Pro Gly Thr Gln Ser Leu 100
105 110 Ile Gln Pro Ile Asp Gly Met Lys Met Glu Lys Ser Pro Leu Arg
Glu 115 120 125 Glu Ala Lys Lys Phe Trp His His Phe Met Phe Arg Lys
Thr Pro Ala 130 135 140 Ser Gln Gly Val Ile Leu Pro Ile Lys Ser His
Glu Val His Trp Glu 145 150 155 160 Thr Cys Arg Thr Val Pro Phe Ser
Gln Thr Ile Thr His Glu Gly Cys 165 170 175 Glu Lys Val Val Val Gln
Asn Asn Leu Cys Phe Gly Lys Cys Gly Ser 180 185 190 Val His Phe Pro
Gly Ala Ala Gln His Ser His Thr Ser Cys Ser His 195 200 205 Cys Leu
Pro Ala Lys Phe Thr Thr Met His Leu Pro Leu Asn Cys Thr 210 215 220
Glu Leu Ser Ser Val Ile Lys Val Val Met Leu Val Glu Glu Cys Gln 225
230 235 240 Cys Lys Val Lys Thr Glu His Glu Asp Gly His Ile Leu His
Ala Gly 245 250 255 Ser Gln Asp Ser Phe Ile Pro Gly Val Ser Ala 260
265 44 180 PRT Homo sapiens 44 Met Leu Arg Val Leu Val Gly Ala Val
Leu Pro Ala Met Leu Leu Ala 1 5 10 15 Ala Pro Pro Pro Ile Asn Lys
Leu Ala Leu Phe Pro Asp Lys Ser Ala 20 25 30 Trp Cys Glu Ala Lys
Asn Ile Thr Gln Ile Val Gly His Ser Gly Cys 35 40 45 Glu Ala Lys
Ser Ile Gln Asn Arg Ala Cys Leu Gly Gln Cys Phe Ser 50 55 60 Tyr
Ser Val Pro Asn Thr Phe Pro Gln Ser Thr Glu Ser Leu Val His 65 70
75 80 Cys Asp Ser Cys Met Pro Ala Gln Ser Met Trp Glu Ile Val Thr
Leu 85 90 95 Glu Cys Pro Gly His Glu Glu Val Pro Arg Val Asp Lys
Leu Val Glu 100 105 110 Lys Ile Leu His Cys Ser Cys Gln Ala Cys Gly
Lys Glu Pro Ser His 115 120 125 Glu Gly Leu Ser Val Tyr Val Gln Gly
Glu Asp Gly Pro Gly Ser Gln 130 135 140 Pro Gly Thr His Pro His Pro
His Pro His Pro His Pro Gly Gly Gln 145 150 155 160 Thr Pro Glu Pro
Glu Asp Pro Pro Gly Ala Pro His Thr Glu Glu Glu 165 170 175 Gly Ala
Glu Asp 180 45 642 DNA Homo sapiens CDS (1)..(639) 45 atg cag ctc
cca ctg gcc ctg tgt ctc gtc tgc ctg ctg gta cac aca 48 Met Gln Leu
Pro Leu Ala Leu Cys Leu Val Cys Leu Leu Val His Thr 1 5 10 15 gcc
ttc cgt gta gtg gag ggc cag ggg tgg cag gcg ttc aag aat gat 96 Ala
Phe Arg Val Val Glu Gly Gln Gly Trp Gln Ala Phe Lys Asn Asp 20 25
30 gcc acg gaa atc atc ccc gag ctc gga gag tac ccc gag cct cca ccg
144 Ala Thr Glu Ile Ile Pro Glu Leu Gly Glu Tyr Pro Glu Pro Pro Pro
35 40 45 gag ctg gag aac aac aag acc atg aac cgg gcg gag aac gga
ggg cgg 192 Glu Leu Glu Asn Asn Lys Thr Met Asn Arg Ala Glu Asn Gly
Gly Arg 50 55 60 cct ccc cac cac ccc ttt gag acc aaa gac gtg tcc
gag tac agc tgc 240 Pro Pro His His Pro Phe Glu Thr Lys Asp Val Ser
Glu Tyr Ser Cys 65 70 75 80 cgc gag ctg cac ttc acc cgc tac gtg acc
gat ggg ccg tgc cgc agc 288 Arg Glu Leu His Phe Thr Arg Tyr Val Thr
Asp Gly Pro Cys Arg Ser 85 90 95 gcc aag ccg gtc acc gag ctg gtg
tgc tcc ggc cag tgc ggc ccg gcg 336 Ala Lys Pro Val Thr Glu Leu Val
Cys Ser Gly Gln Cys Gly Pro Ala 100 105 110 cgc ctg ctg ccc aac gcc
atc ggc cgc ggc aag tgg tgg cga cct agt 384 Arg Leu Leu Pro Asn Ala
Ile Gly Arg Gly Lys Trp Trp Arg Pro Ser 115 120 125 ggg ccc gac ttc
cgc tgc atc ccc gac cgc tac cgc gcg cag cgc gtg 432 Gly Pro Asp Phe
Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gln Arg Val 130 135 140 cag ctg
ctg tgt ccc ggt ggt gag gcg ccg cgc gcg cgc aag gtg cgc 480 Gln Leu
Leu Cys Pro Gly Gly Glu Ala Pro Arg Ala Arg Lys Val Arg 145 150 155
160 ctg gtg gcc tcg tgc aag tgc aag cgc ctc acc cgc ttc cac aac cag
528 Leu Val Ala Ser Cys Lys Cys Lys Arg Leu Thr Arg Phe His Asn Gln
165 170 175 tcg gag ctc aag gac ttc ggg acc gag gcc gct cgg ccg cag
aag ggc 576 Ser Glu Leu Lys Asp Phe Gly Thr Glu Ala Ala Arg Pro Gln
Lys Gly 180 185 190 cgg aag ccg cgg ccc cgc gcc cgg agc gcc aaa gcc
aac cag gcc gag 624 Arg Lys Pro Arg Pro Arg Ala Arg Ser Ala Lys Ala
Asn Gln Ala Glu 195 200 205
ctg gag aac gcc tac tag 642 Leu Glu Asn Ala Tyr 210 46 190 PRT Homo
sapiens 46 Gln Gly Trp Gln Ala Phe Lys Asn Asp Ala Thr Glu Ile Ile
Pro Glu 1 5 10 15 Leu Gly Glu Tyr Pro Glu Pro Pro Pro Glu Leu Glu
Asn Asn Lys Thr 20 25 30 Met Asn Arg Ala Glu Asn Gly Gly Arg Pro
Pro His His Pro Phe Glu 35 40 45 Thr Lys Asp Val Ser Glu Tyr Ser
Cys Arg Glu Leu His Phe Thr Arg 50 55 60 Tyr Val Thr Asp Gly Pro
Cys Arg Ser Ala Lys Pro Val Thr Glu Leu 65 70 75 80 Val Cys Ser Gly
Gln Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile 85 90 95 Gly Arg
Gly Lys Trp Trp Arg Pro Ser Gly Pro Asp Phe Arg Cys Ile 100 105 110
Pro Asp Arg Tyr Arg Ala Gln Arg Val Gln Leu Leu Cys Pro Gly Gly 115
120 125 Glu Ala Pro Arg Ala Arg Lys Val Arg Leu Val Ala Ser Cys Lys
Cys 130 135 140 Lys Arg Leu Thr Arg Phe His Asn Gln Ser Glu Leu Lys
Asp Phe Gly 145 150 155 160 Thr Glu Ala Ala Arg Pro Gln Lys Gly Arg
Lys Pro Arg Pro Arg Ala 165 170 175 Arg Ser Ala Lys Ala Asn Gln Ala
Glu Leu Glu Asn Ala Tyr 180 185 190 47 20 PRT Homo sapiens 47 Gln
Gly Trp Gln Ala Phe Lys Asn Asp Ala Thr Glu Ile Ile Pro Glu 1 5 10
15 Leu Gly Glu Tyr 20 48 20 PRT Homo sapiens 48 Thr Glu Ile Ile Pro
Glu Leu Gly Glu Tyr Pro Glu Pro Pro Pro Glu 1 5 10 15 Leu Glu Asn
Asn 20 49 20 PRT Homo sapiens 49 Pro Glu Pro Pro Pro Glu Leu Glu
Asn Asn Lys Thr Met Asn Arg Ala 1 5 10 15 Glu Asn Gly Gly 20 50 20
PRT Homo sapiens 50 Lys Thr Met Asn Arg Ala Glu Asn Gly Gly Arg Pro
Pro His His Pro 1 5 10 15 Phe Glu Thr Lys 20 51 16 PRT Homo sapiens
51 Arg Pro Pro His His Pro Phe Glu Thr Lys Asp Val Ser Glu Tyr Ser
1 5 10 15 52 21 PRT Artificial Sequence Human SOST peptide fragment
with additional cysteine added 52 Gln Gly Trp Gln Ala Phe Lys Asn
Asp Ala Thr Glu Ile Ile Pro Glu 1 5 10 15 Leu Gly Glu Tyr Cys 20 53
21 PRT Artificial Sequence Human SOST peptide fragment with
additional cysteine added 53 Thr Glu Ile Ile Pro Glu Leu Gly Glu
Tyr Pro Glu Pro Pro Pro Glu 1 5 10 15 Leu Glu Asn Asn Cys 20 54 21
PRT Artificial Sequence Human SOST peptide fragment with additional
cysteine added 54 Pro Glu Pro Pro Pro Glu Leu Glu Asn Asn Lys Thr
Met Asn Arg Ala 1 5 10 15 Glu Asn Gly Gly Cys 20 55 21 PRT
Artificial Sequence Human SOST peptide fragment with additional
cysteine added 55 Lys Thr Met Asn Arg Ala Glu Asn Gly Gly Arg Pro
Pro His His Pro 1 5 10 15 Phe Glu Thr Lys Cys 20 56 17 PRT
Artificial Sequence Human SOST peptide fragment with additional
cysteine added 56 Arg Pro Pro His His Pro Phe Glu Thr Lys Asp Val
Ser Glu Tyr Ser 1 5 10 15 Cys 57 24 PRT Rattus norvegicus 57 Gln
Gly Trp Gln Ala Phe Lys Asn Asp Ala Thr Glu Ile Ile Pro Gly 1 5 10
15 Leu Arg Glu Tyr Pro Glu Pro Pro 20 58 20 PRT Rattus morvegicus
58 Pro Glu Pro Pro Gln Glu Leu Glu Asn Asn Gln Thr Met Asn Arg Ala
1 5 10 15 Glu Asn Gly Gly 20 59 20 PRT Rattus norvegicus 59 Glu Asn
Gly Gly Arg Pro Pro His His Pro Tyr Asp Thr Lys Asp Val 1 5 10 15
Ser Glu Tyr Ser 20 60 20 PRT Rattus norvegicus 60 Thr Glu Ile Ile
Pro Gly Leu Arg Glu Tyr Pro Glu Pro Pro Gln Glu 1 5 10 15 Leu Glu
Asn Asn 20 61 25 PRT Artificial Sequence Rat SOST peptide fragment
with additional cysteine added 61 Gln Gly Trp Gln Ala Phe Lys Asn
Asp Ala Thr Glu Ile Ile Pro Gly 1 5 10 15 Leu Arg Glu Tyr Pro Glu
Pro Pro Cys 20 25 62 21 PRT Artificial Sequence Rat SOST peptide
fragment with additional cysteine added 62 Pro Glu Pro Pro Gln Glu
Leu Glu Asn Asn Gln Thr Met Asn Arg Ala 1 5 10 15 Glu Asn Gly Gly
Cys 20 63 21 PRT Artificial Sequence Rat SOST peptide fragment with
additional cysteine added 63 Glu Asn Gly Gly Arg Pro Pro His His
Pro Tyr Asp Thr Lys Asp Val 1 5 10 15 Ser Glu Tyr Ser Cys 20 64 21
PRT Artificial Sequence Rat SOST peptide fragment with additional
cysteine added 64 Thr Glu Ile Ile Pro Gly Leu Arg Glu Tyr Pro Glu
Pro Pro Gln Glu 1 5 10 15 Leu Glu Asn Asn Cys 20 65 190 PRT Rattus
norvegicus 65 Gln Gly Trp Gln Ala Phe Lys Asn Asp Ala Thr Glu Ile
Ile Pro Gly 1 5 10 15 Leu Arg Glu Tyr Pro Glu Pro Pro Gln Glu Leu
Glu Asn Asn Gln Thr 20 25 30 Met Asn Arg Ala Glu Asn Gly Gly Arg
Pro Pro His His Pro Tyr Asp 35 40 45 Thr Lys Asp Val Ser Glu Tyr
Ser Cys Arg Glu Leu His Tyr Thr Arg 50 55 60 Phe Val Thr Asp Gly
Pro Cys Arg Ser Ala Lys Pro Val Thr Glu Leu 65 70 75 80 Val Cys Ser
Gly Gln Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile 85 90 95 Gly
Arg Val Lys Trp Trp Arg Pro Asn Gly Pro Asp Phe Arg Cys Ile 100 105
110 Pro Asp Arg Tyr Arg Ala Gln Arg Val Gln Leu Leu Cys Pro Gly Gly
115 120 125 Ala Ala Pro Arg Ser Arg Lys Val Arg Leu Val Ala Ser Cys
Lys Cys 130 135 140 Lys Arg Leu Thr Arg Phe His Asn Gln Ser Glu Leu
Lys Asp Phe Gly 145 150 155 160 Pro Glu Thr Ala Arg Pro Gln Lys Gly
Arg Lys Pro Arg Pro Arg Ala 165 170 175 Arg Gly Ala Lys Ala Asn Gln
Ala Glu Leu Glu Asn Ala Tyr 180 185 190 66 20 PRT Homo sapiens 66
Ile Pro Asp Arg Tyr Arg Ala Gln Arg Val Gln Leu Leu Cys Pro Gly 1 5
10 15 Gly Glu Ala Pro 20 67 20 PRT Homo sapiens 67 Gln Leu Leu Cys
Pro Gly Gly Glu Ala Pro Arg Ala Arg Lys Val Arg 1 5 10 15 Leu Val
Ala Ser 20 68 21 PRT Artificial Sequence Human SOST peptide
fragment with additional cysteine added 68 Ile Pro Asp Arg Tyr Arg
Ala Gln Arg Val Gln Leu Leu Cys Pro Gly 1 5 10 15 Gly Glu Ala Pro
Cys 20 69 21 PRT Artificial Sequence Human SOST peptide fragment
with additional cysteine added 69 Gln Leu Leu Cys Pro Gly Gly Glu
Ala Pro Arg Ala Arg Lys Val Arg 1 5 10 15 Leu Val Ala Ser Cys 20 70
17 PRT Rattus Norvegicus 70 Ile Pro Asp Arg Tyr Arg Ala Gln Arg Val
Gln Leu Leu Cys Pro Gly 1 5 10 15 Gly 71 16 PRT Rattus norvegicus
71 Pro Gly Gly Ala Ala Pro Arg Ser Arg Lys Val Arg Leu Val Ala Ser
1 5 10 15 72 18 PRT Artificial Sequence Rat SOST peptide fragment
with additional cysteine added 72 Ile Pro Asp Arg Tyr Arg Ala Gln
Arg Val Gln Leu Leu Ser Pro Gly 1 5 10 15 Gly Cys 73 17 PRT
Artificial Sequence Rat SOST peptide fragment with additional
cysteine added 73 Pro Gly Gly Ala Ala Pro Arg Ser Arg Lys Val Arg
Leu Val Ala Ser 1 5 10 15 Cys 74 20 PRT Homo sapiens 74 Cys Gly Pro
Ala Arg Leu Leu Pro Asn Ala Ile Gly Arg Gly Lys Trp 1 5 10 15 Trp
Arg Pro Ser 20 75 16 PRT Homo sapiens 75 Ile Gly Arg Gly Lys Trp
Trp Arg Pro Ser Gly Pro Asp Phe Arg Cys 1 5 10 15 76 18 PRT Rattus
norvegicus 76 Pro Asn Ala Ile Gly Arg Val Lys Trp Trp Arg Pro Asn
Gly Pro Asp 1 5 10 15 Phe Arg 77 19 PRT Artificial Sequence Rat
SOST peptide fragment with additional cysteine added 77 Pro Asn Ala
Ile Gly Arg Val Lys Trp Trp Arg Pro Asn Gly Pro Asp 1 5 10 15 Phe
Arg Cys 78 20 PRT Homo sapiens 78 Lys Arg Leu Thr Arg Phe His Asn
Gln Ser Glu Leu Lys Asp Phe Gly 1 5 10 15 Thr Glu Ala Ala 20 79 20
PRT Homo sapiens 79 Glu Leu Lys Asp Phe Gly Thr Glu Ala Ala Arg Pro
Gln Lys Gly Arg 1 5 10 15 Lys Pro Arg Pro 20 80 20 PRT Homo sapiens
80 Arg Pro Gln Lys Gly Arg Lys Pro Arg Pro Arg Ala Arg Ser Ala Lys
1 5 10 15 Ala Asn Gln Ala 20 81 16 PRT Homo sapiens 81 Arg Ala Arg
Ser Ala Lys Ala Asn Gln Ala Glu Leu Glu Asn Ala Tyr 1 5 10 15 82 21
PRT Artificial Sequence Human SOST peptide fragment with additional
cysteine added 82 Lys Arg Leu Thr Arg Phe His Asn Gln Ser Glu Leu
Lys Asp Phe Gly 1 5 10 15 Thr Glu Ala Ala Cys 20 83 21 PRT
Artificial Sequence Human SOST peptide fragment with additional
cysteine added 83 Glu Leu Lys Asp Phe Gly Thr Glu Ala Ala Arg Pro
Gln Lys Gly Arg 1 5 10 15 Lys Pro Arg Pro Cys 20 84 21 PRT
Artificial Sequence Human SOST peptide fragment with additional
cysteine added 84 Arg Pro Gln Lys Gly Arg Lys Pro Arg Pro Arg Ala
Arg Ser Ala Lys 1 5 10 15 Ala Asn Gln Ala Cys 20 85 17 PRT
Artificial Sequence Human SOST peptide fragment with additional
cysteine added 85 Arg Ala Arg Ser Ala Lys Ala Asn Gln Ala Glu Leu
Glu Asn Ala Tyr 1 5 10 15 Cys 86 23 PRT Rattus norvegicus 86 Lys
Arg Leu Thr Arg Phe His Asn Gln Ser Glu Leu Lys Asp Phe Gly 1 5 10
15 Pro Glu Thr Ala Arg Pro Gln 20 87 23 PRT Rattus norvegicus 87
Lys Gly Arg Lys Pro Arg Pro Arg Ala Arg Gly Ala Lys Ala Asn Gln 1 5
10 15 Ala Glu Leu Glu Asn Ala Tyr 20 88 24 PRT Rattus norvegicus 88
Ser Glu Leu Lys Asp Phe Gly Pro Glu Thr Ala Arg Pro Gln Lys Gly 1 5
10 15 Arg Lys Pro Arg Pro Arg Ala Arg 20 89 24 PRT Artificial
Sequence Rat SOST peptide fragment with additional cysteine added
89 Lys Arg Leu Thr Arg Phe His Asn Gln Ser Glu Leu Lys Asp Phe Gly
1 5 10 15 Pro Glu Thr Ala Arg Pro Gln Cys 20 90 24 PRT Artificial
Sequence Rat SOST peptide fragment with additional cysteine added
90 Lys Gly Arg Lys Pro Arg Pro Arg Ala Arg Gly Ala Lys Ala Asn Gln
1 5 10 15 Ala Glu Leu Glu Asn Ala Tyr Cys 20 91 25 PRT Artificial
Sequence Rat SOST peptide fragment with additional cysteine added
91 Ser Glu Leu Lys Asp Phe Gly Pro Glu Thr Ala Arg Pro Gln Lys Gly
1 5 10 15 Arg Lys Pro Arg Pro Arg Ala Arg Cys 20 25 92 56 PRT Homo
sapiens 92 Gln Gly Trp Gln Ala Phe Lys Asn Asp Ala Thr Glu Ile Ile
Pro Glu 1 5 10 15 Leu Gly Glu Tyr Pro Glu Pro Pro Pro Glu Leu Glu
Asn Asn Lys Thr 20 25 30 Met Asn Arg Ala Glu Asn Gly Gly Arg Pro
Pro His His Pro Phe Glu 35 40 45 Thr Lys Asp Val Ser Glu Tyr Ser 50
55 93 56 PRT Rattus norvegicus 93 Gln Gly Trp Gln Ala Phe Lys Asn
Asp Ala Thr Glu Ile Ile Pro Gly 1 5 10 15 Leu Arg Glu Tyr Pro Glu
Pro Pro Gln Glu Leu Glu Asn Asn Gln Thr 20 25 30 Met Asn Arg Ala
