U.S. patent application number 11/488375 was filed with the patent office on 2007-02-22 for wisp polypeptides and nucleic acids encoding same.
Invention is credited to David Botstein, Robert L. Cohen, Audrey D. Goddard, Austin L. Gurney, Kenneth J. Hillan, David A. Lawrence, Arnold J. Levine, Diane Pennica, Margaret Ann Roy, William I. Wood.
Application Number | 20070041964 11/488375 |
Document ID | / |
Family ID | 27370521 |
Filed Date | 2007-02-22 |
United States Patent
Application |
20070041964 |
Kind Code |
A1 |
Botstein; David ; et
al. |
February 22, 2007 |
WISP polypeptides and nucleic acids encoding same
Abstract
Wnt-1-Induced Secreted Proteins (WISPs) are provided, whose
genes are induced at least by Wnt-1. Also provided are nucleic acid
molecules encoding those polypeptides, as well as vectors and host
cells comprising those nucleic acid sequences, chimeric polypeptide
molecules comprising the polypeptides fused to heterologous
polypeptide sequences, antibodies which bind to the polypeptides,
and methods for producing the polypeptides.
Inventors: |
Botstein; David; (Belmont,
CA) ; Cohen; Robert L.; (San Mateo, CA) ;
Goddard; Audrey D.; (San Francisco, CA) ; Gurney;
Austin L.; (Belmont, CA) ; Hillan; Kenneth J.;
(San Francisco, CA) ; Lawrence; David A.; (San
Francisco, CA) ; Levine; Arnold J.; (New York,
NY) ; Pennica; Diane; (Burlingame, CA) ; Roy;
Margaret Ann; (San Francisco, CA) ; Wood; William
I.; (Hillsborough, CA) |
Correspondence
Address: |
HELLER EHRMAN LLP
275 MIDDLEFIELD ROAD
MENLO PARK
CA
94025-3506
US
|
Family ID: |
27370521 |
Appl. No.: |
11/488375 |
Filed: |
July 17, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10112267 |
Mar 27, 2002 |
7101850 |
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11488375 |
Jul 17, 2006 |
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09182145 |
Oct 29, 1998 |
6387657 |
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10112267 |
Mar 27, 2002 |
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60063704 |
Oct 29, 1997 |
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60073612 |
Feb 4, 1998 |
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60081695 |
Apr 14, 1998 |
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Current U.S.
Class: |
424/131.1 ;
424/146.1; 530/387.2; 530/388.26 |
Current CPC
Class: |
C07K 14/475 20130101;
C07K 16/32 20130101; A61P 31/10 20180101; A61P 17/02 20180101; A61P
35/00 20180101; C07K 14/47 20130101; A61P 17/00 20180101; C07K
16/22 20130101; A61P 15/08 20180101; A61P 7/00 20180101; C12Q
2600/136 20130101; A61P 13/12 20180101; A61P 35/02 20180101; A61P
43/00 20180101; A61P 9/10 20180101; C07K 2317/73 20130101; C07K
2317/74 20130101; A61P 37/02 20180101; A61P 29/00 20180101; C07K
16/30 20130101; C12Q 1/6886 20130101; A61P 31/04 20180101; A61P
19/00 20180101; C07K 16/18 20130101; A61P 39/00 20180101 |
Class at
Publication: |
424/131.1 ;
424/146.1; 530/387.2; 530/388.26 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/40 20070101 C07K016/40; C07K 16/42 20070101
C07K016/42 |
Goverment Interests
GOVERNMENT LICENSE RIGHTS
[0002] This invention was made with government support under grant
no. 5P01 CA41086, awarded by the National Institutes of Health,
National Cancer Institute. The government has certain rights in the
invention.
Claims
1.-11. (canceled)
12. A method for inhibiting the growth of tumor cells comprising
exposing a cell that overexpresses a Wnt-1-induced gene to an
effective amount of an antagonist that inhibits the expression or
activity of a WISP-1 polypeptide.
13. The method of claim 12 wherein said antagonist is an antibody
specifically binding a WISP-1 polypeptide.
14. The method of claim 12 or claim 13 wherein the tumor cells are
colon cancer cells, the antibody is against human WISP-1 and is a
humanized or human monoclonal antibody, and the tumor cells are
those of a human.
Description
RELATED APPLICATIONS
[0001] This is a divisional application of U.S. patent application
Ser. No. 10/112,267, filed on Mar. 27, 2002, now U.S. Pat. No.
7,101,850, which is a divisional application of U.S. patent
application Ser. No. 09/182,145, filed on Oct. 29, 1998, now U.S.
Pat. No. 6,387,657, which claims priority to U.S. Provisional
Patent Application Ser. No. 60/063,704, filed Oct. 29, 1997, and to
U.S. Provisional Patent Application Ser. No. 60/081,695, filed on
Apr. 14, 1998, and to U.S. Provisional Patent Application Ser. No.
60/073,612, filed Feb. 4, 1998, the entire disclosures of which
applications are hereby incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the
identification and isolation of novel DNA and to the recombinant
production of novel polypeptides having homology to connective
tissue growth factor, designated herein as Wnt-1-Induced Secreted
Proteins (WISPs).
BACKGROUND OF THE INVENTION
[0004] Malignant tumors (cancers) are the second leading cause of
death in the United States, after heart disease. Boring et al., CA
Cancer J. Clin., 43:7 (1993).
[0005] Cancer is characterized by the increase in the number of
abnormal, or neoplastic, cells derived from a normal tissue which
proliferate to form a tumor mass, the invasion of adjacent tissues
by these neoplastic tumor cells, and the generation of malignant
cells which eventually spread via the blood or lymphatic system to
regional lymph nodes and to distant sites (metastasis). In a
cancerous state a cell proliferates under conditions in which
normal cells would not grow. Cancer manifests itself in a wide
variety of forms, characterized by different degrees of
invasiveness and aggressiveness.
[0006] Alteration of gene expression is intimately related to the
uncontrolled cell growth and de-differentiation which are a common
feature of all cancers.
[0007] The genomes of certain well studied tumors have been found
to show decreased expression of recessive genes, usually referred
to as tumor suppression genes, which would normally function to
prevent malignant cell growth, and/or overexpression of certain
dominant genes, such as oncogenes, that act to promote malignant
growth. Each of these genetic changes appears to be responsible for
importing some of the traits that, in aggregate, represent the full
neoplastic phenotype. Hunter, Cell, 64:1129 (1991); Bishop, Cell,
64:35-248 (1991).
[0008] A well-known mechanism of gene (e.g., oncogene)
overexpression in cancer cells is gene amplification. This is a
process where in the chromosome of the ancestral cell multiple
copies of a particular gene are produced. The process involves
unscheduled replication of the region of chromosome comprising the
gene, followed by recombination of the replicated segments back
into the chromosome. Alitalo et al., Adv. Cancer Res., 47:235-281
(1986). It is believed that the overexpression of the gene
parallels gene amplification, i.e., is proportionate to the number
of copies made.
[0009] Proto-oncogenes that encode growth factors and growth factor
receptors have been identified to play important roles in the
pathogenesis of various human malignancies, including breast
cancer. For example, it has been found that the human ErbB2 gene
(erbB2, also known as her2, or c-erbB-2), which encodes a 185-kd
transmembrane glycoprotein receptor (p185.sup.HER2; HER2) related
to the epidermal growth factor receptor (EGFR), is overexpressed in
about 25% to 30% of human breast cancer. Slamon et al., Science,
235:177-182 (1987); Slamon et al., Science, 244:707-712 (1989). It
has been reported that gene amplification of a protooncogen is an
event typically involved in the more malignant forms of cancer, and
could act as a predictor of clinical outcome. Schwab et al., Genes
Chromosomes Cancer, 1:181-193 (1990); Alitalo et al., supra. Thus,
erbB2 overexpression is commonly regarded as a predictor of a poor
prognosis, especially in patients with primary disease that
involves axillary lymph nodes (Slamon et al., (1987) and (1989),
supra; Ravdin and Chamness, Gene, 159:19-27 (1995); and Hynes and
Stern, Biochim Biophys Acta, 1198:165-184 (1994)), and has been
linked to sensitivity and/or resistance to hormone therapy and
chemotherapeutic regimens, including CMF (cyclophosphamide,
methotrexate, and fluoruracil) and anthracyclines. Baselga et al.,
Oncology, 11(3 Suppl 1):43-48 (1997). However, despite the
association of erbB2 overexpression with poor prognosis, the odds
of HER2-positive patients responding clinically to treatment with
taxanes were greater than three times those of HER2-negative
patients. Baselga et al., supra. A recombinant humanized anti-ErbB2
(anti-HER2) monoclonal antibody (a humanized version of the murine
anti-ErbB2 antibody 4D5, referred to as rhuMAb HER2 or
HERCEPTIN.RTM.) has been clinically active in patients with
ErbB2-overexpressing metastatic breast cancers that had received
extensive prior anticancer therapy. Baselga et al., J. Clin.
Oncol., 14:737-744 (1996).
[0010] Cytokines have been implicated in the pathogenesis of a
number of brain diseases in which neurological dysfunction has been
attributed to a change in amino acid neurotransmitter metabolism.
In particular, members of the transforming growth factor-.beta.
(TGF-.beta.) family have been implicated. TGF peptides are small
polypeptides that were first identified by their ability to induce
proliferation and transformation in noncancerous cells in culture.
Although initially defined as a growth factor, TGF-.beta. also
inhibits proliferation of epithelial, endothelial, lymphoid, and
hematopoietic cells. This cytokine is thought to play an important
role in regulating the duration of the inflammatory response,
allowing the healing process to proceed. It is also a potent
immunomodulator, which has many pleiotrophic effects, including
regulating many other cytokines.
[0011] The TGF-.beta. superfamily includes bone morphogenetic
proteins (BMP-2, BMP-4, BMP-5, BMP-6, BMP-7), activins A & B,
decapentaplegic (dpp), 60A, OP-2, dorsalin, growth differentiation
factors (GDFs), nodal, MIS, Inhibin-.A-inverted., TGF-.beta.1,
TGF-.beta.2, TGF-.beta.3, TGF-.beta.5, and glial-derived
neurotrophic factor (GDNF). Atrisano, et al., J. Biochemica et
Biophysica Acta, 1222:71-80 (1994), of particular interest are the
growth differentiation factors, for as their name implies, these
factors are implicated in the differentiation of cells.
[0012] Connective tissue growth factor (CTGF) is a growth factor
induced in fibroblasts by many factors, including TGF-.beta. and is
essential for the ability of TGF-.beta. to induce
anchorage-independent growth (AIG), a property of transformed
cells. CTGF was discovered in an attempt to identify the type of
platelet derived growth factor (PDGF) dimers present in the growth
media of cultured endothelial cells, and is related immunologically
and biologically to PDGF. See U.S. Pat. No. 5,408,040. CTGF also is
mitogenic and chemotactic for cells, and hence growth factors in
this family are believed to play a role in the normal development,
growth, and repair of human tissue.
[0013] Seven proteins related to CTGF, including the chicken
ortholog for Cyr61, CEF10, human, mouse, and Xenopus laevis CTGF,
and human, chicken, and Xenopus laevis Nov have been isolated,
cloned, sequenced, and characterized as belonging to the CTGF gene
family. Oemar and Luescher, Arterioscler. Thromb. Vasc. Biol.,
17:1483-1489 (1997). The gene encoding Cyr61 has been found to
promote angiogenesis, tumor growth, and vascularization. Babic et
al., Proc. Natl. Acad. Sci. USA, 95:6355-6360 (1998). The nov gene
is expressed in the kidney essentially at the embryonic stage, and
alterations of nov expression, relative to the normal kidney, have
been detected in both avian nephroblastomas and human Wilms'
tumors. Martinerie et al., Oncogene, 9:2729-2732 (1994). Wt1 down
regulates human nov expression, which down regulation might
represent a key element in normal and tumoral nephrogenesis.
Martinerie et al., Oncogene, 12:1479-1492 (1996). It has recently
been proposed that the CTGF, nov, and cyr61 genes, which encode
secreted proteins that contain conserved sequences and IGFBP motifs
in their N-termini and bind IGFs with low affinity, represent more
members of the IGFBP superfamily, along with the low-affinity
mac25/IGFBP-7 (Yamanaka et al., J. Biol. Chem., 272:30729-30734
(1997)) and the high-affinity IGFBPs 1-6. CTGF under this proposal
would be designated IGFBP-8. Kim et al., Proc. Natl., Acad. Sci.
USA, 94:12981-12986 (1997).
[0014] Recently, a protein was found in the mouse designated ELM1
that is expressed in low metastatic cells. Hashimoto et al., J.
Exp. Med., 187:289-296 (1998). The elm1 gene, a mouse homologue of
WISP-1 disclosed below, is another member of the CTGF, Cyr61/Cef10,
and neuroblastoma overexpressed-gene family and suppresses in vivo
tumor growth and metastasis of K-1735 murine melanoma cells.
Another recent article on rCop-1, the rat orthologue of WISP-2
described below describes the loss of expression of this gene after
cell transformation. Zhang et al., Mol. Cell. Biol., 18:6131-6141
(1998).
[0015] CTGF family members (with the exception of nov) are
immediate early growth-responsive genes that are thought to
regulate cell proliferation, differentiation, embryo genesis, and
wound healing. Sequence homology among members of the CTGF gene
family is high; however, functions of these proteins in vitro range
from growth stimulatory (i.e., human CTGF) to growth inhibitory
(i.e., chicken Nov and also possibly hCTGF). Further, some
molecules homologous to CTGF are indicated to be useful in the
prevention of desmoplasia, the formation of highly cellular,
excessive connective tissue stroma associated with some cancers,
and fibrotic lesions associated with various skin disorders such as
scleroderma, keloid, eosinophilic fasciitis, nodular fasciitis, and
Dupuytren's contracture. Moreover, CTGF expression has recently
been demonstrated in the fibrous stoma of mammary tumors,
suggesting cancer stroma formation involves the induction of
similar fibroproliferative growth factors as wound repair. Human
CTGF is also expressed at very high levels in advanced
atherosclerotic lesions, but not in normal arteries, suggesting it
may play a role in atherosclerosis. Oemar and Luescher, supra.
Therefore, molecules homologous to CTGF are of importance.
[0016] Extracellular and membrane-bound proteins play important
roles in the formation, differentiation, and maintenance of
multicellular organisms. The fate of many individual cells, e.g.,
proliferation, migration, differentiation, or interaction with
other cells, is typically governed by information received from
other cells and/or the immediate environment. This information is
often transmitted by secreted polypeptides (for instance, mitogenic
factors, survival factors, cytotoxic factors, differentiation
factors, neuropeptides, and hormones), which are, in turn, received
and interpreted by diverse cell receptors or membrane-bound
proteins. These secreted polypeptides or signaling molecules
normally pass through the cellular secretory pathway to reach their
site of action in the extracellular environment, usually at a
membrane-bound receptor protein.
[0017] Secreted proteins have various industrial applications,
including use as pharmaceuticals, diagnostics, biosensors, and
bioreactors. In fact, most protein drugs available at present, such
as thrombolytic agents, interferons, interleukins, erythropoietins,
colony stimulating factors, and various other cytokines, are
secreted proteins. Their receptors, which are membrane-bound
proteins, also have potential as therapeutic or diagnostic agents.
Receptor immunoadhesins, for instance, can be employed as
therapeutic agents to block receptor-ligand interaction.
Membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction. Such membrane-bound proteins and cell
receptors include, but are not limited to, cytokine receptors,
receptor kinases, receptor phosphatases, receptors involved in
cell-cell interactions, and cellular adhesin molecules like
selectins and integrins. Transduction of signals that regulate cell
growth and differentiation is regulated in part by phosphorylation
of various cellular proteins. Protein tyrosine kinases, enzymes
that catalyze that process, can also act as growth factor
receptors. Examples include fibroblast growth factor receptor and
nerve growth factor receptor.
[0018] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound receptor
proteins, particularly those having homology to CTGF. Many efforts
are focused on the screening of mammalian recombinant DNA libraries
to identify the coding sequences for novel secreted and
membrane-bound receptor proteins. Examples of screening methods and
techniques are described in the literature. See, for example, Klein
et al., Proc. Natl. Acad. Sci., 93:7108-7113 (1996); and U.S. Pat.
No. 5,536,637.
[0019] Wnts are encoded by a large gene family whose members have
been found in round worms, insects, cartilaginous fish, and
vertebrates. Holland et al., Dev. Suppl., 125-133 (1994). Wnts are
thought to function in a variety of developmental and physiological
processes since many diverse species have multiple conserved Wnt
genes. McMahon, Trends Genet., 8:236-242 (1992); Nusse and Varmus,
Cell, 69:1073-1087 (1992). Wnt genes encode secreted glycoproteins
that are thought to function as paracrine or autocrine signals
active in several primitive cell types. McMahon, supra (1992);
Nusse and Varmus, supra (1992). The Wnt growth factor family
includes more than ten genes identified in the mouse (Wnt-1, -2,
-3A, -3B, -4, -5A, -5B, -6, -7A, -7B, -8A, -8B, -10B, -11, -12, and
-13) (see, e.g., Gavin et al., Genes Dev., 4:2319-2332 (1990); Lee
et al., Proc. Natl. Acad. Sci. USA, 92:2268-2272 (1995);
Christiansen et al., Mech. Dev., 51:341-350 (1995)) and at least
nine genes identified in the human (Wnt-1, -2, -3, -5A, -7A, -7B,
-8B, -10B, and -11) by cDNA cloning. See, e.g., Vant Veer et al.,
Mol. Cell. Biol., 4:2532-2534 (1984).
[0020] The Wnt-1 proto-oncogene (int-1) was originally identified
from mammary tumors induced by mouse mammary tumor virus (MMTV) due
to an insertion of viral DNA sequence. Nusse and Varmus, Cell,
31:99-109 (1982). In adult mice, the expression level of Wnt-1 mRNA
is detected only in the testis during later stages of sperm
development. Wnt-1 protein is about 42 KDa and contains an amino
terminal hydrophobic region, which may function as a signal
sequence for secretion (Nusse and Varmus, supra, 1992). The
expression of WnL-2/irp is detected in mouse fetal and adult
tissues and its distribution does not overlap with the expression
pattern for Wnt-1. Wnt-3 is associated with mouse mammary
tumorigenesis. The expression of Wnt-3 in Mouse embryos is detected
in the neural tubes and in the limb buds. Wnt-5a transcripts are
detected in the developing fore and hind limbs at 9.5 through 14.5
days and highest levels are concentrated in apical ectoderm at the
distal tip of limbs. Nusse and Varmus, supra (1992). Recently, a
Wnt growth factor, termed Wnt-x, was described (WO95/17416) along
with the detection of Wnt-x expression in bone tissues and in
bone-derived cells. Also described was the role of Wnt-x in the
maintenance of mature osteoblasts and the use of the Wnt-x growth
factor as a therapeutic agent or in the development of other
therapeutic agents to treat bone-related diseases.
[0021] Wnts may play a role in local cell signaling. Biochemical
studies have shown that much of the secreted Wnt protein can be
found associated with the cell surface or extracellular matrix
rather than freely diffusible in the medium. Papkoff and Schryver,
Mol. Cell. Biol., 10:2723-2730 (1990); Bradley and Brown, EMBO J.,
9:1569-1575 (1990).
[0022] Studies of mutations in Wnt genes have indicated a role for
Wnts in growth control and tissue patterning. In Drosophila,
wingless (wg) encodes a Wnt-related gene (Rijsewik et al., Cell,
50:649-657 (1987)) and wg mutations alter the pattern of embryonic
ectoderm, neurogenesis, and imaginal disc outgrowth. Morata and
Lawrence, Dev. Biol., 56:227-240 (1977); Baker, Dev. Biol.,
125:96-108 (1988); Klingensmith and Nusse, Dev. Biol., 166:396-414
(1994). In Caenorhabditis elegans, lin-44 encodes a Wnt homolog
which is required for asymmetric cell divisions. Herman and
Horvitz, Development, 120:1035-1047 (1994). Knock-out mutations in
mice have shown Wnts to be essential for brain development (McMahon
and Bradley, Cell, 62:1073-1085 (1990); Thomas and Cappechi,
Nature, 346:847-850 (1990)), and the outgrowth of embryonic
primordia for kidney (Stark et al., Nature, 372:679-683 (1994)),
tail bud (Takada et al., Genes Dev., 8:174-189 (1994)), and limb
bud. Parr and McMahon, Nature, 374:350-353 (1995) Overexpression of
Wnts in the mammary gland can result in mammary hyperplasia
(McMahon, supra (1992); Nusse and Varmus, supra (1992)), and
precocious alveolar development. Bradbury et al., Dev. Biol.,
170:553-563 (1995).
[0023] Wnt-5a and Wnt-5b are expressed in the posterior and lateral
mesoderm and the extraembryonic mesoderm of the day 7-8 murine
embryo. Gavin et al., supra (1990). These embryonic domains
contribute to the AGM region and yolk sac tissues from which
multipotent hematopoietic precursors and HSCs are derived. Dzierzak
and Medvinsky, Trends Genet., 11:359-366 (1995); Zon et al., in
Gluckman and Coulombel, ed., Colloque, INSERM, 235:17-22 (1995),
presented at the Joint International Workshop on Foetal and
Neonatal Hematopoiesis and Mechanism of Bone Marrow Failure, Paris
France, Apr. 3-6, 1995; Kanatsu and Nishikawa, Development,
122:823-830 (1996). Wnt-5a, Wnt-10b, and other Wnts have been
detected in limb buds, indicating possible roles in the development
and patterning of the early bone microenvironment as shown for
Wnt-7b. Gavin et al., supra (1990); Christiansen et al., Mech.
Devel., 51:341-350 (1995); Parr and McMahon, supra (1995).
[0024] The Wnt/Wg signal transduction pathway plays an important
role in the biological development of the organism and has been
implicated in several human cancers. This pathway also includes the
tumor suppressor gene, APC. Mutations in the APC gene are
associated with the development of sporadic and inherited forms of
human colorectal cancer. The Wnt/Wg signal leads to the
accumulation of beta-catenin/Armadillo in the cell, resulting in
the formation of a bipartite transcription complex consisting of
beta-catenin and a member of the lymphoid enhancer binding factor/T
cell factor (LEF/TCF)HMG box transcription factor family. This
complex translocates to the nucleus where it can activate
expression of genes downstream of the Wnt/Wg signal, such as the
engrailed and Ultrabithorax genes in Drosophila. The downstream
target genes of Wnt-1 signaling in vertebrates that presumably
function in tumorigenesis, however, are currently unknown.
[0025] For a most recent review on Wnt, see Cadigan and Nusse,
Genes & Dev., 11:3286-3305 (1997).
[0026] There is a need to elucidate the further members of the
above families, including cell-surface molecules that may be
tumor-specific antigens or proteins that serve a regulatory
function in initiating the Wnt pathway of tumorigenesis. These
would also include downstream components of the Wnt signaling
pathway that are important to the transformed phenotype and the
development of cancer.
SUMMARY OF THE INVENTION
[0027] Several putative Wnt-1-induced genes have been identified at
the mRNA level in a high-throughput cDNA substraction experiment.