Glu Asn Gly Gly Arg Pro Pro His His Pro Tyr Asp 35 40 45 Thr Lys
Asp Val Ser Glu Tyr Ser 50 55 94 32 PRT Homo sapiens 94 Cys Ile Pro
Asp Arg Tyr Arg Ala Gln Arg Val Gln Leu Leu Cys Pro 1 5 10 15 Gly
Gly Glu Ala Pro Arg Ala Arg Lys Val Arg Leu Val Ala Ser Cys 20 25
30 95 32 PRT Rattus norvegicus 95 Cys Ile Pro Asp Arg Tyr Arg Ala
Gln Arg Val Gln Leu Leu Cys Pro 1 5 10 15 Gly Gly Ala Ala Pro Arg
Ser Arg Lys Val Arg Leu Val Ala Ser Cys 20 25 30 96 44 PRT Homo
sapiens 96 Leu Thr Arg Phe His Asn Gln Ser Glu Leu Lys Asp Phe Gly
Thr Glu 1 5 10 15 Ala Ala Arg Pro Gln Lys Gly Arg Lys Pro Arg Pro
Arg Ala Arg Ser 20 25 30 Ala Lys Ala Asn Gln Ala Glu Leu Glu Asn
Ala Tyr 35 40 97 44 PRT Rattus norvegicus 97 Leu Thr Arg Phe His
Asn Gln Ser Glu Leu Lys Asp Phe Gly Pro Glu 1 5 10 15 Thr Ala Arg
Pro Gln Lys Gly Arg Lys Pro Arg Pro Arg Ala Arg Gly 20 25 30 Ala
Lys Ala Asn Gln Ala Glu Leu Glu Asn Ala Tyr 35 40 98 26 PRT Homo
sapiens 98 Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile Gly Arg Gly
Lys Trp 1 5 10 15 Trp Arg Pro Ser Gly Pro Asp Phe Arg Cys 20 25 99
26 PRT Rattus norvegicus 99 Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala
Ile Gly Arg Val Lys Trp 1 5 10 15 Trp Arg Pro Asn Gly Pro Asp Phe
Arg Cys 20 25 100 570 DNA Homo sapiens 100 caggggtggc aggcgttcaa
gaatgatgcc acggaaatca tccccgagct cggagagtac 60 cccgagcctc
caccggagct ggagaacaac aagaccatga accgggcgga gaacggaggg 120
cggcctcccc accacccctt tgagaccaaa gacgtgtccg agtacagctg ccgcgagctg
180 cacttcaccc gctacgtgac cgatgggccg tgccgcagcg ccaagccggt
caccgagctg 240 gtgtgctccg gccagtgcgg cccggcgcgc ctgctgccca
acgccatcgg ccgcggcaag 300 tggtggcgac ctagtgggcc cgacttccgc
tgcatccccg accgctaccg cgcgcagcgc 360 gtgcagctgc tgtgtcccgg
tggtgaggcg ccgcgcgcgc gcaaggtgcg cctggtggcc 420 tcgtgcaagt
gcaagcgcct cacccgcttc cacaaccagt cggagctcaa ggacttcggg 480
accgaggccg ctcggccgca gaagggccgg aagccgcggc cccgcgcccg gagcgccaaa
540 gccaaccagg ccgagctgga gaacgcctac 570 101 570 DNA Rattus
norvegicus 101 caggggtggc aagccttcaa gaatgatgcc acagaaatca
tcccgggact cagagagtac 60 ccagagcctc ctcaggaact agagaacaac
cagaccatga accgggccga gaacggaggc 120 agaccccccc accatcctta
tgacaccaaa gacgtgtccg agtacagctg ccgcgagctg 180 cactacaccc
gcttcgtgac cgacggcccg tgccgcagtg ccaagccggt caccgagttg 240
gtgtgctcgg gccagtgcgg ccccgcgcgg ctgctgccca acgccatcgg gcgcgtgaag
300 tggtggcgcc cgaacggacc cgacttccgc tgcatcccgg atcgctaccg
cgcgcagcgg 360 gtgcagctgc tgtgccccgg cggcgcggcg ccgcgctcgc
gcaaggtgcg tctggtggcc 420 tcgtgcaagt gcaagcgcct cacccgcttc
cacaaccagt cggagctcaa ggacttcgga 480 cctgagaccg cgcggccgca
gaagggtcgc aagccgcggc cccgcgcccg gggagccaaa 540 gccaaccagg
cggagctgga gaacgcctac 570 102 532 PRT Homo sapiens 102 Met Thr Gln
Leu Tyr Ile Tyr Ile Arg Leu Leu Gly Ala Tyr Leu Phe 1 5 10 15 Ile
Ile Ser Arg Val Gln Gly Gln Asn Leu Asp Ser Met Leu His Gly 20 25
30 Thr Gly Met Lys Ser Asp Ser Asp Gln Lys Lys Ser Glu Asn Gly Val
35 40 45 Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys Cys Tyr
Cys Ser 50 55 60 Gly His Cys Pro Asp Asp Ala Ile Asn Asn Thr Cys
Ile Thr Asn Gly 65 70 75 80 His Cys Phe Ala Ile Ile Glu Glu Asp Asp
Gln Gly Glu Thr Thr Leu 85 90 95 Ala Ser Gly Cys Met Lys Tyr Glu
Gly Ser Asp Phe Gln Cys Lys Asp 100 105 110 Ser Pro Lys Ala Gln Leu
Arg Arg Thr Ile Glu Cys Cys Arg Thr Asn 115 120 125 Leu Cys Asn Gln
Tyr Leu Gln Pro Thr Leu Pro Pro Val Val Ile Gly 130 135 140 Pro Phe
Phe Asp Gly Ser Ile Arg Trp Leu Val Leu Leu Ile Ser Met 145 150 155
160 Ala Val Cys Ile Ile Ala Met Ile Ile Phe Ser Ser Cys Phe Cys Tyr
165 170 175 Lys His Tyr Cys Lys Ser Ile Ser Ser Arg Arg Arg Tyr Asn
Arg Asp 180 185 190 Leu Glu Gln Asp Glu Ala Phe Ile Pro Val Gly Glu
Ser Leu Lys Asp 195 200 205 Leu Ile Asp Gln Ser Gln Ser Ser Gly Ser
Gly Ser Gly Leu Pro Leu 210
215 220 Leu Val Gln Arg Thr Ile Ala Lys Gln Ile Gln Met Val Arg Gln
Val 225 230 235 240 Gly Lys Gly Arg Tyr Gly Glu Val Trp Met Gly Lys
Trp Arg Gly Glu 245 250 255 Lys Val Ala Val Lys Val Phe Phe Thr Thr
Glu Glu Ala Ser Trp Phe 260 265 270 Arg Glu Thr Glu Ile Tyr Gln Thr
Val Leu Met Arg His Glu Asn Ile 275 280 285 Leu Gly Phe Ile Ala Ala
Asp Ile Lys Gly Thr Gly Ser Trp Thr Gln 290 295 300 Leu Tyr Leu Ile
Thr Asp Tyr His Glu Asn Gly Ser Leu Tyr Asp Phe 305 310 315 320 Leu
Lys Cys Ala Thr Leu Asp Thr Arg Ala Leu Leu Lys Leu Ala Tyr 325 330
335 Ser Ala Ala Cys Gly Leu Cys His Leu His Thr Glu Ile Tyr Gly Thr
340 345 350 Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser Lys
Asn Ile 355 360 365 Leu Ile Lys Lys Asn Gly Ser Cys Cys Ile Ala Asp
Leu Gly Leu Ala 370 375 380 Val Lys Phe Asn Ser Asp Thr Asn Glu Val
Asp Val Pro Leu Asn Thr 385 390 395 400 Arg Val Gly Thr Lys Arg Tyr
Met Ala Pro Glu Val Leu Asp Glu Ser 405 410 415 Leu Asn Lys Asn His
Phe Gln Pro Tyr Ile Met Ala Asp Ile Tyr Ser 420 425 430 Phe Gly Leu
Ile Ile Trp Glu Met Ala Arg Arg Cys Ile Thr Gly Gly 435 440 445 Ile
Val Glu Glu Tyr Gln Leu Pro Tyr Tyr Asn Met Val Pro Ser Asp 450 455
460 Pro Ser Tyr Glu Asp Met Arg Glu Val Val Cys Val Lys Arg Leu Arg
465 470 475 480 Pro Ile Val Ser Asn Arg Trp Asn Ser Asp Glu Cys Leu
Arg Ala Val 485 490 495 Leu Lys Leu Met Ser Glu Cys Trp Ala His Asn
Pro Ala Ser Arg Leu 500 505 510 Thr Ala Leu Arg Ile Lys Lys Thr Leu
Ala Lys Met Val Glu Ser Gln 515 520 525 Asp Val Lys Ile 530 103 502
PRT Homo sapiens 103 Met Leu Leu Arg Ser Ala Gly Lys Leu Asn Val
Gly Thr Lys Lys Glu 1 5 10 15 Asp Gly Glu Ser Thr Ala Pro Thr Pro
Arg Pro Lys Val Leu Arg Cys 20 25 30 Lys Cys His His His Cys Pro
Glu Asp Ser Val Asn Asn Ile Cys Ser 35 40 45 Thr Asp Gly Tyr Cys
Phe Thr Met Ile Glu Glu Asp Asp Ser Gly Leu 50 55 60 Pro Val Val
Thr Ser Gly Cys Leu Gly Leu Glu Gly Ser Asp Phe Gln 65 70 75 80 Cys
Arg Asp Thr Pro Ile Pro His Gln Arg Arg Ser Ile Glu Cys Cys 85 90
95 Thr Glu Arg Asn Glu Cys Asn Lys Asp Leu His Pro Thr Leu Pro Pro
100 105 110 Leu Lys Asn Arg Asp Phe Val Asp Gly Pro Ile His His Arg
Ala Leu 115 120 125 Leu Ile Ser Val Thr Val Cys Ser Leu Leu Leu Val
Leu Ile Ile Leu 130 135 140 Phe Cys Tyr Phe Arg Tyr Lys Arg Gln Glu
Thr Arg Pro Arg Tyr Ser 145 150 155 160 Ile Gly Leu Glu Gln Asp Glu
Thr Tyr Ile Pro Pro Gly Glu Ser Leu 165 170 175 Arg Asp Leu Ile Glu
Gln Ser Gln Ser Ser Gly Ser Gly Ser Gly Leu 180 185 190 Pro Leu Leu
Val Gln Arg Thr Ile Ala Lys Gln Ile Gln Met Val Lys 195 200 205 Gln
Ile Gly Lys Gly Arg Tyr Gly Glu Val Trp Met Gly Lys Trp Arg 210 215
220 Gly Glu Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu Ala Ser
225 230 235 240 Trp Phe Arg Glu Thr Glu Ile Tyr Gln Thr Val Leu Met
Arg His Glu 245 250 255 Asn Ile Leu Gly Phe Ile Ala Ala Asp Ile Lys
Gly Thr Gly Ser Trp 260 265 270 Thr Gln Leu Tyr Leu Ile Thr Asp Tyr
His Glu Asn Gly Ser Leu Tyr 275 280 285 Asp Tyr Leu Lys Ser Thr Thr
Leu Asp Ala Lys Ser Met Leu Lys Leu 290 295 300 Ala Tyr Ser Ser Val
Ser Gly Leu Cys His Leu His Thr Glu Ile Phe 305 310 315 320 Ser Thr
Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser Lys 325 330 335
Asn Ile Leu Val Lys Lys Asn Gly Thr Cys Cys Ile Ala Asp Leu Gly 340
345 350 Leu Ala Val Lys Phe Ile Ser Asp Thr Asn Glu Val Asp Ile Pro
Pro 355 360 365 Asn Thr Arg Val Gly Thr Lys Arg Tyr Met Pro Pro Glu
Val Leu Asp 370 375 380 Glu Ser Leu Asn Arg Asn His Phe Gln Ser Tyr
Ile Met Ala Asp Met 385 390 395 400 Tyr Ser Phe Gly Leu Ile Leu Trp
Glu Val Ala Arg Arg Cys Val Ser 405 410 415 Gly Gly Ile Val Glu Glu
Tyr Gln Leu Pro Tyr His Asp Leu Val Pro 420 425 430 Ser Asp Pro Ser
Tyr Glu Asp Met Arg Glu Ile Val Cys Ile Lys Lys 435 440 445 Leu Arg
Pro Ser Phe Pro Asn Arg Trp Ser Ser Asp Glu Cys Leu Arg 450 455 460
Gln Met Gly Lys Leu Met Thr Glu Cys Trp Ala His Asn Pro Ala Ser 465
470 475 480 Arg Leu Thr Ala Leu Arg Val Lys Lys Thr Leu Ala Lys Met
Ser Glu 485 490 495 Ser Gln Asp Ile Lys Leu 500 104 502 PRT Homo
sapiens 104 Met Leu Leu Arg Ser Ala Gly Lys Leu Asn Val Gly Thr Lys
Lys Glu 1 5 10 15 Asp Gly Glu Ser Thr Ala Pro Thr Pro Arg Pro Lys
Val Leu Arg Cys 20 25 30 Lys Cys His His His Cys Pro Glu Asp Ser
Val Asn Asn Ile Cys Ser 35 40 45 Thr Asp Gly Tyr Cys Phe Thr Met
Ile Glu Glu Asp Asp Ser Gly Leu 50 55 60 Pro Val Val Thr Ser Gly
Cys Leu Gly Leu Glu Gly Ser Asp Phe Gln 65 70 75 80 Cys Arg Asp Thr
Pro Ile Pro His Gln Arg Arg Ser Ile Glu Cys Cys 85 90 95 Thr Glu
Arg Asn Glu Cys Asn Lys Asp Leu His Pro Thr Leu Pro Pro 100 105 110
Leu Lys Asn Arg Asp Phe Val Asp Gly Pro Ile His His Arg Ala Leu 115
120 125 Leu Ile Ser Val Thr Val Cys Ser Leu Leu Leu Val Leu Ile Ile
Leu 130 135 140 Phe Cys Tyr Phe Arg Tyr Lys Arg Gln Glu Thr Arg Pro
Arg Tyr Ser 145 150 155 160 Ile Gly Leu Glu Gln Asp Glu Thr Tyr Ile
Pro Pro Gly Glu Ser Leu 165 170 175 Arg Asp Leu Ile Glu Gln Ser Gln
Ser Ser Gly Ser Gly Ser Gly Leu 180 185 190 Pro Leu Leu Val Gln Arg
Thr Ile Ala Lys Gln Ile Gln Met Val Lys 195 200 205 Gln Ile Gly Lys
Gly Arg Tyr Gly Glu Val Trp Met Gly Lys Trp Arg 210 215 220 Gly Glu
Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu Ala Ser 225 230 235
240 Trp Phe Arg Glu Thr Glu Ile Tyr Gln Thr Val Leu Met Arg His Glu
245 250 255 Asn Ile Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly
Ser Trp 260 265 270 Thr Gln Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn
Gly Ser Leu Tyr 275 280 285 Asp Tyr Leu Lys Ser Thr Thr Leu Asp Ala
Lys Ser Met Leu Lys Leu 290 295 300 Ala Tyr Ser Ser Val Ser Gly Leu
Cys His Leu His Thr Glu Ile Phe 305 310 315 320 Ser Thr Gln Gly Lys
Pro Ala Ile Ala His Arg Asp Leu Lys Ser Lys 325 330 335 Asn Ile Leu
Val Lys Lys Asn Gly Thr Cys Cys Ile Ala Asp Leu Gly 340 345 350 Leu
Ala Val Lys Phe Ile Ser Asp Thr Asn Glu Val Asp Ile Pro Pro 355 360
365 Asn Thr Arg Val Gly Thr Lys Arg Tyr Met Pro Pro Glu Val Leu Asp
370 375 380 Glu Ser Leu Asn Arg Asn His Phe Gln Ser Tyr Ile Met Ala
Asp Met 385 390 395 400 Tyr Ser Phe Gly Leu Ile Leu Trp Glu Val Ala
Arg Arg Cys Val Ser 405 410 415 Gly Gly Ile Val Glu Glu Tyr Gln Leu
Pro Tyr His Asp Leu Val Pro 420 425 430 Ser Asp Pro Ser Tyr Glu Asp
Met Arg Glu Ile Val Cys Ile Lys Lys 435 440 445 Leu Arg Pro Ser Phe
Pro Asn Arg Trp Ser Ser Asp Glu Cys Leu Arg 450 455 460 Gln Met Gly
Lys Leu Met Thr Glu Cys Trp Ala His Asn Pro Ala Ser 465 470 475 480
Arg Leu Thr Ala Leu Arg Val Lys Lys Thr Leu Ala Lys Met Ser Glu 485
490 495 Ser Gln Asp Ile Lys Leu 500 105 532 PRT Rattus sp. 105 Met
Thr Gln Leu Tyr Thr Tyr Ile Arg Leu Leu Gly Ala Cys Leu Phe 1 5 10
15 Ile Ile Ser His Val Gln Gly Gln Asn Leu Asp Ser Met Leu His Gly
20 25 30 Thr Gly Met Lys Ser Asp Val Asp Gln Lys Lys Pro Glu Asn
Gly Val 35 40 45 Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys
Cys Tyr Cys Ser 50 55 60 Gly His Cys Pro Asp Asp Ala Ile Asn Asn
Thr Cys Ile Thr Asn Gly 65 70 75 80 His Cys Phe Ala Ile Ile Glu Glu
Asp Asp Gln Gly Glu Thr Thr Leu 85 90 95 Thr Ser Gly Cys Met Lys
Tyr Glu Gly Ser Asp Phe Gln Cys Lys Asp 100 105 110 Ser Pro Lys Ala
Gln Leu Arg Arg Thr Ile Glu Cys Cys Arg Thr Asn 115 120 125 Leu Cys
Asn Gln Tyr Leu Gln Pro Thr Leu Pro Pro Val Val Ile Gly 130 135 140
Pro Phe Phe Asp Gly Ser Val Arg Trp Leu Ala Val Leu Ile Ser Met 145
150 155 160 Ala Val Cys Ile Val Ala Met Ile Val Phe Ser Ser Cys Phe
Cys Tyr 165 170 175 Lys His Tyr Cys Lys Ser Ile Ser Ser Arg Gly Arg
Tyr Asn Arg Asp 180 185 190 Leu Glu Gln Asp Glu Ala Phe Ile Pro Val
Gly Glu Ser Leu Lys Asp 195 200 205 Leu Ile Asp Gln Ser Gln Ser Ser
Gly Ser Gly Ser Gly Leu Pro Leu 210 215 220 Leu Val Gln Arg Thr Ile
Ala Lys Gln Ile Gln Met Val Arg Gln Val 225 230 235 240 Gly Lys Gly
Arg Tyr Gly Glu Val Trp Met Gly Lys Trp Arg Gly Glu 245 250 255 Lys
Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu Ala Ser Trp Phe 260 265
270 Arg Glu Thr Glu Ile Tyr Gln Thr Val Leu Met Arg His Glu Asn Ile
275 280 285 Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly Ser Trp
Thr Gln 290 295 300 Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn Gly Ser
Leu Tyr Asp Phe 305 310 315 320 Leu Lys Cys Ala Thr Leu Asp Thr Arg
Ala Leu Leu Lys Leu Ala Tyr 325 330 335 Ser Ala Ala Cys Gly Leu Cys
His Leu His Thr Glu Ile Tyr Gly Thr 340 345 350 Gln Gly Lys Pro Ala
Ile Ala His Arg Asp Leu Lys Ser Lys Asn Ile 355 360 365 Leu Ile Lys
Lys Asn Gly Ser Cys Cys Ile Ala Asp Leu Gly Leu Ala 370 375 380 Val
Lys Phe Asn Ser Asp Thr Asn Glu Val Asp Ile Pro Leu Asn Thr 385 390
395 400 Arg Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Asp Glu
Ser 405 410 415 Leu Ser Lys Asn His Phe Gln Pro Tyr Ile Met Ala Asp
Ile Tyr Ser 420 425 430 Phe Gly Leu Ile Ile Trp Glu Met Ala Arg Arg
Cys Ile Thr Gly Gly 435 440 445 Ile Val Glu Glu Tyr Gln Leu Pro Tyr
Tyr Asn Met Val Pro Ser Asp 450 455 460 Pro Ser Tyr Glu Asp Met Arg
Glu Val Val Cys Val Lys Arg Leu Arg 465 470 475 480 Pro Ile Val Ser
Asn Arg Trp Asn Ser Asp Glu Cys Leu Arg Ala Val 485 490 495 Leu Lys
Leu Met Ser Glu Cys Trp Ala His Asn Pro Ala Ser Arg Leu 500 505 510
Thr Ala Leu Arg Ile Lys Lys Thr Leu Ala Lys Met Val Glu Ser Gln 515