Thus, applicants have identified novel cDNA clones (WISP1, WISP2,
and WISP3) that encode novel polypeptides of the WISP family,
designated as WISP-1, WISP-2, and WISP-3, respectively. This class
of polypeptides was formerly referred to as Wnt-1-induced Gene
(WIG) polypeptides, with WISP-1 and WISP-2 formerly designated as
WIG-1 and WIG-2, respectively. One of the cDNA clones encodes a
novel polypeptide, human WISP-2, having homology to CTGF, wherein
the polypeptide is designated in the present application as "human
WISP-2" or "PR0261". The WISP-1 and WISP-3 molecules also have
homology to CTGF.
[0028] In one embodiment, this invention provides isolated nucleic
acid comprising DNA having at least about 600 nucleotides and at
least about a 75% sequence identity to (a) a DNA molecule encoding
a human WISP-1 polypeptide comprising the sequence of amino acids
23 to 367 of FIGS. 3A-3C (SEQ ID NO:3), or (b) the complement of
the DNA molecule of (a). Preferably, this nucleic acid has at least
one WISP biological activity. In a more preferred embodiment, this
nucleic acid has at least about a 95% sequence identity to (a) a
DNA molecule encoding a human WISP-1 polypeptide comprising the
sequence of amino acids 23 to 367 of FIGS. 3A-3C (SEQ ID NO:3), or
(b) the complement of the DNA molecule of (a).
[0029] More preferred is the nucleic acid comprising DNA encoding a
human WISP-1 polypeptide having amino acid residues 23 to 367 of
FIGS. 3A-3C (SEQ ID NO:3), or DNA encoding a human WISP-1
polypeptide having amino acid residues 1 to 367 of FIGS. 3A-3C (SEQ
ID NO:4), or the complement of either of the encoding DNAs. Further
preferred is this nucleic acid comprising DNA encoding a human
WISP-1 polypeptide having amino acid residues 23 to 367 or 1 to 367
of FIGS. 3A-3C except for an isoleucine residue at position 184
rather than a valine residue or a serine residue at position 202
rather than an alanine residue (SEQ ID NOS:5-8, respectively).
Further preferred also is this nucleic acid comprising DNA encoding
a human WISP-1 polypeptide having amino acid residues 23 to 367 or
1 to 367 of FIGS. 3A-3C except for an isoleucine residue at
position 184 rather than a valine residue and a serine residue at
position 202 rather than an alanine residue (SEQ ID NOS:21-22,
respectively).
[0030] Also preferred is this nucleic acid comprising DNA encoding
a mouse WISP-1 polypeptide having amino acid residues 23 to 367 of
FIGS. 1A-1B (SEQ ID NO:11), or DNA encoding a mouse WISP-1
polypeptide having amino acid residues 1 to 367 of FIGS. 1A-1B (SEQ
ID NO:12), or the complement of either of the encoding DNAs.
[0031] Also provided by this invention is isolated nucleic acid
comprising DNA having at least about 600 nucleotides and at least
about a 85% sequence identity to (a) a DNA molecule encoding a
mouse WISP-1 polypeptide comprising the sequence of amino acids 23
to 367 of FIGS. 1A-1B (SEQ ID NO:11), or (b) the complement of the
DNA molecule of (a). Preferably, this nucleic acid has at least one
WISP biological activity. More preferably, this nucleic acid
comprises DNA having at least about a 95% sequence identity to (a)
a DNA molecule encoding a mouse WISP-1 polypeptide comprising the
sequence of amino acids 23 to 367 of FIGS. 1A-1B (SEQ ID NO:11), or
(b) the complement of the DNA molecule of (a).
[0032] In another preferred embodiment, the invention provides an
isolated nucleic acid comprising DNA having at least about 600
nucleotides and at least about a 75% sequence identity to (a) a DNA
molecule encoding the same full-length polypeptide encoded by the
human WISP-1 polypeptide cDNA in ATCC Deposit No. 209533
(pRK5E.h.WISP-1.568.38), or (b) the complement of the DNA molecule
of (a). This nucleic acid preferably comprises DNA having at least
about 600 nucleotides and at least about a 95% sequence identity to
(a) a DNA molecule encoding the same full-length polypeptide
encoded by the human WISP-1 polypeptide cDNA in ATCC Deposit No.
209533 (pRK5E.h.WISP-1.568.38), or (b) the complement of the DNA
molecule of (a).
[0033] In another aspect, the invention provides a process for
producing a WISP-1 polypeptide comprising culturing a host cell
comprising the above nucleic acid under conditions suitable for
expression of the WISP-1 polypeptide and recovering the WISP-1
polypeptide from the cell culture. Additionally provided is an
isolated WISP-1 polypeptide encoded by the above nucleic acid,
including where the polypeptide is human WISP-1 or mouse
WISP-1.
[0034] In another embodiment, the invention provides isolated
nucleic acid comprising SEQ ID NO:23, 24, 25, 26, 27, 28, or 29,
and an isolated WISP-1 polypeptide encoded by such a nucleic
acid.
[0035] Also provided by this invention is an isolated nucleic acid
having at least about 600 nucleotides and produced by hybridizing a
test DNA molecule under stringent conditions with (a) a DNA
molecule encoding a human WISP-1 polypeptide comprising the
sequence of amino acids 23 to 367 of FIGS. 3A-3C (SEQ ID NO:3), or
(b) the complement of the DNA molecule of (a), and, if the test DNA
molecule has at least about a 75% sequence identity to (a) or (b),
isolating the test DNA molecule.
[0036] Further provided is a polypeptide produced by (i)
hybridizing a test DNA molecule under stringent conditions with (a)
a DNA molecule encoding a human WISP-1 polypeptide comprising the
sequence of amino acids 23 to 367 of FIGS. 3A-3C (SEQ ID NO:3), or
(b) the complement of the DNA molecule of (a), and if the test DNA
molecule has at least about a 75% sequence identity to (a) or (b),
(ii) culturing a host cell comprising the test DNA molecule under
conditions suitable for expression of the polypeptide, and (iii)
recovering the polypeptide from the cell culture.
[0037] In another aspect, the invention provides isolated nucleic
acid comprising DNA having at least about an 80% sequence identity
to (a) a DNA molecule encoding a human WISP-2 polypeptide
comprising the sequence of amino acids 24 to 250 of FIGS. 4A-4B
(SEQ ID NO:15), or (b) the complement of the DNA molecule of (a).
Preferably, this nucleic acid has at least one WISP biological
activity. Also, preferably this nucleic acid comprises DNA having
at least about a 95% sequence identity to (a) a DNA molecule
encoding a human WISP-2 polypeptide comprising the sequence of
amino acids 24 to 250 of FIGS. 4A-4B (SEQ ID NO:15), or (b) the
complement of the DNA molecule of (a) In another preferred
embodiment, this nucleic acid comprises DNA encoding a human WISP-2
polypeptide having amino acid residues 24 to 250 of FIGS. 4A-4B
(SEQ ID NO:15), or DNA encoding a human WISP-2 polypeptide having
amino acid residues 1 to 250 of FIGS. 4A-4B (SEQ ID NO:16), or a
complement of either of the encoding DNAs.
[0038] In another aspect, the invention provides isolated nucleic
acid comprising DNA having at least about an 80% sequence identity
to (a) a DNA molecule encoding a human WISP-2 polypeptide
comprising the sequence of amino acids 1 to 250 of FIGS. 4A-4B (SEQ
ID NO:16), or (b) the complement of the DNA molecule of (a).
[0039] In another aspect, the invention provides isolated nucleic
acid comprising DNA having at least about 500 nucleotides and at
least about an 80% sequence identity to (a) a DNA molecule encoding
a mouse WISP-2 polypeptide comprising the sequence of amino acids
24 to 251 of FIGS. 2A-2B (SEQ ID NO:19), or (b) the complement of
the DNA molecule of (a). In a preferred embodiment, this nucleic
acid comprises DNA having at least about a 95% sequence identity to
(a) a DNA molecule encoding a mouse WISP-2 polypeptide comprising
the sequence of amino acids 24 to 251 of FIGS. 2A-2B (SEQ ID
NO:19), or (b) the complement of the DNA molecule of (a). More
preferably, the nucleic acid comprises DNA encoding a mouse WISP-2
polypeptide having amino acid residues 24 to 251 of FIGS. 2A-2B
(SEQ ID NO:19), or DNA encoding a mouse WISP-2 polypeptide having
amino acid residues 1 to 251 of FIGS. 2A-2B (SEQ ID NO:20), or the
complement of either of these encoding DNAs.
[0040] In a further aspect, the invention provides isolated nucleic
acid comprising DNA having at least about 500 nucleotides and at
least about an 80% sequence identity to (a) a DNA molecule encoding
a mouse WISP-2 polypeptide comprising the sequence of amino acids 1
to 251 of FIGS. 2A-2B (SEQ ID NO:20), or (b) the complement of the
DNA molecule of (a).
[0041] In yet another aspect, the invention provides an isolated
nucleic acid comprising DNA having at least about 400 nucleotides
and at least about a 75% sequence identity to (a) a DNA molecule
encoding the same full-length polypeptide encoded by the human
WISP-2 polypeptide cDNA in ATCC Deposit No. 209391 (DNA33473), or
(b) the complement of the DNA molecule of (a). Preferably, this
nucleic acid comprises DNA having at least about a 95% sequence
identity to (a) a DNA molecule encoding the same full-length
polypeptide encoded by the human WISP-2 polypeptide cDNA in ATCC
Deposit No. 209391 (DNA33473), or (b) the complement of the DNA
molecule of (a).
[0042] In another embodiment, this invention provides an isolated
nucleic acid comprising the nucleotide sequence of the full-length
coding sequence of clone UNQ228 (DNA33473) deposited under
accession number ATCC 209391.
[0043] In another aspect, the invention provides a process for
producing a WISP-2 polypeptide comprising culturing a host cell
comprising the above nucleic acid under conditions suitable for
expression of the WISP-2 polypeptide and recovering the WISP-2
polypeptide from the cell culture. Additionally provided is a
WISP-2 polypeptide encoded by the isolated nucleic acid, including
where the polypeptide is human WISP-2 or mouse WISP-2. In a
specific embodiment of this, the invention provides isolated
native-sequence human WISP-2 polypeptide comprising amino acid
residues 1 to 250 of FIGS. 4A-4B (SEQ ID NO:16) or comprising amino
acid residues 24 to 250 of FIGS. 4A-4B (SEQ ID NO:15).
[0044] In a further embodiment, the invention provides an isolated
nucleic acid having at least about 400 nucleotides and produced by
hybridizing a test DNA molecule under stringent conditions with (a)
a DNA molecule encoding a human WISP-2 polypeptide comprising the
sequence of amino acids 24 to 250 of FIGS. 4A-4B (SEQ ID NO:15), or
(b) the complement of the DNA molecule of (a), and, if the test DNA
molecule has at least about a 75% sequence identity to (a) or (b),
isolating the test DNA molecule.
[0045] In a still further embodiment, the invention provides a
polypeptide produced by (i) hybridizing a test DNA molecule under
stringent conditions with (a) a DNA molecule encoding a human
WISP-2 polypeptide comprising the sequence of amino acids 24 to 250
of FIGS. 4A-4B (SEQ ID NO:15), or (b) the complement of the DNA
molecule of (a), and if the test DNA molecule has at least about a
75% sequence identity to (a) or (b), (ii) culturing a host cell
comprising the test DNA molecule under conditions suitable for
expression of the polypeptide, and (iii) recovering the polypeptide
from the cell culture.
[0046] In yet another embodiment, the invention provides isolated
nucleic acid comprising DNA having a 100% sequence identity in more
than about 500 nucleotides to (a) a DNA molecule encoding a human
WISP-3 polypeptide comprising the sequence of amino acids 34 to 372
of FIGS. 6A-6C (SEQ ID NO:32), or (b) the complement of the DNA
molecule of (a). Preferably, this nucleic acid has at least one
WISP biological activity. Preferably, this nucleic acid comprises
DNA encoding a human WISP-3 polypeptide having amino acid residues
34 to 372 of FIGS. 6A-6C (SEQ ID NO:32) or amino acids 1 to 372 of
FIGS. 6A-6C (SEQ ID NO:33), or the complement thereof.
[0047] In a still further, embodiment, the invention provides an
isolated nucleic acid comprising DNA having a 100% sequence
identity in more than about 500 nucleotides to (a) a DNA molecule
encoding the same full-length polypeptide encoded by the human
WISP-3 polypeptide cDNA in ATCC Deposit No. 209706
(DNA56350-1176-2), or (b) the complement of the DNA molecule of
(a). A still further aspect of the invention involves a process for
producing a WISP-3 polypeptide comprising culturing a host cell
comprising WISP-3-encoding nucleic acid under conditions suitable
for expression of the WISP-3 polypeptide and recovering the WISP-3
polypeptide from the cell culture.
[0048] Further provided is an isolated WISP-3 polypeptide encoded
by the WISP-3-encoding nucleic acid. Preferably, this polypeptide
is human WISP-3.
[0049] In another embodiment, the invention provides an isolated
nucleic acid produced by hybridizing a test DNA molecule under
stringent conditions with (a) a DNA molecule encoding a human
WISP-3 polypeptide comprising the sequence of amino acids 34 to 372
of FIGS. 6A-6C (SEQ ID NO:32), or (b) the complement of the DNA
molecule of (a), and, if the test DNA molecule has a 100% sequence
identity to (a) or (b) in more than about 500 nucleotides,
isolating the test DNA molecule.
[0050] Also provided is a polypeptide produced by (i) hybridizing a
test DNA molecule under stringent conditions with (a) a DNA
molecule encoding a human WISP-3 polypeptide comprising the
sequence of amino acids 34 to 372 of FIGS. 6A-6C (SEQ ID NO:32), or
(b) the complement of the DNA molecule of (a), and if the test DNA
molecule has a 100% sequence identity to (a) or (b) in more than
about 500 nucleotides, (ii) culturing a host cell comprising the
test DNA molecule under conditions suitable for expression of the
polypeptide, and (iii) recovering the polypeptide from the cell
culture.
[0051] In yet another embodiment, the invention provides isolated
nucleic acid comprising DNA having a 100% sequence identity in more
than about 400 nucleotides to (a) a DNA molecule encoding a human
WISP-3 polypeptide comprising the sequence of amino acids 16 to 355
of FIGS. 7A-7C (SEQ ID NO:36), or (b) the complement of the DNA
molecule of (a). Preferably, this nucleic acid has at least one
WISP biological activity. Preferably, this nucleic acid comprises
DNA encoding a human WISP-3 polypeptide having amino acid residues
16 to 355 of FIGS. 7A-7C (SEQ ID NO:36), or amino acid residues 1
to 355 of FIGS. 7A-7C (SEQ ID NO:37) or the complement thereof.
[0052] In a still further embodiment, the invention provides an
isolated nucleic acid comprising DNA having a 100% sequence
identity in more than about 400 nucleotides to (a) a DNA molecule
encoding the same full-length polypeptide encoded by the human
WISP-3 polypeptide cDNA in ATCC Deposit No. 209707
(DNA58800-1176-2), or (b) the complement of the DNA molecule of
(a).
[0053] A still further aspect of the invention involves a process
for producing a WISP-3 polypeptide of FIG. 7A-7C comprising
culturing a host cell comprising WISP-3-encoding nucleic acid under
conditions suitable for expression of the WISP-3 polypeptide and
recovering the WISP-3 polypeptide from the cell culture.
[0054] Further provided is an isolated WISP-3 polypeptide of FIG.
7A-7C encoded by the WISP-3-encoding nucleic acid. Preferably, this
polypeptide is human WISP-3.
[0055] In another embodiment, the invention provides an isolated
nucleic acid produced by hybridizing a test DNA molecule under
stringent conditions with (a) a DNA molecule encoding a human
WISP-3 polypeptide comprising the sequence of amino acids 16 to 355
of FIGS. 7A-7C (SEQ ID NO:36), or (b) the complement of the DNA
molecule of (a), and, if the test DNA molecule has a 100% sequence
identity to (a) or (b) in more than about 400 nucleotides,
isolating the test DNA molecule.
[0056] Also provided is a polypeptide produced by (i) hybridizing a
test DNA molecule under stringent conditions with (a) a DNA
molecule encoding a human WISP-3 polypeptide comprising the
sequence of amino acids 16 to 355 of FIGS. 7A-7C (SEQ ID NO:36), or
(b) the complement of the DNA molecule of (a), and if the test DNA
molecule has a 100% sequence identity to (a) or (b) in more than
about 400 nucleotides, (ii) culturing a host cell comprising the
test DNA molecule under conditions suitable for expression of the
polypeptide, and (iii) recovering the polypeptide from the cell
culture.
[0057] Preferably the complements of the DNA molecules herein
remain stably bound to the primary sequence under at least
moderate, and optionally, under high stringency conditions. Also
provided are vectors comprising the above nucleic acids, host cells
comprising the vector, preferably wherein the cell is a Chinese
hamster ovary (CHO) cell, an E. coli cell, a baculovirus-infected
cell, or a yeast cell.
[0058] Additionally provided are a chimeric molecule comprising one
of the above polypeptides or an inactivated variant thereof, fused
to a heterologous amino acid sequence, wherein the heterologous
amino acid sequence may be, for example, an epitope tag sequence, a
polyamino acid such as poly-histidine, or an immunoglobulin
constant region (Fc). Also provided is an antibody which
specifically binds to one of the above polypeptides, wherein the
antibody can be a monoclonal antibody.
[0059] Further provided are a composition comprising one of the
above polypeptides and a carrier therefor, and a composition
comprising an antagonist to one of the polypeptides and a carrier
therefor. In one such embodiment, the invention provides a
composition comprising a WISP-1, WISP-2, or WISP-3 polypeptide and
a pharmaceutically acceptable carrier. Preferably, the polypeptide
is a human polypeptide. Also, preferably, these compositions may
also comprise a chemotherapeutic agent or growth-inhibitory
agent.
[0060] In another aspect, the invention provides a pharmaceutical
product comprising:
[0061] (a) the composition comprising WISP-1, WISP-2, or WISP-3
polypeptide and a pharmaceutically acceptable carrier;
[0062] (b) a container containing said composition; and
[0063] (c) a label affixed to said container, or a package insert
included in said pharmaceutical product referring to the use of
said WISP-1, WISP-2, or WISP-3 polypeptide in the treatment of a
WISP-related disorder.
[0064] In yet another embodiment, the invention provides a method
for treating a WISP-related disorder in a mammal comprising
administering to the mammal an effective amount of any of the above
compositions, including the composition of a WISP-1, WISP-2, or
WISP-3 polypeptide in a pharmaceutically acceptable carrier, and
including the composition of an antagonist to a WISP-1, WISP-2, or
WISP-3 polypeptide in a pharmaceutically acceptable carrier.
Preferably, the disorder is a malignant disorder or
arteriosclerosis. More preferably, the malignant disorder is breast
cancer, ovarian cancer, colon cancer, or melanoma. Also, preferably
the mammal is human. In another preferred embodiment, the WISP-1,
WISP-2, or WISP-3 polypeptide is administered in combination with a
chemotherapeutic agent, a growth inhibitory agent, or a cytotoxic
agent.
[0065] In another embodiment, the invention supplies a process for
diagnosing a disease or a susceptibility to a disease related to a
mutation in a nucleic acid sequence encoding a WISP-1, WISP-2, or
WISP-3 polypeptide comprising:
[0066] (a) isolating a nucleic acid sequence encoding a WISP-1,
WISP-2, or WISP-3 polypeptide from a sample derived from a host;
and
[0067] (b) determining a mutation in the nucleic acid sequence
encoding a WISP-1, WISP-2, or WISP-3 polypeptide.
[0068] In another embodiment, the invention provides a method of
diagnosing a WISP-related disorder in a mammal comprising detecting
the level of expression of a gene encoding a WISP-1, WISP-2, or
WISP-3 polypeptide (a) in a test sample of tissue cells obtained
from the mammal, and (b) in a control sample of known normal tissue
cells of the same cell type, wherein a higher or lower expression
level in the test sample indicates the presence of a WISP-related
dysfunction in the mammal from which the test tissue cells were
obtained. Preferably, such a disorder is a type of cancer and a
higher expression level in the test sample indicates the presence
of a tumor in the mammal.
[0069] In a still further embodiment, the invention provides an
isolated antibody binding a WISP-1, WISP-2, or WISP-3 polypeptide.
Preferably, the antibody induces death of a cell overexpressing a
WISP-1, WISP-2, or WISP-3 polypeptide, more preferably a cancer
cell. Also preferred is an antibody that binds to a human WISP-1,
WISP-2, or WISP-3 polypeptide, and is a human or humanized
antibody. More preferred is a monoclonal antibody, still more
preferred, a monoclonal antibody that has complementary-determining
regions and constant immunoglobulin regions, and in other
embodiments is an antibody fragment, a single-chain antibody, or an
anti-idiotypic antibody. In addition, the antibody is suitably
labeled with a detectable label or immobilized on a solid
support.
[0070] Also provided is a composition comprising an antibody to a
WISP-1, WISP-2, or WISP-3 polypeptide in admixture with a
pharmaceutically acceptable carrier. Preferably, the antibody is to
a human WISP-1, WISP-2, or WISP-3 polypeptide, and is a human or
humanized antibody, most preferably a monoclonal antibody against
human WISP-1. Further, the composition may comprise a
growth-inhibitory amount of said antibody. In another embodiment,
the invention provides a method for treating cancer in a mammal
comprising administering to the mammal an effective amount of the
above antibody composition. In a preferred aspect of this method,
the cancer is colon cancer, the antibody is against human WISP-1
and is a humanized or human monoclonal antibody, and the mammal is
human.
[0071] In another aspect, the invention provides a method for
treating a WISP related disorder in a mammal comprising
administering to the mammal an effective amount of a composition
comprising an antagonist to a WISP-1, WISP-2, or WISP-3 polypeptide
in a pharmaceutically acceptable carrier.
[0072] In a further aspect, the invention provides a method for
inhibiting the growth of tumor cells comprising exposing a cell
that overexpresses a Wnt-1-induced gene to an effective amount of
an antagonist that inhibits the expression or activity of a WISP-1,
WISP-2, or WISP-3 polypeptide.
[0073] A further aspect entails a method for inhibiting the growth
of tumor cells comprising exposing said cells to an effective
amount of the composition with the growth-inhibiting amount of an
anti-WISP-1, anti-WISP-2, or anti-WISP-3 antibody in admixture with
the carrier. In a preferred aspect of this method, the tumor cells
are colon cancer cells, the antibody is against human WISP-1 and is
a humanized or human monoclonal antibody, and the mammal is
human.
[0074] Also provided herein is a kit comprising one of the above
WISP polypeptides or WISP antagonists, such as anti-WISP
antibodies, and instructions for using the polypeptide or
antagonist to detect or treat a WISP-related disorder, such as
cancer induced by Wnt, one such preferred kit is a cancer
diagnostic kit comprising an anti-WISP-1, anti-WISP-2, or
anti-WISP-3 antibody and a carrier in suitable packaging.
Preferably, this kit further comprises instructions for using said
antibody to detect the WISP-1, WISP-2, or WISP-3 polypeptide.
[0075] Also provided is a method for inducing cell death comprising
exposing a cell which is induced by Wnt to an effective amount of
one of the above WISP polypeptides or WISP antagonists, such as
anti-WISP antibodies. Preferably, such cell is a cancer cell. More
preferably, the cell is in a mammal, more preferably a human. In
addition, an effective amount of another chemotherapeutic antibody
is used in the exposure of the cell, such as an anti-ErbB2
antibody. Further, optionally the method comprises exposing the
cell to a chemotherapeutic agent, a growth-inhibitory agent, or
radiation. Optionally, the cell is exposed to the growth-inhibitory
agent prior to exposure to the antibody.
[0076] In a further aspect, the invention provides an article of
manufacture, comprising:
[0077] a container;
[0078] a label on the container; and
[0079] a composition comprising an active agent contained within
the container; wherein the composition is effective for inducing
cell death or inhibiting the growth of tumor cells, the label on
the container indicates that the composition can be used for
treating conditions characterized by overinduction of Wnt or a
WISP-related disorder or by overexpression of a WISP-1, WISP-2, or
WISP-3 polypeptide, and the active agent in the composition is an
antagonist to one of the polypeptides, that is, an agent that
inhibits the expression and/or activity of the WISP-1, WISP-2, or
WISP-3 polypeptide. Preferably, the active agent in such article of
manufacture is an anti-WISP-1, anti-WISP-2, or anti-WISP-3
antibody, and the label on the container indicates that the
composition can be used for treating a WISP-related disorder.
[0080] In another embodiment, the invention provides a process for
identifying agonists to a WISP-1, WISP-2, or WISP-3 polypeptide
comprising:
[0081] (a) contacting cells and a compound to be screened under
conditions suitable for the stimulation of cell proliferation by
the polypeptide; and
[0082] (b) measuring the proliferation of the cells to determine if
the compound is an effective agonist.
[0083] Additionally, the invention provides an agonist to a WISP-1,
WISP-2, or WISP-3 polypeptide identified by the above process.
[0084] Further, the invention provides a method for identifying a
compound that inhibits the expression or activity of a WISP-1,
WISP-2, or WISP-3 polypeptide, comprising contacting a candidate
compound with a WISP-1, WISP-2, or WISP-3 polypeptide under
conditions and for a time sufficient to allow the compound and
polypeptide to interact. In a preferred embodiment, this method
comprises the steps of:
[0085] (a) contacting cells and a compound to be screened in the
presence of the WISP-1, WISP-2, or WISP-3 polypeptide under
conditions suitable for the stimulation of cell proliferation by
polypeptide; and (b) measuring the proliferation of the cells to
determine if the compound is an effective antagonist.
[0086] Further, a compound identified by this method is
provided.
[0087] In another aspect, this invention provides a compound that
inhibits the expression or activity of a WISP-1, WISP-2, or WISP-3
polypeptide.
[0088] In another embodiment, the invention provides a method for
determining the presence of a WISP-1, WISP-2, or WISP-3 polypeptide
comprising exposing a cell suspected of containing the WISP-1,
WISP-2, or WISP-3 polypeptide to an anti-WISP-1, anti-WISP-2, or
anti-WISP-3 antibody and determining binding of said antibody to
said cell.
[0089] In another preferred embodiment, the invention provides a
method of diagnosing a WISP-related disorder in a mammal comprising
(a) contacting an anti-WISP-1, anti-WISP-2, or anti-WISP-3 antibody
with a test sample of tissue cells obtained from the mammal, and
(b) detecting the formation of a complex between the anti-WISP-1,
anti-WISP-2, or anti-WISP-3 antibody and the WISP-1, WISP-2, or
WISP-3 polypeptide in the test sample. Preferably, said test sample
is obtained from an individual suspected to have neoplastic cell
growth or proliferation. Also, preferably the antibody is labeled
with a detectable label and/or is immobilized on a solid
support.
BRIEF DESCRIPTION OF THE DRAWINGS
[0090] FIGS. 1A-1B show the derived amino acid sequence of a
native-sequence mouse WISP-1 protein from amino acids 1 to 367 (SEQ
ID NO:12) and the nucleotide sequence (and complementary sequence)
encoding the protein (SEQ ID NOS:9 and 10, respectively). There is
a 1104-bp coding region and 584 bp of 3' untranslated region. In
the Figure, amino acids 1 through 22 form a putative signal
sequence, amino acids 23 through 367 are the putative mature
protein (SEQ ID NO:11), with amino acids 86 to 88, 143 to 145, 284
to 286, and 343 to 345 being potential glycosylation sites.
Potential protein kinase C phosphorylation sites are at amino acids
43-45, 159-161, 235-237, 292-294, 295-297, and 345-347. Potential
casein kinase II phosphorylation sites are at amino acids 44-47,
131-134, 145-148, and 358-361. Potential N-myristoylation sites are
at amino acids 18-23, 72-77, 127-132, 149-154, 231-236, and
289-294. A potential amidation site, is at amino acids 269-272. A
potential prokaryotic membrane lipoprotein lipid attachment site is
at amino acids 113-123. A potential von Willebrand C1 domain is at
amino acids 130-146. A potential thrombospondin 1 domain is at
amino acids 223-237. A potential CT module is at amino acids
301-312. A potential IGF binding protein consensus site is at amino
acids 72-80.
[0091] FIGS. 2A-2B show the derived amino acid sequence of a
native-sequence mouse WISP-2 protein from amino acids 1 to 251 (SEQ
ID NO:20) and the nucleotide sequence (and complementary sequence)
encoding the protein (SEQ ID NOS:17 and 18, respectively) from a
clone 1367.3. There are 756 bp of coding nucleotides and 722 bp of
3' untranslated region. In the Figure, amino acids 1 through 23
form a putative signal sequence; amino acids 24 through 251 are the
putative mature protein (SEQ ID NO:19). A potential N-glycosylation
site is at amino acids 197-200. A potential glycosaminoglycan
attachment site is at amino acids 85-88. Potential protein kinase C
phosphorylation sites are at amino acids 85-87 and 112-114.
Potential N-myristoylation sites are at amino acids 49-54, 81-86,
126-131, 210-215, and 245-250. A potential amidation site is at
amino acids 103-106. A potential phospholipase A2 aspartic acid
active site is at amino acids 120-130. A potential IGF binding
protein consensus signature is at amino acids 49-64. A potential
von Willebrand C1 domain is at amino acids 107-123. A potential
thrombospondin 1 domain is at amino acids 202-216. A potential IGF
binding protein consensus site is at amino acids 49-57.
[0092] FIGS. 3A-3C show the derived amino acid sequence of a
native-sequence human WISP-1 protein from amino acids 1 to 367 (SEQ
ID NO:4) and the nucleotide sequence (and complementary sequence)
encoding the protein (SEQ ID NOS:1 and 2, respectively). There are
1104 bp of coding region in this human clone 568.38, and 1638 bp of
3' untranslated region. In the Figure, amino acids 1 through 22
form a putative signal sequence, amino acids 23 through 367 are the
putative mature protein (SEQ ID NO:3), with amino acids 85 to 87,
143 to 145, 284 to 286, and 343 to 345 being potential
glycosylation sites. A potential cAMP- and cGMP-dependent protein
kinase phosphorylation site is from amino acids 171 to 174;
potential protein kinase C phosphorylation sites are at amino acids
43-45, 235-237, 292-294, and 345-347. Potential casein kinase II
phosphorylation sites are at amino acids 30-33, 145-148, and
358-361. Potential N-myristoylation sites are at amino acids 72-77,
127-132, 149-154, 201-206, 231-236, 289-294, and 327-332. A
potential amidation site is at amino acids 269-272. A potential
prokaryotic membrane lipoprotein lipid attachment site is at amino
acids 113-123. A potential von Willebrand C1 domain is at amino
acids 130-146. A potential thrombospondin I domain is at amino
acids 223-237. A potential CT (C-Terminal) module is at amino acids
301-312. A potential IGF binding protein consensus site is at amino
acids 72-80.
[0093] FIGS. 4A-4B show the derived amino acid sequence of a
native-sequence human WISP-2 protein from amino acids 1 to 250 (SEQ
ID NO:16) and the nucleotide sequence (and complementary sequence)
encoding the protein (SEQ ID NOS:13 and 14, respectively). The
coding region is 753 bp and the 3' untranslated region is 519 bp.
The putative signal sequence is from amino acid residues 1 through
23 and the putative mature region is from 24 through 250 (SEQ ID
NO:15). The clone designated herein as "UNQ228" and/or
"DNA33473-seq min" (SEQ ID NO:38) begins at nucleotide 34 of SEQ ID
NO:13. Potential protein kinase C phosphorylation sites are at
amino acids 4-6, 118-120, and 227-229. A potential casein-kinase II
phosphorylation site is at amino acids 98-101. A potential
N-myristoylation site is at amino acids 3-8, 49-54, 81-86, 85-90,
126-131, 164-169, 166-171, 167-172, 183-188, and 209-214. A
potential IGF binding protein consensus signature is at amino acids
49-64. A potential von Willebrand C1 domain is at amino acids
107-123. A potential thrombospondin 1 domain is at amino acids
2,01-215. A potential IGF binding protein consensus site is at
amino acids 49-57.
[0094] FIG. 5 shows a 841-bp consensus nucleotide sequence
designated "DNA30843" (SEQ ID NO:39) derived from the nucleotide
sequences of twenty different expressed sequence tags from Incyte.
When aligned with the other sequences, DNA30843 has 3 gaps. It has
441 bp orf (+I). DNA30843 was used to design probes for isolation
of human WISP-2.
[0095] FIGS. 6A-6C show the derived amino acid sequence of a
native-sequence human WISP-3 protein from amino acids 1 to 372 (SEQ
ID NO:33) and the nucleotide sequence (and complementary sequence)
encoding the protein (SEQ ID NOS:30 and 31, respectively). In the
Figure, amino acids 1 through 33 form a putative signal sequence,
amino acids 34 through 372 are the putative mature protein (SEQ ID
NO:32), with amino acids 196 to 198 and 326 to 328 being potential
glycosylation sites. Potential protein kinase C phosphorylation
sites are at amino acids 209-211, 246-248, 277-279, 308-310, and
342-344. Potential casein kinase II phosphorylation sites are at
amino acids 47-50, 254-257, and 293-296. Potential N-myristoylation
sites are at amino acids 21-26, 89-94, 139-144, 166-171, 180-185,
185-190, 188-193, 242-247, and 302-307. A potential amidation site
is at amino acids 188-191. Potential prokaryotic membrane
lipoprotein lipid attachment sites are at amino acids 130-140 and
160-170. A potential IGF binding protein signature site is at amino
acids 89-104. A potential IGF binding protein site (less stringent
than prosite's) is at amino acids 89-97.
[0096] FIGS. 7A-7C show the derived amino acid sequence of a
native-sequence human WISP-3 protein from amino acids 1 to 355 (SEQ
ID NO:37) and the nucleotide sequence (and complementary sequence)
encoding the protein (SEQ ID NOS:34 and 35, respectively). This
protein is believed to be a splice variant of the nucleotide
sequence shown in FIG. 6 with a shorter 5' end. In the Figure,
amino acids 1 through 15 form a putative signal sequence, amino
acids 16 through 355 are the putative mature protein (SEQ ID
NO:36), with amino acids 178 to 180 and 308 to 310 being potential
glycosylation sites. Potential protein kinase C phosphorylation
sites are at amino acids 191-193, 228-230, 259-261, 290-292, and
324-326. Potential casein kinase II phosphorylation sites are at
amino acids 29-32, 236-239, and 275-278. Potential N-myristoylation
sites are at amino acids 3-8, 71-76, 121-126, 148-153, 162-167,
167-172, 170-175, 224-229, and 284-289. A potential amidation site
is at amino acids 170-173. Potential prokaryotic membrane
lipoprotein lipid attachment sites are at amino acids 112-122 and
142-152. A potential IGF binding protein signature site is at amino
acids 71-87. A potential IGF binding protein site (less stringent
than prosite's) is at amino acids 71-79.
[0097] FIG. 8 shows an alignment of the full-length amino acid
sequences of the human and mouse WISP-1 (SEQ ID NOS:4 and 12,
respectively).
[0098] FIG. 9 shows an alignment of the full-length amino acid
sequences of the human and mouse WISP-2 (SEQ ID NOS:16 and 20,
respectively).
[0099] FIG. 10 shows an alignment of the amino acid sequences of
the two clones of human WISP-3 (SEQ ID NOS:33 and 37,
respectively).
[0100] FIG. 11A-11C show an alignment of the nucleotide sequences
of human WISP-1 (nucleotides 89-1188 of SEQ ID NO:1) and the human
WISP-3 (SEQ ID NO:30).
[0101] FIG. 12 shows an alignment of the amino acid sequences of
human WISP-1 (SEQ ID NO:4) and the human WISP-3 (SEQ ID NO:33).
[0102] FIG. 13 shows a map of the vector pBabe puro (5.1 kb) used
to transform cells for purposes of differential expression. The
vector includes both unique restriction sites and multiple
restriction sites. It is shown here in modified form for Wnt-1
cloning wherein the HindIII site after the SV40 promoter in the
original pBabe puro vector has been removed and a HindIII site
added to the multiple cloning site of the original pBabe puro
vector. Wnt-1 is cloned from EcoRI-HindIII in the multiple cloning
site. Constructs derived from this vector are selected in
ampicillin (100 .mu.g/ml) and the cells infected in culture are
selected in 1.0-2.5 .mu.g/ml puromycin.
[0103] FIG. 14 shows the sequences of the PCR-Select@ cDNA
synthesis primer (SEQ ID NO:40), adaptors 1 and 2 (SEQ ID NOS:41
and 42, respectively) and complementary sequences for the adaptors
(SEQ ID NOS:43 and 44, respectively), PCR primer 1 (SEQ ID NO:45),
PCR primer 2 (SEQ ID NO:46), nested PCR primer 1 (SEQ ID NO:47),
nested PCR primer 2 (SEQ ID NO:48), control primer G3PDH 51 primer
(SEQ ID NO:49), and control primer G3PDH 3' primer (SEQ ID NO:50)
used for suppression subtractive hybridization for identifying WISP
clones. When the adaptors are ligated to RsaI-digested cDNA, the
RsaI site is restored.
[0104] FIG. 15 shows the cloning site region of the plasmid pGEM-T
used to clone all of the WISP sequences herein (SEQ ID NOS:51 and
52 for 5' and 3' sequences, respectively).
[0105] FIGS. 16A-16D show the sequence (SEQ ID NO:53) of a plasmid
that is used to prepare an expression plasmid for expression of
mouse WISP-1 in mammalian cells, the latter being designated
pRK5.CMV.puro-dhfR.mWISP-1.6His.
[0106] FIGS. 17A-17D show the sequence (SEQ ID NO:54) of plasmid
pb.PH.IgG, which is used to prepare an expression plasmid for
expression of mouse WISP-1 DNA in baculovirus-infected insect
cells.
[0107] FIGS. 18A-18D show the sequence (SEQ ID NO:55) of plasmid
pbPH.His.c, which is used to prepare an expression plasmid for
expression of mouse WISP-1 DNA in baculovirus-infected insect
cells, the latter being designated pbPH.mu.568.8his.baculo.
[0108] FIGS. 19A-19D show graphs of the delta CT in nine colon
cancer cell lines and DNA from the blood of ten normal human donors
(Nor Hu) as control, for human TNF, human WISP-1. Lyra, and human
Apo2 ligand, respectively, using the ABI Prism 7700.TM. Sequence
Detection System procedure for testing genomic amplification.
[0109] FIGS. 20A-20D show graphs of the delta CT in nine colon
cancer cell lines and Nor Hu as control, for human DCR1, huFAS,
human WISP-2, and Apo3, respectively, using the ABI Prism 7700.TM.
Sequence Detection System procedure for testing genomic
amplification.
[0110] FIGS. 21A-21D show graphs of the delta CT in nine colon
cancer cell lines and Nor Hu as control, for three different runs
of human WISP-1 (designated in the figure as huWISP-1c, -1b, and
-1a) and the average of these three runs of human WISP-1,
respectively, using the ABI Prism 7700.TM. Sequence Detect-Ion
System procedure for testing genomic amplification.
[0111] FIGS. 22A-22D show graphs of the delta CT in nine colon
cancer cell lines and Nor Hu as control, for three different runs
of human WISP-2 (designated in the figure as huWISP-2c, -2b, and
-2a; FIGS. 22A, C, and D, respectively) and the average of these
three runs of human WISP-2 (FIG. 22B), using the ABI Prism 7700.TM.
Sequence Detection System procedure for testing genomic
amplification.
[0112] FIGS. 23A-23C show graphs of the delta CT in nine colon
cancer cell lines and Nor Hu as control, for two different runs of
human DR5 (DR5a and DR5b) and the average of these two runs of DR5,
respectively, using the ABI Prism 7700.TM., Sequence Detection
System procedure for testing genomic amplification.
[0113] FIGS. 24A-24D show graphs of the delta CT in nine colon
cancer cell lines and Nor Hu as control, for four different runs of
c-myc (c-myc(a1), c-myc(b1), c-myc (b), and c-myc (a)),
respectively, using the ABI Prism 7700.TM. Sequence Detection
System procedure for testing genomic amplification.
[0114] FIGS. 25A-25D show graphs of the delta CT in nine colon
cancer cell lines and Nor Hu as control, for two different runs of
human WISP-1 (designated in the figure as huWISP-1(a) and
huWISP-1(b)) and for two different runs of human WISP-2 (designated
in the figure as huWISP-2(a) and huWISP-2(b)), respectively, using
the ABI Prism 7700.TM. Sequence Detection System procedure for
testing genomic amplification.
[0115] FIG. 26 shows the sequence (SEQ ID NO:23) of clone 568.13, a
potential splice variant of human WISP-1 obtained by screening with
a probe derived from clone 568.15A, which is the initial clone
isolated from a human lung library in the process to obtain
full-length human WISP-1 DNA.
[0116] FIG. 27 shows the sequence (SEQ ID NO:24) of clone 568.1A, a
potential human WISP-1 splice variant, 51 end only, obtained by
screening with a probe derived from clone 568.15A.
[0117] FIG. 28 shows the sequence (SEQ ID NO:25) of clone 568.39, a
potential human WISP-1 splice variant, 5' end only, obtained by
screening with a probe derived from clone 568.15A.
[0118] FIG. 29 shows the sequence (SEQ ID NO:26) of clone 568.4A, a
potential human WISP-1 splice variant obtained by screening with a
probe derived from clone 568.15A.
[0119] FIG. 30 shows the sequence (SEQ ID NO:27) of clone 568.5A, a
potential human WISP-1 splice variant, 51 end only, obtained by
screening with a probe derived from clone 568.15A.
[0120] FIG. 31 shows the sequence (SEQ ID NO:28) of clone 568.6B, a
potential human WISP-1 splice variant, 51 end only, obtained by
screening with a probe derived from clone 568.15A.
[0121] FIG. 32 shows the sequence (SEQ ID NO:29) of clone 568.7, a
potential human WISP-1 splice variant, 51 end only, obtained by
screening with a probe derived from clone 568.15A.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0122] The term "WISP polypeptide" refers to the family of
native-sequence human and mouse WISP proteins and variants
described herein whose genes are induced at least by Wnt-1. This
term includes WISP-1, WISP-2, and WISP-3.
[0123] The terms "WISP-1 polypeptide", "WISP-1 homologue" and
grammatical variants thereof, as used herein, encompass
native-sequence WISP-1 protein and variants (which are further
defined herein). The WISP-1 polypeptide may be isolated from a
variety of sources, such as from human tissue types or from another
source, or prepared by recombinant or synthetic methods, or by any
combination of these and similar techniques.
[0124] The terms "WISP-2 polypeptide", "WISP-2 homologue",
"PR0261", and "PR0261 polypeptide" and grammatical variants
thereof, as used herein, encompass native-sequence WISP-2 protein
and variants (which are further defined herein). The WISP-2
polypeptide may be isolated from a variety of sources, such as from
human tissue types or from another source, or prepared by
recombinant or synthetic methods, or by any combination of these
and similar techniques.
[0125] The terms "WISP-3 polypeptide", "WISP-3 homologue", and
grammatical variants thereof, as used herein, encompass
native-sequence WISP-3 protein and variants (which are further
defined herein) The WISP-3 polypeptide may be isolated from a
variety of sources, such as from human tissue types or from another
source, or prepared by recombinant or synthetic methods, or by any
combination of these and similar techniques.
[0126] A "native-sequence WISP-1 polypeptide" comprises a
polypeptide having the same amino acid sequence as a WISP-1
polypeptide derived from nature. Such native-sequence WISP-1
polypeptides can be isolated from nature or can be produced by
recombinant or synthetic means. The term "native-sequence WISP-1
polypeptide" specifically encompasses naturally occurring truncated
or secreted forms of a WISP-1 polypeptide disclosed herein,
naturally occurring variant forms (e.g., alternatively spliced
forms or splice variants), and naturally occurring allelic variants
of a WISP-1 polypeptide. In one embodiment of the invention, the
native-sequence WISP-1 polypeptide is a mature or full-length
native-sequence human WISP-1 polypeptide comprising amino acids 23
to 367 of FIGS. 3A-3C (SEQ ID NO: 3) or amino acids 1 to 367 of
FIGS. 3A-3C (SEQ ID NO: 4), respectively, with or without the
N-terminal methionine.
[0127] In another embodiment of the invention, the native-sequence
WISP-1 polypeptide is the full-length or mature native-sequence
human WISP-1 polypeptide comprising amino acids 23 to 367 or 1 to
367 of FIGS. 3A-3C wherein the valine residue at position 184 or
the alanine residue at position 202 has/have been changed to an
isoleucine or serine residue, respectively (SEQ ID NOS: 5-8), with
or without the N-terminal methionine. In another embodiment of the
invention, the native-sequence WISP-1 polypeptide is the
full-length or mature native-sequence human WISP-1 polypeptide
comprising amino acids 23 to 367 or 1 to 367 of FIGS. 3A-3C wherein
the valine residue at position 184 and the alanine residue at
position 202 has/have been changed to an isoleucine or serine
residue, respectively (SEQ ID NOS: 21 and 22, respectively), with
or without the N-terminal methionine. In another embodiment of the
invention, the native sequence WISP-1 polypeptide is a mature or
full-length native-sequence mouse WISP-1 polypeptide comprising
amino acids 23 to 367 of FIGS. 1A-1B (SEQ ID NO: 11), or amino
acids 1 to 367 of FIGS. 1A-1B (SEQ ID NO: 12), respectively, with
or without the N-terminal methionine.
[0128] In another embodiment of the invention, the native-sequence
WISP-1 polypeptide is one which is encoded by a nucleotide sequence
comprising one of the human WISP-1 splice or other native-sequence
variants, including SEQ ID NOS:23, 24, 25, 26, 27, 28, or 29, with
or without an N-terminal methionine.