520 525 Asp Val Lys Ile 530 106 532 PRT Rattus norvegicus 106 Met
Thr Gln Leu Tyr Thr Tyr Ile Arg Leu Leu Gly Ala Cys Leu Phe 1 5 10
15 Ile Ile Ser His Val Gln Gly Gln Asn Leu Asp Ser Met Leu His Gly
20 25 30 Thr Gly Met Lys Ser Asp Val Asp Gln Lys Lys Pro Glu Asn
Gly Val 35 40 45 Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys
Cys Tyr Cys Ser 50 55 60 Gly His Cys Pro Asp Asp Ala Ile Asn Asn
Thr Cys Ile Thr Asn Gly 65 70 75 80 His Cys Phe Ala Ile Ile Glu Glu
Asp Asp Gln Gly Glu Thr Thr Leu 85 90 95 Thr Ser Gly Cys Met Lys
Tyr Glu Gly Ser Asp Phe Gln Cys Lys Asp 100 105 110 Ser Pro Lys Ala
Gln Leu Arg Arg Thr Ile Glu Cys Cys Arg Thr Asn 115 120 125 Leu Cys
Asn Gln Tyr Leu Gln Pro Thr Leu Pro Pro Val Val Ile Gly 130 135 140
Pro Phe Phe Asp Gly Ser Val Arg Trp Leu Ala Val Leu Ile Ser Met 145
150 155 160 Ala Val Cys Ile Val Ala Met Ile Val Phe Ser Ser Cys Phe
Cys Tyr 165 170 175 Lys His Tyr Cys Lys Ser Ile Ser Ser Arg Gly Arg
Tyr Asn Arg Asp 180 185 190 Leu Glu Gln Asp Glu Ala Phe Ile Pro Val
Gly Glu Ser Leu Lys Asp 195 200 205 Leu Ile Asp Gln Ser Gln Ser Ser
Gly Ser Gly Ser Gly Leu Pro Leu 210 215 220 Leu Val Gln Arg Thr Ile
Ala Lys Gln Ile Gln Met Val Arg Gln Val 225 230 235 240 Gly Lys Gly
Arg Tyr Gly Glu Val Trp Met Gly Lys Trp Arg Gly Glu 245 250 255 Lys
Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu Ala Ser Trp Phe 260 265
270 Arg Glu Thr Glu Ile Tyr Gln Thr Val Leu Met Arg His Glu Asn Ile
275 280 285 Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly Ser Trp
Thr Gln 290 295 300 Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn Gly Ser
Leu Tyr Asp Phe 305 310 315 320 Leu Lys Cys Ala Thr Leu Asp Thr Arg
Ala Leu Leu Lys Leu Ala Tyr 325 330 335 Ser Ala Ala Cys Gly Leu Cys
His Leu His Thr Glu Ile Tyr Gly Thr 340 345 350 Gln Gly Lys Pro Ala
Ile Ala His Arg Asp Leu Lys Ser Lys Asn Ile 355 360 365 Leu Ile Lys
Lys Asn Gly Ser Cys Cys Ile Ala Asp Leu Gly Leu Ala 370 375 380 Val
Lys Phe Asn Ser Asp Thr Asn Glu Val Asp Ile Pro Leu Asn Thr 385 390
395 400 Arg Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Asp Glu
Ser 405 410 415 Leu Ser Lys Asn His Phe Gln Pro Tyr Ile Met Ala Asp
Ile Tyr Ser 420 425 430 Phe Gly Leu Ile Ile Trp Glu Met Ala Arg Arg
Cys Ile Thr Gly Gly 435 440 445 Ile Val Glu Glu Tyr Gln Leu Pro Tyr
Tyr Asn Met Val Pro Ser Asp 450 455 460 Pro Ser Tyr Glu Asp Met Arg
Glu Val Val Cys Val Lys Arg Leu Arg 465 470 475 480 Pro Ile Val Ser
Asn Arg Trp Asn Ser Asp Glu Cys Leu Arg Ala Val 485 490 495 Leu Lys
Leu Met Ser Glu Cys Trp Ala His Asn Pro Ala Ser Arg Leu 500 505 510
Thr Ala Leu Arg Ile Lys Lys Thr Leu Ala Lys Met Val Glu Ser Gln 515
520 525 Asp Val Lys Ile 530 107 532 PRT Rattus norvegicus 107 Met
Thr Gln Leu Tyr Thr Tyr Ile Arg Leu Leu Gly Ala Cys Leu Phe 1 5 10
15 Ile Ile Ser His Val Gln Gly Gln Asn Leu Asp Ser Met Leu His Gly
20 25 30 Thr Gly Met Lys Ser Asp Val Asp Gln Lys Lys Pro Glu Asn
Gly Val 35 40 45 Thr Leu Ala Pro Glu Asp Thr
Leu Pro Phe Leu Lys Cys Tyr Cys Ser 50 55 60 Gly His Cys Pro Asp
Asp Ala Ile Asn Asn Thr Cys Ile Thr Asn Gly 65 70 75 80 His Cys Phe
Ala Ile Ile Glu Glu Asp Asp Gln Gly Glu Thr Thr Leu 85 90 95 Thr
Ser Gly Cys Met Lys Tyr Glu Gly Ser Asp Phe Gln Cys Lys Asp 100 105
110 Ser Pro Lys Ala Gln Leu Arg Arg Thr Ile Glu Cys Cys Arg Thr Asn
115 120 125 Leu Cys Asn Gln Tyr Leu Gln Pro Thr Leu Pro Pro Val Val
Ile Gly 130 135 140 Pro Phe Phe Asp Gly Ser Val Arg Trp Leu Ala Val
Leu Ile Ser Met 145 150 155 160 Ala Val Cys Ile Val Ala Met Ile Val
Phe Ser Ser Cys Phe Cys Tyr 165 170 175 Lys His Tyr Cys Lys Ser Ile
Ser Ser Arg Gly Arg Tyr Asn Arg Asp 180 185 190 Leu Glu Gln Asp Glu
Ala Phe Ile Pro Val Gly Glu Ser Leu Lys Asp 195 200 205 Leu Ile Asp
Gln Ser Gln Ser Ser Gly Ser Gly Ser Gly Leu Pro Leu 210 215 220 Leu
Val Gln Arg Thr Ile Ala Lys Gln Ile Gln Met Val Arg Gln Val 225 230
235 240 Gly Lys Gly Arg Tyr Gly Glu Val Trp Met Gly Lys Trp Arg Gly
Glu 245 250 255 Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu Ala
Ser Trp Phe 260 265 270 Arg Glu Thr Glu Ile Tyr Gln Thr Val Leu Met
Arg His Glu Asn Ile 275 280 285 Leu Gly Phe Ile Ala Ala Asp Ile Lys
Gly Thr Gly Ser Trp Thr Gln 290 295 300 Leu Tyr Leu Ile Thr Asp Tyr
His Glu Asn Gly Ser Leu Tyr Asp Phe 305 310 315 320 Leu Lys Cys Ala
Thr Leu Asp Thr Arg Ala Leu Leu Lys Leu Ala Tyr 325 330 335 Ser Ala
Ala Cys Gly Leu Cys His Leu His Thr Glu Ile Tyr Gly Thr 340 345 350
Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser Lys Asn Ile 355
360 365 Leu Ile Lys Lys Asn Gly Ser Cys Cys Ile Ala Asp Leu Gly Leu
Ala 370 375 380 Val Lys Phe Asn Ser Asp Thr Asn Glu Val Asp Ile Pro
Leu Asn Thr 385 390 395 400 Arg Val Gly Thr Arg Arg Tyr Met Ala Pro
Glu Val Leu Asp Glu Ser 405 410 415 Leu Ser Lys Asn His Phe Gln Pro
Tyr Ile Met Ala Asp Ile Tyr Ser 420 425 430 Phe Gly Leu Ile Ile Trp
Glu Met Ala Arg Arg Cys Ile Thr Gly Gly 435 440 445 Ile Val Glu Glu
Tyr Gln Leu Pro Tyr Tyr Asn Met Val Pro Ser Asp 450 455 460 Pro Ser
Tyr Glu Asp Met Arg Glu Val Val Cys Val Lys Arg Leu Arg 465 470 475
480 Pro Ile Val Ser Asn Arg Trp Asn Ser Asp Glu Cys Leu Arg Ala Val
485 490 495 Leu Lys Leu Met Ser Glu Cys Trp Ala His Asn Pro Ala Ser
Arg Leu 500 505 510 Thr Ala Leu Arg Ile Lys Lys Thr Leu Ala Lys Met
Val Glu Ser Gln 515 520 525 Asp Val Lys Ile 530 108 502 PRT Homo
sapiens 108 Met Leu Leu Arg Ser Ala Gly Lys Leu Asn Val Gly Thr Lys
Lys Glu 1 5 10 15 Asp Gly Glu Ser Thr Ala Pro Thr Pro Arg Pro Lys
Val Leu Arg Cys 20 25 30 Lys Cys His His His Cys Pro Glu Asp Ser
Val Asn Asn Ile Cys Ser 35 40 45 Thr Asp Gly Tyr Cys Phe Thr Met
Ile Glu Glu Asp Asp Ser Gly Leu 50 55 60 Pro Val Val Thr Ser Gly
Cys Leu Gly Leu Glu Gly Ser Asp Phe Gln 65 70 75 80 Cys Arg Asp Thr
Pro Ile Pro His Gln Arg Arg Ser Ile Glu Cys Cys 85 90 95 Thr Glu
Arg Asn Glu Cys Asn Lys Asp Leu His Pro Thr Leu Pro Pro 100 105 110
Leu Lys Asn Arg Asp Phe Val Asp Gly Pro Ile His His Arg Ala Leu 115
120 125 Leu Ile Ser Val Thr Val Cys Ser Leu Leu Leu Val Leu Ile Ile
Leu 130 135 140 Phe Cys Tyr Phe Arg Tyr Lys Arg Gln Glu Thr Arg Pro
Arg Tyr Ser 145 150 155 160 Ile Gly Leu Glu Gln Asp Glu Thr Tyr Ile
Pro Pro Gly Glu Ser Leu 165 170 175 Arg Asp Leu Ile Glu Gln Ser Gln
Ser Ser Gly Ser Gly Ser Gly Leu 180 185 190 Pro Leu Leu Val Gln Arg
Thr Ile Ala Lys Gln Ile Gln Met Val Lys 195 200 205 Gln Ile Gly Lys
Gly Arg Tyr Gly Glu Val Trp Met Gly Lys Trp Arg 210 215 220 Gly Glu
Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu Ala Ser 225 230 235
240 Trp Phe Arg Glu Thr Glu Ile Tyr Gln Thr Val Leu Met Arg His Glu
245 250 255 Asn Ile Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly
Ser Trp 260 265 270 Thr Gln Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn
Gly Ser Leu Tyr 275 280 285 Asp Tyr Leu Lys Ser Thr Thr Leu Asp Ala
Lys Ser Met Leu Lys Leu 290 295 300 Ala Tyr Ser Ser Val Ser Gly Leu
Cys His Leu His Thr Glu Ile Phe 305 310 315 320 Ser Thr Gln Gly Lys
Pro Ala Ile Ala His Arg Asp Leu Lys Ser Lys 325 330 335 Asn Ile Leu
Val Lys Lys Asn Gly Thr Cys Cys Ile Ala Asp Leu Gly 340 345 350 Leu
Ala Val Lys Phe Ile Ser Asp Thr Asn Glu Val Asp Ile Pro Pro 355 360
365 Asn Thr Arg Val Gly Thr Lys Arg Tyr Met Pro Pro Glu Val Leu Asp
370 375 380 Glu Ser Leu Asn Arg Asn His Phe Gln Ser Tyr Ile Met Ala
Asp Met 385 390 395 400 Tyr Ser Phe Gly Leu Ile Leu Trp Glu Val Ala
Arg Arg Cys Val Ser 405 410 415 Gly Gly Ile Val Glu Glu Tyr Gln Leu
Pro Tyr His Asp Leu Val Pro 420 425 430 Ser Asp Pro Ser Tyr Glu Asp
Met Arg Glu Ile Val Cys Ile Lys Lys 435 440 445 Leu Arg Pro Ser Phe
Pro Asn Arg Trp Ser Ser Asp Glu Cys Leu Arg 450 455 460 Gln Met Gly
Lys Leu Met Thr Glu Cys Trp Ala His Asn Pro Ala Ser 465 470 475 480
Arg Leu Thr Ala Leu Arg Val Lys Lys Thr Leu Ala Lys Met Ser Glu 485
490 495 Ser Gln Asp Ile Lys Leu 500 109 502 PRT Homo sapiens 109
Met Leu Leu Arg Ser Ala Gly Lys Leu Asn Val Gly Thr Lys Lys Glu 1 5
10 15 Asp Gly Glu Ser Thr Ala Pro Thr Pro Arg Pro Lys Val Leu Arg
Cys 20 25 30 Lys Cys His His His Cys Pro Glu Asp Ser Val Asn Asn
Ile Cys Ser 35 40 45 Thr Asp Gly Tyr Cys Phe Thr Met Ile Glu Glu
Asp Asp Ser Gly Leu 50 55 60 Pro Val Val Thr Ser Gly Cys Leu Gly
Leu Glu Gly Ser Asp Phe Gln 65 70 75 80 Cys Arg Asp Thr Pro Ile Pro
His Gln Arg Arg Ser Ile Glu Cys Cys 85 90 95 Thr Glu Arg Asn Glu
Cys Asn Lys Asp Leu His Pro Thr Leu Pro Pro 100 105 110 Leu Lys Asn
Arg Asp Phe Val Asp Gly Pro Ile His His Arg Ala Leu 115 120 125 Leu
Ile Ser Val Thr Val Cys Ser Leu Leu Leu Val Leu Ile Ile Leu 130 135
140 Phe Cys Tyr Phe Arg Tyr Lys Arg Gln Glu Thr Arg Pro Arg Tyr Ser
145 150 155 160 Ile Gly Leu Glu Gln Asp Glu Thr Tyr Ile Pro Pro Gly
Glu Ser Leu 165 170 175 Arg Asp Leu Ile Glu Gln Ser Gln Ser Ser Gly
Ser Gly Ser Gly Leu 180 185 190 Pro Leu Leu Val Gln Arg Thr Ile Ala
Lys Gln Ile Gln Met Val Lys 195 200 205 Gln Ile Gly Lys Gly Arg Tyr
Gly Glu Val Trp Met Gly Lys Trp Arg 210 215 220 Gly Glu Lys Val Ala
Val Lys Val Phe Phe Thr Thr Glu Glu Ala Ser 225 230 235 240 Trp Phe
Arg Glu Thr Glu Ile Tyr Gln Thr Val Leu Met Arg His Glu 245 250 255
Asn Ile Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly Ser Trp 260
265 270 Thr Gln Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn Gly Ser Leu
Tyr 275 280 285 Asp Tyr Leu Lys Ser Thr Thr Leu Asp Ala Lys Ser Met
Leu Lys Leu 290 295 300 Ala Tyr Ser Ser Val Ser Gly Leu Cys His Leu
His Thr Glu Ile Phe 305 310 315 320 Ser Thr Gln Gly Lys Pro Ala Ile
Ala His Arg Asp Leu Lys Ser Lys 325 330 335 Asn Ile Leu Val Lys Lys
Asn Gly Thr Cys Cys Ile Ala Asp Leu Gly 340 345 350 Leu Ala Val Lys
Phe Ile Ser Asp Thr Asn Glu Val Asp Ile Pro Pro 355 360 365 Asn Thr
Arg Val Gly Thr Lys Arg Tyr Met Pro Pro Glu Val Leu Asp 370 375 380
Glu Ser Leu Asn Arg Asn His Phe Gln Ser Tyr Ile Met Ala Asp Met 385
390 395 400 Tyr Ser Phe Gly Leu Ile Leu Trp Glu Val Ala Arg Arg Cys
Val Ser 405 410 415 Gly Gly Ile Val Glu Glu Tyr Gln Leu Pro Tyr His
Asp Leu Val Pro 420 425 430 Ser Asp Pro Ser Tyr Glu Asp Met Arg Glu
Ile Val Cys Ile Lys Lys 435 440 445 Leu Arg Pro Ser Phe Pro Asn Arg
Trp Ser Ser Asp Glu Cys Leu Arg 450 455 460 Gln Met Gly Lys Leu Met
Thr Glu Cys Trp Ala His Asn Pro Ala Ser 465 470 475 480 Arg Leu Thr
Ala Leu Arg Val Lys Lys Thr Leu Ala Lys Met Ser Glu 485 490 495 Ser
Gln Asp Ile Lys Leu 500 110 532 PRT Rattus sp. 110 Met Thr Gln Leu
Tyr Thr Tyr Ile Arg Leu Leu Gly Ala Cys Leu Phe 1 5 10 15 Ile Ile
Ser His Val Gln Gly Gln Asn Leu Asp Ser Met Leu His Gly 20 25 30
Thr Gly Met Lys Ser Asp Val Asp Gln Lys Lys Pro Glu Asn Gly Val 35
40 45 Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys Cys Tyr Cys
Ser 50 55 60 Gly His Cys Pro Asp Asp Ala Ile Asn Asn Thr Cys Ile
Thr Asn Gly 65 70 75 80 His Cys Phe Ala Ile Ile Glu Glu Asp Asp Gln
Gly Glu Thr Thr Leu 85 90 95 Thr Ser Gly Cys Met Lys Tyr Glu Gly
Ser Asp Phe Gln Cys Lys Asp 100 105 110 Ser Pro Lys Ala Gln Leu Arg
Arg Thr Ile Glu Cys Cys Arg Thr Asn 115 120 125 Leu Cys Asn Gln Tyr
Leu Gln Pro Thr Leu Pro Pro Val Val Ile Gly 130 135 140 Pro Phe Phe
Asp Gly Ser Val Arg Trp Leu Ala Val Leu Ile Ser Met 145 150 155 160
Ala Val Cys Ile Val Ala Met Ile Val Phe Ser Ser Cys Phe Cys Tyr 165
170 175 Lys His Tyr Cys Lys Ser Ile Ser Ser Arg Gly Arg Tyr Asn Arg
Asp 180 185 190 Leu Glu Gln Asp Glu Ala Phe Ile Pro Val Gly Glu Ser
Leu Lys Asp 195 200 205 Leu Ile Asp Gln Ser Gln Ser Ser Gly Ser Gly
Ser Gly Leu Pro Leu 210 215 220 Leu Val Gln Arg Thr Ile Ala Lys Gln
Ile Gln Met Val Arg Gln Val 225 230 235 240 Gly Lys Gly Arg Tyr Gly
Glu Val Trp Met Gly Lys Trp Arg Gly Glu 245 250 255 Lys Val Ala Val
Lys Val Phe Phe Thr Thr Glu Glu Ala Ser Trp Phe 260 265 270 Arg Glu
Thr Glu Ile Tyr Gln Thr Val Leu Met Arg His Glu Asn Ile 275 280 285
Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly Ser Trp Thr Gln 290
295 300 Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn Gly Ser Leu Tyr Asp
Phe 305 310 315 320 Leu Lys Cys Ala Thr Leu Asp Thr Arg Ala Leu Leu
Lys Leu Ala Tyr 325 330 335 Ser Ala Ala Cys Gly Leu Cys His Leu His
Thr Glu Ile Tyr Gly Thr 340 345 350 Gln Gly Lys Pro Ala Ile Ala His
Arg Asp Leu Lys Ser Lys Asn Ile 355 360 365 Leu Ile Lys Lys Asn Gly
Ser Cys Cys Ile Ala Asp Leu Gly Leu Ala 370 375 380 Val Lys Phe Asn
Ser Asp Thr Asn Glu Val Asp Ile Pro Leu Asn Thr 385 390 395 400 Arg
Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Asp Glu Ser 405 410
415 Leu Ser Lys Asn His Phe Gln Pro Tyr Ile Met Ala Asp Ile Tyr Ser
420 425 430 Phe Gly Leu Ile Ile Trp Glu Met Ala Arg Arg Cys Ile Thr
Gly Gly 435 440 445 Ile Val Glu Glu Tyr Gln Leu Pro Tyr Tyr Asn Met
Val Pro Ser Asp 450 455 460 Pro Ser Tyr Glu Asp Met Arg Glu Val Val
Cys Val Lys Arg Leu Arg 465 470 475 480 Pro Ile Val Ser Asn Arg Trp
Asn Ser Asp Glu Cys Leu Arg Ala Val 485 490 495 Leu Lys Leu Met Ser
Glu Cys Trp Ala His Asn Pro Ala Ser Arg Leu 500 505 510 Thr Ala Leu
Arg Ile Lys Lys Thr Leu Ala Lys Met Val Glu Ser Gln 515 520 525 Asp