[0129] A "native-sequence WISP-2 polypeptide" or a "native-sequence
PRO261 polypeptide" comprises a polypeptide having the same amino
acid sequence as a WISP-2 polypeptide derived from nature. Such
native-sequence WISP-2 polypeptides can be isolated from nature or
can be produced by recombinant or synthetic means. The term
"native-sequence WISP-2 polypeptide" specifically encompasses
naturally occurring truncated or secreted forms of a WISP-2
polypeptide disclosed herein, naturally occurring variant forms
(e.g., alternatively spliced forms or splice variants), and
naturally occurring allelic variants of a WISP-2 polypeptide. In
one embodiment of the invention, the native-sequence WISP-2
polypeptide is a mature or full-length native-sequence human WISP2
polypeptide comprising amino acids 1-24 up to 250 of FIGS. 4A-4B
(SEQ ID NOS:15, 16, and 56-77), including amino acids 24 to 250 and
amino acids 1 to 250 of FIGS. 4A-4B (SEQ ID NOS:15 and 16,
respectively), with or without the N-terminal methionine. In
another embodiment of the invention, the native-sequence WISP-2
polypeptide is a mature or full-length native-sequence mouse WISP-2
polypeptide comprising amino acids 1-24 up to 251 of FIGS. 2A-2B
(SEQ ID NOS:19, 20, and 78-99), including amino acids 24 to 251 and
amino acids 1 to 251 of FIGS. 2A-2B (SEQ ID NOS:19 and 20,
respectively), with or without the N-terminal methionine.
[0130] A "native-sequence WISP-3 polypeptide" comprises a
polypeptide having the same amino acid sequence as a WISP-3
polypeptide derived from nature. Such native-sequence WISP-3
polypeptides can be isolated from nature or can be produced by
recombinant or synthetic means. The term "native-sequence WISP-3
polypeptide" specifically encompasses naturally occurring truncated
or other forms of a WISP-3 polypeptide disclosed herein, naturally
occurring variant forms (e.g., alternatively spliced forms or
splice variants), and naturally occurring allelic variants of a
WISP-3 polypeptide. In one embodiment of the invention, the
native-sequence WISP-3 polypeptide is a mature or full-length,
native-sequence human WISP-3 polypeptide comprising amino acids 34
to 372 of FIGS. 6A-6C (SEQ ID NO:32) or amino acids 1 to 372 of
FIGS. 6A-6C (SEQ ID NO:33), respectively, with or without the
N-terminal methionine. In another embodiment of the invention, the
native-sequence WISP-3 polypeptide is a mature or full-length,
native-sequence human WISP-3 polypeptide comprising amino acids 16
to 355 of FIGS. 7A-7C (SEQ ID NO:36) or amino acids 1 to 355 of
FIGS. 7A-7C (SEQ ID NO:37) respectively, with or without the
N-terminal methionine.
[0131] The term "WISP-1 variant" means an active WISP-1 polypeptide
as defined below having at least about 80%, preferably at least
about 85%, more preferably at least about 90%, most preferably at
least about 95% amino acid sequence identity with human mature
WISP-1 having the deduced amino acid sequence shown in FIGS. 3A-3C
(SEQ ID NO: 3), and/or with human full-length WISP-1 having the
deduced amino acid sequence shown in FIGS. 3A-3C (SEQ ID NO: 4),
and/or with mouse mature WISP-1 having the deduced amino acid
sequence shown in FIG. 1A-1B (SEQ ID NO: 11), and/or with mouse
full-length WISP-1 having the deduced amino acid sequence shown in
FIG. 1A-1B (SEQ ID NO: 12). Such variants include, for example,
WISP-1 polypeptides wherein one or more amino acid residues are
added to, or deleted from, the N- or C-terminus of the full-length
or mature sequences of FIGS. 3A-3C and 1A-1B (SEQ ID NOS:4, 3, 12,
and 11, respectively), including variants from other species, but
excludes a native-sequence WISP-1 polypeptide.
[0132] The term "WISP-2 variant" or "PR0261 variant" means an
active WISP-2 polypeptide as defined below having at least about
80%, preferably at least about 85%, more preferably at least about
90%, most preferably at least about 95% amino acid sequence
identity with human mature WISP-2 having the putative deduced amino
acid sequence shown in FIG. 4A-4B (SEQ ID NO:15), and/or with human
full-length WISP-2 having the deduced amino acid sequence shown in
FIG. 4A-4B (SEQ ID NO:16), and/or with mouse mature WISP-2 having
the putative deduced amino acid sequence shown in FIG. 2A-2B (SEQ
ID NO:19), and/or with mouse full-length WISP-2 having the deduced
amino acid sequence shown in FIG. 2A-2B (SEQ ID NO:20).
[0133] Such variants include, for instance, WISP-2 polypeptides
wherein one or more amino acid residues are added to, or deleted
from, the N- or C-terminus of the full-length and putative mature
sequences of FIGS. 4A-4B and 2A-2B (SEQ ID NOS:16, 15, 20, and 19,
respectively), including variants from other species, but excludes
a native-sequence WISP-2 polypeptide.
[0134] The term "WISP-3 variant" means an active WISP-3 polypeptide
as defined below having at least about 80%, preferably at least
about 85%, more preferably at least about 90%, most preferably at
least about 95% amino acid sequence identity with human mature
WISP-3 having the deduced amino acid sequence shown in FIGS. 6A-6C
(SEQ ID NO:32), and/or with human full-length WISP-3 having the
deduced amino acid sequence shown in FIGS. 6A-6C (SEQ ID NO:33),
and/or with human mature WISP-3 having the deduced amino acid
sequence shown in FIGS. 7A-7C (SEQ ID NO:36), or with human
full-length WISP-3 having the deduced amino acid sequence shown in
FIGS. 7A-7C (SEQ ID NO:37). Such variants include, for instance,
WISP-3 polypeptides wherein one or more amino acid residues are
added to, or deleted from, the N- or C-terminus of the full-length
or mature sequences of FIGS. 6A-6C and 7A-7C (SEQ ID NOS:32, 33,
36, and 37, respectively), including variants from other species,
but excludes a native-sequence WISP-3 polypeptide.
[0135] "Percent (%) amino acid sequence identity" with respect to
the WISP sequences identified herein is defined as the percentage
of amino acid residues in a candidate sequence that are identical
with the amino acid residues in a WISP polypeptide sequence, after
aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, ALIGN, or Megalign (DNASTAR.TM.) software.
Those skilled in the art can determine appropriate parameters for
measuring alignment, including any algorithms needed to achieve
maximal alignment over the full length of the sequences being
compared.
[0136] "Percent (%) nucleic acid sequence identity" with respect to
the coding region of the WISP sequences identified herein,
including UNQ228 (DNA34387-seq min) sequence, and the coding region
therein, is defined as the percentage of nucleotides in a candidate
sequence that are identical with the nucleotides in the coding
region of the WISP sequence of interest, e.g., in the UNQ228
(DNA34387-seq min) sequence (SEQ ID NO:38) or coding region therein
(SEQ ID NO:16), after aligning the sequences and introducing gaps,
if necessary, to achieve the maximum percent sequence identity.
Alignment for purposes of determining percent nucleic acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, ALIGN, or Megalign (DNASTAR) software.
Those skilled in the art can determine appropriate parameters for
measuring alignment, including any algorithms needed to achieve
maximal alignment over the full length of the sequences being
compared.
[0137] "Stringent conditions" are those that (1) employ low ionic
strength and high temperature for washing, for example, 0.015 M
sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate
at 50.degree. C.; (2) employ during hybridization a denaturing
agent, such as formamide, for example, 50% (vol/vol) formamide with
0.1%; bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50
mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride,
75 mm sodium citrate at 42.degree. C.; (3) employ 50% formamide,
5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8) 0.1% sodium pyrophosphate, 5.times.Denhardt's
solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and
10% dextran sulfate at 42.degree. C., with washes at 42.degree. C.
in 0.2.times.SSC and 0.1% SDS; or (4) employ a buffer of 109
dextran sulfate, 2.times.SSC (sodium chloride/sodium citrate), and
50% formamide at 55.degree. C., followed by a high-stringency wash
consisting of 0.1.times.SSC containing EDTA at 55.degree. C.
[0138] "Moderately stringent conditions" are described in Sambrook
et al., Molecular Cloning: A Laboratory Manual (New York: Cold
Spring Harbor Laboratory Press, 1989), and include the use of a
washing solution and hybridization conditions (e.g., temperature,
ionic strength, and percent SDS) less stringent than described
above. An example of moderately stringent conditions is a condition
such as overnight incubation at 370C. in a solution comprising: 20%
formamide, 5.times.SSC (150 mM NaCl, is mm trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, lot
dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,
followed by washing the filters in 1.times.SSC at about
37-50.degree. C. The skilled artisan will recognize how to adjust
the temperature, ionic strength, etc., as necessary to accommodate
factors such as probe length and the like.
[0139] "Isolated," when used to describe the various polypeptides
disclosed herein, means polypeptide that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would typically interfere with diagnostic or
therapeutic uses for the polypeptide, and may include enzymes,
hormones, and other proteinaceous or non-proteinaceous solutes. In
preferred embodiments, the polypeptide will be purified (1) to a
degree sufficient to obtain at least 15 residues of N-terminal or
internal amino acid sequence by use of a spinning cup sequenator,
or (2) to homogeneity by SDS-PAGE under non-reducing or reducing
conditions using Coomassie blue or, preferably, silver stain.
Isolated polypeptide includes polypeptide in situ within
recombinant cells, since at least one component of the WISP natural
environment will not be present. Ordinarily, however, isolated
polypeptide will be prepared by at least one purification step. An
"isolated" nucleic acid encoding a WISP polypeptide or "isolated"
DNA33473 or "isolated" PR0261 polypeptide-encoding nucleic acid
molecule is a nucleic acid molecule that is identified and
separated from at least one contaminant nucleic acid molecule with
which it is ordinarily associated in the natural source of the
respective nucleic acid. Isolated DNA33473 or an isolated
WISP-encoding nucleic acid molecule is other than in the form or
setting in which it is found in nature. An isolated WISP-encoding
or DNA33473 nucleic acid molecule therefore is distinguished from
the WISP-encoding or DNA33473 nucleic acid molecule, respectively,
as it exists in natural cells. However, an isolated WISP-encoding
or DNA33473 nucleic acid molecule includes a nucleic acid molecule
contained in cells that ordinarily express WISP-encoding DNA or
DNA33473, respectively, where, for example, the nucleic acid
molecule is in a chromosomal location different from that of
natural cells.
[0140] The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0141] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptides; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0142] The term "antibody" is used in the broadest sense and
specifically covers single anti-WISP polypeptide, such as
anti-PR0261, monoclonal antibodies (including agonist, antagonist,
and neutralizing antibodies), and anti-WISP polypeptide, such as
anti-PR0261, and antibody compositions with polyepitopic
specificity. The term "monoclonal antibody" as used herein refers
to an antibody obtained from a population of substantially
homogeneous antibodies, i.e., the individual antibodies comprising
the population are identical except for possible naturally
occurring mutations that may be present in minor amounts.
[0143] "Active" or "activity" or "WISP biological activity", for
purposes herein, describes form(s) of a WISP polypeptide, such as
PR0261, including its variants, or its antagonists, which retain
the biologic and/or immunologic activities of a native or naturally
occurring (native-sequence) WISP polypeptide, such as PR0261, or
its antagonist. Preferred "activities" for a WISP polypeptide or
its antagonist include the ability to inhibit proliferation of
tumor cells or to stimulate proliferation of normal cells and to
treat arteriosclerosis, including atherosclerosis, as well as to
induce wound repair and hematopoiesis, prevent desmoplasia, prevent
fibrotic lesions associated with skin disorders such as
scleroderma, keloid, eosinophilic fasciitis, nodular fasciitis, and
Dupuytren's contracture, to treat bone-related diseases such as
osteoporosis, to regulate anabolism including promotion of growth,
to treat immune disorders, to treat Wilms' tumor and kidney-related
disorders, to treat testis-related disorders, to treat lung-related
disorders, and to treat cardiac disorders.
[0144] An "antagonist" of a WISP polypeptide is a molecule that
inhibits an activity of a WISP polypeptide. Preferred antagonists
are those which interfere with or block an undesirable biological
activity of a WISP polypeptide, such as where a WISP polypeptide
might act to stimulate cancer cells and the antagonist would serve
to inhibit the growth of those cells. In some cases, such as with
WISP-1, WISP-2, and WISP-3, the antagonist may be useful to inhibit
the binding of a WISP polypeptide to an IGF. Such molecules include
anti-bodies and small molecules that have such inhibitory
capability, as well as WISP polypeptide variants of, and receptors
for, WISP polypeptide (if available) or portions thereof that bind
to WISP. For example, antagonists can be derived from receptors of
WISP-1, WISP-2, and WISP-3 using the predicted family of receptors
for WISPs-1, -2, and -3 (the CTGF receptors). Thus, the receptor
can be expression cloned from the family; then a soluble form of
the receptor is made by identifying the extracellular domain and
excising the transmembrane domain therefrom. The soluble form of
the receptor can then be used as an antagonist, or the receptor can
be used to screen for small molecules that would antagonize WISP
polypeptide activity.
[0145] Alternatively, using the murine sequences shown in FIGS.
1A-1B and 2A-2B (SEQ ID NOS:11, 12, 19, and 20, respectively) or
the human sequences shown in FIGS. 3A-3C, 4A-4B, (SEQ ID NOS:3, 4,
15, and 16, respectively), 6A-6C, and 7A-7C, variants of native
WISP-1, WISP-2, or WISP-3, are made that act as antagonists. Using
knowledge from the CTGF receptor family, the receptor binding sites
of WISP-1, WISP-2, and WISP-3 polypeptides can be determined by
binding studies and one of them eliminated by standard techniques
(deletion or radical substitution) so that the molecule acts as an
antagonist.
[0146] Antagonist activity can be determined by several means,
including standard assays for induction of cell death such as that
described herein, e.g., .sup.3H-thymidine proliferation assays, or
other mitogenic assays, such as an assay measuring the capability
of the candidate antagonist of inducing EGF-potentiated anchorage
independent growth of target cell lines (Volckaert et al., Gene,
15:215-223 (1981)) and/or growth inhibition of neoplastic cell
lines. Roberts et al., Proc. Natl. Acad. Sci. USA, 82:119-123
(1985). Anchorage-independent growth refers to the ability of WISP
polypeptide-treated, or TGF-.beta.-treated and EGF-treated
non-neoplastic target cells to form colonies in soft agar, a
characteristic ascribed to transformation of the cells. In this
assay, the candidate is incubated together with an equimolar amount
of a WISP polypeptide otherwise detectable in the EGF-potentiated
anchorage-independent target cell growth assay, and the culture
observed for failure to induce anchorage independent growth. In
addition, an antagonist may be an IGF such as IGF-I or a peptide
mimic of IGF-I or a receptor to IGF or a receptor to an IGFBP.
[0147] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disorder or condition as well as
those in which the disorder or condition is to be prevented.
[0148] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic, and farm
animals, and zoo, sports, or pet animals, such as dogs, horses,
cats, sheep, pigs, cows, etc. Preferably, the mammal is human.
[0149] A "disorder" or "WISP-related disorder" is any condition
that would benefit from treatment with the WISP polypeptides or
WISP antagonists herein. This includes chronic and acute disorders,
as well as those pathological conditions which predispose the
mammal to the disorder in question. Non-limiting examples of
disorders to be treated herein include benign and malignant tumors;
leukemias and lymphoid malignancies; neuronal, glial, astrocytal,
hypothalamic and other glandular, macrophagal, epithelial, stromal,
and blastocoelic disorders; hematopoiesis-related disorders;
tissue-growth disorders; skin disorders; desmoplasia, fibrotic
lesions; kidney disorders; bone-related disorders; trauma such as
bums, incisions, and other wounds; catabolic states;
testicular-related disorders; and inflammatory, angiogenic, and
immunologic disorders, including arteriosclerosis. A "Wnt-related
disorder" is one caused at least by the upregulation of the Wnt
gene pathway, including Wnt-1 and Wnt-4, but preferably Wnt-1, and
may include cancer.
[0150] The terms "cancer", "cancerous", and "malignant" refer to or
describe the physiological condition in mammals that is typically
characterized by unregulated cell growth. Examples of cancer
include but are not limited to, carcinoma including adenocarcinoma,
lymphoma, blastoma, melanoma, sarcoma, and leukemia. More
particular examples of such cancers include squamous cell cancer,
small-cell lung cancer, non-small cell lung cancer,
gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer such as hepatic carcinoma and hepatoma, bladder
cancer, breast cancer, colon cancer, colorectal cancer, endometriat
carcinoma, salivary gland carcinoma, kidney cancer such as renal
cell carcinoma and Wilms' tumors, basal cell carcinoma, melanoma,
prostate cancer, vulval cancer, thyroid cancer, testicular cancer,
esophageal cancer, and various types of head and neck cancer. The
preferred cancers for treatment herein are breast, colon, lung, and
melanoma.
[0151] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., .sup.131I, .sup.125I, .sup.90Y, and
.sup.186Re), chemotherapeutic agents, and toxins such as
enzymatically active toxins of bacterial, fungal, plant, or animal
origin, or fragments thereof.
[0152] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine
arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Busulfan,
Cytoxin, Taxol, Toxotere, Methotrexate, Cisplatin, Melphalan,
Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C,
Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide,
Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins,
Esperamicins (see U.S. Pat. No. 4,675,187), Melphalan, and other
related nitrogen mustards. Also included in this definition are
hormonal agents that act to regulate or inhibit hormone action on
tumors, such as tamoxifen and onapristone.
[0153] A "growth-inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, such as an
Wnt-overexpressing cancer cell, either in vitro or in vivo. Thus,
the growth-inhibitory agent is one which significantly reduces the
percentage of malignant cells in S phase. Examples of
growth-inhibitory agents include agents that block cell cycle
progression (at a place other than S phase), such as agents that
induce G1 arrest and M-phase arrest. Classical M-phase blockers
include the vincas (vincristine and vinblastine), taxol, and topo
II inhibitors such as doxorubicin, daunorubicin, etoposide, and
bleomycin. Those agents that arrest G1 also spill over into S-phase
arrest, for example, DNA alkylating agents such as tamoxifen,
prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate,
5-fluorouracil, and ara-C. Further information can be found in The
Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1,
entitled "Cell cycle regulation, oncogenes, and antineoplastic
drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995),
especially p. 13. The 4D5 antibody (and functional equivalents
thereof) can also be employed for this purpose if the cancer
involves ErbB2-overexpressing cancer cells. See, e.g., WO
92/22653.
[0154] "Northern analysis" or "Northern blot" is a method used to
identify RNA sequences that hybridize to a known probe such as an
oligonucleotide, DNA fragment, cDNA or fragment thereof, or RNA
fragment. The probe is labeled with a radioisotope such as
.sup.32p, or by biotinylation, or with an enzyme. The RNA to be
analyzed is usually electrophoretically separated on an agarose or
polyacrylamide gel, transferred to nitrocellulose, nylon, or other
suitable membrane, and hybridized with the probe, using standard
techniques well known in the art such as those described in
sections 7.39-7.52 of Sambrook et al., supra.
[0155] The technique of "polymerase chain reaction." or "PCR." as
used herein generally refers to a procedure wherein minute amounts
of a specific piece of nucleic acid, RNA and/or DNA, are amplified
as described in U.S. Pat. No. 4,683,195 issued 28 Jul. 1987.
Generally, sequence information from the ends of the region of
interest or beyond needs to be available, such that oligonucleotide
primers can be designed-, these primers, will be identical or
similar in sequence to opposite strands of the template to be
amplified. The 5' terminal nucleotides of the two primers may
coincide with the ends of the amplified material. PCR can be used
to amplify specific RNA sequences, specific DNA sequences from
total genomic DNA, and cDNA transcribed from total cellular RNA,
bacteriophage, or plasmid sequences, etc. See generally Mullis et
al., Cold Spring Harbor Symp. Quant. Biol., 51:263 (1987): Erlich.
ed. PCR Technology. (Stockton Press, NY, 1989). As used herein, PCR
is considered to be one, but not the only example of a nucleic acid
polymerase reaction method for amplifying a nucleic acid test
sample comprising the use of a known nucleic acid as a primer and a
nucleic acid polymerase to amplify or generate a specific piece of
nucleic acid.
II. Compositions and Methods of the Invention
[0156] A. Full-Length WISP Polypeptide
[0157] The present invention provides newly-identified and isolated
nucleotide sequences encoding a polypeptide referred to in the
present application as a WISP polypeptide, including a WISP-1,
WISP-2, or WISP-3 polypeptide. In particular, cDNAs have been
identified and isolated encoding novel murine and human WISP-1 and
WISP-2, and human WISP-3 splice variants as disclosed in further
detail in the Examples below.
[0158] Using BLAST and FastA sequence alignment computer programs,
it was found that the coding sequences of mouse and human WISP-1
and -2, as well as the two coding sequences of human WISP-3
disclosed herein, show significant homology to DNA sequences
disclosed in the GenBank database, including those published by
Adams et al., Nature, 377:3-174 (1995).
[0159] Further, using BLAST and FastA sequence alignment computer
programs, it was found that various portions of the coding
sequences of mouse and human WISP-1 and WISP-2 show significant
homology to CTGF, cef-10, Cyr61, and/or Nov protein. In this
regard, mouse WISP-1 is 47% homologous to mouse CTGF and 46%
homologous to human CTGF, mouse WISP-2 is 46% homologous to chick
cef-10 protein precursor and 42% homologous to human Cyr61 protein,
human WISP-1 is 47% homologous to mouse CTGF and 48% homologous to
human CTGF, and human WISP-2 is 48% homologous to mouse CTGF, 49%
homologous to human CTGF precursor, 46% homologous to mouse Nov
protein homolog precursor, 49% homologous to human CTGF, and 48%
homologous to mouse CTGF precursor. Further, apparently the amino
acid sequences of mouse WISP-1 and mouse ELM 1 (Hashimoto et al.,
supra) are identical, and the amino acid sequences of human WISP-1
and mouse ELM1 are 84% identical.
[0160] Since these factors have also been correlated with IGFBPs,
it is presently believed that the WISP-1 and WISP-2 polypeptides
disclosed in the present application are newly identified members
of the CTGF or IGFBP family and possess activity relating to
development of normal, injured, and cancerous cells and tissue.
More specifically, WISP-1 and WISP-2 may be involved in breast
cancer, lung cancer, melanoma, and colon cancer, as well as in
wound repair. Further, they may be involved in atherosclerosis.
[0161] Further, using BLAST and FastA sequence alignment computer
programs, it was found that various portions of the coding
sequences of the two splice variants of human WISP-3 show
significant homology to mouse ELM1 and CTGF proteins. In this
regard, both splice variants of WISP-3 are 45% homologous to mouse
ELM1 and 42% homologous to mouse and human CTGF and its precursor,
with the longer variant of FIG. 6 being 43% homologous to Xenopus
CTGF and the shorter variant of FIG. 7 being 42% homologous to
Xenopus CTGF.
[0162] B. WISP Polypeptide Variants
[0163] In addition to the full-length native-sequence WISP
polypeptides described herein, it is contemplated that variants of
these sequences can be prepared. WISP variants can be prepared by
introducing appropriate nucleotide changes into the WISP-encoding
DNA, or by synthesis of the desired variant WISP polypeptides.
Those skilled in the art will appreciate that amino acid changes
may alter posttranslational processes of the WISP polypeptide, such
as changing the number or position of glycosylation sites or
altering the membrane-anchoring characteristics, if the native WISP
polypeptide is membrane bound.