Val Lys Ile 530 111 530 PRT Homo sapiens 111 Met Thr Ser Ser Leu
Gln Arg Pro Trp Arg Val Pro Trp Leu Pro Trp 1 5 10 15 Thr Ile Leu
Leu Val Ser Thr Ala Ala Ala Ser Gln Asn Gln Glu Arg 20 25 30 Leu
Cys Ala Phe Lys Asp Pro Tyr Gln Gln Asp Leu Gly Ile Gly Glu 35 40
45 Ser Arg Ile Ser His Glu Asn Gly Thr Ile Leu Cys Ser Lys Gly Ser
50 55 60 Thr Cys Tyr Gly Leu Trp Glu Lys Ser Lys Gly Asp Ile Asn
Leu Val 65 70 75 80 Lys Gln Gly Cys Trp Ser His Ile Gly Asp Pro Gln
Glu Cys His Tyr 85 90 95 Glu Glu Cys Val Val Thr Thr Thr Pro Pro
Ser Ile Gln Asn Gly Thr 100 105 110 Tyr Arg Phe Cys Cys Cys Ser Thr
Asp Leu Cys Asn Val Asn Phe Thr 115 120 125 Glu Asn Phe Pro Pro Pro
Asp Thr Thr Pro Leu Ser Pro Pro His Ser 130 135 140 Phe Asn Arg Asp
Glu Thr Ile Ile Ile Ala Leu Ala Ser Val Ser Val 145 150 155 160 Leu
Ala Val Leu Ile Val Ala Leu Cys Phe Gly Tyr Arg Met Leu Thr 165 170
175 Gly Asp Arg Lys Gln Gly Leu His Ser Met Asn Met Met Glu Ala Ala
180 185 190 Ala Ser Glu Pro Ser Leu Asp Leu Asp Asn Leu Lys Leu Leu
Glu Leu 195 200 205 Ile Gly Arg Gly Arg Tyr Gly Ala Val Tyr Lys Gly
Ser Leu Asp Glu 210 215 220 Arg Pro Val Ala Val Lys Val Phe Ser Phe
Ala Asn Arg Gln Asn Phe 225 230 235 240 Ile Asn Glu Lys Asn Ile Tyr
Arg Val Pro Leu Met Glu His Asp Asn 245 250 255 Ile Ala Arg Phe Ile
Val Gly Asp Glu Arg Val Thr Ala Asp Gly Arg 260 265 270 Met Glu Tyr
Leu Leu Val Met Glu Tyr Tyr Pro Asn Gly Ser Leu Cys 275 280 285 Lys
Tyr Leu Ser Leu His Thr Ser Asp Trp Val Ser Ser Cys Arg Leu 290 295
300 Ala His Ser Val Thr Arg Gly Leu Ala Tyr Leu His Thr Glu Leu Pro
305 310 315 320 Arg Gly Asp His Tyr Lys Pro Ala Ile Ser His Arg Asp
Leu Asn Ser 325 330 335 Arg Asn Val Leu Val Lys Asn Asp Gly Thr Cys
Val Ile Ser Asp Phe 340 345 350 Gly Leu Ser Met Arg Leu Thr Gly Asn
Arg Leu Val Arg Pro Gly Glu 355 360 365 Glu Asp Asn Ala Ala Ile Ser
Glu Val Gly Thr Ile Arg Tyr Met Ala 370 375 380 Pro Glu Val Leu Glu
Gly Ala Val Asn Leu Arg Asp Cys Glu Ser Ala 385 390 395 400 Leu Lys
Gln Val Asp Met Tyr Ala Leu Gly Leu Ile Tyr Trp Glu Ile 405 410 415
Phe Met Arg Cys Thr Asp Leu Phe Pro Gly Glu Ser
Val Pro Glu Tyr 420 425 430 Gln Met Ala Phe Gln Thr Glu Val Gly Asn
His Pro Thr Phe Glu Asp 435 440 445 Met Gln Val Leu Val Ser Arg Glu
Lys Gln Arg Pro Lys Phe Pro Glu 450 455 460 Ala Trp Lys Glu Asn Ser
Leu Ala Val Arg Ser Leu Lys Glu Thr Ile 465 470 475 480 Glu Asp Cys
Trp Asp Gln Asp Ala Glu Ala Arg Leu Thr Ala Gln Cys 485 490 495 Ala
Glu Glu Arg Met Ala Glu Leu Met Met Ile Trp Glu Arg Asn Lys 500 505
510 Ser Val Ser Pro Thr Val Asn Pro Met Ser Thr Ala Met Gln Asn Glu
515 520 525 Arg Arg 530 112 530 PRT Homo sapiens 112 Met Thr Ser
Ser Leu Gln Arg Pro Trp Arg Val Pro Trp Leu Pro Trp 1 5 10 15 Thr
Ile Leu Leu Val Ser Thr Ala Ala Ala Ser Gln Asn Gln Glu Arg 20 25
30 Leu Cys Ala Phe Lys Asp Pro Tyr Gln Gln Asp Leu Gly Ile Gly Glu
35 40 45 Ser Arg Ile Ser His Glu Asn Gly Thr Ile Leu Cys Ser Lys
Gly Ser 50 55 60 Thr Cys Tyr Gly Leu Trp Glu Lys Ser Lys Gly Asp
Ile Asn Leu Val 65 70 75 80 Lys Gln Gly Cys Trp Ser His Ile Gly Asp
Pro Gln Glu Cys His Tyr 85 90 95 Glu Glu Cys Val Val Thr Thr Thr
Pro Pro Ser Ile Gln Asn Gly Thr 100 105 110 Tyr Arg Phe Cys Cys Cys
Ser Thr Asp Leu Cys Asn Val Asn Phe Thr 115 120 125 Glu Asn Phe Pro
Pro Pro Asp Thr Thr Pro Leu Ser Pro Pro His Ser 130 135 140 Phe Asn
Arg Asp Glu Thr Ile Ile Ile Ala Leu Ala Ser Val Ser Val 145 150 155
160 Leu Ala Val Leu Ile Val Ala Leu Cys Phe Gly Tyr Arg Met Leu Thr
165 170 175 Gly Asp Arg Lys Gln Gly Leu His Ser Met Asn Met Met Glu
Ala Ala 180 185 190 Ala Ser Glu Pro Ser Leu Asp Leu Asp Asn Leu Lys
Leu Leu Glu Leu 195 200 205 Ile Gly Arg Gly Arg Tyr Gly Ala Val Tyr
Lys Gly Ser Leu Asp Glu 210 215 220 Arg Pro Val Ala Val Lys Val Phe
Ser Phe Ala Asn Arg Gln Asn Phe 225 230 235 240 Ile Asn Glu Lys Asn
Ile Tyr Arg Val Pro Leu Met Glu His Asp Asn 245 250 255 Ile Ala Arg
Phe Ile Val Gly Asp Glu Arg Val Thr Ala Asp Gly Arg 260 265 270 Met
Glu Tyr Leu Leu Val Met Glu Tyr Tyr Pro Asn Gly Ser Leu Cys 275 280
285 Lys Tyr Leu Ser Leu His Thr Ser Asp Trp Val Ser Ser Cys Arg Leu
290 295 300 Ala His Ser Val Thr Arg Gly Leu Ala Tyr Leu His Thr Glu
Leu Pro 305 310 315 320 Arg Gly Asp His Tyr Lys Pro Ala Ile Ser His
Arg Asp Leu Asn Ser 325 330 335 Arg Asn Val Leu Val Lys Asn Asp Gly
Thr Cys Val Ile Ser Asp Phe 340 345 350 Gly Leu Ser Met Arg Leu Thr
Gly Asn Arg Leu Val Arg Pro Gly Glu 355 360 365 Glu Asp Asn Ala Ala
Ile Ser Glu Val Gly Thr Ile Arg Tyr Met Ala 370 375 380 Pro Glu Val
Leu Glu Gly Ala Val Asn Leu Arg Asp Cys Glu Ser Ala 385 390 395 400
Leu Lys Gln Val Asp Met Tyr Ala Leu Gly Leu Ile Tyr Trp Glu Ile 405
410 415 Phe Met Arg Cys Thr Asp Leu Phe Pro Gly Glu Ser Val Pro Glu
Tyr 420 425 430 Gln Met Ala Phe Gln Thr Glu Val Gly Asn His Pro Thr
Phe Glu Asp 435 440 445 Met Gln Val Leu Val Ser Arg Glu Lys Gln Arg
Pro Lys Phe Pro Glu 450 455 460 Ala Trp Lys Glu Asn Ser Leu Ala Val
Arg Ser Leu Lys Glu Thr Ile 465 470 475 480 Glu Asp Cys Trp Asp Gln
Asp Ala Glu Ala Arg Leu Thr Ala Gln Cys 485 490 495 Ala Glu Glu Arg
Met Ala Glu Leu Met Met Ile Trp Glu Arg Asn Lys 500 505 510 Ser Val
Ser Pro Thr Val Asn Pro Met Ser Thr Ala Met Gln Asn Glu 515 520 525
Arg Arg 530 113 1038 PRT Homo sapiens 113 Met Thr Ser Ser Leu Gln
Arg Pro Trp Arg Val Pro Trp Leu Pro Trp 1 5 10 15 Thr Ile Leu Leu
Val Ser Thr Ala Ala Ala Ser Gln Asn Gln Glu Arg 20 25 30 Leu Cys
Ala Phe Lys Asp Pro Tyr Gln Gln Asp Leu Gly Ile Gly Glu 35 40 45
Ser Arg Ile Ser His Glu Asn Gly Thr Ile Leu Cys Ser Lys Gly Ser 50
55 60 Thr Cys Tyr Gly Leu Trp Glu Lys Ser Lys Gly Asp Ile Asn Leu
Val 65 70 75 80 Lys Gln Gly Cys Trp Ser His Ile Gly Asp Pro Gln Glu
Cys His Tyr 85 90 95 Glu Glu Cys Val Val Thr Thr Thr Pro Pro Ser
Ile Gln Asn Gly Thr 100 105 110 Tyr Arg Phe Cys Cys Cys Ser Thr Asp
Leu Cys Asn Val Asn Phe Thr 115 120 125 Glu Asn Phe Pro Pro Pro Asp
Thr Thr Pro Leu Ser Pro Pro His Ser 130 135 140 Phe Asn Arg Asp Glu
Thr Ile Ile Ile Ala Leu Ala Ser Val Ser Val 145 150 155 160 Leu Ala
Val Leu Ile Val Ala Leu Cys Phe Gly Tyr Arg Met Leu Thr 165 170 175
Gly Asp Arg Lys Gln Gly Leu His Ser Met Asn Met Met Glu Ala Ala 180
185 190 Ala Ser Glu Pro Ser Leu Asp Leu Asp Asn Leu Lys Leu Leu Glu
Leu 195 200 205 Ile Gly Arg Gly Arg Tyr Gly Ala Val Tyr Lys Gly Ser
Leu Asp Glu 210 215 220 Arg Pro Val Ala Val Lys Val Phe Ser Phe Ala
Asn Arg Gln Asn Phe 225 230 235 240 Ile Asn Glu Lys Asn Ile Tyr Arg
Val Pro Leu Met Glu His Asp Asn 245 250 255 Ile Ala Arg Phe Ile Val
Gly Asp Glu Arg Val Thr Ala Asp Gly Arg 260 265 270 Met Glu Tyr Leu
Leu Val Met Glu Tyr Tyr Pro Asn Gly Ser Leu Cys 275 280 285 Lys Tyr
Leu Ser Leu His Thr Ser Asp Trp Val Ser Ser Cys Arg Leu 290 295 300
Ala His Ser Val Thr Arg Gly Leu Ala Tyr Leu His Thr Glu Leu Pro 305
310 315 320 Arg Gly Asp His Tyr Lys Pro Ala Ile Ser His Arg Asp Leu
Asn Ser 325 330 335 Arg Asn Val Leu Val Lys Asn Asp Gly Thr Cys Val
Ile Ser Asp Phe 340 345 350 Gly Leu Ser Met Arg Leu Thr Gly Asn Arg
Leu Val Arg Pro Gly Glu 355 360 365 Glu Asp Asn Ala Ala Ile Ser Glu
Val Gly Thr Ile Arg Tyr Met Ala 370 375 380 Pro Glu Val Leu Glu Gly
Ala Val Asn Leu Arg Asp Cys Glu Ser Ala 385 390 395 400 Leu Lys Gln
Val Asp Met Tyr Ala Leu Gly Leu Ile Tyr Trp Glu Ile 405 410 415 Phe
Met Arg Cys Thr Asp Leu Phe Pro Gly Glu Ser Val Pro Glu Tyr 420 425
430 Gln Met Ala Phe Gln Thr Glu Val Gly Asn His Pro Thr Phe Glu Asp
435 440 445 Met Gln Val Leu Val Ser Arg Glu Lys Gln Arg Pro Lys Phe
Pro Glu 450 455 460 Ala Trp Lys Glu Asn Ser Leu Ala Val Arg Ser Leu
Lys Glu Thr Ile 465 470 475 480 Glu Asp Cys Trp Asp Gln Asp Ala Glu
Ala Arg Leu Thr Ala Gln Cys 485 490 495 Ala Glu Glu Arg Met Ala Glu
Leu Met Met Ile Trp Glu Arg Asn Lys 500 505 510 Ser Val Ser Pro Thr
Val Asn Pro Met Ser Thr Ala Met Gln Asn Glu 515 520 525 Arg Asn Leu
Ser His Asn Arg Arg Val Pro Lys Ile Gly Pro Tyr Pro 530 535 540 Asp
Tyr Ser Ser Ser Ser Tyr Ile Glu Asp Ser Ile His His Thr Asp 545 550
555 560 Ser Ile Val Lys Asn Ile Ser Ser Glu His Ser Met Ser Ser Thr
Pro 565 570 575 Leu Thr Ile Gly Glu Lys Asn Arg Asn Ser Ile Asn Tyr
Glu Arg Gln 580 585 590 Gln Ala Gln Ala Arg Ile Pro Ser Pro Glu Thr
Ser Val Thr Ser Leu 595 600 605 Ser Thr Asn Thr Thr Thr Thr Asn Thr
Thr Gly Leu Thr Pro Ser Thr 610 615 620 Gly Met Thr Thr Ile Ser Glu
Met Pro Tyr Pro Asp Glu Thr Asn Leu 625 630 635 640 His Thr Thr Asn
Val Ala Gln Ser Ile Gly Pro Thr Pro Val Cys Leu 645 650 655 Gln Leu
Thr Glu Glu Asp Leu Glu Thr Asn Lys Leu Asp Pro Lys Glu 660 665 670
Val Asp Lys Asn Leu Lys Glu Ser Ser Asp Glu Asn Leu Met Glu His 675
680 685 Ser Leu Lys Gln Phe Ser Gly Pro Asp Pro Leu Ser Ser Thr Ser
Ser 690 695 700 Ser Leu Leu Tyr Pro Leu Ile Lys Leu Ala Val Glu Ala
Thr Gly Gln 705 710 715 720 Gln Asp Phe Thr Gln Thr Ala Asn Gly Gln
Ala Cys Leu Ile Pro Asp 725 730 735 Val Leu Pro Thr Gln Ile Tyr Pro
Leu Pro Lys Gln Gln Asn Leu Pro 740 745 750 Lys Arg Pro Thr Ser Leu
Pro Leu Asn Thr Lys Asn Ser Thr Lys Glu 755 760 765 Pro Arg Leu Lys
Phe Gly Ser Lys His Lys Ser Asn Leu Lys Gln Val 770 775 780 Glu Thr
Gly Val Ala Lys Met Asn Thr Ile Asn Ala Ala Glu Pro His 785 790 795
800 Val Val Thr Val Thr Met Asn Gly Val Ala Gly Arg Asn His Ser Val
805 810 815 Asn Ser His Ala Ala Thr Thr Gln Tyr Ala Asn Gly Thr Val
Leu Ser 820 825 830 Gly Gln Thr Thr Asn Ile Val Thr His Arg Ala Gln
Glu Met Leu Gln 835 840 845 Asn Gln Phe Ile Gly Glu Asp Thr Arg Leu
Asn Ile Asn Ser Ser Pro 850 855 860 Asp Glu His Glu Pro Leu Leu Arg
Arg Glu Gln Gln Ala Gly His Asp 865 870 875 880 Glu Gly Val Leu Asp
Arg Leu Val Asp Arg Arg Glu Arg Pro Leu Glu 885 890 895 Gly Gly Arg
Thr Asn Ser Asn Asn Asn Asn Ser Asn Pro Cys Ser Glu 900 905 910 Gln
Asp Val Leu Ala Gln Gly Val Pro Ser Thr Ala Ala Asp Pro Gly 915 920
925 Pro Ser Lys Pro Arg Arg Ala Gln Arg Pro Asn Ser Leu Asp Leu Ser
930 935 940 Ala Thr Asn Val Leu Asp Gly Ser Ser Ile Gln Ile Gly Glu
Ser Thr 945 950 955 960 Gln Asp Gly Lys Ser Gly Ser Gly Glu Lys Ile
Lys Lys Arg Val Lys 965 970 975 Thr Pro Tyr Ser Leu Lys Arg Trp Arg
Pro Ser Thr Trp Val Ile Ser 980 985 990 Thr Glu Ser Leu Asp Cys Glu
Val Asn Asn Asn Gly Ser Asn Arg Ala 995 1000 1005 Val His Ser Lys
Ser Ser Thr Ala Val Tyr Leu Ala Glu Gly Gly Thr 1010 1015 1020 Ala
Thr Thr Met Val Ser Lys Asp Ile Gly Met Asn Cys Leu 1025 1030 1035
114 1038 PRT Homo sapiens 114 Met Thr Ser Ser Leu Gln Arg Pro Trp
Arg Val Pro Trp Leu Pro Trp 1 5 10 15 Thr Ile Leu Leu Val Ser Thr
Ala Ala Ala Ser Gln Asn Gln Glu Arg 20 25 30 Leu Cys Ala Phe Lys
Asp Pro Tyr Gln Gln Asp Leu Gly Ile Gly Glu 35 40 45 Ser Arg Ile
Ser His Glu Asn Gly Thr Ile Leu Cys Ser Lys Gly Ser 50 55 60 Thr
Cys Tyr Gly Leu Trp Glu Lys Ser Lys Gly Asp Ile Asn Leu Val 65 70
75 80 Lys Gln Gly Cys Trp Ser His Ile Gly Asp Pro Gln Glu Cys His
Tyr 85 90 95 Glu Glu Cys Val Val Thr Thr Thr Pro Pro Ser Ile Gln
Asn Gly Thr 100 105 110 Tyr Arg Phe Cys Cys Cys Ser Thr Asp Leu Cys
Asn Val Asn Phe Thr 115 120 125 Glu Asn Phe Pro Pro Pro Asp Thr Thr
Pro Leu Ser Pro Pro His Ser 130 135 140 Phe Asn Arg Asp Glu Thr Ile
Ile Ile Ala Leu Ala Ser Val Ser Val 145 150 155 160 Leu Ala Val Leu
Ile Val Ala Leu Cys Phe Gly Tyr Arg Met Leu Thr 165 170 175 Gly Asp
Arg Lys Gln Gly Leu His Ser Met Asn Met Met Glu Ala Ala 180 185 190
Ala Ser Glu Pro Ser Leu Asp Leu Asp Asn Leu Lys Leu Leu Glu Leu 195
200 205 Ile Gly Arg Gly Arg Tyr Gly Ala Val Tyr Lys Gly Ser Leu Asp
Glu 210 215 220 Arg Pro Val Ala Val Lys Val Phe Ser Phe Ala Asn Arg
Gln Asn Phe 225 230 235 240 Ile Asn Glu Lys Asn Ile Tyr Arg Val Pro
Leu Met Glu His Asp Asn 245 250 255 Ile Ala Arg Phe Ile Val Gly Asp
Glu Arg Val Thr Ala Asp Gly Arg 260 265 270 Met Glu Tyr Leu Leu Val
Met Glu Tyr Tyr Pro Asn Gly Ser Leu Cys 275 280 285 Lys Tyr Leu Ser
Leu His Thr Ser Asp Trp Val Ser Ser Cys Arg Leu 290 295 300 Ala His
Ser Val Thr Arg Gly Leu Ala Tyr Leu His Thr Glu Leu Pro 305 310 315
320 Arg Gly Asp His Tyr Lys Pro Ala Ile Ser His Arg Asp Leu Asn Ser
325 330 335 Arg Asn Val Leu Val Lys Asn Asp Gly Thr Cys Val Ile Ser
Asp Phe 340 345 350 Gly Leu Ser Met Arg Leu Thr Gly Asn Arg Leu Val
Arg Pro Gly Glu 355 360 365 Glu Asp Asn Ala Ala Ile Ser Glu Val Gly
Thr Ile Arg Tyr Met Ala 370 375 380 Pro Glu Val Leu Glu Gly Ala Val
Asn Leu Arg Asp Cys Glu Ser Ala 385 390 395 400 Leu Lys Gln Val Asp
Met Tyr Ala Leu Gly Leu Ile Tyr Trp Glu Ile 405 410 415 Phe Met Arg
Cys Thr Asp Leu Phe Pro Gly Glu Ser Val Pro Glu Tyr 420 425 430 Gln
Met Ala Phe Gln Thr Glu Val Gly Asn His Pro Thr Phe Glu Asp 435 440
445 Met Gln Val Leu Val Ser Arg Glu Lys Gln Arg Pro Lys Phe Pro Glu
450 455 460 Ala Trp Lys Glu Asn Ser Leu Ala Val Arg Ser Leu Lys Glu
Thr Ile 465 470 475 480 Glu Asp Cys Trp Asp Gln Asp Ala Glu Ala Arg
Leu Thr Ala Gln Cys 485 490 495 Ala Glu Glu Arg Met Ala Glu Leu Met
Met Ile Trp Glu Arg Asn Lys 500 505 510 Ser Val Ser Pro Thr Val Asn
Pro Met Ser Thr Ala Met Gln Asn Glu 515 520 525 Arg Asn Leu Ser His
Asn Arg Arg Val Pro Lys Ile Gly Pro Tyr Pro 530 535 540 Asp Tyr Ser
Ser Ser Ser Tyr Ile Glu Asp Ser Ile His His Thr Asp 545 550 555 560
Ser Ile Val Lys Asn Ile Ser Ser Glu His Ser Met Ser Ser Thr Pro 565
570 575 Leu Thr Ile Gly Glu Lys Asn Arg Asn Ser Ile Asn Tyr Glu Arg
Gln 580 585 590 Gln Ala Gln Ala Arg Ile Pro Ser Pro Glu Thr Ser Val
Thr Ser Leu 595 600 605 Ser Thr Asn Thr Thr Thr Thr Asn Thr Thr Gly
Leu Thr Pro Ser Thr 610 615 620 Gly Met Thr Thr Ile Ser Glu Met Pro
Tyr Pro Asp Glu Thr Asn Leu 625 630 635 640 His Thr Thr Asn Val Ala
Gln Ser Ile Gly Pro Thr Pro Val Cys Leu 645 650 655 Gln Leu Thr Glu
Glu Asp Leu Glu Thr Asn Lys Leu Asp Pro Lys Glu 660 665 670 Val Asp
Lys Asn Leu Lys Glu Ser Ser Asp Glu Asn Leu Met Glu His 675 680 685
Ser Leu Lys Gln Phe Ser Gly Pro Asp Pro Leu Ser Ser Thr Ser Ser 690
695 700 Ser Leu Leu Tyr Pro Leu Ile Lys Leu Ala Val Glu Ala Thr Gly
Gln 705 710 715 720 Gln Asp Phe Thr Gln Thr Ala Asn Gly Gln Ala Cys
Leu Ile Pro Asp 725 730 735 Val Leu Pro Thr Gln Ile Tyr Pro Leu Pro
Lys Gln Gln Asn Leu Pro 740 745 750 Lys Arg Pro Thr Ser Leu Pro Leu
Asn Thr Lys Asn Ser Thr Lys Glu 755
760 765 Pro Arg Leu Lys Phe Gly Ser Lys His Lys Ser Asn Leu Lys Gln
Val 770 775 780 Glu Thr Gly Val Ala Lys Met Asn Thr Ile Asn Ala Ala
Glu Pro His 785 790 795 800 Val Val Thr Val Thr Met Asn Gly Val Ala
Gly Arg Asn His Ser Val 805 810 815 Asn Ser His Ala Ala Thr Thr Gln
Tyr Ala Asn Arg Thr Val Leu Ser 820 825 830 Gly Gln Thr Thr Asn Ile
Val Thr His Arg Ala Gln Glu Met Leu Gln 835 840 845 Asn Gln Phe Ile
Gly Glu Asp Thr Arg Leu Asn Ile Asn Ser Ser Pro 850 855 860 Asp Glu
His Glu Pro Leu Leu Arg Arg Glu Gln Gln Ala Gly His Asp 865 870 875
880 Glu Gly Val Leu Asp Arg Leu Val Asp Arg Arg Glu Arg Pro Leu Glu
885 890 895 Gly Gly Arg Thr Asn Ser Asn Asn Asn Asn Ser Asn Pro Cys
Ser Glu 900 905 910 Gln Asp Val Leu Ala Gln Gly Val Pro Ser Thr Ala
Ala Asp Pro Gly 915 920 925 Pro Ser Lys Pro Arg Arg Ala Gln Arg Pro
Asn Ser Leu Asp Leu Ser 930 935 940 Ala Thr Asn Val Leu Asp Gly Ser
Ser Ile Gln Ile Gly Glu Ser Thr 945 950 955 960 Gln Asp Gly Lys Ser
Gly Ser Gly Glu Lys Ile Lys Lys Arg Val Lys 965 970 975 Thr Pro Tyr
Ser Leu Lys Arg Trp Arg Pro Ser Thr Trp Val Ile Ser 980 985 990 Thr
Glu Ser Leu Asp Cys Glu Val Asn Asn Asn Gly Ser Asn Arg Ala 995
1000 1005 Val His Ser Lys Ser Ser Thr Ala Val Tyr Leu Ala Glu Gly
Gly Thr 1010 1015 1020 Ala Thr Thr Met Val Ser Lys Asp Ile Gly Met
Asn Cys Leu 1025 1030 1035 115 1038 PRT Homo sapiens 115 Met Thr
Ser Ser Leu Gln Arg Pro Trp Arg Val Pro Trp Leu Pro Trp 1 5 10 15
Thr Ile Leu Leu Val Ser Thr Ala Ala Ala Ser Gln Asn Gln Glu Arg 20
25 30 Leu Cys Ala Phe Lys Asp Pro Tyr Gln Gln Asp Leu Gly Ile Gly
Glu 35 40 45 Ser Arg Ile Ser His Glu Asn Gly Thr Ile Leu Cys Ser
Lys Gly Ser 50 55 60 Thr Cys Tyr Gly Leu Trp Glu Lys Ser Lys Gly
Asp Ile Asn Leu Val 65 70 75 80 Lys Gln Gly Cys Trp Ser His Ile Gly
Asp Pro Gln Glu Cys His Tyr 85 90 95 Glu Glu Cys Val Val Thr Thr
Thr Pro Pro Ser Ile Gln Asn Gly Thr 100 105 110 Tyr Arg Phe Cys Cys
Cys Ser Thr Asp Leu Cys Asn Val Asn Phe Thr 115 120 125 Glu Asn Phe
Pro Pro Pro Asp Thr Thr Pro Leu Ser Pro Pro His Ser 130 135 140 Phe
Asn Arg Asp Glu Thr Ile Ile Ile Ala Leu Ala Ser Val Ser Val 145 150
155 160 Leu Ala Val Leu Ile Val Ala Leu Cys Phe Gly Tyr Arg Met Leu
Thr 165 170 175 Gly Asp Arg Lys Gln Gly Leu His Ser Met Asn Met Met
Glu Ala Ala 180 185 190 Ala Ser Glu Pro Ser Leu Asp Leu Asp Asn Leu
Lys Leu Leu Glu Leu 195 200 205 Ile Gly Arg Gly Arg Tyr Gly Ala Val
Tyr Lys Gly Ser Leu Asp Glu 210 215 220 Arg Pro Val Ala Val Lys Val
Phe Ser Phe Ala Asn Arg Gln Asn Phe 225 230 235 240 Ile Asn Glu Lys
Asn Ile Tyr Arg Val Pro Leu Met Glu His Asp Asn 245 250 255 Ile Ala
Arg Phe Ile Val Gly Asp Glu Arg Val Thr Ala Asp Gly Arg 260 265 270
Met Glu Tyr Leu Leu Val Met Glu Tyr Tyr Pro Asn Gly Ser Leu Cys 275
280 285 Lys Tyr Leu Ser Leu His Thr Ser Asp Trp Val Ser Ser Cys Arg
Leu 290 295 300 Ala His Ser Val Thr Arg Gly Leu Ala Tyr Leu His Thr
Glu Leu Pro 305 310 315 320 Arg Gly Asp His Tyr Lys Pro Ala Ile Ser
His Arg Asp Leu Asn Ser 325 330 335 Arg Asn Val Leu Val Lys Asn Asp
Gly Thr Cys Val Ile Ser Asp Phe 340 345 350 Gly Leu Ser Met Arg Leu
Thr Gly Asn Arg Leu Val Arg Pro Gly Glu 355 360 365 Glu Asp Asn Ala
Ala Ile Ser Glu Val Gly Thr Ile Arg Tyr Met Ala 370 375 380 Pro Glu
Val Leu Glu Gly Ala Val Asn Leu Arg Asp Cys Glu Ser Ala 385 390 395
400 Leu Lys Gln Val Asp Met Tyr Ala Leu Gly Leu Ile Tyr Trp Glu Ile
405 410 415 Phe Met Arg Cys Thr Asp Leu Phe Pro Gly Glu Ser Val Pro
Glu Tyr 420 425 430 Gln Met Ala Phe Gln Thr Glu Val Gly Asn His Pro
Thr Phe Glu Asp 435 440 445 Met Gln Val Leu Val Ser Arg Glu Lys Gln
Arg Pro Lys Phe Pro Glu 450 455 460 Ala Trp Lys Glu Asn Ser Leu Ala
Val Arg Ser Leu Lys Glu Thr Ile 465 470 475 480 Glu Asp Cys Trp Asp
Gln Asp Ala Glu Ala Arg Leu Thr Ala Gln Cys 485 490 495 Ala Glu Glu
Arg Met Ala Glu Leu Met Met Ile Trp Glu Arg Asn Lys 500 505 510 Ser
Val Ser Pro Thr Val Asn Pro Met Ser Thr Ala Met Gln Asn Glu 515 520
525 Arg Asn Leu Ser His Asn Arg Arg Val Pro Lys Ile Gly Pro Tyr Pro
530 535 540 Asp Tyr Ser Ser Ser Ser Tyr Ile Glu Asp Ser Ile His His
Thr Asp 545 550 555 560 Ser Ile Val Lys Asn Ile Ser Ser Glu His Ser
Met Ser Ser Thr Pro 565 570 575 Leu Thr Ile Gly Glu Lys Asn Arg Asn
Ser Ile Asn Tyr Glu Arg Gln 580 585 590 Gln Ala Gln Ala Arg Ile Pro
Ser Pro Glu Thr Ser Val Thr Ser Leu 595 600 605 Ser Thr Asn Thr Thr
Thr Thr Asn Thr Thr Gly Leu Thr Pro Ser Thr 610 615 620 Gly Met Thr
Thr Ile Ser Glu Met Pro Tyr Pro Asp Glu Thr Asn Leu 625 630 635 640
His Thr Thr Asn Val Ala Gln Ser Ile Gly Pro Thr Pro Val Cys Leu 645
650 655 Gln Leu Thr Glu Glu Asp Leu Glu Thr Asn Lys Leu Asp Pro Lys
Glu 660 665 670 Val Asp Lys Asn Leu Lys Glu Ser Ser Asp Glu Asn Leu
Met Glu His 675 680 685 Ser Leu Lys Gln Phe Ser Gly Pro Asp Pro Leu
Ser Ser Thr Ser Ser 690 695 700 Ser Leu Leu Tyr Pro Leu Ile Lys Leu
Ala Val Glu Ala Thr Gly Gln 705 710 715 720 Gln Asp Phe Thr Gln Thr
Ala Asn Gly Gln Ala Cys Leu Ile Pro Asp 725 730 735 Val Leu Pro Thr
Gln Ile Tyr Pro Leu Pro Lys Gln Gln Asn Leu Pro 740 745 750 Lys Arg
Pro Thr Ser Leu Pro Leu Asn Thr Lys Asn Ser Thr Lys Glu 755 760 765
Pro Arg Leu Lys Phe Gly Ser Lys His Lys Ser Asn Leu Lys Gln Val 770
775 780 Glu Thr Gly Val Ala Lys Met Asn Thr Ile Asn Ala Ala Glu Pro
His 785 790 795 800 Val Val Thr Val Thr Met Asn Gly Val Ala Gly Arg
Asn His Ser Val 805 810 815 Asn Ser His Ala Ala Thr Thr Gln Tyr Ala
Asn Arg Thr Val Leu Ser 820 825 830 Gly Gln Thr Thr Asn Ile Val Thr
His Arg Ala Gln Glu Met Leu Gln 835 840 845 Asn Gln Phe Ile Gly Glu
Asp Thr Arg Leu Asn Ile Asn Ser Ser Pro 850 855 860 Asp Glu His Glu
Pro Leu Leu Arg Arg Glu Gln Gln Ala Gly His Asp 865 870 875 880 Glu
Gly Val Leu Asp Arg Leu Val Asp Arg Arg Glu Arg Pro Leu Glu 885 890
895 Gly Gly Arg Thr Asn Ser Asn Asn Asn Asn Ser Asn Pro Cys Ser Glu
900 905 910 Gln Asp Val Leu Ala Gln Gly Val Pro Ser Thr Ala Ala Asp
Pro Gly 915 920 925 Pro Ser Lys Pro Arg Arg Ala Gln Arg Pro Asn Ser
Leu Asp Leu Ser 930 935 940 Ala Thr Asn Val Leu Asp Gly Ser Ser Ile
Gln Ile Gly Glu Ser Thr 945 950 955 960 Gln Asp Gly Lys Ser Gly Ser
Gly Glu Lys Ile Lys Lys Arg Val Lys 965 970 975 Thr Pro Tyr Ser Leu
Lys Arg Trp Arg Pro Ser Thr Trp Val Ile Ser 980 985 990 Thr Glu Ser
Leu Asp Cys Glu Val Asn Asn Asn Gly Ser Asn Arg Ala 995 1000 1005
Val His Ser Lys Ser Ser Thr Ala Val Tyr Leu Ala Glu Gly Gly Thr
1010 1015 1020 Ala Thr Thr Met Val Ser Lys Asp Ile Gly Met Asn Cys
Leu 1025 1030 1035 116 2932 DNA Homo sapiens 116 gctccgcgcc
gagggctgga ggatgcgttc cctggggtcc ggacttatga aaatatgcat 60
cagtttaata ctgtcttgga attcatgaga tggaagcata ggtcaaagct gtttggagaa
120 aatcagaagt acagttttat ctagccacat cttggaggag tcgtaagaaa
gcagtgggag 180 ttgaagtcat tgtcaagtgc ttgcgatctt ttacaagaaa
atctcactga atgatagtca 240 tttaaattgg tgaagtagca agaccaatta
ttaaaggtga cagtacacag gaaacattac 300 aattgaacaa tgactcagct
atacatttac atcagattat tgggagccta tttgttcatc 360 atttctcgtg
ttcaaggaca gaatctggat agtatgcttc atggcactgg gatgaaatca 420
gactccgacc agaaaaagtc agaaaatgga gtaaccttag caccagagga taccttgcct
480 tttttaaagt gctattgctc agggcactgt ccagatgatg ctattaataa
cacatgcata 540 actaatggac attgctttgc catcatagaa gaagatgacc
agggagaaac cacattagct 600 tcagggtgta tgaaatatga aggatctgat
tttcagtgca aagattctcc aaaagcccag 660 ctacgccgga caatagaatg
ttgtcggacc aatttatgta accagtattt gcaacccaca 720 ctgccccctg
ttgtcatagg tccgtttttt gatggcagca ttcgatggct ggttttgctc 780
atttctatgg ctgtctgcat aattgctatg atcatcttct ccagctgctt ttgttacaaa
840 cattattgca agagcatctc aagcagacgt cgttacaatc gtgatttgga
acaggatgaa 900 gcatttattc cagttggaga atcactaaaa gaccttattg
accagtcaca aagttctggt 960 agtgggtctg gactaccttt attggttcag
cgaactattg ccaaacagat tcagatggtc 1020 cggcaagttg gtaaaggccg
atatggagaa gtatggatgg gcaaatggcg tggcgaaaaa 1080 gtggcggtga
aagtattctt taccactgaa gaagccagct ggtttcgaga aacagaaatc 1140
taccaaactg tgctaatgcg ccatgaaaac atacttggtt tcatagcggc agacattaaa
1200 ggtacaggtt cctggactca gctctatttg attactgatt accatgaaaa
tggatctctc 1260 tatgacttcc tgaaatgtgc tacactggac accagagccc
tgcttaaatt ggcttattca 1320 gctgcctgtg gtctgtgcca cctgcacaca
gaaatttatg gcacccaagg aaagcccgca 1380 attgctcatc gagacctaaa
gagcaaaaac atcctcatca agaaaaatgg gagttgctgc 1440 attgctgacc
tgggccttgc tgttaaattc aacagtgaca caaatgaagt tgatgtgccc 1500
ttgaatacca gggtgggcac caaacgctac atggctcccg aagtgctgga cgaaagcctg
1560 aacaaaaacc acttccagcc ctacatcatg gctgacatct acagcttcgg
cctaatcatt 1620 tgggagatgg ctcgtcgttg tatcacagga gggatcgtgg
aagaatacca attgccatat 1680 tacaacatgg taccgagtga tccgtcatac
gaagatatgc gtgaggttgt gtgtgtcaaa 1740 cgtttgcggc caattgtgtc
taatcggtgg aacagtgatg aatgtctacg agcagttttg 1800 aagctaatgt
cagaatgctg ggcccacaat ccagcctcca gactcacagc attgagaatt 1860
aagaagacgc ttgccaagat ggttgaatcc caagatgtaa aaatctgatg gttaaaccat
1920 cggaggagaa actctagact gcaagaactg tttttaccca tggcatgggt
ggaattagag 1980 tggaataagg atgttaactt ggttctcaga ctctttcttc
actacgtgtt cacaggctgc 2040 taatattaaa cctttcagta ctcttattag
gatacaagct gggaacttct aaacacttca 2100 ttctttatat atggacagct
ttattttaaa tgtggttttt gatgcctttt tttaagtggg 2160 tttttatgaa
ctgcatcaag acttcaatcc tgattagtgt ctccagtcaa gctctgggta 2220
ctgaattgcc tgttcataaa acggtgcttt ctgtgaaagc cttaagaaga taaatgagcg
2280 cagcagagat ggagaaatag actttgcctt ttacctgaga cattcagttc
gtttgtattc 2340 tacctttgta aaacagccta tagatgatga tgtgtttggg
atactgctta ttttatgata 2400 gtttgtcctg tgtccttagt gatgtgtgtg
tgtctccatg cacatgcacg ccgggattcc 2460 tctgctgcca tttgaattag
aagaaaataa tttatatgca tgcacaggaa gatattggtg 2520 gccggtggtt
ttgtgcttta aaaatgcaat atctgaccaa gattcgccaa tctcatacaa 2580
gccatttact ttgcaagtga gatagcttcc ccaccagctt tattttttaa catgaaagct
2640 gatgccaagg ccaaaagaag tttaaagcat ctgtaaattt ggactgtttt
ccttcaacca 2700 ccattttttt tgtggttatt atttttgtca cggaaagcat
cctctccaaa gttggagctt 2760 ctattgccat gaaccatgct tacaaagaaa
gcacttctta ttgaagtgaa ttcctgcatt 2820 tgatagcaat gtaagtgcct
ataaccatgt tctatattct ttattctcag taacttttaa 2880 aagggaagtt
atttatattt tgtgtataat gtgctttatt tgcaaatcac cc 2932 117 1575 DNA
Homo sapiens 117 gcaaacttcc ttgataacat gcttttgcga agtgcaggaa
aattaaatgt gggcaccaag 60 aaagaggatg gtgagagtac agcccccacc
ccccgtccaa aggtcttgcg ttgtaaatgc 120 caccaccatt gtccagaaga
ctcagtcaac aatatttgca gcacagacgg atattgtttc 180 acgatgatag
aagaggatga ctctgggttg cctgtggtca cttctggttg cctaggacta 240
gaaggctcag attttcagtg tcgggacact cccattcctc atcaaagaag atcaattgaa
300 tgctgcacag aaaggaacga atgtaataaa gacctacacc ctacactgcc
tccattgaaa 360 aacagagatt ttgttgatgg acctatacac cacagggctt
tacttatatc tgtgactgtc 420 tgtagtttgc tcttggtcct tatcatatta
ttttgttact tccggtataa aagacaagaa 480 accagacctc gatacagcat
tgggttagaa caggatgaaa cttacattcc tcctggagaa 540 tccctgagag
acttaattga gcagtctcag agctcaggaa gtggatcagg cctccctctg 600
ctggtccaaa ggactatagc taagcagatt cagatggtga aacagattgg aaaaggtcgc
660 tatggggaag tttggatggg aaagtggcgt ggcgaaaagg tagctgtgaa
agtgttcttc 720 accacagagg aagccagctg gttcagagag acagaaatat
atcagacagt gttgatgagg 780 catgaaaaca ttttgggttt cattgctgca
gatatcaaag ggacagggtc ctggacccag 840 ttgtacctaa tcacagacta
tcatgaaaat ggttcccttt atgattatct gaagtccacc 900 accctagacg
ctaaatcaat gctgaagtta gcctactctt ctgtcagtgg cttatgtcat 960
ttacacacag aaatctttag tactcaaggc aaaccagcaa ttgcccatcg agatctgaaa
1020 agtaaaaaca ttctggtgaa gaaaaatgga acttgctgta ttgctgacct
gggcctggct 1080 gttaaattta ttagtgatac aaatgaagtt gacataccac
ctaacactcg agttggcacc 1140 aaacgctata tgcctccaga agtgttggac
gagagcttga acagaaatca cttccagtct 1200 tacatcatgg ctgacatgta
tagttttggc ctcatccttt gggaggttgc taggagatgt 1260 gtatcaggag
gtatagtgga agaataccag cttccttatc atgacctagt gcccagtgac 1320
ccctcttatg aggacatgag ggagattgtg tgcatcaaga agttacgccc ctcattccca
1380 aaccggtgga gcagtgatga gtgtctaagg cagatgggaa aactcatgac