[0164] Variations in the native full-length WISP sequences, or in
various domains of the WISP polypeptides described herein, can be
made, for example, using any of the techniques and guidelines for
conservative and non-conservative mutations set forth, for
instance, in U.S. Pat. No. 5,364,934. Variations may be a
substitution, deletion, or insertion of one or more codons encoding
the WISP polypeptide that results in a change in the amino acid
sequence as compared with the native-sequence WISP polypeptide.
Optionally the variation is by substitution of at least one amino
acid with any other amino acid in any portion of the WISP
polypeptide. Guidance in determining which amino acid residue may
be inserted, substituted, or deleted without adversely affecting
the desired activity may be found by comparing the sequence of the
WISP polypeptide with that of homologous known CTGF protein
molecules, in the case of WISP-1, WISP-2, and WISP-3, and
minimizing the number of amino acid sequence changes made in
regions of high homology. Amino acid substitutions can be the
result of replacing one amino acid with another amino acid having
similar structural and/or chemical properties, such as the
replacement of a leucine with a serine, i.e., conservative amino
acid replacements. Insertions or deletions may optionally be in the
range of 1 to about 5 amino acids. The variation allowed may be
determined by systematically making insertions, deletions, or
substitutions of amino acids in the sequence and testing the
resulting variants for activity in in vitro assays for gene
upregulation or down regulation and in transgenic or knockout
animals.
[0165] The variations can be made on the cloned DNA to produce the
WISP DNA or WISP polypeptide variant DNA using methods known in the
art such as oligonucleotide-mediated (site-directed) mutagenesis
(Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al.,
Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et
al., Gene, 34:315-(1985)), alanine scanning, PCR mutagenesis,
restriction selection mutagenesis (Wells et al., Philos. Trans. R.
Soc. London SerA, 317:415 (1986)), or other known techniques.
[0166] Scanning amino acid analysis can also be employed to
identify one or more amino acids along a contiguous sequence. Among
the preferred scanning amino acids are relatively small, neutral
amino acids. Such amino acids include alanine, glycine, serine, and
cysteine. Alanine is typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the
beta-carbon and is less likely to alter the main-chain conformation
of the variant. Alanine is also typically preferred because it is
the most common amino acid. Further, it is frequently found in both
buried and exposed positions. T. E. Creighton, Proteins: Structure
and Molecular Properties (W.H. Freeman & Co., San Francisco,
1983); Chothia, J. Mol. Biol., 150:1 (1976). If alanine
substitution does not yield adequate amounts of variant, an
isoteric amino acid can be used.
[0167] Further deletional variants of the full-length WISP
polypeptide include variants from which the N-terminal signal
peptide, if any (such as, for example, those putatively identified
as amino acids 1 to 22 for WISP-1, 1 to 23 for WISP2, 1-33 for the
WISP-3 of FIG. 6 and 1-15 for the WISP-3 of FIG. 7), and/or the
initiating methionine has been deleted.
[0168] C. Modifications of the WISP Polypeptide
[0169] Covalent modifications of the WISP polypeptides are included
within the scope of this invention. One type of covalent
modification includes reacting targeted amino acid residues of a
WISP polypeptide with an organic derivatizing agent that is capable
of reacting with selected side chains or the N- or C-terminal
residues. Derivatization with bifunctional agents is useful, for
instance, for crosslinking a WISP polypeptide to a water-insoluble
support matrix or surface for use in the method for purifying
anti-WISP antibodies, and viceversa. Commonly used crosslinking
agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,
glutaraldehyde, N-hydroxysuccinimide esters, for example, esters
with 4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane, and agents such as
methyl-3-((p-azidophenyl)dithio)propioimidate.
[0170] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the .A-inverted.-amino groups of lysine, arginine,
and histidine side chains (Creighton, supra, pp. 79-86),
acetylation of the N-terminal amine, and amidation of any
C-terminal carboxyl group.
[0171] Another type of covalent modification of the WISP
polypeptide included within the scope of this invention comprises
altering the native glycosylation pattern of the polypeptide.
"Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in the native sequence (either by deleting the underlying
glycosylation site or by removing the glycosylation moieties by
chemical and/or enzymatic means) and/or adding one or more
glycosylation sites that are not present in the native sequence. In
addition, the phrase includes qualitative changes in the
glycosylation of the native proteins, involving a change in the
nature and proportion of the various sugar residues present.
[0172] Addition of glycosylation sites to the WISP polypeptide
herein may be accomplished by altering the amino acid sequence. The
alteration may be made, for example, by the addition of, or
substitution by, one or more serine or threonine residues to the
native sequence (for O-linked glycosylation sites). The amino acid
sequence may optionally be altered through changes at the DNA
level, particularly by mutating the DNA encoding the WISP
polypeptide at preselected bases such that codons are generated
that will translate into the desired amino acids. The DNA
mutation(s) may be made using methods described above.
[0173] Another means of increasing the number of carbohydrate
moieties on the WISP polypeptide is by chemical or enzymatic
coupling of glycosides to the polypeptide. Such methods are
described in the art, e.g., in WO 87/05330 published 11 Sep. 1987,
and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306
(1981).
[0174] Removal of carbohydrate moieties present on the WISP
polypeptide may be accomplished chemically or enzymatically or by
mutational substitution of codons encoding amino acid residues that
serve as targets for glycosylation. Chemical deglycosylation
techniques are known in the art and described, for instance, by
Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by
Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of
carbohydrate moieties on polypeptides can be achieved by the use of
a variety of endo- and exoglycosidases as described by Thotakura et
al., Meth. Enzymol., 138:350 (1987).
[0175] Another type of covalent modification comprises linking the
WISP polypeptide to one of a variety of nonproteinaceous polymers,
e.g., polyethylene glycol, polypropylene glycol, or
polyoxyalkylenes, in the manner set forth, e.g., in U.S. Pat. Nos.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0176] The WISP polypeptide of the present invention may also be
modified in a way to form a chimeric molecule comprising a WISP
polypeptide, or a fragment thereof, fused to a heterologous
polypeptide or amino acid sequence. In one embodiment, such a
chimeric molecule comprises a fusion of the WISP polypeptide with a
tag polypeptide which provides an epitope to which an anti-tag
antibody can selectively bind. The epitope tag is generally placed
at the amino- or carboxyl-terminus of a native or variant WISP
molecule. The presence of such epitope-tagged forms can be detected
using an antibody against the tag polypeptide. Also, provision of
the epitope tag enables the WISP polypeptides to be readily
purified by affinity purification using an anti-tag antibody or
another type of affinity matrix that binds to the epitope tag. In
an alternative embodiment, the chimeric molecule may comprise a
fusion of the WISP polypeptides, or fragments thereof, with an
immunoglobulin or a particular region of an immunoglobulin. For a
bivalent form of the chimeric molecule, such a fusion could be to
the Fc region of an Ig, such as an IgG molecule.
[0177] Various tag polypeptides and their respective antibodies are
well known in the art. Examples include poly-histidine (poly-His)
or poly-histidine-glycine (poly-His-Gly) tags; the flu HA tag
polypeptide and its antibody 12CA5 (Field et al., Mol. Cell. Biol.,
8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10r G4, B7,
and 9E10 antibodies thereto (Evan et al., Molecular and Cellular
Biology, 5:3610-3616 (1985)); and the Herpes Simplex virus
glycoprotein D (gD) tag and its antibody. Paborsky et al., Protein
Engineering, 2 (6):547-553 (1990). Other tag polypeptides include
the Flag-peptide (Hopp et al., BioTechnology, 6:1204-1210 (1988));
the KT3 epitope peptide (Martin et al., Science, 255:192-194
(1992)); an a-tubulin epitope peptide (Skinner et al., J. Biol.
Chem., 266:15163-15166 (1991)); and the T7 gene 10 protein peptide
tag. Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,
87:6393-6397 (1990).
[0178] D. Preparation of WISP Polypeptide
[0179] The description below relates primarily to production of
WISP polypeptides by culturing cells transformed or transfected
with a vector containing at least DNA encoding the mature or
full-length sequences of human or mouse WISP-1 (SEQ ID NOS:3, 4,
11, or 12, respectively), or containing at least DNA encoding the
mature or full-length sequences of human or mouse WISP-2 (SEQ ID
NOS:15, 16, 19, or 20, respectively), or containing at least DNA
encoding the mature or full-length sequences of human WISP-3 of
FIG. 6A-6C (SEQ ID NOS:32 or 33, respectively), or containing at
least DNA encoding the mature or full-length sequences of human
WISP-3 of FIG. 7A-7C (SEQ ID NOS:36 or 37, respectively).
[0180] It is, of course, contemplated that alternative methods,
which are well known in the art, may be employed to prepare WISP
polypeptides. For instance, the WISP polypeptide sequence, or
portions thereof, may be produced by direct peptide synthesis using
solid-phase techniques. See, e.g., Stewart et al., Solid-Phase
Peptide Synthesis (W.H. Freeman Co.: San Francisco, Calif., 1969);
Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963). In vitro
protein synthesis may be performed using manual techniques or by
automation. Automated synthesis may be accomplished, for instance,
using an Applied Biosystems peptide synthesizer (Foster City,
Calif.) in accordance with manufacturer's instructions various
portions of WISP polypeptides may be chemically synthesized
separately and combined using chemical or enzymatic methods to
produce a full-length WISP polypeptide.
[0181] 1. Isolation of DNA Encoding WISP Polypeptide
[0182] DNA encoding a WISP polypeptide may be obtained from a cDNA
library prepared from tissue believed to possess the mRNA for WISP
polypeptide and to express it at a detectable level. Accordingly,
DNA encoding human WISP polypeptide can be conveniently obtained
from a cDNA library prepared from human tissue, such as a human
fetal liver library or as otherwise described in the Examples. The
gene encoding WISP polypeptide may also be obtained from a genomic
library or by oligonucleotide synthesis.
[0183] A still alternative method of cloning WISP polypeptide is
suppressive subtractive hybridization which is a method for
generating differentially regulated or tissue-specific cDNA probes
and libraries. This is described, for example, in Diatchenko et al.
Proc. Natl. Acad. Sci. USA, 93:6025-6030 (1996). The procedure is
based primarily on a technique called suppression, PCR and combines
normalization and subtraction in a single procedure, The
normalization step equalizes the abundance of cDNAs within the
target population and the subtraction step excludes the common
sequences between the target and driver populations.
[0184] Libraries can be screened with probes (such as antibodies to
a WISP polypeptide or oligonucleotides of at least about 20-80
bases) designed to identify the gene of interest or the protein
encoded by it. Screening the cDNA or genomic library with the
selected probe may be conducted using standard procedures, such as
described in Sambrook et al., supra. An alternative means to
isolate the gene encoding WISP polypeptide is to use PCR
methodology. Sambrook et al., supra; Dieffenbach et al., PCR
Primer: A Laboratory Manual (New York: Cold Spring Harbor
Laboratory Press, 1995).
[0185] The Examples below describe techniques for screening a cDNA
library. The oligonucleotide sequences selected as probes should be
of sufficient length and sufficiently unambiguous that false
positives are minimized. The oligonucleotide is preferably labeled
such that it can be detected upon hybridization to DNA in the
library being screened. Methods of labeling are well known in the
art, and include the use of radiolabels like 3-P-labeled ATP,
biotinylation, or enzyme labeling. Hybridization conditions,
including moderate stringency and high stringency, are provided in
Sambrook et al., supra.
[0186] Sequences identified in such library screening methods can
be compared and aligned to other known sequences deposited and
available in public databases such as GenBank or other private
sequence databases. Sequence identity (at either the amino acid or
nucleotide level) within defined regions of the molecule or across
the full-length sequence can be determined through sequence
alignment using computer software programs such as ALIGN, DNAstar,
and INHERIT which employ various algorithms to measure
homology.
[0187] Nucleic acid having polypeptide-coding sequence may be
obtained by screening selected cDNA or genomic libraries using the
deduced amino acid sequences disclosed herein for the first time,
and, if necessary, using conventional primer extension procedures
as described in Sambrook et al., supra, to detect precursors and
processing intermediates of mRNA that may not have been
reverse-transcribed into cDNA.
[0188] 2. Selection and Transformation of Host Cells
[0189] Host cells are transfected or transformed with expression or
cloning vectors described herein for WISP polypeptide production
and cultured in conventional nutrient media modified as appropriate
for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences. The culture conditions, such
as media, temperature, pH, and the like, can be selected by the
skilled artisan without undue experimentation. In general,
principles, protocols, and practical techniques for maximizing the
productivity of cell cultures can be found in Mammalian Cell
Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press,
1991) and Sambrook et al., supra.
[0190] Methods of transfection are known to the ordinarily skilled
artisan, for example, CaPO-4 and electroporation. Depending on the
host cell used, transformation is performed using standard
techniques appropriate to such cells. The calcium treatment
employing calcium chloride, as described in Sambrook et al., supra,
or electroporation is generally used for prokaryotes or other cells
that contain substantial cell-wall barriers. Infection with
Agrobacterium tumefaciens is used for transformation of certain
plant cells, as described by Shaw et al., Gene, 23:315 (1983) and
WO 89/05859 published 29 Jun. 1989. For mammalian cells without
such cell walls, the calcium phosphate precipitation method of
Graham and van der Eb, Virology, 52:456-457 (1978) can be employed.
General aspects of mammalian cell host system trans format ions
have been described in U.S. Pat. No. 4,399,216. Transformations
into yeast are typically carried out according to the method of Van
Solingen et al., J. Bact., 130:946 (19-77) and Hsiao et al., Proc.
Natl. Acad. Sci. (USA), 76:3829 (1979) However, other methods for
introducing DNA into cells, such as by nuclear microinjection,
electroporation, bacterial protoplast fusion with intact cells, or
polycations, e.g., polybrene or polyornithine, may also be used.
For various techniques for transforming mammalian cells, see Keown
et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et
al., Nature, 336:348-352 (1988).
[0191] Suitable host cells for cloning or expressing the DNA in the
vectors herein include prokaryote, yeast, or higher eukaryote
cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989). Pseudomonas such as P. aeruginosa, and
Streptomyces. Various E. coli strains are publicly available, such
as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC
31,537); E. coli strain W3110 (ATCC 27,325); and K5772 (ATCC
53,635). These examples are illustrative rather than limiting.
Strain W3110 is one particularly preferred host or parent host
because it is a common host strain for recombinant DNA product
fermentations. Preferably, the host cell secretes minimal amounts
of proteolytic enzymes. For example, strain W311G may be modified
to effect a genetic mutation in the genes encoding proteins
endogamous to the host, with examples of such hosts including E.
coli W3110 strain 1A2, which has the complete genotype tonA; E.
coli W3110 strain 9E4, which has the complete genotype tonA ptr3;
E. coli W3110 strain 27C7 (AT-CC 55,244), which has the complete
genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kan.sup.r; E.
coli W3110 strain 37D6, which has the complete genotype tonA ptr3
phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan.sup.r; E. coli W3110
strain 40B4, which is strain 37D6 with a non-kanamycin resistant
degP deletion mutation; and an E. coli strain having mutant
periplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued 7
Aug. 1990. Alternatively, in vitro methods of cloning, e.g., PCR or
other nucleic acid polymerase reactions, are suitable.
[0192] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for vectors containing nucleic acid encoding WISP polypeptide.
Saccharomyces cerevisiae is a commonly used lower eukaryotic host
microorganism. However, a number of other genera, species, and
strains are commonly available and useful herein, such as
Schizosaccharomyces pombe (Beach and Nurse, Nature, 290:140 (1981);
EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat.
No. 4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such
as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al.,
J. Bacteriol., 737 (1983)), K. fragilis (ATCC 12,424), K.
bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii
(ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al.,
Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus;
yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et
al., J. Basic Microbiol., 28:265-278 (1988) Candida; Trichoderma
reesia (EP 244, 234); Neurospora crassa (Case et al., Proc. Natl.
Acad. Sci. USA, 76:5259-5263 (1979)); Schwanniomyces such as
Schwanniomyces occidentalis (EP 394, 538 published 31 Oct. 1990);
and filamentous fungi such as, e.g., Neurospora, Penicillium,
Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillus
hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res.
Commun., 112:284-289 (1983); Tilburn et al., Gene, 26:205-221
(1983); Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474
(1984)) and A. niger Kelly and Hynes, EMBO J., 4:475-479 (1985).
Methylotropic yeasts are suitable herein and include, but are not
limited to, yeast capable of growth on methanol selected from the
genera consisting of Hansenula, Candida, Kloeckera, Pichia,
Saccharomyces, Torulopsis, and Rhodotorula. A list of specific
species that are exemplary of this class of yeasts may be found in
C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).
[0193] Suitable host cells for the expression of glycosylated WISP
are derived from multicellular organisms. Examples of invertebrate
cells include insect cells such as Drosophila S2 and Spodoptera
Sf9, as well as plant cells. Examples of useful mammalian host cell
lines include Chinese hamster ovary (CHO) and COS cells. More
specific examples include monkey kidney CV1 line transformed by
SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or
293 cells subcloned for growth in suspension culture (Graham et
al., J. Gen Virol. 36:59 (1977)); Chinese hamster ovary cells/-DHFR
(CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216
(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,
23:243-251 (1980); human lung cells (W138, ATCC CCL 75); human
liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562,
ATCC CCL51). The selection of the appropriate host cell is deemed
to be within the skill in the art.
[0194] 3. Selection and Use of a Replicable Vector
[0195] The nucleic acid (e.g., cDNA or genomic DNA) encoding the
desired WISP polypeptide may be inserted into a replicable vector
for cloning (amplification of the DNA) or for expression. Various
vectors are publicly available. The vector may, for example, be in
the form of a plasmid, cosmid, viral particle, or phage. The
appropriate nucleic acid sequence may be inserted into the vector
by a variety of procedures. In general, DNA is inserted into an
appropriate restriction endonuclease site(s) using techniques known
in the art. Vector components generally include, but are not
limited to, one or more of a signal sequence, an origin of
replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination sequence. Construction of
suitable vectors containing one or more of these components employs
standard ligation techniques which are known to the skilled
artisan.
[0196] The desired WISP polypeptide may be produced recombinantly
not only directly, but also as a fusion polypeptide with a
heterologous polypeptide, which may be a signal sequence, if the
WISP polypeptide is conducive to being secreted, or other
polypeptide having a specific cleavage site at the N-terminus of
the mature or full-length protein or polypeptide. In general, the
signal sequence may be a component of the vector, or it may be a
part of the DNA encoding the WISP polypeptide that is inserted into
the vector. The signal sequence may be a prokaryotic signal
sequence such as, for example, the alkaline phosphatase,
penicillinase, lpp, or heat-stable enterotoxin II leaders. For
yeast secretion the signal sequence may be, e.g., the yeast
invertase leader, alpha factor leader (including Saccharomyces and
Kluyveromyces a-factor leaders, the latter described in U.S. Pat.
No. 5,010,182), or acid phosphatase leader, the C. albicans
glucoamylase leader (EP 362,179 published 4 Apr. 1990), or the
signal described in WO 90/13646 published 15 Nov. 1990. In
mammalian cell expression, mammalian signal sequences may be used
to direct secretion of the protein, such as signal sequences from
secreted polypeptides of the same or related species, as well as
viral secretory leaders, and including signals from WISP
polypeptides.
[0197] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Such sequences are well known for a variety of
bacteria, yeast, and viruses. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative bacteria, the 2u
plasmid origin is suitable for yeast, and various viral origins
(SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning
vectors in mammalian cells.
[0198] Expression and cloning vectors will typically contain a
selection gene, also termed a selectable marker. Typical selection
genes encode proteins that (a) confer resistance to antibiotics or
other toxins, e.g., ampicillin, neomycin, methotrexate, or
tetracycline, (b) complement auxotrophic deficiencies, or (c)
supply critical nutrients not available from complex media, e.g.,
the gene encoding D-alanine racemase for Bacilli.
[0199] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the nucleic acid encoding WISP polypeptide, such as DHFR
or thymidine kinase. An appropriate host cell when wild-type DHFR
is employed is the CHO cell line deficient in DHFR activity,
prepared and propagated as described by Urlaub et al., Proc. Natl.
Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use
in yeast is the trp1 gene present in the yeast plasmid YRp7.
Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene,
7:141 (1979); Tschemper et al., Gene, 10:157 (1980). The trp1 gene
provides a selection marker for a mutant strain of yeast lacking
the ability to grow in tryptophan, for example, ATCC No. 44076 or
PEP4-1. Jones, Genetics, 85:12 (1977).
[0200] Expression and cloning vectors usually contain a promoter
operably linked to the nucleic acid sequence encoding WISP
polypeptide to direct mRNA synthesis. Promoters recognized by a
variety of potential host cells are well known. Promoters suitable
for use with prokaryotic hosts include the B-lactamase and lactose
promoter systems (Chang et al., Nature, 275:615 (1978); Goeddel et
al., Nature, 281:544 (1979)), alkaline phosphatase, a tryptophan
(trp) promoter system (Goeddel, Nucleic Acids Res., 8:4057 (1980);
EP 36,776), and hybrid promoters such as the tac promoter. deBoer
et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983). Promoters for
use in bacterial systems also will contain a Shine-Dalgamo (S.D.)
sequence operably linked to the DNA encoding the WISP
polypeptide.
[0201] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase (Hitzeman
et al., J. Biol. Chem., 255:2073 (1980)) or other glycolytic
enzymes (Hess et al., J. Adv. Enzyme Rea., 7:149 (1968); Holland,
Biochemistry, 17:4900 (1978)), such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0202] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657.
[0203] WISP transcription from vectors in mammalian host cells is
controlled, for example, by promoters obtained from the genomes of
viruses such as polyoma virus, fowlpox virus (UK 2,211,504
published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine
papilloma virus, avian sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus, and Simian Virus 40 (SV40); from
heterologous mammalian promoters, e.g., the actin promoter or an
immunoglobulin promoter; and from heat-shock promoters, provided
such promoters are compatible with the host cell systems.
[0204] Transcription of a DNA encoding a WISP polypeptide by higher
eukaryotes may be increased by inserting an enhancer'sequence into
the vector. Enhancers are cis-acting elements of DNA, usually about
from 10 to 300 bp, that act on a promoter to increase its
transcription. Many enhancer sequences are now known from mammalian
genes (globin, elastase, albumin, .alpha.-fetoprotein, and
insulin). Typically, however, one will use an enhancer from a
eukaryotic cell virus. Examples include the SV40 enhancer on the
late side of the replication origin (bp 100-270), the
cytomegalovirus early promoter enhancer, the polyoma,enhancer on
the late side of the replication origin, and adenovirus enhancers.
The enhancer may be spliced into the vector at a position 5' or 3'
to the sequence coding for a WISP polypeptide, but is preferably
located at a site 5' from the promoter.
[0205] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding WISP
polypeptide.
[0206] Still other methods, vectors, and host cells suitable for
adaptation to the synthesis of WISP polypeptides in recombinant
vertebrate cell culture are described in Gething et al., Nature,
293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP
117,060; and EP 117,058.
[0207] 4. Detecting Gene Amplification/Expression
[0208] Gene amplification and/or expression may be measured in a
sample directly, for example, by conventional Southern blotting,
Northern blotting to quantitate the transcription of mRNA (Thomas,
Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Alternatively,
antibodies may be employed that can recognize specific duplexes,
including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes
or DNA-protein duplexes. The antibodies in turn may be labeled and
the assay may be carried out where the duplex is bound to a
surface, so that upon the formation of duplex on the surface, the
presence of antibody bound to the duplex can be detected.