agaatgctgg 1440 gctcacaatc ctgcatcaag gctgacagcc ctgcgggtta
agaaaacact tgccaaaatg 1500 tcagagtccc aggacattaa actctgatag
gagaggaaaa gtaagcatct ctgcagaaag 1560 ccaacaggta ccctt 1575 118
2032 DNA Homo sapiens 118 cgcggggcgc ggagtcggcg gggcctcgcg
ggacgcgggc agtgcggaga ccgcggcgct 60 gaggacgcgg gagccgggag
cgcacgcgcg gggtggagtt cagcctactc tttcttagat 120 gtgaaaggaa
aggaagatca tttcatgcct tgttgataaa ggttcagact tctgctgatt 180
cataaccatt tggctctgag ctatgacaag agaggaaaca aaaagttaaa cttacaagcc
240 tgccataagt gagaagcaaa cttccttgat aacatgcttt tgcgaagtgc
aggaaaatta 300 aatgtgggca ccaagaaaga ggatggtgag agtacagccc
ccaccccccg tccaaaggtc 360 ttgcgttgta aatgccacca ccattgtcca
gaagactcag tcaacaatat ttgcagcaca 420 gacggatatt gtttcacgat
gatagaagag gatgactctg ggttgcctgt ggtcacttct 480 ggttgcctag
gactagaagg ctcagatttt cagtgtcggg acactcccat tcctcatcaa 540
agaagatcaa ttgaatgctg cacagaaagg aacgaatgta ataaagacct acaccctaca
600 ctgcctccat tgaaaaacag agattttgtt gatggaccta tacaccacag
ggctttactt 660 atatctgtga ctgtctgtag tttgctcttg gtccttatca
tattattttg ttacttccgg 720 tataaaagac aagaaaccag acctcgatac
agcattgggt tagaacagga tgaaacttac 780 attcctcctg gagaatccct
gagagactta attgagcagt ctcagagctc aggaagtgga 840 tcaggcctcc
ctctgctggt ccaaaggact atagctaagc agattcagat ggtgaaacag 900
attggaaaag gtcgctatgg ggaagtttgg atgggaaagt ggcgtggcga aaaggtagct
960 gtgaaagtgt tcttcaccac agaggaagcc agctggttca gagagacaga
aatatatcag 1020 acagtgttga tgaggcatga aaacattttg ggtttcattg
ctgcagatat caaagggaca 1080 gggtcctgga cccagttgta cctaatcaca
gactatcatg aaaatggttc cctttatgat 1140 tatctgaagt ccaccaccct
agacgctaaa tcaatgctga agttagccta ctcttctgtc 1200 agtggcttat
gtcatttaca cacagaaatc tttagtactc aaggcaaacc agcaattgcc 1260
catcgagatc tgaaaagtaa aaacattctg gtgaagaaaa atggaacttg ctgtattgct
1320 gacctgggcc tggctgttaa atttattagt gatacaaatg aagttgacat
accacctaac 1380 actcgagttg gcaccaaacg ctatatgcct ccagaagtgt
tggacgagag cttgaacaga 1440 aatcacttcc agtcttacat catggctgac
atgtatagtt ttggcctcat cctttgggag 1500 gttgctagga gatgtgtatc
aggaggtata gtggaagaat accagcttcc ttatcatgac 1560 ctagtgccca
gtgacccctc ttatgaggac atgagggaga ttgtgtgcat caagaagtta 1620
cgcccctcat tcccaaaccg gtggagcagt gatgagtgtc taaggcagat gggaaaactc
1680 atgacagaat gctgggctca caatcctgca tcaaggctga cagccctgcg
ggttaagaaa 1740 acacttgcca aaatgtcaga gtcccaggac attaaactct
gataggagag gaaaagtaag 1800 catctctgca gaaagccaac aggtactctt
ctgtttgtgg gcagagcaaa agacatcaaa 1860 taagcatcca cagtacaagc
cttgaacatc gtcctgcttc ccagtgggtt cagacctcac 1920 ctttcaggga
gcgacctggg caaagacaga gaagctccca gaaggagaga ttgatccgtg 1980
tctgtttgta ggcggagaaa ccgttgggta acttgttcaa gatatgatgc at 2032 119
3167 DNA Rattus sp. 119 gaattcatga gatggaaaca taggtcaaag ctgtttggag
aaattggaac tacagtttta 60 tctagccaca tctctgagaa gtctgaagaa
agcagcaggt gaaagtcatt gtcaagtgat 120 tttgttcttc tgtaaggaaa
cctcgttcag taaggccgtt tacttcagtg aaacagcagg 180 accagtaatc
aaggtggccc ggacaggaca cgtgcgaatt ggacaatgac tcagctatac 240
acttacatca gattactggg agcctgtctg ttcatcattt
ctcatgttca agggcagaat 300 ctagatagta tgctccatgg tactggtatg
aaatcagacg tggaccagaa gaagccggaa 360 aatggagtga cgttagcacc
agaggacacc ttacctttct taaaatgcta ttgctcagga 420 cactgcccag
atgacgctat taataacaca tgcataacta atggccattg ctttgccatt 480
atagaagaag atgatcaggg agaaaccacg ttaacttctg ggtgtatgaa gtatgaaggc
540 tctgattttc aatgcaagga ttcaccaaaa gcccagctac gcaggacaat
agaatgttgt 600 cggaccaatt tgtgcaacca atatttgcag cctacactgc
cccctgtcgt tataggccca 660 ttctttgatg gcagcgtccg atggctggct
gtgctcatct ctatggctgt ctgtattgtc 720 gccatgatcg tcttctccag
ctgcttctgt tacaaacatt actgtaagag tatctcaagc 780 agaggtcgtt
acaaccgtga cttggaacag gatgaagcat ttattccagt aggagaatca 840
ctgaaagacc tgattgacca gtcacaaagc tctggtagtg gatctggatt acctttattg
900 gttcagcgaa ctattgccaa acagattcag atggttcggc aggttggtaa
aggccggtat 960 ggagaagtat ggatgggtaa atggcgtggt gaaaaagtgg
ctgtcaaagt attttttacc 1020 actgaagaag ctagctggtt tagagaaaca
gaaatctacc agacggtgtt aatgcgtcat 1080 gaaaatatac ttggttttat
agctgcagac attaaaggca ccggttcctg gactcagctg 1140 tatttgatta
ctgattacca tgagaatggg tctctctatg acttcctgaa atgtgccacc 1200
ctggacacca gagccctact caagttagct tattctgctg cctgtggtct gtgccacctc
1260 cacacagaaa tttatggcac gcaaggcaag cctgcaattg ctcatcgaga
cctgaagagc 1320 aaaaacatcc ttattaagaa aaatggtagt tgctgtattg
ctgacctggg cctagctgtt 1380 aaattcaaca gtgacacaaa tgaagttgac
atacccttga acaccagggt gggcaccagg 1440 cggtacatgg ctccagaagt
gctggacgag agcctgagta aaaaccattt ccagccctac 1500 atcatggctg
acatctacag ctttggtttg atcatttggg agatggcccg tcgctgtatt 1560
acaggaggaa tcgtggagga atatcaatta ccatattaca acatggtgcc tagtgaccca
1620 tcttatgaag acatgcgtga ggtcgtgtgt gtgaaacgct tgcggccaat
cgtctctaac 1680 cgctggaaca gtgatgaatg tcttcgagcc gttttgaagc
tgatgtcaga atgctgggcc 1740 cataatccag catccagact cacagctttg
agaatcaaga agacgctcgc aaagatggtt 1800 gaatcccagg atgtaaagat
ttgacaaaca gttttgagaa agaatttaga ctgcaagaaa 1860 ttcacccgag
gaagggtgga gttagcatgg actaggatgt cggcttggtt tccagactct 1920
ctcctctaca tcttcacagg ctgctaacag taaactttca ggactctgca gaatgcaggg
1980 ttggagcttc agacatagga cttcagacat gctgttcttt gcgtatggac
agctttgttt 2040 taaatgtggg cttttgatgc ctttttggtt tttatgaatt
gcatcaagac tccaatcctg 2100 ataagaagtc tctggtcaaa ctctggttac
tcactatcct gtccataaag tggtgctttc 2160 tgtgaaagcc ttaaggaaat
tagtgagctc agcagagatg gagaaaggca tatttgccct 2220 ctacagagaa
aatatctgtc tgtgttctgt ctctgtaaac agcctggact atgatctctt 2280
tgggatgctg cctggttgat gatggtgcat catgcctctg atatgcatac cagacttcct
2340 ctgctgccat gggcttacaa gacaagaatg tgaaggttgc acaggacggt
atttgtggcc 2400 agtggtttaa atatgcaata tctaatcgac attcgccaat
ctcataaaag ccatctacct 2460 tgtaactgaa gtaacttctc taccaacttt
atttttagca taatagttgt aaaggccaaa 2520 ctatgtataa agtgtccata
gactcgaact gttttcctcc agtcaccatt ttgttttcct 2580 tttggtaatt
atttttgtta tataattcct cctatccaga attggcgctc actgtcttga 2640
accatacttt gaaagaaatg cctcttcctg gagtctgcct tactgcatct gatcaccatg
2700 tgcatacctc tgatcaaatt ctggagtctt tgttctcggt acctcttaaa
aagggaaatt 2760 gtgtatcatg tgtagtgtgc ttttattttc aaaatcttca
tagcctttat tctagccatt 2820 tttacctaca tactcattct gtacaaaaca
gctcactcgg tctcacggct gatcctcagt 2880 ggaaatgatt taaagtagag
ctgtgtacga atttcagaat tcatgtattt aaaaacttca 2940 cactaacact
ttactaagat attgtctcat atcttttatg aggatgtcag ctgattttca 3000
atgactataa atgtatctta gctatctaaa tcttttgaaa tttggtttta taatttctgg
3060 tccctaactt gtgaagacaa agaggcagaa gtacccagtc taccacattt
acactgtaca 3120 ttattaaata aaaaaatgta tattttaaaa aaaaaaaaaa aaaaaaa
3167 120 3167 DNA Rattus norvegicus 120 gaattcatga gatggaaaca
taggtcaaag ctgtttggag aaattggaac tacagtttta 60 tctagccaca
tctctgagaa gtctgaagaa agcagcaggt gaaagtcatt gtcaagtgat 120
tttgttcttc tgtaaggaaa cctcgttcag taaggccgtt tacttcagtg aaacagcagg
180 accagtaatc aaggtggccc ggacaggaca cgtgcgaatt ggacaatgac
tcagctatac 240 acttacatca gattactggg agcctgtctg ttcatcattt
ctcatgttca agggcagaat 300 ctagatagta tgctccatgg tactggtatg
aaatcagacg tggaccagaa gaagccggaa 360 aatggagtga cgttagcacc
agaggacacc ttacctttct taaaatgcta ttgctcagga 420 cactgcccag
atgacgctat taataacaca tgcataacta atggccattg ctttgccatt 480
atagaagaag atgatcaggg agaaaccacg ttaacttctg ggtgtatgaa gtatgaaggc
540 tctgattttc aatgcaagga ttcaccaaaa gcccagctac gcaggacaat
agaatgttgt 600 cggaccaatt tgtgcaacca atatttgcag cctacactgc
cccctgtcgt tataggccca 660 ttctttgatg gcagcgtccg atggctggct
gtgctcatct ctatggctgt ctgtattgtc 720 gccatgatcg tcttctccag
ctgcttctgt tacaaacatt actgtaagag tatctcaagc 780 agaggtcgtt
acaaccgtga cttggaacag gatgaagcat ttattccagt aggagaatca 840
ctgaaagacc tgattgacca gtcacaaagc tctggtagtg gatctggatt acctttattg
900 gttcagcgaa ctattgccaa acagattcag atggttcggc aggttggtaa
aggccggtat 960 ggagaagtat ggatgggtaa atggcgtggt gaaaaagtgg
ctgtcaaagt attttttacc 1020 actgaagaag ctagctggtt tagagaaaca
gaaatctacc agacggtgtt aatgcgtcat 1080 gaaaatatac ttggttttat
agctgcagac attaaaggca ccggttcctg gactcagctg 1140 tatttgatta
ctgattacca tgagaatggg tctctctatg acttcctgaa atgtgccacc 1200
ctggacacca gagccctact caagttagct tattctgctg cctgtggtct gtgccacctc
1260 cacacagaaa tttatggcac gcaaggcaag cctgcaattg ctcatcgaga
cctgaagagc 1320 aaaaacatcc ttattaagaa aaatggtagt tgctgtattg
ctgacctggg cctagctgtt 1380 aaattcaaca gtgacacaaa tgaagttgac
atacccttga acaccagggt gggcaccagg 1440 cggtacatgg ctccagaagt
gctggacgag agcctgagta aaaaccattt ccagccctac 1500 atcatggctg
acatctacag ctttggtttg atcatttggg agatggcccg tcgctgtatt 1560
acaggaggaa tcgtggagga atatcaatta ccatattaca acatggtgcc tagtgaccca
1620 tcttatgaag acatgcgtga ggtcgtgtgt gtgaaacgct tgcggccaat
cgtctctaac 1680 cgctggaaca gtgatgaatg tcttcgagcc gttttgaagc
tgatgtcaga atgctgggcc 1740 cataatccag catccagact cacagctttg
agaatcaaga agacgctcgc aaagatggtt 1800 gaatcccagg atgtaaagat
ttgacaaaca gttttgagaa agaatttaga ctgcaagaaa 1860 ttcacccgag
gaagggtgga gttagcatgg actaggatgt cggcttggtt tccagactct 1920
ctcctctaca tcttcacagg ctgctaacag taaactttca ggactctgca gaatgcaggg
1980 ttggagcttc agacatagga cttcagacat gctgttcttt gcgtatggac
agctttgttt 2040 taaatgtggg cttttgatgc ctttttggtt tttatgaatt
gcatcaagac tccaatcctg 2100 ataagaagtc tctggtcaaa ctctggttac
tcactatcct gtccataaag tggtgctttc 2160 tgtgaaagcc ttaaggaaat
tagtgagctc agcagagatg gagaaaggca tatttgccct 2220 ctacagagaa
aatatctgtc tgtgttctgt ctctgtaaac agcctggact atgatctctt 2280
tgggatgctg cctggttgat gatggtgcat catgcctctg atatgcatac cagacttcct
2340 ctgctgccat gggcttacaa gacaagaatg tgaaggttgc acaggacggt
atttgtggcc 2400 agtggtttaa atatgcaata tctaatcgac attcgccaat
ctcataaaag ccatctacct 2460 tgtaactgaa gtaacttctc taccaacttt
atttttagca taatagttgt aaaggccaaa 2520 ctatgtataa agtgtccata
gactcgaact gttttcctcc agtcaccatt ttgttttcct 2580 tttggtaatt
atttttgtta tataattcct cctatccaga attggcgctc actgtcttga 2640
accatacttt gaaagaaatg cctcttcctg gagtctgcct tactgcatct gatcaccatg
2700 tgcatacctc tgatcaaatt ctggagtctt tgttctcggt acctcttaaa
aagggaaatt 2760 gtgtatcatg tgtagtgtgc ttttattttc aaaatcttca
tagcctttat tctagccatt 2820 tttacctaca tactcattct gtacaaaaca
gctcactcgg tctcacggct gatcctcagt 2880 ggaaatgatt taaagtagag
ctgtgtacga atttcagaat tcatgtattt aaaaacttca 2940 cactaacact
ttactaagat attgtctcat atcttttatg aggatgtcag ctgattttca 3000
atgactataa atgtatctta gctatctaaa tcttttgaaa tttggtttta taatttctgg
3060 tccctaactt gtgaagacaa agaggcagaa gtacccagtc taccacattt
acactgtaca 3120 ttattaaata aaaaaatgta tattttaaaa aaaaaaaaaa aaaaaaa
3167 121 3003 DNA Rattus norvegicus 121 cgttcagtaa ggccgtttac
ttcagtgaaa cagcaggacc agtaatcaag gtggcccgga 60 caggacacgt
gcgaattgga caatgactca gctatacact tacatcagat tactgggagc 120
ctgtctgttc atcatttctc atgttcaagg gcagaatcta gatagtatgc tccatggtac
180 tggtatgaaa tcagacgtgg accagaagaa gccggaaaat ggagtgacgt
tagcaccaga 240 ggacacctta cctttcttaa aatgctattg ctcaggacac
tgcccagatg acgctattaa 300 taacacatgc ataactaatg gccattgctt
tgccattata gaagaagatg atcagggaga 360 aaccacgtta acttctgggt
gtatgaagta tgaaggctct gattttcaat gcaaggattc 420 accaaaagcc
cagctacgca ggacaataga atgttgtcgg accaatttgt gcaaccaata 480
tttgcagcct acactgcccc ctgtcgttat aggcccattc tttgatggca gcgtccgatg
540 gctggctgtg ctcatctcta tggctgtctg tattgtcgcc atgatcgtct
tctccagctg 600 cttctgttac aaacattact gtaagagtat ctcaagcaga
ggtcgttaca accgtgactt 660 ggaacaggat gaagcattta ttccagtagg
agaatcactg aaagacctga ttgaccagtc 720 acaaagctct ggtagtggat
ctggattacc tttattggtt cagcgaacta ttgccaaaca 780 gattcagatg
gttcggcagg ttggtaaagg ccggtatgga gaagtatgga tgggtaaatg 840
gcgtggtgaa aaagtggctg tcaaagtatt ttttaccact gaagaagcta gctggtttag
900 agaaacagaa atctaccaga cggtgttaat gcgtcatgaa aatatacttg
gttttatagc 960 tgcagacatt aaaggcaccg gttcctggac tcagctgtat
ttgattactg attaccatga 1020 gaatgggtct ctctatgact tcctgaaatg
tgccaccctg gacaccagag ccctactcaa 1080 gttagcttat tctgctgcct
gtggtctgtg ccacctccac acagaaattt atggcacgca 1140 aggcaagcct
gcaattgctc atcgagacct gaagagcaaa aacatcctta ttaagaaaaa 1200
tggtagttgc tgtattgctg acctgggcct agctgttaaa ttcaacagtg acacaaatga
1260 agttgacata cccttgaaca ccagggtggg caccaggcgg tacatggctc
cagaagtgct 1320 ggacgagagc ctgagtaaaa accatttcca gccctacatc
atggctgaca tctacagctt 1380 tggtttgatc atttgggaga tggcccgtcg
ctgtattaca ggaggaatcg tggaggaata 1440 tcaattacca tattacaaca
tggtgcctag tgacccatct tatgaagaca tgcgtgaggt 1500 cgtgtgtgtg
aaacgcttgc ggccaatcgt ctctaaccgc tggaacagtg atgaatgtct 1560
tcgagccgtt ttgaagctga tgtcagaatg ctgggcccat aatccagcat ccagactcac
1620 agctttgaga atcaagaaga cgctcgcaaa gatggttgaa tcccaggatg
taaagatttg 1680 acaaacagtt ttgagaaaga atttagactg caagaaattc
acccgaggaa gggtggagtt 1740 agcatggact aggatgtcgg cttggtttcc
agactctctc ctctacatct tcacaggctg 1800 ctaacagtaa actttcagga
ctctgcagaa tgcagggttg gagcttcaga cataggactt 1860 cagacatgct
gttctttgcg tatggacagc tttgttttaa atgtgggctt ttgatgcctt 1920
tttggttttt atgaattgca tcaagactcc aatcctgata agaagtctct ggtcaaactc
1980 tggttactca ctatcctgtc cataaagtgg tgctttctgt gaaagcctta
aggaaattag 2040 tgagctcagc agagatggag aaaggcatat ttgccctcta
cagagaaaat atctgtctgt 2100 gttctgtctc tgtaaacagc ctggactatg
atctctttgg gatgctgcct ggttgatgat 2160 ggtgcatcat gcctctgata
tgcataccag acttcctctg ctgccatggg cttacaagac 2220 aagaatgtga
aggttgcaca ggacggtatt tgtggccagt ggtttaaata tgcaatatct 2280
aatcgacatt cgccaatctc ataaaagcca tctaccttgt aactgaagta acttctctac
2340 caactttatt tttagcataa tagttgtaaa ggccaaacta tgtataaagt
gtccatagac 2400 tcgaactgtt ttcctccagt caccattttg ttttcctttt
ggtaattatt tttgttatat 2460 aattcctcct atccagaatt ggcgctcact
gtcttgaacc atactttgaa agaaatgcct 2520 cttcctggag tctgccttac
tgcatctgat caccatgtgc atacctctga tcaaattctg 2580 gagtctttgt
tctcggtacc tcttaaaaag ggaaattgtg tatcatgtgt agtgtgcttt 2640
tattttcaaa atcttcatag cctttattct agccattttt acctacatac tcattctgta
2700 caaaacagct cactcggtct cacggctgat cctcagtgga aatgatttaa
agtagagctg 2760 tgtacgaatt tcagaattca tgtatttaaa aacttcacac
taacacttta ctaagatatt 2820 gtctcatatc ttttatgagg atgtcagctg
attttcaatg actataaatg tatcttagct 2880 atctaaatct tttgaaattt
ggttttataa tttctggtcc ctaacttgtg aagacaaaga 2940 ggcagaagta
cccagtctac cacatttaca ctgtacatta ttaaataaaa aaatgtatat 3000 ttt
3003 122 2063 DNA Homo sapiens 122 gaattccggt gatgatgatg gtgatggtga
tgatggtgat gaggatgatg gtgatgatga 60 tgatggtgtt ggtgatggtt
tttgcatctt ccattcatga actaagtact cttattagtg 120 aatttctttt
ctttgccctc ctgattcttg gctggcccag ggatgacttc ctcgctgcag 180
cggccctggc gggtgccctg gctaccatgg accatcctgc tggtcagcac tgcggctgct
240 tcgcagaatc aagaacggct atgtgcgttt aaagatccgt atcagcaaga
ccttgggata 300 ggtgagagta gaatctctca tgaaaatggg acaatattat
gctcgaaagg tagcacctgc 360 tatggccttt gggagaaatc aaaaggggac
ataaatcttg taaaacaagg atgttggtct 420 cacattggag atccccaaga
gtgtcactat gaagaatgtg tagtaactac cactcctccc 480 tcaattcaga
atggaacata ccgtttctgc tgttgtagca cagatttatg taatgtcaac 540
tttactgaga attttccacc tcctgacaca acaccactca gtccacctca ttcatttaac
600 cgagatgaga caataatcat tgctttggca tcagtctctg tattagctgt
tttgatagtt 660 gccttatgct ttggatacag aatgttgaca ggagaccgta
aacaaggtct tcacagtatg 720 aacatgatgg aggcagcagc atccgaaccc
tctcttgatc tagataatct gaaactgttg 780 gagctgattg gccgaggtcg
atatggagca gtatataaag gctccttgga tgagcgtcca 840 gttgctgtaa
aagtgttttc ctttgcaaac cgtcagaatt ttatcaacga aaagaacatt 900
tacagagtgc ctttgatgga acatgacaac attgcccgct ttatagttgg agatgagaga
960 gtcactgcag atggacgcat ggaatatttg cttgtgatgg agtactatcc
caatggatct 1020 ttatgcaagt atttaagtct ccacacaagt gactgggtaa
gctcttgccg tcttgctcat 1080 tctgttacta gaggactggc ttatcttcac
acagaattac cacgaggaga tcattataaa 1140 cctgcaattt cccatcgaga
tttaaacagc agaaatgtcc tagtgaaaaa tgatggaacc 1200 tgtgttatta
gtgactttgg actgtccatg aggctgactg gaaatagact ggtgcgccca 1260
ggggaggaag ataatgcagc cataagcgag gttggcacta tcagatatat ggcaccagaa
1320 gtgctagaag gagctgtgaa cttgagggac tgtgaatcag ctttgaaaca
agtagacatg 1380 tatgctcttg gactaatcta ttgggagata tttatgagat
gtacagacct cttcccaggg 1440 gaatccgtac cagagtacca gatggctttt
cagacagagg ttggaaacca tcccactttt 1500 gaggatatgc aggttctcgt
gtctagggaa aaacagagac ccaagttccc agaagcctgg 1560 aaagaaaata
gcctggcagt gaggtcactc aaggagacaa tcgaagactg ttgggaccag 1620
gatgcagagg ctcggcttac tgcacagtgt gctgaggaaa ggatggctga acttatgatg
1680 atttgggaaa gaaacaaatc tgtgagccca acagtcaatc caatgtctac
tgctatgcag 1740 aatgaacgta ggtgagtcaa cacaagatgg caaatcagga
tcaggtgaaa agatcaagaa 1800 acgtgtgaaa actccctatt ctcttaagcg
gtggcgcccc tccacctggg tcatctccac 1860 tgaatcgctg gactgtgaag
tcaacaataa tggcagtaac agggcagttc attccaaatc 1920 cagcactgct
gtttaccttg cagaaggagg cactgctaca accatggtgt ctaaagatat 1980
aggaatgaac tgtctgtgaa atgttttcaa gcctatggag tgaaattatt ttttgcatca
2040 tttaaacatg cagaagatgt tta 2063 123 1964 DNA Homo sapiens 123
atttcttttc tttgccctcc tgattcttgg ctggcccagg gatgacttcc tcgctgcagc
60 ggccctggcg ggtgccctgg ctaccatgga ccatcctgct ggtcagcact
gcggctgctt 120 cgcagaatca agaacggcta tgtgcgttta aagatccgta
tcagcaagac cttgggatag 180 gtgagagtag aatctctcat gaaaatggga
caatattatg ctcgaaaggt agcacctgct 240 atggcctttg ggagaaatca
aaaggggaca taaatcttgt aaaacaagga tgttggtctc 300 acattggaga
tccccaagag tgtcactatg aagaatgtgt agtaactacc actcctccct 360
caattcagaa tggaacatac cgtttctgct gttgtagcac agatttatgt aatgtcaact
420 ttactgagaa ttttccacct cctgacacaa caccactcag tccacctcat
tcatttaacc 480 gagatgagac aataatcatt gctttggcat cagtctctgt
attagctgtt ttgatagttg 540 ccttatgctt tggatacaga atgttgacag
gagaccgtaa acaaggtctt cacagtatga 600 acatgatgga ggcagcagca
tccgaaccct ctcttgatct agataatctg aaactgttgg 660 agctgattgg
ccgaggtcga tatggagcag tatataaagg ctccttggat gagcgtccag 720
ttgctgtaaa agtgttttcc tttgcaaacc gtcagaattt tatcaacgaa aagaacattt
780 acagagtgcc tttgatggaa catgacaaca ttgcccgctt tatagttgga
gatgagagag 840 tcactgcaga tggacgcatg gaatatttgc ttgtgatgga
gtactatccc aatggatctt 900 tatgcaagta tttaagtctc cacacaagtg
actgggtaag ctcttgccgt cttgctcatt 960 ctgttactag aggactggct
tatcttcaca cagaattacc acgaggagat cattataaac 1020 ctgcaatttc
ccatcgagat ttaaacagca gaaatgtcct agtgaaaaat gatggaacct 1080
gtgttattag tgactttgga ctgtccatga ggctgactgg aaatagactg gtgcgcccag
1140 gggaggaaga taatgcagcc ataagcgagg ttggcactat cagatatatg
gcaccagaag 1200 tgctagaagg agctgtgaac ttgagggact gtgaatcagc
tttgaaacaa gtagacatgt 1260 atgctcttgg actaatctat tgggagatat
ttatgagatg tacagacctc ttcccagggg 1320 aatccgtacc agagtaccag
atggcttttc agacagaggt tggaaaccat cccacttttg 1380 aggatatgca
ggttctcgtg tctagggaaa aacagagacc caagttccca gaagcctgga 1440
aagaaaatag cctggcagtg aggtcactca aggagacaat cgaagactgt tgggaccagg
1500 atgcagaggc tcggcttact gcacagtgtg ctgaggaaag gatggctgaa
cttatgatga 1560 tttgggaaag aaacaaatct gtgagcccaa cagtcaatcc
aatgtctact gctatgcaga 1620 atgaacgtag gtgagtcaac acaagatggc
aaatcaggat caggtgaaaa gatcaagaaa 1680 cgtgtgaaaa ctccctattc
tcttaagcgg tggcgcccct ccacctgggt catctccact 1740 gaatcgctgg
actgtgaagt caacaataat ggcagtaaca gggcagttca ttccaaatcc 1800
agcactgctg tttaccttgc agaaggaggc actgctacaa ccatggtgtc taaagatata
1860 ggaatgaact gtctgtgaaa tgttttcaag cctatggagt gaaattattt
tttgcatcat 1920 ttaaacatgc agaagatgtt taaaaataaa aaaaaaactg cttt
1964 124 3611 DNA Homo sapiens 124 cgccccccga ccccggatcg aatccccgcc
ctccgcaccc tggatatgtt ttctcccaga 60 cctggatatt tttttgatat
cgtgaaacta cgagggaaat aatttggggg atttcttctt 120 ggctccctgc
tttccccaca gacatgcctt ccgtttggag ggccgcggca ccccgtccga 180
ggcgaaggaa cccccccagc cgcgagggag agaaatgaag ggaatttctg cagcggcatg
240 aaagctctgc agctaggtcc tctcatcagc catttgtcct ttcaaactgt
attgtgatac 300 gggcaggatc agtccacggg agagaagacg agcctcccgg
ctgtttctcc gccggtctac 360 ttcccatatt tcttttcttt gccctcctga
ttcttggctg gcccagggat gacttcctcg 420 ctgcagcggc cctggcgggt
gccctggcta ccatggacca tcctgctggt cagcactgcg 480 gctgcttcgc
agaatcaaga acggctatgt gcgtttaaag atccgtatca gcaagacctt 540
gggataggtg agagtagaat ctctcatgaa aatgggacaa tattatgctc gaaaggtagc
600 acctgctatg gcctttggga gaaatcaaaa ggggacataa atcttgtaaa
acaaggatgt 660 tggtctcaca ttggagatcc ccaagagtgt cactatgaag
aatgtgtagt aactaccact 720 cctccctcaa ttcagaatgg aacataccgt
ttctgctgtt gtagcacaga tttatgtaat 780 gtcaacttta ctgagaattt
tccacctcct gacacaacac cactcagtcc acctcattca 840 tttaaccgag
atgagacaat aatcattgct ttggcatcag tctctgtatt agctgttttg 900
atagttgcct tatgctttgg atacagaatg ttgacaggag accgtaaaca aggtcttcac
960 agtatgaaca tgatggaggc agcagcatcc gaaccctctc ttgatctaga
taatctgaaa 1020 ctgttggagc tgattggccg aggtcgatat ggagcagtat
ataaaggctc cttggatgag 1080 cgtccagttg ctgtaaaagt gttttccttt
gcaaaccgtc agaattttat caacgaaaag 1140 aacatttaca gagtgccttt
gatggaacat gacaacattg cccgctttat agttggagat 1200 gagagagtca
ctgcagatgg acgcatggaa tatttgcttg tgatggagta ctatcccaat 1260
ggatctttat gcaagtattt aagtctccac acaagtgact gggtaagctc ttgccgtctt
1320 gctcattctg ttactagagg actggcttat cttcacacag aattaccacg
aggagatcat 1380 tataaacctg caatttccca tcgagattta aacagcagaa
atgtcctagt gaaaaatgat 1440 ggaacctgtg ttattagtga ctttggactg
tccatgaggc tgactggaaa tagactggtg 1500 cgcccagggg aggaagataa
tgcagccata agcgaggttg gcactatcag atatatggca 1560 ccagaagtgc
tagaaggagc tgtgaacttg agggactgtg aatcagcttt gaaacaagta 1620
gacatgtatg ctcttggact aatctattgg gagatattta tgagatgtac agacctcttc
1680
ccaggggaat ccgtaccaga gtaccagatg gcttttcaga cagaggttgg aaaccatccc
1740 acttttgagg atatgcaggt tctcgtgtct agggaaaaac agagacccaa
gttcccagaa 1800 gcctggaaag aaaatagcct ggcagtgagg tcactcaagg
agacaatcga agactgttgg 1860 gaccaggatg cagaggctcg gcttactgca
cagtgtgctg aggaaaggat ggctgaactt 1920 atgatgattt gggaaagaaa
caaatctgtg agcccaacag tcaatccaat gtctactgct 1980 atgcagaatg
aacgcaacct gtcacataat aggcgtgtgc caaaaattgg tccttatcca 2040
gattattctt cctcctcata cattgaagac tctatccatc atactgacag catcgtgaag
2100 aatatttcct ctgagcattc tatgtccagc acacctttga ctatagggga
aaaaaaccga 2160 aattcaatta actatgaacg acagcaagca caagctcgaa
tccccagccc tgaaacaagt 2220 gtcaccagcc tctccaccaa cacaacaacc
acaaacacca caggactcac gccaagtact 2280 ggcatgacta ctatatctga
gatgccatac ccagatgaaa caaatctgca taccacaaat 2340 gttgcacagt
caattgggcc aacccctgtc tgcttacagc tgacagaaga agacttggaa 2400
accaacaagc tagacccaaa agaagttgat aagaacctca aggaaagctc tgatgagaat
2460 ctcatggagc actctcttaa acagttcagt ggcccagacc cactgagcag
tactagttct 2520 agcttgcttt acccactcat aaaacttgca gtagaagcaa
ctggacagca ggacttcaca 2580 cagactgcaa atggccaagc atgtttgatt
cctgatgttc tgcctactca gatctatcct 2640 ctccccaagc agcagaacct
tcccaagaga cctactagtt tgcctttgaa caccaaaaat 2700 tcaacaaaag
agccccggct aaaatttggc agcaagcaca aatcaaactt gaaacaagtc 2760
gaaactggag ttgccaagat gaatacaatc aatgcagcag aacctcatgt ggtgacagtc
2820 accatgaatg gtgtggcagg tagaaaccac agtgttaact cccatgctgc
cacaacccaa 2880 tatgccaatg ggacagtact atctggccaa acaaccaaca
tagtgacaca tagggcccaa 2940 gaaatgttgc agaatcagtt tattggtgag
gacacccggc tgaatattaa ttccagtcct 3000 gatgagcatg agcctttact
gagacgagag caacaagctg gccatgatga aggtgttctg 3060 gatcgtcttg
tggacaggag ggaacggcca ctagaaggtg gccgaactaa ttccaataac 3120
aacaacagca atccatgttc agaacaagat gttcttgcac agggtgttcc aagcacagca
3180 gcagatcctg ggccatcaaa gcccagaaga gcacagaggc ctaattctct
ggatctttca 3240 gccacaaatg tcctggatgg cagcagtata cagataggtg
agtcaacaca agatggcaaa 3300 tcaggatcag gtgaaaagat caagaaacgt
gtgaaaactc cctattctct taagcggtgg 3360 cgcccctcca cctgggtcat
ctccactgaa tcgctggact gtgaagtcaa caataatggc 3420 agtaacaggg
cagttcattc caaatccagc actgctgttt accttgcaga aggaggcact 3480
gctacaacca tggtgtctaa agatatagga atgaactgtc tgtgaaatgt tttcaagcct
3540 atggagtgaa attatttttt gcatcattta aacatgcaga agatgtttaa
aaataaaaaa 3600 aaaactgctt t 3611 125 3871 DNA Homo sapiens 125
ggcctccgca ccctggatat gttttctccc agacctggat atttttttga tatcgtgaaa
60 ctacgaggga aataatttgg gggatttctt cttggctccc tgctttcccc
acagacatac 120 cttccgtttg gagggccgcg gcaccccgtc cgaggcgaag
gaaccccccc atccgcgagg 180 gagagaaatg aagggaattt ctgcagcggc
atgaaagctc tgcagctagg tcctctcatc 240 agccatttgt cctttcaaac
tgtattgtga tacgggcagg atcagtccac gggagagaag 300 acgagcctcc
cggctgtttc tccgccggtc tacttcccat atttcttttc tttgccctcc 360
tgattcttgg ctggcccagg gatgacttcc tcgctgcagc ggccctggcg ggtgccctgg
420 ctaccatgga ccatcctgct ggtcagcact gcggctgctt cgcagaatca
agaacggcta 480 tgtgcgttta aagatccgta tcagcaagac cttgggatag
gtgagagtag aatctctcat 540 gaaaatggga caatattatg ctcgaaaggt
agcacctgct atggcctttg ggagaaatca 600 aaaggggaca taaatcttgt
aaaacaagga tgttggtctc acattggaga tccccaagag 660 tgtcactatg
aagaatgtgt agtaactacc actcctccct caattcagaa tggaacatac 720
cgtttctgct gttgtagcac agatttatgt aatgtcaact ttactgagaa ttttccacct
780 cctgacacaa caccactcag tccacctcat tcatttaacc gagatgagac
aataatcatt 840 gctttggcat cagtctctgt attagctgtt ttgatagttg
ccttatgctt tggatacaga 900 atgttgacag gagaccgtaa acaaggtctt
cacagtatga acatgatgga ggcagcagca 960 tccgaaccct ctcttgatct
agataatctg aaactgttgg agctgattgg ccgaggtcga 1020 tatggagcag
tatataaagg ctccttggat gagcgtccag ttgctgtaaa agtgttttcc 1080
tttgcaaacc gtcagaattt tatcaacgaa aagaacattt acagagtgcc tttgatggaa
1140 catgacaaca ttgcccgctt tatagttgga gatgagagag tcactgcaga
tggacgcatg 1200 gaatatttgc ttgtgatgga gtactatccc aatggatctt
tatgcaagta tttaagtctc 1260 cacacaagtg actgggtaag ctcttgccgt
cttgctcatt ctgttactag aggactggct 1320 tatcttcaca cagaattacc
acgaggagat cattataaac ctgcaatttc ccatcgagat 1380 ttaaacagca
gaaatgtcct agtgaaaaat gatggaacct gtgttattag tgactttgga 1440
ctgtccatga ggctgactgg aaatagactg gtgcgcccag gggaggaaga taatgcagcc
1500 ataagcgagg ttggcactat cagatatatg gcaccagaag tgctagaagg
agctgtgaac 1560 ttgagggact gtgaatcagc tttgaaacaa gtagacatgt
atgctcttgg actaatctat 1620 tgggagatat ttatgagatg tacagacctc
ttcccagggg aatccgtacc agagtaccag 1680 atggcttttc agacagaggt
tggaaaccat cccacttttg aggatatgca ggttctcgtg 1740 tctagggaaa
aacagagacc caagttccca gaagcctgga aagaaaatag cctggcagtg 1800
aggtcactca aggagacaat cgaagactgt tgggaccagg atgcagaggc tcggcttact
1860 gcacagtgtg ctgaggaaag gatggctgaa cttatgatga tttgggaaag
aaacaaatct 1920 gtgagcccaa cagtcaatcc aatgtctact gctatgcaga
atgaacgcaa cctgtcacat 1980 aataggcgtg tgccaaaaat tggtccttat
ccagattatt cttcctcctc atacattgaa 2040 gactctatcc atcatactga
cagcatcgtg aagaatattt cctctgagca ttctatgtcc 2100 agcacacctt