[0209] Gene expression, alternatively, may be measured by
immunological methods, such as immunohistochemical staining of
cells or tissue sections and assay of cell culture or body fluids,
to quantitate directly the expression of gene product. Antibodies
useful for immunohistochemical staining and/or assay of sample
fluids may be either monoclonal or polyclonal, and may be prepared
in any mammal. Conveniently, the antibodies may be prepared against
a native-sequence WISP polypeptide or against a synthetic peptide
based on the DNA sequences provided herein or against exogenous
sequence fused to DNA encoding WISP polypeptide and encoding a
specific antibody epitope.
[0210] 5. Purification of Polypeptide
[0211] Forms of WISP polypeptide may be recovered from culture
medium or from host cell lysates. If membrane-bound, it can be
released from the membrane using a suitable detergent solution
(e.g., Triton-X 100) or by enzymatic cleavage. Cells employed in
expression of WISP polypeptides can be disrupted by various
physical or chemical means, such as freeze-thaw cycling,
sonication, mechanical disruption, or cell lysing agents.
[0212] It may be desired to purify WISP polypeptide from
recombinant cell proteins or polypeptides. The following procedures
are exemplary of suitable purification procedures: by fractionation
on an ion-exchange column; ethanol precipitation; reverse phase
HPLC; chromatography on silica or on a cation-exchange resin such
as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate
precipitation; gel filtration using, for example, SEPHADEX.TM.
G-75; protein A SEPHAROSE.TM. columns to remove contaminants such
as IgG; and metal chelating columns to bind epitope-tagged forms of
the WISP polypeptide. Various methods of protein purification may
be employed, and such methods are known in the art and described,
for example, in Deutscher, Methods in Enzymology, 182 (1990); and
Scopes, Protein Purification: Principles and Practice
(Springer-Veriag: New York, 1982).
[0213] In one specific example of purification, either a poly-His
tag or the Fc portion of human IgG is added to the C-terminal
coding region of the cDNA for WISP-1, WISP-2, or WISP-3 before
expression. The conditioned media from the transfected cells are
harvested by centrifrigation to remove the cells and filtered. For
the poly-His-tagged constructs, the protein may be purified using a
Ni-NTA column. After loading, the column may be washed with
additional equilibration buffer and the protein eluted with
equilibration buffer containing 0.25 M imidazole. The highly
purified protein may then be desalted into a storage buffer if
desired.
[0214] Immunoadhesin (Fc-containing) constructs of the WISP-1,
WISP-2, and WISP-3 proteins may be purified from the conditioned
media by pumping them onto a 5-ml Protein A column which had been
equilibrated in a phosphate buffer. After loading, the column may
be washed extensively with equilibration buffer before elution with
citric acid. The eluted protein may be immediately neutralized by
collecting 1-ml fractions into tubes containing TRIS buffer. The
highly purified protein may be subsequently desalted into storage
buffer as described above for the poly-His-tagged proteins. The
homogeneity of the protein may be assessed by SDS polyacrylamide
gels and by N-terminal amino acid sequencing by Edman
degradation.
[0215] The purification step(s) selected will depend, for example,
on the nature of the production process used and the particular
WISP polypeptide produced.
[0216] E. Uses for WISP Polypeptide and its Nucleic Acid
[0217] Nucleotide sequences (or their complement) encoding WISP
polypeptides have various applications in the art of molecular
biology, including uses as hybridization probes, in chromosome and
gene mapping, and in the generation of anti-sense RNA and DNA.
Nucleic acid encoding WISP polypeptide will also be useful for the
preparation of WISP polypeptides by the recombinant techniques
described herein.
[0218] The full-length nucleotide sequences for mouse or human
WTSP-1 or WISP-2 (SEQ ID NOS: 9, 1, 17, and 13, respectively), or
portions thereof, or the full-length nucleotide sequences for human
WISP-3 of FIG. 6 (SEQ ID NO:30) or for WISP-3 of FIG. 7 (SEQ ID
NO:34) may be used as hybridization probes for a cDNA library to
isolate or detect the full-length gene encoding the WISP
polypeptide of interest or to isolate or detect still other genes
(for instance, those encoding naturally occurring variants of WISP
polypeptide, other WISP polypeptide family members, or WISP
polypeptides from other species) which have a desired sequence
identity to the WISP polypeptide sequences disclosed in FIGS. 1, 2,
3A and 3B, 4, 6A and 6B, and 7A and 7B (SEQ ID NOS: 3, 4, 11, 12,
15, 16, 19, 20, 32, 33, 36, or 37). For example, such procedures as
in situ hybridization, Northern and Southern blotting, and PCR
analysis may be used to determine whether DNA and/or RNA encoding a
different WISP is present in the cell type(s) being evaluated.
Optionally, the length of the probes will be about 20 to about 50
bases. For example, the hybridization probes may be derived from
the UNQ228 (DNA33473-seq min) nucleotide sequence (SEQ ID NO:38) or
the full-length human WISP-2 nucleotide sequence (SEQ ID NO:13) as
shown in FIG. 4 or from genomic sequences including promoters,
enhancer elements, and introns of DNA encoding native-sequence WISP
polypeptide.
[0219] By way of example, a screening method will comprise
isolating the coding region of the WISP gene using the known DNA
sequence to synthesize a selected probe of about 40 bases.
Hybridization probes may be labeled by a variety of labels,
including radionucleotides such as .sup.32P or .sup.35S, or
enzymatic labels such as alkaline phosphatase coupled to the probe
via avidin/biotin coupling systems. Labeled probes having a
sequence complementary to that of any of the genes encoding WISP
polypeptides of the present invention can be used to screen
libraries of human cDNA, genomic DNA, or mRNA to determine to which
members of such libraries the probe hybridizes. Hybridization
techniques are described in further detail in the Examples
below.
[0220] The probes may also be employed in PCR techniques to
generate a pool of sequences for identification of closely related
WISP sequences.
[0221] Nucleotide sequences encoding a WISP polypeptide can also be
used to construct hybridization probes for mapping the gene which
encodes that WISP polypeptide and for the genetic analysis of
individuals with genetic disorders. The nucleotide sequences
provided herein may be mapped to a chromosome and specific regions
of a chromosome using known techniques, such as in situ
hybridization, linkage analysis against known chromosomal markers,
and hybridization screening with libraries. If the amplification of
a given gene is functionally relevant, then that gene should be
amplified more than neighboring genomic regions which are not
important for tumor survival. To test this, the gene can be mapped
to a particular chromosome, e.g. by radiation-hybrid analysis. The
amplification level is then determined at the location identified,
and at neighboring genomic region. Selective or preferential
amplification at the genomic region to which to gene has been
mapped is consistent with the possibility that the gene
amplification observed promotes tumor growth or survival.
Chromosome mapping includes both framework and epicenter mapping.
For further details see e.g., Stewart et al., Genome Research 7,
422-433 (1997)
[0222] Nucleic acid encoding a WISP polypeptide may be used as a
diagnostic to determine the extent and rate of the expression of
the DNA encoding the WISP polypeptide in the cells of a patient. To
accomplish such an assay, a sample of a patient's cells is treated,
via in situ hybridization, or by other suitable means, and analyzed
to determine whether the sample contains mRNA molecules capable of
hybridizing with the nucleic acid molecule.
[0223] Nucleic acids which encode WISP polypeptides or any of their
modified forms can also be used to generate either transgenic
animals or "knock-out" animals which, in turn, are useful in the
development and screening of therapeutically useful reagents. A
transgenic animal (e.g., a mouse or rat) is an animal having cells
that contain a transgene, which transgene was introduced into the
animal or an ancestor of the animal at a prenatal, e.g., an
embryonic stage. A transgene is a DNA which is integrated into the
genome of a cell from which a transgenic animal develops. In one
embodiment, cDNA encoding a WISP polypeptide can be used to clone
genomic DNA encoding the WISP polypeptide in accordance with
established techniques and the genomic sequences used to generate
transgenic animals that contain cells which express DNA encoding
the WISP polypeptide.
[0224] Methods for generating transgenic animals, particularly
animals such as mice or rats, have become conventional in the art
and are described, for example, in U.S. Pat. Nos. 4,736,866 and
4,870,009 and WO 97/38086. Typically, particular cells would be
targeted for WISP transgene incorporation with tissue-specific
enhancers. Transgenic animals that include a copy of a transgene
encoding the WISP polypeptide introduced into the germ line of the
animal at an embryonic stage can be used to examine the effect of
increased expression of DNA encoding the WISP polypeptide. Such
animals can be used as tester animals for reagents thought to
confer protection from, for example, pathological conditions
associated with its overexpression. In accordance with this facet
of the invention, an animal is treated with the Teagent and a
reduced incidence of the pathological condition, compared to
untreated animals bearing the transgene, would indicate a potential
therapeutic intervention for the pathological condition.
[0225] Alternatively, non-human homologues of WISP polypeptides can
be used to construct a WISP polypeptide "knock-out" animal which
has a defective or altered gene encoding a WISP polypeptide as a
result of homologous recombination between the endogenous gene
encoding the WISP polypeptide and altered genomic DNA encoding the
WISP polypeptide introduced into an embryonic cell of the animal.
For example, cDNA encoding the WISP polypeptide can be used to
clone genomic DNA encoding the WISP polypeptide in accordance with
established techniques. A portion of the genomic DNA encoding the
WISP polypeptide can be deleted or replaced with another gene, such
as a gene encoding a selectable marker which can be used to monitor
integration. Typically, several kilobases of unaltered flanking DNA
(both at the 5' and 3' ends) are included in the vector. See e.g.,
Thomas and Capecchi, Cell, 51:503 (1987) for a description of
homologous recombination vectors. The vector is introduced into an
embryonic stem cell line (e.g., by electroporation) and cells in
which the introduced DNA has homologously recombined with the
endogenous DNA are selected. See e.g., Li et al., Cell, 69:915
(1992). The selected cells are then injected into a blastocyst of
an animal (e.g., a mouse or rat) to form aggregation chimeras. See
e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp.
113-152. A chimeric embryo can then be implanted into a suitable
pseudopregnant female foster animal and the embryo brought to term
to create a "knock-out" animal. Progeny harboring the homologously
recombined DNA in their germ cells can be identified by standard
techniques and used to breed animals in which all cells of the
animal contain the homologously recombined DNA. Knockout animals
can be characterized, for instance, by their ability to defend
against certain pathological conditions and by their development of
pathological conditions due to absence of the WISP polypeptide.
[0226] In particular, assays in which CTGF, IGFBPs, and other
members of the CTGF superfamily and other growth factors are
usually used are preferably performed with the WISP-1 and WISP-2
polypeptides. For example, an assay to determine whether TGF-.beta.
induces the WISP polypeptide, indicating a role in cancer, may be
performed as known in the art, as well as assays involving
induction of cell death and .sup.3H-thymidine proliferation assays.
Mitogenic and tissue growth assays are also performed with the WISP
polypeptide as set forth above. The results are applied
accordingly.
[0227] The WISP polypeptides of the present invention may also be
used to induce the formation of anti-WISP polypeptide antibodies,
which are identified by routine screening as detailed below.
[0228] In addition to their uses above the WISP-1, WISP-2, and
WISP-3 polypeptides of the present invention are useful as the
basis for assays of IGF activity. Importantly, since such an as say
measures a physiologically significant binding event, i.e., that of
an IGF to its IGFBP, triggering a detectable change (such as
phosphorylation, cleavage, chemical modification, etc.) It is
unlikely to be both more sensitive and more accurate than
immunoassays which detect the physiologically non-significant
binding of an IGF to anti-WISP polypeptide antibody. Although more
sensitive and accurate than antibodies, the WISP-1, WISP-2, and
WISP-3 molecules of the invention can be used to assay IGF (such as
IGF-I or IGF-II) levels in a sample in the same ways in which
antibodies are used.
[0229] For diagnostic purposes, the WISP-1, WISP-2, or WISP-3
polypeptide can be used in accordance with immunoassay technology.
Examples of immunoassays are provided by Wide at pages 199-206 of
Radioimmune Assay Method, Kirkham and Huner. ed. E & S.
Livingstone. Edinburgh. 1970.
[0230] Thus, in one embodiment. WISP-1, WISP-2, and WISP-3
polypeptides can be detectably labeled and incubated with a test
sample containing IGF molecules (such as biological fluids, e.g.,
serum, sputum, urine, etc. ), and the amount of WISP-1, WISP-2, or
WISP-3 molecule bound to the sample ascertained.
[0231] Immobilization of reagents is required for certain assay
methods. Immobilization entails separating the WISP-1, WISP-2, or
WISP-3 polypeptide from any analyte that remains free in solution.
This conventionally is accomplished by either insolubilizing the
WISP-1, WISP-2, or WISP-3 polypeptide before the assay procedure,
as by adsorption to a water insoluble matrix or surface (Bennich et
al. U.S. Pat. No. 3,720,760), by covalent coupling (for example,
using glutaraldehyde cross-linking), or by insolubilizing the
molecule afterward, e.g. by immunoprecipitation.
[0232] The foregoing are merely exemplary diagnostic assays for
IGF. Other methods now or hereafter developed for the determination
of these analytes are included within the scope hereof.
[0233] WISP-1, WISP-2, and WISP-3 polypeptides are also useful in
radioimmunoassays to measure IGFs such as IGF-I or IGF-II. Such a
radioimmunoassay would be conducted as described in the literature
using the naturally purified or recombinant WISP-1, WISP-2 or
WISP-3 as the WISP element.
[0234] In addition, WISP polypeptides are useful for screening for
compounds that bind to them as defined above. Preferably, these
compounds are small molecules such as organic or peptide molecules
that exhibit one or more of the desired activities. Screening
assays of this kind are conventional in the art, and any such
screening procedure may be employed, whereby the test sample is
contacted with the WISP polypeptide herein and the extent of
binding and biological activity of the bound molecule are
determined.
[0235] More specifically, this invention encompasses methods of
screening compounds to identify those that mimic the WISP
polypeptide (agonists) or prevent the effect of the WISP
polypeptide (antagonists). Screening assays for antagonist drug
candidates are designed to identify compounds that bind or complex
with the WISP polypeptides encoded by the genes identified herein,
or otherwise interfere with the interaction of the encoded
polypeptides with other cellular proteins. Such screening assays
will include assays amenable to high-throughput screening of
chemical libraries, making them particularly suitable for
identifying small molecule drug candidates.
[0236] The assays can be performed in a variety of formats,
including protein-protein binding assays, biochemical screening
assays, immunoassays, and cell-based assays, which are well
characterized in the art.
[0237] All assays for antagonists are common in that they call for
contacting the drug candidate with a WISP polypeptide encoded by a
nucleic acid identified herein under conditions and for a time
sufficient to allow these two components to interact.
[0238] In binding assays, the interaction is binding and the
complex formed can be isolated or detected in the reaction mixture.
In a particular embodiment, the WISP polypeptide encoded by the
gene identified herein or the drug candidate is immobilized on a
solid phase, e.g., on a microtiter plate, by covalent or
non-covalent attachments. Non-covalent attachment generally is
accomplished by coating the solid surface with a solution of the
WISP polypeptide and drying. Alternatively, an immobilized
antibody, e.g., a monoclonal antibody, specific for the WISP
polypeptide to be immobilized can be used to anchor it to a solid
surface. The assay is performed by adding the non-immobilized
component, which may be labeled by a detectable label, to the
immobilized component, e.g., the coated surface containing the
anchored component. When the reaction is complete, the non-reacted
components are removed, e.g., by washing, and complexes anchored on
the solid surface are detected. When the originally non-immobilized
component carries a detectable label, the detection of label
immobilized on the surface indicates that complexing occurred.
Where the originally non-immobilized component does not carry a
label, complexing can be detected, for example, by using a labeled
antibody specifically binding the immobilized complex.
[0239] If the candidate compound interacts with but does not bind
to a particular WISP polypeptide encoded by a gene identified
herein, its interaction with that polypeptide can be assayed by
methods well known for detecting protein-protein interactions. Such
assays include traditional approaches, such as, e.g., cross-linking
co-immunoprecipitation, and co-purification through gradients or
chromatographic columns. In addition, protein-protein interactions
can be monitored by using a yeast-based genetic system described by
Fields and co-workers (Fields and Song, Nature (London), 340:
245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA, 88:
9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc. Natl.
Acad. Sci. USA, 89: 5789-5793 (1991). Many transcriptional
activators, such as yeast GAL4, consist of two physically discrete
modular domains, one acting as the DNA-binding domain, the other
one functioning as the transcription-activation domain. The yeast
expression system described in the foregoing publications
(generally referred to as the "two-hybrid system") takes advantage
of this property, and employs two hybrid proteins, one in which the
target protein is fused to the DNA-binding domain of GAL4, and
another, in which candidate activating proteins are fused to the
activation domain. The expression of a GAL1-lacZ reporter gene
under control of a GAL4-activated promoter depends on
reconstitution of GAL4 activity via protein-protein interaction.
Colonies containing interacting polypeptides are detected with a
chromogenic substrate for .beta.-galactosidase. A complete kit
(MATCHMAKER.TM.) for identifying protein-protein interactions
between two specific proteins using the two-hybrid technique is
commercially available from Clontech. This system can also be
extended to map protein domains involved in specific protein
interactions as well as to pinpoint amino acid residues that are
crucial for these interactions.
[0240] Compounds that interfere with the interaction of a gene
encoding a WISP polypeptide identified herein and other intra- or
extracellular components can be tested as follows: usually a
reaction mixture is prepared containing the product of the gene and
the intra- or extracellular component under conditions and for a
time allowing for the interaction and binding of the two products.
To test the ability of a candidate compound to inhibit binding, the
reaction is run in the absence and in the presence of the test
compound. In addition, a placebo may be added to a third reaction
mixture, to serve as positive control. The binding (complex
formation) between the test compound and the intra- or
extracellular component present in the mixture is monitored as
described hereinabove. The formation of a complex in the control
reaction(s) but not in the reaction mixture containing the test
compound indicates that the test compound interferes with the
interaction of the test compound and its reaction partner.
[0241] If the WISP polypeptide has the ability to stimulate the
proliferation of endothelial cells in the presence of the
co-mitogen ConA, then one example of a screening method takes
advantage of this ability. Specifically, in the proliferation
assay, human umbilical vein endothelial cells are obtained and
cultured in 96-well flat-bottomed culture plates (Costar,
Cambridge, Mass.) and supplemented with a reaction mixture
appropriate for facilitating proliferation of the cells, the
mixture containing Con-A (Calbiochem, La Jolla, Calif.). Con-A and
the compound to be screened are added and after incubation at
37.degree. C., cultures are pulsed with .sup.3-H-thymidine and
harvested onto glass fiber filters (phD; Cambridge Technology,
Watertown, Mass.). Mean .sup.3-(H)-thymidine incorporation (cpm) of
triplicate cultures is determined using a liquid scintillation
counter (Beckman Instruments, Irvine, Calif.). Significant
.sup.3-(H)thymidine incorporation indicates stimulation of
endothelial cell proliferation.
[0242] To assay for antagonists, the assay described above is
performed; however, in this assay the WISP polypeptide is added
along with the compound to be screened and the ability of the
compound to inhibit .sup.3-(H)-thymidine incorporation in the
presence of the WISP polypeptide indicates that the compound is an
antagonist to the WISP polypeptide. Alternatively, antagonists may
be detected by combining the WISP polypeptide and a potential
antagonist with membrane-bound WISP polypeptide receptors or
recombinant receptors under appropriate conditions for a
competitive inhibition assay. The WISP polypeptide can be labeled,
such as by radioactivity, such that the number of WISP polypeptide
molecules bound to the receptor can be used to determine the
effectiveness of the potential antagonist. The gene encoding the
receptor can be identified by numerous methods known to those of
skill in the art, for example, ligand panning and FACS sorting.
Coligan et al., Current Protocols in Immun., 1 (2): Chapter 5
(1991). Preferably, expression cloning is employed wherein
polyadenylated RNA is prepared from a cell responsive to the WISP
polypeptide and a cDNA library created from this RNA is divided
into pools and used to transfect COS cells or other cells that are
not responsive to the WISP polypeptide. Transfected cells that are
grown on glass slides are exposed to labeled WISP polypeptide. The
WISP polypeptide can be labeled by a variety of means including
iodination or inclusion of a recognition site for a site-specific
protein kinase. Following fixation and incubation, the slides are
subjected to autoradiographic analysis. Positive pools are
identified and sub-pools are prepared and re-transfected using an
interactive sub-pooling and re-screening process, eventually
yielding a single clone that encodes the putative receptor.
[0243] As an alternative approach for receptor identification,
labeled WISP polypeptide can be photo affinity-linked with cell
membrane or extract preparations that express the receptor
molecule. Cross-linked material is resolved by PAGE and exposed to
X-ray film. The labeled complex containing the receptor can be
excised, resolved into peptide fragments, and subjected to protein
micro-sequencing. The amino acid sequence obtained from
micro-sequencing would be used to design a set of degenerate
oligonucleotide probes to screen a cDNA library to identify the
gene encoding the putative receptor.
[0244] In another assay for antagonists, mammalian cells or a
membrane preparation expressing the receptor would be incubated
with labeled WISP polypeptide in the presence of the candidate
compound. The ability of the compound to enhance or block this
interaction could then be measured.
[0245] The compositions useful in the treatment of WISP-related
disorders include, without limitation, antibodies, small organic
and inorganic molecules, peptides, phosphopeptides, antisense and
ribozyme molecules, triple-helix molecules, etc-, that inhibit the
expression and/or activity of the target gene product.
[0246] More specific examples of potential antagonists include an
oligonucleotide that binds to the WISP polypeptide,
(poly)peptide-immunoglobulin fusions, and, in particular,
antibodies including, without limitation, poly- and monoclonal
antibodies and antibody fragments, single-chain antibodies,
anti-idiotypic antibodies, and chimeric or humanized versions of
such antibodies or fragments, as well as human antibodies and
antibody fragments. Alternatively, a potential antagonist may be a
closely related protein, for example, a mutated form of the WISP
polypeptide that recognizes the receptor but imparts no effect,
thereby competitively inhibiting the action of the WISP
polypeptide.
[0247] Another potential WISP polypeptide antagonist is an
antisense RNA or DNA construct prepared using antisense technology,
where, e.g., an antisense RNA or DNA molecule acts to block
directly the translation of mRNA by hybridizing to targeted mRNA
and preventing protein translation. Antisense technology can be
used to control gene expression through triple-helix formation or
antisense DNA or RNA, both of which methods are based on binding of
a polynucleotide to D13A or RNA. For example, the 5' coding portion
of the polynucleotide sequence, which encodes the mature WISP
polypeptides herein, is used to design an antisense RNA
oligonucleotide of from about 10 to 40 base pairs in length. A DNA
oligonucleotide is designed to be complementary to a region of the
gene involved in transcription (triple helix--see Lee et al., Nucl.
Acids Res., 6: 3073 (1979); Cooney et al., Science, 241: 456
(1988); Dervan et al., Science, 251: 1360 (1991)), thereby
preventing transcription and the production of the WISP
polypeptide. The antisense RNA oligonucleotide hybridizes to the
mRNA in vivo and blocks translation of the mRNA molecule into the
WISP polypeptide (anti-sense,--Okano, Neurochem., 56: 560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression
(CRC Press: Boca Raton, Fla., 1988). The oligonucleotides described
above can also be delivered to cells such that the antisense RNA or
DNA may be expressed in vivo to inhibit production of the WISP
polypeptide. When antisense DNA is used, oligodeoxyribonucleotides
derived from the translation-initiation site, e.g., between about
-10 and +10 positions of the target gene nucleotide sequence, are
preferred.
[0248] Potential antagonists include small molecules that bind to
the active site, the receptor binding site, or growth factor or
other relevant binding site of the WISP polypeptide, thereby
blocking the normal biological activity of the WISP polypeptide.
Examples of small molecules include, but are not limited to, small
peptides or peptide-like molecules, preferably soluble peptides,
and synthetic non-peptidyl organic or inorganic compounds.
[0249] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. Ribozymes act by sequence-specific
hybridization to the complementary target RNA, followed by
endonucleolytic cleavage. Specific ribozyme cleavage sites within a
potential RNA target can be identified by known techniques. For
further details see, e.g., Rossi, Current Biology, 4: 469-471
(1994), and PCT publication No. WO 97/33551 (published Sep. 18,
1997).
[0250] Nucleic acid molecules in triple-helix formation used to
inhibit transcription should be single-stranded and composed of
deoxynucleotides. The base composition of these oligonucleotides is
designed such that it promotes triple-helix formation via Hoogsteen
base-pairing rules, which generally require sizeable stretches of
purines or pyrimidines on one strand of a duplex. For further
details see, e.g., PCT publication No. WO 97/33551, supra.
[0251] These small molecules can be identified by any one or more
of the screening assays discussed hereinabove and/or by any other
screening techniques well known for those skilled in the art.
[0252] WISP-1, WISP-2, and WISP-3 polypeptides are additionally
useful in affinity purification of an 1GF that binds to WISP-1,
WISP-2, or WISP-3 (such as, for example, IGF-I) and in purifying
antibodies thereto. The WISP-1, WISP-2, or WISP-3 is typically
coupled to an immobilized resin such as Affi-Gel 10.TM. (Bio-Rad.
Richmond, Calif.) or other such resins (support matrices) by means
well known in the art. The resin is equilibrated in a buffer (such
as one containing 150 mM NaCl, 20 mM HEPES, pH 7.4 supplemented to
contain 20% glycerol and 0.5% NP-40) and the preparation to be
purified is placed in contact with the resin, whereby the molecules
are selectively adsorbed to the WISP-1, WISP-2, or WISP-3 on the
resin.
[0253] The resin is then sequentially washed with suitable buffers
to remove non-adsorbed material, including unwanted contaminants,
from the mixture to be purified, using, e.g. 150 mM NaCl, 20 mM
HEPES, pH 7.4, containing 0.5% NP-40: 150 mM NaCl, 20 mM HEPES, pH
7.4 containing 0.5 M NaCl and 0.1% NP-40: 150 mM NaCl, 20 mM HEPES,
pH 7.4 containing 0.1% deoxycholate: 150 mM NaCl, 20 mM HEPES, pH
7.4 containing 0.1% NP-40: and a solution of 0.1% NP-40, 20%
glycerol and 50 mM glycine, pH 3. The resin is then treated so as
to elute the 1GF using a buffer that will break the bond between
the 1GF and WISP-1, WISP-2, or WISP-3 (using, e.g. 50 mM glycine,
pH 3, 0.1% NP-40, 20% glycerol, and 100 mM NaCl).
[0254] It is contemplated that the WISP polypeptides of the present
invention may be used to treat various conditions, including those
characterized by overexpression and/or activation of at least the
Wnt pathway. Further, since the WISP-1, WISP-2, and WISP-3
molecules respond to hormone-expressed breast cancer in mice and
are abnormally expressed in human cancer, and are over-amplified in
various colon cancer cell lines, they are useful in diagnosing
cancer, for example, as a marker for increased susceptibility to
cancer or for having cancer. Exemplary conditions or disorders to
be treated with the WISP polypeptides include benign or malignant
tumors (e.g., renal, liver, kidney, bladder, testicular, breast,
gastric, ovarian, colorectal, prostate, pancreatic, lung,
esophageal, vulval, thyroid, hepatic carcinomas; sarcomas;
glioblastomas; and various head and neck tumors); leukemias and
lymphoid malignancies; other disorders such as neuronal, glial,
astrocytal, hypothalamic, and other glandular, macrophagal,
epithelial, stromal, and blastocoelic disorders; cardiac disorders;
renal disorders; catabolic disorders; bone-related disorders such
as osteoporosis; and inflammatory, angiogenic, and immunologic
disorders, such as arteriosclerosis; as well as connective tissue
disorders, including wound healing.
[0255] The WISP polypeptides of the invention are administered to a
mammal, preferably a human, in accord with known methods, such as
intravenous administration as a bolus or by continuous infusion
over a period of time, by intramuscular, intraperitoneal,
intracerebrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, oral, topical, or inhalation routes. Intravenous or
subcutaneous administration of the polypeptide is preferred.
[0256] Therapeutic formulations of the WISP polypeptide are
prepared for storage by mixing the polypeptide having the desired
degree of purity with optional pharmaceutically acceptable
carriers, excipients, or stabilizers (Remington's Pharmaceutical
Sciences 16th edition, Osol, A. Ed. (1980)), in the form of
lyophilized formulations or aqueous solutions. Acceptable carriers,
excipients, or stabilizers are nontoxic to recipients at the
dosages and concentrations employed, and include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccha rides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose, or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.,
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM., or polyethylene glycol (PEG).
[0257] Other therapeutic regimens may be combined with the
administration of the WISP polypeptides of the instant invention.
For example, the patient to be treated with the polypeptides
disclosed herein may also receive radiation therapy if the disorder
is cancer. Alternatively, or in addition, a chemotherapeutic agent
may be administered to the patient with cancer. Preparation and
dosing schedules for such chemotherapeutic agents may be used
according to manufacturers' instructions or as determined
empirically by the skilled practitioner. Preparation and dosing
schedules for such chemotherapy are also described in Chemotherapy
Service, Ed., M. C. Perry (Williams & Wilkins: Baltimore, Md.,
1992). The chemotherapeutic agent may precede or follow
administration of the polypeptide or may be given simultaneously
therewith. The polypeptide may be combined with an anti-oestrogeri
compound such as tamoxifen or an anti-progesterone such as
onapristone (see, EP 616812) in dosages known for such
molecules.
[0258] It may be desirable also to co-administer with the WISP
polypeptide (or anti-WISP polypeptide) antibodies against other
tumor-associated antigens, such as antibodies which bind to HER-2,
EGFR, ErbB2, ErbB3, ErbB4, or vascular endothelial factor (VEGF).
Alternatively, or in addition, two or more different anti-cancer
antibodies, such as anti-ErbB2 antibodies, may be co-administered
to the patient with the WISP polypeptide (or anti-WISP polypeptide
antibody). Sometimes, it may be beneficial also to administer one
or more cytokines to the patient.
[0259] In a preferred embodiment, the WISP polypeptide is
co-administered with a growth-inhibitory agent to the cancer
patient. For example, the growth-inhibitory agent may be
administered first, followed by the WISP polypeptide. However,
simultaneous administration or administration of the WISP
polypeptide first is also contemplated. Suitable dosages for the
growth-inhibitory agent are those presently used and may be lowered
due to the combined action (synergy) of the growth-inhibitory agent
and polypeptide. The antibodies, cytotoxic agents, cytokines, or
growth-inhibitory agents are suitably present in combination in
amounts that are effective for the purpose intended.
[0260] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethyl cellulose
or gelatin microcapsules and poly-(methylmethacylate)microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nanoparticles, and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed.
(1980), supra.
[0261] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0262] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the polypeptide,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene7vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated polypeptides remain in
the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0263] For the prevention or treatment of disease or disorder, the
appropriate dosage of WISP polypeptide will depend on the type of
disorder to be treated, as defined above, the severity and course
of the disorder, whether the polypeptide is administered for
preventive or therapeutic purposes, previous therapy, the patient's
clinical history and response to the polypeptide, the route of
administrazion, the condition of the patient, and the discretion of
the attending physician. The polypeptide is suitably administered
to the patient at one time or over a series of treatments.
[0264] Depending on the type and severity of the disease, about 1
.mu.g/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of WISP polypeptide is an
initial candidate dosage for administration to the patient,
whether, for example, by one or more separate administrations, or
by continuous infusion. A typical daily dosage might range from
about 1 .mu.g/kg to 100 mg/kg or more, depending on the factors
mentioned above. For repeated administrations over several days or
longer, depending on the condition, the treatment is sustained
until a desired suppression of symptoms of the disorder occurs.
However, other dosage regimens may be useful. The progress of this
therapy is easily monitored by conventional techniques and assays.
In another embodiment of the invention, an article of manufacture
containing materials useful for the treatment of the disorders
described above is provided. The article of manufacture comprises a
container and a label. Suitable containers include, for example,
bottles, vials, syringes, and test tubes. The containers may be
formed from a variety of materials such as glass or plastic. The
container holds a composition which is effective for treating the
condition and may have a sterile access port (for example, the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The active
agent in the composition is the WISP polypeptide. The label on, or
associated with, the container indicates that the composition is
used for treating the condition or disorder of choice. The article
of manufacture may further comprise a second container comprising a
pharmaceutically acceptable buffer, such as phosphate-buffered
saline, Ringer's solution, and dextrose solution. It may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use.
[0265] F. Anti-WISP Polypeptide Antibodies
[0266] The present invention further provides anti-WISP polypeptide
antibodies. Exemplary antibodies include polyclonal, monoclonal,
humanized, bispecific, and heteroconjugate antibodies.
[0267] 1. Polyclonal Antibodies
[0268] The anti-WISP polypeptide antibodies of the present
invention may comprise polyclonal antibodies. Methods of preparing
polyclonal antibodies are known to the skilled artisan. Polyclonal
antibodies can be raised in a mammal, for example, by one or more
injections of an immunizing agent and, if desired, an adjuvant.
Typically, the immunizing agent and/or adjuvant will be injected in
the mammal by multiple subcutaneous or intraperitoneal injections.
The immunizing agent may include the WISP polypeptide or a fusion
protein thereof. It may be useful to conjugate the immunizing agent
to a protein known to be immunogenic in the mammal being immunized.
Examples of such immunogenic proteins include but are not limited
to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin,
and soybean trypsin inhibitor. Examples of adjuvants which may be
employed include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The
immunization protocol may be selected by one skilled in the art
without undue experimentation.
[0269] 2. Monoclonal Antibodies
[0270] The anti-WISP polypeptide antibodies may, alternatively, be
monoclonal antibodies. Monoclonal antibodies may be prepared using
hybridoma methods, such as those described by Kohler and Milstein,
Nature, 256: 4 95 (1975). In a hybridoma method, a mouse, hamster,
or other appropriate host animal is typically immunized with an
immunizing agent to elicit lymphocytes that produce or are capable
of producing antibodies that will specifically bind to the
immunizing agent. Alternatively, the lymphocytes may be immunized
in vitro.
[0271] The immunizing agent will typically include the WISP
polypeptide or a fusion protein thereof. Generally, either
peripheral blood lymphocytes ("PBLs") are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if
non-human mammalian sources are desired. The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent,
such as PEG, to form a hybridoma cell. Goding, Monoclonal
Antibodies: Principles and Practice (Academic Press: New York,
1986) pp. 59-103. Immortalized cell lines are usually transformed
mammalian cells, particularly myeloma cells of rodent, bovine, and
human origin. Usually, rat or mouse myeloma cell lines are
employed. The hybridoma cells may be cultured in a suitable culture
medium that preferably contains one or more substances that inhibit
the growth or survival of the unfused, immortalized cells. For
example, if the parental cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for
the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine ("HAT medium"), which substances prevent the growth
of HGPRT-deficient cells.
[0272] Preferred immortalized cell lines are those that fuse
efficiently, support stable high-level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif., and
the American Type Culture Collection, Manassas, Va. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies. Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications (Marcel Dekker, Inc.: New
York, 1987) pp. 51-63.
[0273] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against a WISP polypeptide. Preferably, the binding
specificity of monoclonal antibodies produced by the hybridoma
cells is determined by immunoprecipitation or by an in vitro
binding assay, such as radioimmunoassay (RIA) or enzyme-linked
immunoabsorbent assay (ELISA). Such techniques and assays are known
in the art. The binding affinity of the monoclonal antibody can,
for example, be determined by the Scatchard analysis of Munson and
Pollard, Anal. Biochem., 107: 220 (1980).
[0274] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods. Goding, supra. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells may be
grown in vivo as ascites in a mammal.
[0275] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0276] The monoclonal antibodies may also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA may be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, CHO cells, or myeloma cells that do not
otherwise produce immunoglobulin protein, to obtain the synthesis
of monoclonal antibodies in the recombinant host cells. The DNA
also may be modified, for example, by substituting the coding
sequence for human heavy and light chain constant domains in place
of the homologous murine sequences (U.S. Pat. No. 4,816,567;
Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984))
or by covalently joining to the immunoglobulin coding sequence all
or part of the coding sequence for a non-immunoglobulin
polypeptide. Such a non-immunoglobulin polypeptide can be
substituted for the constant domains of an antibody of the
invention, or can be substituted for the variable domains of one
antigen-combining site of an antibody of the invention to create a
chimeric bivalent antibody.
[0277] The antibodies may be monovalent antibodies. Methods for
preparing monovalent antibodies are well known in the art. For
example, one method involves recombinant expression of
immunoglobulin light chain and modified heavy chain. The heavy
chain is truncated generally at any point in the Fc region so as to
prevent heavy-chain crosslinking. Alternatively, the relevant
cysteine residues are substituted with another amino acid residue
or are deleted so as to prevent crosslinking.
[0278] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly Fab fragments, can be accomplished using routine
techniques known in the art.
[0279] 3. Humanized Antibodies
[0280] The anti-WISP antibodies of the invention may further
comprise humanized antibodies or human antibodies. Humanized forms
of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains, or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2, or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived
from non-human immunoglobulin. Humanized antibodies include human
immunoglobulins (recipient antibody) in which residues from a
complementary-determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat, or rabbit having the desired
specificity, affinity, and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human
immunoglobulin, and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody preferably also will comprise at least a portion of an Fc,
typically that of a human immunoglobulin. Jones et al., Nature,
321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988);
Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).
[0281] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0282] Human antibodies can also be produce a using various
techniques known in the art, including phage-display libraries.
Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al.,
J. Mol. Biol., 222:581 (1991). The techniques of Cole et al. and
Boemer et al. are also available for the preparation of human
monoclonal antibodies. Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 7? (1985); Boemer et al., J.
Immunol., 147(1):86-95 (1991).
[0283] 4. Bispecific Antibodies
[0284] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for a WISP polypeptide; the other one is for any
other antigen, and preferably for a cell-surface protein or
receptor or receptor subunit.
[0285] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities. Milstein and Cuello, Nature, 305:537-539
(1983). Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published 13 May
1993, and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).
[0286] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant-domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions, it is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology,
121-210 (1986).
[0287] 5. Heteroconjugate Antibodies
[0288] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells (U.S.
Pat. No. 4,676,980), and for treatment of HIV infection. WO
91/00360; WO 92/200373; EP 03089. It is contemplated that the
antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving
cross-linking agents. For example, immunotoxins may be constructed
using a disulfide-exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0289] G. Uses for Anti-WISP Polypeptide Antibodies
[0290] The antibodies of the invention may be used as affinity
purification agents. In this process, the antibodies are
immobilized on a solid phase such a SEPHADEX.TM. resin or filter
paper, using methods well known in the art. The immobilized
antibody is contacted with a sample containing the WISP polypeptide
(or fragment thereof) to be purified, and thereafter the support is
washed with a suitable solvent that will remove substantially all
the material in the sample except the WISP protein, which is bound
to the immobilized antibody. Finally, the support is washed with
another suitable solvent, such as glycine buffer, pH 5.0, that will
release the WISP polypeptide from the antibody.
[0291] Anti-WISP polypeptide antibodies may also be useful in
diagnostic assays for WISP polypeptide, e.g., detecting its
expression in specific cells, tissues, or serum. Thus, the
antibodies may be used in the diagnosis of human malignancies (see,
for example, U.S. Pat. No. 5,183,884).
[0292] For diagnostic applications, the antibody typically will be
labeled with a detectable moiety. Numerous labels are available
which can be preferably grouped into the following categories:
[0293] (a) Radioisotopes, such as .sup.35s, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I. The antibody can be labeled with the
radioisotope using the techniques described in Current Protocols in
Immunology, Volumes 1 and 2. Coligen et al, Ed.,
(Wiley-Interscience: New York, 1991), for example, and
radioactivity can be measured using scintillation counting.
[0294] (b) Fluorescent labels such as rare earth chelates (europium
chelates) or fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, Lissamine, phycoerythrin, and Texas Red are
available. The fluorescent labels can be conjugated to the antibody
using the techniques disclosed in Current Protocols in Immunology,
supra, Coligen, ed., for example. Fluorescence can be quantified
using a fluorimeter.
[0295] (c) Various enzyme-substrate labels are available, and U.S.
Pat. No. 4,275,149 provides a review of some of these. The enzyme
preferably catalyzes a chemical alteration of the chromogenic
substrate which can be measured using various techniques. For
example, the enzyme may catalyze a color change in a substrate,
which can be measured spectrophotometrically. Alternatively, the
enzyme may alter the fluorescence or chemiluminescence of the
substrate. Techniques for quantifying a change in fluorescence are
described above. The chemiluminescent substrate becomes
electronically excited by a chemical reaction and may then emit
light which can be measured (using a chemiluminometer, for example)
or donates energy to a fluorescent acceptor. Examples of enzymatic
labels include luciferases (e.g., firefly luciferase and bacterial
luciferase; U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, malate dehydrogenase, urease,
peroxidase such as horseradish peroxidase (HRPO), alkaline
phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and
glucoge-6-phosphate dehydrogenase), heterocyclic oxidases (such as
uricase and xanthine oxidase), lactoperoxidase, microperoxidase,
and the like. Techniques for conjugating enzymes to antibodies are
described in O'Sullivan et al., Methods for the Preparation of
Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in
Methods in Enzym., Vol. 73, Langone and Van Vunakis, eds. (New
York: Academic Press, 1981), pp. 147-166.
[0296] Examples of enzyme-substrate combinations include:
[0297] (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase
as a substrate, wherein the hydrogen peroxidase oxidizes a dye
precursor (e.g., orthophenylene diamine (OPD) or
3,3',5,5'-tetramethyl benzidine hydrochloride (TMB));
[0298] (ii) alkaline phosphatase (AP) with para-nitrophenyl
phosphate as chromogenic substrate; and
[0299] (iii) .beta.-D-galactosidase (.beta.-D-Gal) with a
chromogenic substrate (e.g., p-nitrophenyl-.beta.-D-galactosidase)
or fluorogenic substrate
(4-methylumbelliferyl-.beta.-D-galactosidase).
[0300] Numerous other enzyme-substrate combinations are available
to those skilled in the art. For a general review of these, see,
for example, U.S. Pat. Nos. 4,275,149 and 4,318,980.
[0301] Sometimes, the label is indirectly conjugated with the
antibody. The skilled artisan will be aware of various techniques
for achieving this. For example, the antibody can be conjugated
with biotin and any of the three broad categories of labels
mentioned above can be conjugated with avidin, or vice versa.
Biotin binds selectively to avidin, and thus, the label can be
conjugated with the antibody in this indirect manner.
Alternatively, to achieve indirect conjugation of the label with
the antibody, the antibody is conjugated with a small hapten (e.g.,
digoxin) and one of the different types of labels mentioned above
is conjugated with an anti-hapten antibody (e.g., anti-digoxin
antibody). Thus, indirect conjugation of the label with the
antibody can be achieved.
[0302] In another embodiment of the invention, the anti-WISP
polypeptide antibody need not be labeled, and the presence thereof
can be detected using a labeled antibody which binds to the
anti-WISP polypeptide antibody.
[0303] The antibodies of the present invention may be employed in
any known assay method, such as competitive binding assays, direct
and indirect sandwich assays, and immunoprecipitation assays. Zola,
Monoclonal Antibodies: A Manual of Techniques (New York: CRC Press,
Inc., 1987), pp.147-158.
[0304] Competitive binding assays rely on the ability of a labeled
standard to compete with the test sample analyte for binding with a
limited amount of antibody. The amount of WISP protein in the test
sample is inversely proportional to the amount of standard that
becomes bound to the antibodies. To facilitate determining the
amount of standard that becomes bound, the antibodies preferably
are insolubilized before or after the competition, so that the
standard and analyte that are bound to the antibodies may
conveniently be separated from the standard and analyte which
remain unbound.
[0305] Sandwich assays involve the use of two antibodies, each
capable of bincting to a different immunogenic portion, or epitope,
of the protein to be detected. In a sandwich assay, the test sample
analyte is bound by a first antibody which is immobilized on a
solid support, and thereafter a second antibody binds to the
analyte, thus forming an insoluble three-part complex. See, e.g.,
U.S. Pat No. 4,376,110. The second antibody may itself be labeled
with a detectable moiety (direct sandwich assays) or may be
measured using an anti-immunoglobulin antibody that is labeled with
a detectable moiety (indirect sandwich assay). For example, one
type of sandwich assay is an ELISA assay, in which case the
detectable moiety is an enzyme.
[0306] For immunohistochemistry, the tumor sample may be fresh or
frozen or may be embedded in paraffin and fixed with a preservative
such as formalin, for example.
[0307] The antibodies may also be used for in vivo diagnostic
assays. Preferably, the antibody is labeled with a radionuclide
(such as .sup.111In, .sup.99Tc, .sup.14C, .sup.131I, .sup.125I,
.sup.3H, .sup.32P or .sup.35S) so that the tumor can be localized
using immunoscintiography.
[0308] Additionally, anti-WISP polypeptide antibodies may be useful
as antagonists to WISP polypeptide functions where WISP polypeptide
is upregulated in cancer cells or stimulates their prolilferation
or is upregulated in atheroscierotie-tissue. Hence, for example,
the anti-WISP polypeptide antibodies may by themselves or with a
chemotherapeutic agent or other cancer treatment or drug such as
anti-HER-2 antibodies be effective in treating certain forms of
cancer such as breast cancer, colon cancer, lung cancer, and
melanoma. Further uses for the antibodies include inhibiting the
binding of a WISP polypeptide to its receptor, if applicable, or to
an IGF, if applicable. For therapeutic use, the antibodies can be
used in the formulations, schedules, routes, and doses indicated
above under uses for the WISP polypeptides. In addition, anti-WISP
polypeptide antibody may be administered into the lymph as well as
the blood stream.
[0309] As a matter of convenience, the anti-WISP antibody of the
present invention can be provided as an article of manufacture such
as a kit. An article of manufacture containing a WISP polypeptide
or antagonists thereof useful for the diagnosis or treatment of the
disorders described above comprises at least a container and a
label. Suitable containers include, for example, bottles, vials,
syringes, and test tubes. The containers may be formed from a
variety of materials such as glass or plastic. The container holds
a composition that is effective for diagnosing or treating the
condition and may have a sterile access port (for example, the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle).