tgactatagg ggaaaaaaac cgaaattcaa ttaactatga acgacagcaa 2160
gcacaagctc gaatccccag ccctgaaaca agtgtcacca gcctctccac caacacaaca
2220 accacaaaca ccacaggact cacgccaagt actggcatga ctactatatc
tgagatgcca 2280 tacccagatg aaacaaatct gcataccaca aatgttgcac
agtcaattgg gccaacccct 2340 gtctgcttac agctgacaga agaagacttg
gaaaccaaca agctagaccc aaaagaagtt 2400 gataagaacc tcaaggaaag
ctctgatgag aatctcatgg agcactctct taaacagttc 2460 agtggcccag
acccactgag cagtactagt tctagcttgc tttacccact cataaaactt 2520
gcagtagaag caactggaca gcaggacttc acacagactg caaatggcca agcatgtttg
2580 attcctgatg ttctgcctac tcagatctat cctctcccca agcagcagaa
ccttcccaag 2640 agacctacta gtttgccttt gaacaccaaa aattcaacaa
aagagccccg gctaaaattt 2700 ggcagcaagc acaaatcaaa cttgaaacaa
gtcgaaactg gagttgccaa gatgaataca 2760 atcaatgcag cagaacctca
tgtggtgaca gtcaccatga atggtgtggc aggtagaaac 2820 cacagtgtta
actcccatgc tgccacaacc caatatgcca ataggacagt actatctggc 2880
caaacaacca acatagtgac acatagggcc caagaaatgt tgcagaatca gtttattggt
2940 gaggacaccc ggctgaatat taattccagt cctgatgagc atgagccttt
actgagacga 3000 gagcaacaag ctggccatga tgaaggtgtt ctggatcgtc
ttgtggacag gagggaacgg 3060 ccactagaag gtggccgaac taattccaat
aacaacaaca gcaatccatg ttcagaacaa 3120 gatgttcttg cacagggtgt
tccaagcaca gcagcagatc ctgggccatc aaagcccaga 3180 agagcacaga
ggcctaattc tctggatctt tcagccacaa atgtcctgga tggcagcagt 3240
atacagatag gtgagtcaac acaagatggc aaatcaggat caggtgaaaa gatcaagaaa
3300 cgtgtgaaaa ctccctattc tcttaagcgg tggcgcccct ccacctgggt
catctccact 3360 gaatcgctgg actgtgaagt caacaataat ggcagtaaca
gggcagttca ttccaaatcc 3420 agcactgctg tttaccttgc agaaggaggc
actgctacaa ccatggtgtc taaagatata 3480 ggaatgaact gtctgtgaaa
tgttttcaag cctatggagt gaaattattt tttgcatcat 3540 ttaaacatgc
agaagatgtt taccgggcgg ggtgacagga gagagcgtca gcggcaagct 3600
gtggaggatg gggctcagaa tgcagacctg ggctggccgc atggcctctc cctgagccct
3660 gatttgtggt agggaagcag tatgggtgca gtcccctcct aggcctccct
ctggggtccc 3720 ccgatcctat cccacctctt cagggtgagc cagcctcacc
tcttcctagt cctgagggtg 3780 agggcaggct gaggcaacga gtgggaggtt
caaacaagag tgggctggag ccaagggaaa 3840 atagagatga tgtaatttct
ttccggaatt c 3871 126 88 PRT Homo sapiens 126 Cys Arg Glu Leu His
Phe Thr Arg Tyr Val Thr Asp Gly Pro Cys Arg 1 5 10 15 Ser Ala Lys
Pro Val Thr Glu Leu Val Cys Ser Gly Gln Cys Gly Pro 20 25 30 Ala
Arg Leu Leu Pro Asn Ala Ile Gly Arg Gly Lys Trp Trp Arg Pro 35 40
45 Ser Gly Pro Asp Phe Arg Cys Ile Pro Asp Arg Tyr Arg Ala Gln Arg
50 55 60 Val Gln Leu Leu Cys Pro Gly Gly Glu Ala Pro Arg Ala Arg
Lys Val 65 70 75 80 Arg Leu Val Ala Ser Cys Lys Cys 85 127 82 PRT
Homo sapiens 127 Cys Arg Pro Ile Asn Ala Thr Leu Ala Val Glu Lys
Glu Gly Cys Pro 1 5 10 15 Val Cys Ile Thr Val Asn Thr Thr Ile Cys
Ala Gly Tyr Cys Pro Thr 20 25 30 Met Thr Arg Val Leu Gln Gly Val
Leu Pro Ala Leu Pro Gln Val Val 35 40 45 Cys Asn Tyr Arg Asp Val
Arg Phe Glu Ser Ile Arg Leu Pro Gly Cys 50 55 60 Pro Arg Gly Val
Asn Pro Val Val Ser Tyr Ala Val Ala Leu Ser Cys 65 70 75 80 Gln Cys
128 82 PRT Homo sapiens 128 Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile
Glu Lys Glu Glu Cys Arg 1 5 10 15 Phe Cys Ile Ser Ile Asn Thr Thr
Trp Cys Ala Gly Tyr Cys Tyr Thr 20 25 30 Arg Asp Leu Val Tyr Lys
Asp Pro Ala Arg Pro Lys Ile Gln Lys Thr 35 40 45 Cys Thr Phe Lys
Glu Leu Val Tyr Glu Thr Val Arg Val Pro Gly Cys 50 55 60 Ala His
His Ala Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr Gln Cys 65 70 75 80
His Cys 129 84 PRT Homo sapiens 129 Cys Ile Pro Thr Glu Tyr Thr Met
His Ile Glu Arg Arg Glu Cys Ala 1 5 10 15 Tyr Cys Leu Thr Ile Asn
Thr Thr Ile Cys Ala Gly Tyr Cys Met Thr 20 25 30 Arg Asp Ile Asn
Gly Lys Leu Phe Leu Pro Lys Tyr Ala Leu Ser Gln 35 40 45 Asp Val
Cys Thr Tyr Arg Asp Phe Ile Tyr Arg Thr Val Glu Ile Pro 50 55 60
Gly Cys Pro Leu His Val Ala Pro Tyr Phe Ser Tyr Pro Val Ala Leu 65
70 75 80 Ser Cys Lys Cys 130 83 PRT Homo sapiens 130 Cys Asn Asp
Ile Thr Ala Arg Leu Gln Tyr Val Lys Val Gly Ser Cys 1 5 10 15 Lys
Ser Glu Val Glu Val Asp Ile His Tyr Cys Gln Gly Lys Cys Ala 20 25
30 Ser Lys Ala Met Tyr Ser Ile Asp Ile Asn Asp Val Gln Asp Gln Cys
35 40 45 Ser Cys Cys Ser Pro Thr Arg Thr Glu Pro Met Gln Val Ala
Leu His 50 55 60 Cys Thr Asn Gly Ser Val Val Tyr His Glu Val Leu
Asn Ala Met Glu 65 70 75 80 Cys Lys Cys 131 80 PRT Homo sapiens 131
Cys Ser Thr Val Pro Val Thr Thr Glu Val Ser Tyr Ala Gly Cys Thr 1 5
10 15 Lys Thr Val Leu Met Asn His Cys Ser Gly Ser Cys Gly Thr Phe
Val 20 25 30 Met Tyr Ser Ala Lys Ala Gln Ala Leu Asp His Ser Cys
Ser Cys Cys 35 40 45 Lys Glu Glu Lys Thr Ser Gln Arg Glu Val Val
Leu Ser Cys Pro Asn 50 55 60 Gly Gly Ser Leu Thr His Thr Tyr Thr
His Ile Glu Ser Cys Gln Cys 65 70 75 80 132 80 PRT Homo sapiens 132
Cys Arg Thr Val Pro Phe Ser Gln Thr Ile Thr His Glu Gly Cys Glu 1 5
10 15 Lys Val Val Val Gln Asn Asn Leu Cys Phe Gly Lys Cys Gly Ser
Val 20 25 30 His Phe Pro Gly Ala Ala Gln His Ser His Thr Ser Cys
Ser His Cys 35 40 45 Leu Pro Ala Lys Phe Thr Thr Met His Leu Pro
Leu Asn Cys Thr Glu 50 55 60 Leu Ser Ser Val Ile Lys Val Val Met
Leu Val Glu Glu Cys Gln Cys 65 70 75 80 133 85 PRT Homo sapiens 133
Cys Lys Thr Gln Pro Leu Lys Gln Thr Ile His Glu Glu Gly Cys Asn 1 5
10 15 Ser Arg Thr Ile Ile Asn Arg Phe Cys Tyr Gly Gln Cys Asn Ser
Phe 20 25 30 Tyr Ile Pro Arg His Ile Arg Lys Glu Glu Gly Ser Phe
Gln Ser Cys 35 40 45 Ser Phe Cys Lys Pro Lys Lys Phe Thr Thr Met
Met Val Thr Leu Asn 50 55 60 Cys Pro Glu Leu Gln Pro Pro Thr Lys
Lys Lys Arg Val Thr Arg Val 65 70 75 80 Lys Gln Cys Arg Cys 85 134
86 PRT Homo sapiens 134 Cys Glu Ala Lys Asn Ile Thr Gln Ile Val Gly
His Ser Gly Cys Glu 1 5 10 15 Ala Lys Ser Ile Gln Asn Arg Ala Cys
Leu Gly Gln Cys Phe Ser Tyr 20 25 30 Ser Val Pro Asn Thr Phe Pro
Gln Ser Thr Glu Ser Leu Val His Cys 35 40 45 Asp Ser Cys Met Pro
Ala Gln Ser Met Trp Glu Ile Val Thr Leu Glu 50 55 60 Cys Pro Gly
His Glu Glu Val Pro Arg Val Asp Lys Leu Val Glu Lys 65 70 75 80 Ile
Leu His Cys Ser Cys 85 135 70 PRT Homo sapies 135 Cys Ile Arg Thr
Pro Lys Ile Ser Lys Pro Ile Lys Phe Glu Leu Ser 1 5 10 15 Gly Cys
Thr Ser Met Lys Thr Tyr Arg Ala Lys Phe Cys Gly Val Cys 20 25 30
Thr Asp Gly Arg Cys Cys Thr Pro His Arg Thr Thr Thr Leu Pro Val 35
40 45 Glu Phe Lys Cys Pro Asp Gly Glu Val Met Lys Lys Asn Met Met
Phe 50 55 60 Ile Lys Thr Cys Ala Cys 65 70 136 70 PRT Homo sapiens
136 Cys Leu Arg Thr Lys Lys Ser Leu Lys Ala Ile His Leu Gln Phe Lys
1 5 10 15 Asn Cys Thr Ser Leu His Thr Tyr Lys Pro Arg Phe Cys Gly
Val Cys 20 25 30 Ser Asp Gly Arg Cys Cys Thr Pro His Asn Thr Lys
Thr Ile Gln Ala 35 40 45 Glu Phe Gln Cys Ser Pro Gly Gln Ile Val
Lys Lys Pro Val Met Val 50 55 60 Ile Gly Thr Cys Thr Cys 65 70 137
70 PRT Homo sapiens 137 Cys Ser Lys Thr Lys Lys Ser Pro Glu Pro Val
Arg Phe Thr Tyr Ala 1 5 10 15 Gly Cys Leu Ser Val Lys Lys Tyr Arg
Pro Lys Tyr Cys Gly Ser Cys 20 25 30 Val Asp Gly Arg Cys Cys Thr
Pro Gln Leu Thr Arg Thr Val Lys Met 35 40 45 Arg Phe Arg Cys Glu
Asp Gly Glu Thr Phe Ser Lys Asn Val Met Met 50 55 60 Ile Gln Ser
Cys Lys Cys 65 70 138 205 PRT Homo sapiens 138 Gln His Tyr Leu His
Ile Arg Pro Ala Pro Ser Asp Asn Leu Pro Leu 1 5 10 15 Val Asp Leu
Ile Glu His Pro Asp Pro Ile Phe Asp Pro Lys Glu Lys 20 25 30 Asp
Leu Asn Glu Thr Leu Leu Arg Ser Leu Leu Gly Gly His Tyr Asp 35 40
45 Pro Gly Phe Met Ala Thr Ser Pro Pro Glu Asp Arg Pro Gly Gly Gly
50 55 60 Gly Gly Ala Ala Gly Gly Ala Glu Asp Leu Ala Glu Leu Asp
Gln Leu 65 70 75 80 Leu Arg Gln Arg Pro Ser Gly Ala Met Pro Ser Glu
Ile Lys Gly Leu 85 90 95 Glu Phe Ser Glu Gly Leu Ala Gln Gly Lys
Lys Gln Arg Leu Ser Lys 100 105 110 Lys Leu Arg Arg Lys Leu Gln Met
Trp Leu Trp Ser Gln Thr Phe Cys 115 120 125 Pro Val Leu Tyr Ala Trp
Asn Asp Leu Gly Ser Arg Phe Trp Pro Arg 130 135 140 Tyr Val Lys Val
Gly Ser Cys Phe Ser Lys Arg Ser Cys Ser Val Pro 145 150 155 160 Glu
Gly Met Val Cys Lys Pro Ser Lys Ser Val His Leu Thr Val Leu 165 170
175 Arg Trp Arg Cys Gln Arg Arg Gly Gly Gln Arg Cys Gly Trp Ile Pro
180 185 190 Ile Gln Tyr Pro Ile Ile Ser Glu Cys Lys Cys Ser Cys 195
200 205 139 197 PRT Gallus gallus 139 Gln His Tyr Leu His Ile Arg
Pro Ala Pro Ser Asp Asn Leu Pro Leu 1 5 10 15 Val Asp Leu Ile Glu
His Pro Asp Pro Ile Phe Asp Pro Lys Glu Lys 20 25 30 Asp Leu Asn
Glu Thr Leu Leu Arg Ser Leu Met Gly Gly His Phe Asp 35 40 45 Pro
Asn Phe Met Ala Met Ser Leu Pro Glu Asp Arg Leu Gly Val Asp 50 55
60 Asp Leu Ala Glu Leu Asp Leu Leu Leu Arg Gln Arg Pro Ser Gly Ala
65 70 75 80 Met Pro Gly Glu Ile Lys Gly Leu Glu Phe Tyr Asp Gly Leu
Gln Pro 85 90 95 Gly Lys Lys His Arg Leu Ser Lys Lys Leu Arg Arg
Lys Leu Gln Met 100 105 110 Trp Leu Trp Ser Gln Thr Phe Cys Pro Val
Leu Tyr Thr Trp Asn Asp 115 120 125 Leu Gly Ser Arg Phe Trp Pro Arg
Tyr Val Lys Val Gly Ser Cys Tyr 130 135 140 Ser Lys Arg Ser Cys Ser
Val Pro Glu Gly Met Val Cys Lys Pro Ala 145 150 155 160 Lys Ser Val
His Leu Thr Ile Leu Arg Trp Arg Cys Gln Arg Arg Gly 165 170 175 Gly
Gln Arg Cys Thr Trp Ile Pro Ile Gln Tyr Pro Ile Ile Ala Glu 180 185
190 Cys Lys Cys Ser Cys 195 140 196 PRT Xenopus laevis 140 Gln His
Tyr Leu His Ile Arg Pro Ala Pro Ser Glu Asn Leu Pro Leu 1 5 10 15
Val Asp Leu Ile Glu His Pro Asp Pro Ile Tyr Asp Pro Lys Glu Lys 20
25 30 Asp Leu Asn Glu Thr Leu Leu Arg Thr Leu Met Val Gly His Phe
Asp 35 40 45 Pro Asn Phe Met Ala Thr Ile Leu Pro Glu Glu Arg Leu
Gly Val Glu 50 55 60 Asp Leu Gly Glu Leu Asp Leu Leu Leu Arg Gln
Lys Pro Ser Gly Ala 65 70 75
80 Met Pro Ala Glu Ile Lys Gly Leu Glu Phe Tyr Glu Gly Leu Gln Ser
85 90 95 Lys Lys His Arg Leu Ser Lys Lys Leu Arg Arg Lys Leu Gln
Met Trp 100 105 110 Leu Trp Ser Gln Thr Phe Cys Pro Val Leu Tyr Thr
Trp Asn Asp Leu 115 120 125 Gly Thr Arg Phe Trp Pro Arg Tyr Val Lys
Val Gly Ser Cys Tyr Ser 130 135 140 Lys Arg Ser Cys Ser Val Pro Glu
Gly Met Val Cys Lys Ala Ala Lys 145 150 155 160 Ser Met His Leu Thr
Ile Leu Arg Trp Arg Cys Gln Arg Arg Val Gln 165 170 175 Gln Lys Cys
Ala Trp Ile Thr Ile Gln Tyr Pro Val Ile Ser Glu Cys 180 185 190 Lys
Cys Ser Cys 195 141 195 PRT Takifugu rubripes 141 Gln Pro Tyr Tyr
Leu Leu Arg Pro Ile Pro Ser Asp Ser Leu Pro Ile 1 5 10 15 Val Glu
Leu Lys Glu Asp Pro Gly Pro Val Phe Asp Pro Lys Glu Arg 20 25 30
Asp Leu Asn Glu Thr Glu Leu Lys Ser Val Leu Gly Asp Phe Asp Ser 35
40 45 Arg Phe Leu Ser Val Leu Pro Pro Ala Glu Asp Gly His Ala Gly
Asn 50 55 60 Asp Glu Leu Asp Asp Phe Asp Ala Gln Arg Trp Gly Gly
Ala Leu Pro 65 70 75 80 Lys Glu Ile Arg Ala Val Asp Phe Asp Ala Pro
Gln Leu Gly Lys Lys 85 90 95 His Lys Pro Ser Lys Lys Leu Lys Arg
Arg Leu Gln Gln Trp Leu Trp 100 105 110 Ala Tyr Ser Phe Cys Pro Leu
Ala His Ala Trp Thr Asp Leu Gly Ser 115 120 125 Arg Phe Trp Pro Arg
Phe Val Arg Ala Gly Ser Cys Leu Ser Lys Arg 130 135 140 Ser Cys Ser
Val Pro Glu Gly Met Thr Cys Lys Pro Ala Thr Ser Thr 145 150 155 160
His Leu Thr Ile Leu Arg Trp Arg Cys Val Gln Arg Lys Val Gly Leu 165
170 175 Lys Cys Ala Trp Ile Pro Met Gln Tyr Pro Val Ile Thr Asp Cys
Lys 180 185 190 Cys Ser Cys 195 142 196 PRT Danio rerio 142 Gln His
Tyr Tyr Leu Leu Arg Pro Ile Pro Ser Asp Ser Leu Pro Ile 1 5 10 15
Val Glu Leu Lys Glu Asp Pro Asp Pro Val Leu Asp Pro Lys Glu Arg 20
25 30 Asp Leu Asn Glu Thr Glu Leu Arg Ala Ile Leu Gly Ser His Phe
Glu 35 40 45 Gln Asn Phe Met Ser Ile Asn Pro Pro Glu Asp Lys His
Ala Gly Gln 50 55 60 Asp Glu Leu Asn Glu Ser Glu Leu Met Lys Gln
Arg Pro Asn Gly Ile 65 70 75 80 Met Pro Lys Glu Ile Lys Ala Met Glu
Phe Asp Ile Gln His Gly Lys 85 90 95 Lys His Lys Pro Ser Lys Lys
Leu Arg Arg Arg Leu Gln Leu Trp Leu 100 105 110 Trp Ser Tyr Thr Phe
Cys Pro Val Val His Thr Trp Gln Asp Leu Gly 115 120 125 Asn Arg Phe
Trp Pro Arg Tyr Leu Lys Val Gly Ser Cys Tyr Asn Lys 130 135 140 Arg
Ser Cys Ser Val Pro Glu Gly Met Val Cys Lys Pro Pro Lys Ser 145 150
155 160 Ser His Leu Thr Val Leu Arg Trp Arg Cys Val Gln Arg Lys Gly
Gly 165 170 175 Leu Lys Cys Ala Trp Ile Pro Val Gln Tyr Pro Val Ile
Ser Glu Cys 180 185 190 Lys Cys Ser Cys 195 143 188 PRT Mus
musculus 143 Gln Gly Trp Gln Ala Phe Arg Asn Asp Ala Thr Glu Val
Ile Pro Gly 1 5 10 15 Leu Gly Glu Tyr Pro Glu Pro Pro Pro Glu Asn
Asn Gln Thr Met Asn 20 25 30 Arg Ala Glu Asn Gly Gly Arg Pro Pro
His His Pro Tyr Asp Ala Lys 35 40 45 Gly Val Ser Glu Tyr Ser Cys
Arg Glu Leu His Tyr Thr Arg Phe Leu 50 55 60 Thr Asp Gly Pro Cys
Arg Ser Ala Lys Pro Val Thr Glu Leu Val Cys 65 70 75 80 Ser Gly Gln
Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile Gly Arg 85 90 95 Val
Lys Trp Trp Arg Pro Asn Gly Pro Asp Phe Arg Cys Ile Pro Asp 100 105
110 Arg Tyr Arg Ala Gln Arg Val Gln Leu Leu Cys Pro Gly Gly Ala Ala
115 120 125 Pro Arg Ser Arg Lys Val Arg Leu Val Ala Ser Cys Lys Cys
Lys Arg 130 135 140 Leu Thr Arg Phe His Asn Gln Ser Glu Leu Lys Asp
Phe Gly Pro Glu 145 150 155 160 Thr Ala Arg Pro Gln Lys Gly Arg Lys
Pro Arg Pro Gly Ala Arg Gly 165 170 175 Ala Lys Ala Asn Gln Ala Glu
Leu Glu Asn Ala Tyr 180 185
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