[0310] The active agent in the composition is the WISP polypeptide
or an agonist or antagonist thereto. The label on, or associated
with, the container indicates that the composition is used for
diagnosing or treating the condition of choice. The article of
manufacture may further comprise a second container comprising a
pharmaceutically-acceptable buffer, such as phosphate-buffered
saline, Ringer's solution, and dextrose solution. It may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use. The
article of manufacture may also comprise a second or third
container with another active agent as described above. A kit
format generally is a packaged combination of reagents in
predetermined amounts with instructions for performing the
diagnostic or treatment assay.
[0311] If the active agent is an antibody that is labeled with an
enzyme, the kit will include substrates and cofactors required by
the enzyme (e.g., a substrate precursor which provides the
detectable chromophore or fluorophore). In addition, other
additives may be included such as stabilizers, buffers (e.g., a
block buffer or lysis buffer), and the like. The relative amounts
of the various reagents may be varied widely to provide for
concentrations in solution of the reagents which substantially
maximize the sensitivity of the assay. Particularly, the reagents
may be provided as dry powders, usually lyophilized, including
excipients which on dissolution will provide a reagent solution
having the appropriate concentration.
[0312] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0313] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0314] Commercially available reagents referred to in the examples
were used according to manufacturer's instructions unless otherwise
indicated. The source of those cells identified in the following
examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, 10801 University
Blvd., Manassas, Va.
Example 1
Isolation of cDNA Clones Encoding Mouse WISP-1
[0315] Several putative WISP genes have been identified at the mRNA
level in a high-throughput PCR-select cDNA substraction experiment
carried out using a mouse mammary cell line (C57MG), which has been
transformed by a Wnt-1 retroviral vector and compared with the
parental cell line. The WISP family disclosed herein, including the
mouse WISP-1 gene, was induced only in the transformed cell line
C57MGWnt-1.
[0316] 1. Suppression Subtractive Hybridization
[0317] Mouse WISP-1 was isolated independently by Wnt-1
differential screening using suppression subtractive hybridization
(SSH), as described by Diatchenko et al., Proc. Natl. Acad. Sci.
USA, 93: 6025-6030 (1996). SSH was carried out using the
PCR-SELECT.RTM.R cDNA Subtraction Kit (Clontech Laboratories, Inc.)
according to the manufacturer's protocol. Driver double-stranded
(ds) cDNA was synthesized from 2 micrograms of polyA+ RNA isolated
from a mouse mammary cell line (C57MG), obtainable from a mouse
breast cancer myoepithelial cell line. This cell line is described
in Brown et al., Cell, 46: 1001-1009 (1986); Olson and Papkoff,
Cell Growth and Differentiation, 5: 197-206 (1994); Wong et al.,
Mol. Cell. Biol., 14: 6278-6286 (1994); and Jue et al., Mol. Cell.
Biol., 12: 321-328 (1992), and is responsive to Wnt-1 but not to
Wnt-4. Tester ds cDNA was synthesized from 2 micrograms of polyA+
RNA isolated from a transformed version of C57MG, called
C57MG/wnt-1.
[0318] The C57MG/wnt-1 mouse mammary derivative cell line was
prepared by first transforming the parent line with a Wnt-1
retroviral vector, pBabe Puro (5.1 kb). This vector has a 5' LTR,
packaging elements, a multiple cloning site, the
puromycin-resistance gene driven off the SV40 promoter, a 3' LTR,
and the bacterial elements for replication and ampicillin
selection. The vector was modified slightly for Wnt-1 cloning by
removing the HindIII site after the SV40 promoter and adding a
HindIII site to the multiple cloning site. Wnt-1 is cloned from
EcoRI-HindIII in the multiple cloning site. FIG. 13 shows a map of
the vector.
[0319] The transformed derivative cells were grown up in a
conventional fashion, and the final cell population was selected in
DMEM+10% FCS with 2.5 .mu.g/ml puromycin to stabilize the
expression vector.
[0320] PCR was performed using the Clontech kit, including the cDNA
synthesis primer (SEQ ID NO:40), adaptors 1 and 2 (SEQ ID NOS:41
and 42, respectively) and complementary sequences for the adaptors
(SEQ ID NOS: 43 and 44, respectively), PCR primer 1 (SEQ ID NO:45),
PCR primer 2 (SEQ ID NO:46), nested PCR primer 1 (SEQ ID NO:47),
nested PCR primer 2 (SEQ ID NO:48), control primer G3PDH5' primer
(SEQ ID NO:49), and control primer G3PDH3' primer (SEQ ID NO:50),
shown in FIG. 14.
[0321] Products generated from the secondary PCR reaction were
inserted into the cloning site region of pGEM-T vector (Promega),
shown in FIG. 15 (SEQ ID NOS: 51 and 52 for 5' and 3' sequences,
respectively). Plasmid DNAs were prepared using the WIZARD
MINIPREP.TM. Kit (Promega). DNA sequencing of the subcloned PCR
fragments was performed manually by the chain termination reaction
(SEQUENASE 2.0.TM. Kit, Pharmacia). Nucleic acid homology searches
were performed using the BLAST program noted above.
[0322] A total of 1384 clones were sequenced out of greater than
5000 tound. A total of 1996 DNA templates were prepared. A program
was used to trim the vector off, and a different program used to
cluster the clones into two or more identical clones or with an
overlap of 50 bases the same., Then a BLAST was performed of a
representative clone from the cluster. Primers were designed for
RT-PCR to see if the clones were differentially expressed.
[0323] 2. Semi-Quantitative RT-PCR
[0324] One of the clones was clone 568 having 71 bp, which was
identified as encoding mouse WISP-1. There were six clones in this
cluster. The nucleotide sequence and putative amino acid sequence
of full-length mouse WISP-1 are shown in FIGS. 1A-1B (SEQ ID NOS: 9
and 12, respectively). RT-PCR primers were designed for confirming
differential expression, screening for full-length mouse clone, and
screening for the human clone. These primers were 568.PCR.top1
(nucleotides 909-932 of the full-length nucleotide sequence
encoding mouse WISP-1 (SEQ ID NO:9) of FIGS. 1A-1B) and
568.PCR.bot1 (nucleotides 955-978 of the full-length complementary
nucleotide sequence encoding mouse WISP-1 (SEQ ID NO:10) of FIGS.
1A-1B), which are as follows: TABLE-US-00001 (SEQ ID NO:100)
568.PCR.top1: 5'-CCAGCCAGAGGAGGCCACGAAC (SEQ ID NO:101)
568.PCR.bot1: 3'-TGTGCGTGGATGGCTGGGTTCATG
[0325] For the RT-PCR procedure, cell lines were grown to
subconfluence before extracting the RNA. Total RNA was extracted
using Stat-60.TM. (TEL-TEST.TM. B) per manufacturer's instructions.
First-strand cDNA was prepared from 0.1 .mu.g-3 .mu.g of total RNA
with the SUPERSCRIPT.TM. RT kit (Gibco, BRL). PCR amplification of
5 .mu.l of first-strand cDNA was performed in a 50-.mu.l PCR
reaction. The above primers were used to amplify first-strand cDNA.
As controls, primers corresponding to nucleotide positions 707-729
(sense; 5'-GTGGCCCATGCTCTGGCAGAGGG (SEQ ID NO:102)) or 836-859
(sense; 5'-GACTGGAGCAAGGTCGTCCTCGCC (SEQ ID NO:103)) and 1048-1071
(anti-sense; 5'-GCACCACCCACAAGGAAGCCATCC (SEQ ID NO:104)) of human
triosephosphate isomerase (huTPI) (Maquat et al., J. Biol. Chem.,
260: 3748-3753 (1985); Brown et al., Mol. Cell. Biol., 5: 1694-1706
(1985)) were used to amplify first-strand cDNA. For mouse
triosephosphate isomerase, primers corresponding to nucleotide
positions 433-456 (sense; 51-GACGAAAGGGAAGCCGGCATCACC (SEQ ID
NO:105)) or 457-480 bp (sense; 51-GAGAAGGTCGTGTTCGAGCAAACC (SEQ ID
NO:106)) and 577-600 bp (antisense; 51-CTTCTCGTGTACTTCCTGTGCCTG
(SEQ ID NO:107)) or 694-717 bp (antisense;
5'-CACGTCAGCTGGCGTTGCCAGCTC (SEQ ID NO:108)) were used for
amplification.
[0326] Briefly, 4 .mu.Ci of (.sup.32P-)CTP (3000 Ci/mmol) was added
to each reaction with 2.5 U of TAKARA EX TAQ.TM. (PanVera, Madison,
Wis.) and 0.2 .mu.M of each dNTP. The reactions were amplified in a
480 PCR THERMOCYCLER.TM. (Perkin Elmer) using the following
conditions: 94.degree. C. for 1 min., 62.degree. C. for 30 sec.,
72.degree. C. for 1 min, for 18-25 cycles. 5 .mu.l of PCR products
were electrophoresed on a 6% polyacrylamide gel. The gel was
exposed to film. Densitometry measurements were obtained using
ALPHA EASE VERSION 3.3a.TM. software (Alpha Innotech Corporation)
to quantitate the WISP-or TPI-specific gene products.
[0327] 3. Northern Blot Analysis
[0328] Adult multiple-tissue Northern blots (Clontech) and the
Northern blot of the C57MG parent and C57MG/Wnt-1 derivative
polyA+RNA (2 .mu.g/lane) were hybridized with a 70-bp mouse WISP-1
probe (amino acids 278 through 300 of FIG. 1A-1B;
QPEEATNFTLAGCVSTRTYRPKY; SEQ ID NO:109) generated using the primers
568.PCR.top1 and 568.pcr.bot1 noted above. The membranes were
washed in 0.1.times.SSC at 55-65.degree. C. and exposed for
autoradiography. Blots were rehybridized with a 75-bp synthetic
probe from the human actin gene. See Godowski et al., Proc. Natl.
Acad. Sci. USA, 86: 8083-8087 (1989) for a method for making a
probe with overlapping oligos, which is how the actin probe was
prepared.
[0329] 4. cDNA Library Screening
[0330] Clones encoding the full-length mouse WISP-1 were isolated
by screening a Xgt1O oligodT primed mouse embryo library (Clontech)
with the primers 568.PCR.top1 and 568.PCR.bot1 noted above. The
inserts of 13 of these clones were subcloned into PBLUESCRIPT.TM.
IISK+ and their DNA sequences determined by dideoxy DNA sequencing
on both strands.
[0331] 5. Results
[0332] The recently described technique of SSH combines a high
subtraction efficiency with an equalized representation of
differentially expressed sequences. This method is based on
specific PCR reactions that permit exponential amplification of
cDNAs which differ in abundance, whereas amplification of sequences
of identical abundance in two populations is suppressed. The SSH
technique was used herein to isolate genes expressed in a mouse
mammary myoepithelial cell transformed with Wnt-1 whose expression
is reduced or absent in the parental myoepithelial cell. The polyA
RNA extracted from both types of cells was used to synthesize
tester and driver cDNAs. The degree of subtraction efficiency was
monitored by Southern blot analysis of unsubtracted and subtracted
PCR products using a .beta.-actin probe. No .beta.-actin mRNA was
apparent in the subtracted PCR products, confirming the efficiency
of the subtraction.
[0333] The subtracted cDNA library was subcloned into a pGEM-T
vector for further analysis. A random sample of 1996 clones was
sequenced from the transformed colonies obtained. To determine if
the clones obtained were differentially expressed, PCR primers were
designed for selected clones and semi-quantitative RT-PCR and
Northern analyses were performed using mRNA from the mouse mammary
cell line and its derivative. It was found that expression of Wnt-1
in C57MG cells leads to elongated cell morphology and loss of
contact inhibition.
[0334] One clone (m568.19A) of those that fulfilled the criteria
for differential expression was found to encode full-length mouse
WISP-1. By both RT-PCR analysis and Northern analysis, it was found
that this clone provided an about three-fold induction in the Wnt-1
cell line over the parent cell line.
[0335] The cDNA sequence of this clone and deduced amino acid
sequence of full-length mouse WISP-1 are shown in FIGS. 1A-1B (SEQ
ID NOS:9 and 12, respectively) The sequence alignment of human and
mouse WISP-1 (SEQ ID NOS:4 and 12, respectively) is shown in FIG.
8. In situ analysis of the clone is presented below, along with
thymidine incorporation assay and angiostatic assay results.
[0336] This clone was placed in pRK5E, an E. coli-derived cloning
vector having a human cytomegalovirus intermediate early gene
promoter, an SV40 origin and polyA site, an sp6 transcription
initiation site, a human immunoglobulin splice acceptor, and
XhoI/NotI cDNA cloning sites. It is a progeny of pRK5D that has an
added SceI site. Holmes et al., Science, 253:1278-1280 (1991). Upon
transformation into JM109 cells, the plasmid rendered the cells
ampicillin resistant. Upon digestion with XbaI and BamHI, a 1140-bp
fragment was obtained, and the mouse insert size was 1122 base
pairs, from the ATG to the stop codon, including a 3' tag of six
histidines.
Example 2
Isolation of a cDNA Clone Encoding Mouse WISP-2
[0337] The cDNA for mouse WISP-2 was isolated independently by
Wnt-1 differential screening using the procedure described in
Example 1. The initial clone isolated was 318 bp in length and was
designated clone 1367. There were four clones in this cluster. The
clone was sequenced as described above and RT-PCR primers were
designed as follows: TABLE-US-00002 1367.pcr.top1: nucleotides
1604-1627 of FIGS. 2A-2B: (SEQ ID NO:110)
3'-GGTGTGAAGACCGTCCGGTCCCGG and 1367.pcr.bot1: nucleotides
1438-1461 of FIGS. 2A-2B: (SEQ ID NO:111)
5'-GTGT(3CCTTTCCTGATCTGAGAAC
After RT-PCR and Northern blot procedures were carried out as
described in Example 1 to confirm differential expression, a
five-fold induction in the Wnt-1 cell line was observed.
[0338] Clones encoding full-length mouse WISP-2 were isolated from
RNA library 211: C57MG/Wnt-1. mRNA for construction of this library
was isolated from the C57MG/Wnt-1 cell line described in Example 1.
The RNA was used to generate an oligo-dT-primed cDNA library in the
cloning vector pRK5E using reagents and protocols from Life
Technologies, Gaithersburg, Md. (SUPERSCRIPT PLASMID
SYSTEM.TM.).
[0339] In this procedure, the double-stranded cDNA was primed with
oligo dT containing a NotI site, linked with blunt-to-SalI
hemikinased adaptors, cleaved with NotI, sized to greater than 1000
bp appropriately by gel electrophoresis, and cloned in a defined
orientation into the XhoI/NotI-cleaved pRK5E vector. The library
was screened by colony hybridization with a probe 1367.50mer.1 of
bases 1463-1512 of FIGS. 2A-2B:
3'-GGGACGGGCCGACCCTTCTTAAAAGACCCTTGTACTTCTCTACCTTAGTG (SEQ ID
NO:112). The full-length mouse WISP-2 clone was obtained,
designated clone 1367.3.
[0340] The cDNA for mouse WISP-2, like the mouse WISP-1 molecule,
encodes a novel secreted protein that belongs to the CTGF family
and is the mouse homologue of SST DNA33473 of Example 4. (The
alignment of human and mouse WISP-2 (SEQ ID NOS: 16 and 20,
respectively) is shown in FIG. 9.) The mouse WISP-2 gene is 38%
identical in sequence to mouse wisp-1, disclosed in Example 1, but
lacks the C-terminal 95 amino acids thought to be involved in
dimerization and receptor binding. Mouse WISP-2 was highly
expressed in the lung. In-situ analysis of the clone is noted
below. The nucleotide sequence and putative amino acid sequence of
full-length mouse WISP-2 are shown in FIGS. 2A-2B (SEQ ID NOS:17
and 20, respectively). The putative signal sequence is from amino
acid positions 1 to 23 of SEQ ID:20.
[0341] The clone was inserted into pRK5E, described above. Upon
transformation of JM109 cells, the plasmid rendered the cells
ampicillin resistant, Upon digestion with BamHI and NotI, a 1770-bp
fragment was obtained, having a mouse insert of 756 bp from ATG to
the stop codon.
Example 3
Isolation of a cDNA Clone Encoding Human WISP-1
[0342] To isolate the full-length human clone corresponding to
m568.19A (mouse WISP-1), a human lung cDNA library (Clontech),
treated with the SUPERSCRIPT.TM. kit using the pRKSE vector as
described above, was screened with a 70-bp probe at low stringency
(20% formamide, 1.times.SSC, 55.degree. C. wash). The probe had the
sequence from nucleotides 909-978 of the full-length mouse WISP-1
nucleotide sequence of FIGS. 1A-1B, i.e., the sequence:
TABLE-US-00003 (SEQ ID NO:113)
51-CCAGCCAGAGGAGGCCACGAACTTCACTCTCGCAGGCTGTGTCAGCA
CACGCACCTACCGACCCAAGTAC
[0343] Only one clone was identified, hL.568.15A. The insert to
this clone was subcloned into pBLUESCRIPT.TM. IISK+ and its DNA
sequence determined by dideoxy DNA sequencing on both strands. This
clone was found to be missing about 280 amino acids. Hence, a new
probe (hu.568.50mer.1) was designed from clone 15A having the
nucleotides 750-799 of the full-length human WISP-1 nucleotide
sequence shown in FIGS. 3A and 3B, i.e., TABLE-US-00004 (SEQ ID
NO:114) 51-GCCCCTGGAGCCCTTGCTCCACCAGCTGCGGCCTGGGGGTCTCCACT CGG
This probe was used to screen a human fetal kidney cDNA library
(Clontech) treated with the SUPERSCRIPT.TM. kit using the pRK5E
vector as described above, by colony hybridization. A number of
clones were obtained by screening this human fetal kidney cDNA
library (clones without the A or B designation) or by screening a
human fetal kidney .lamda.gt10 library (clones with the A or B
designation) using the same probes described above. The inserts of
these clones were subcloned into PBLUESCRIPT.TM. IISK+ and their
DNA sequences determined by dideoxy DNA sequencing on both
strands.
[0344] Two of these clones, designated as 568.1A and 568.4A, have
their respective sequences (SEQ ID NOS:24 and 26) shown in FIGS. 27
and 29. These clones are missing the von Willebrand C1 domain, the
variable domain, and the thrombospondin 1 domain, and have a
frameshift, other clones, designated as 568.13, 568.39, 568.5A,
568.6B, and 568.7 (SEQ ID NOS:23, 25, 27, 28, and 29, respectively;
FIGS. 26, 28, and 30-32, respectively), were obtained that lack one
or more domains and/or short amino-acid stretches (e.g., an
8-amino-acid deletion) or contain additional short amino-acid
stretches and may contain introns or alternative splice
variants.
[0345] Two clones sharing a significant amount of sequence with the
full-length clone 568.38 were identified: 568.23 and 568.35. Human
clone 568.38 encoded the full-length human WISP-1. The nucleotide
sequence and putative amino acid sequence for clone 568.38 are
shown in FIGS. 3A-3C (SEQ ID NOS:1 and 4, respectively). The
aligning sequences of clones 568.38 and 568.35 differ from the
corresponding aligning sequences of clones 568.15A and 568.23 in
that the respective sequences of the latter two clones have an
isoleucine residue at amino acid position 184 of FIGS. 3A-3C,
whereas the respective corresponding sequences of clones 568.38 and
568.35 have a valine residue at this position. Further, the
aligning sequences of clones 568.35 and 568.38 differ from each
other in that the sequence of clone 568.35 has a serine residue at
amino acid position 202 of FIGS. 3A-3C, whereas the corresponding
sequence of clone 568.38 has an alanine residue at this
position.
[0346] The human WISP-1 polypeptide, by homology searching, is also
found to be a member of the CTGF family. The clone was placed in a
pRK5E plasmid as described above and deposited with the ATCC. Upon
transformation into JM109 cells, the plasmid rendered the cells
ampicillin resistant. Digestion with ClaI and EcoRV yielded a
1435-bp fragment with an insert size of 1104 basepairs from ATG to
the stop codon.
[0347] In situ hybridization of human WISP-1 was performed, with
the results given below. Northern analysis of human WISP-1 showed
high expression in adult heart tissue and ovary tissue, and in
fetal kidney tissue. Also presented below are thymidine
incorporation assay, gene amplification assay, and angiostatic
assay results.
[0348] The chromosomal location of the human WISP genes was
determined by radiation hybrid mapping using the Stanford G3.TM.
and the MIT Genebridge 4 Radiation Hybrid.TM. panels. WISP-1
resides at approximately 3.48 cR from the meiotic marker AFM259xc5
(LOD score 16.31) on the Genebridge map. This places WISP-1 in band
8q24.1 to 8q24.3, roughly four megabases distal to c-myc located at
chromosome band 8q24.12-8q24.13. Takahashi et al., Cytogenet. Cell
Genet., 57: 109-111 (1991). c-myc is a region that is a recurrent
site of amplification in non-small cell lung carcinoma.
Example 4
Isolation of a cDNA Clone Encoding Human PR0261 (Designated Herein
as Human WISP-2)
[0349] The extracellular domain (ECD) sequences (including the
secretion signal, if any) of from about 950 known secreted proteins
from the SWISS-PROT.TM. public protein database were used to search
expressed sequence tag (EST) databases. The EST databases included
public EST databases (e.g., GenBank) and a proprietary EST DNA
database (LIFESEQ.TM., Incyte Pharmaceuticals. Palo Alto, Calif.).
The search was performed using the computer program BLAST or BLAST2
(Altshul et al., Methods in Enzymology 266:460-480 (1996)) as a
comparison of the ECD protein sequences to a 6-frame translation of
the EST sequence. Those comparisons resulting in a BLAST score of
70 (or in some cases 90) or greater that did not encode known
proteins were clustered and assembled into consensus DNA sequences
with the program "phrap" (Phil Green, University of Washington,
Seattle, Wash.:
http://bozeman.mbt.washington.edu/phrap.docs/phrap.html).
[0350] A consensus DNA sequence was assembled relative to other EST
sequences using phrap. The EST sequences used (from Incyte) were
Nos. 2633736, 2118874, 360014, 2316216, 1985573, 2599326, 1544634,
2659601, 1319684, 783649, 627240, 1962606, 2369125, 939761,
1666205, 692911, 984510, 1985843, 2104709, and 2120142. This
consensus sequence is herein designated DNA30843 (see FIG. 5).
Based on the DNA30843 consensus sequence, oligonucleotides were
synthesized: 1) to identify by PCR a cDNA library that contained
the sequence of interest, and 2) for use as probes to isolate a
clone of the full-length coding sequence for PR0261 (human,
WISP-2). A pair of PCR primers, forward and reverse, were
synthesized having the respective sequences: TABLE-US-00005 (SEQ ID
NO:115) 5'-AAAGGTGCGTACCCAGCTGTGCC and (SEQ ID NO:116)
3'-TCCAGTCGGCAGAAGCGGTTCTGG.
[0351] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30843 sequence, which
probe has the sequence: TABLE-US-00006 (SEQ ID NO:117)
5'-CCTGGTGCTGGATGGCTGTGGCTGCTGCCGGGTATGTGCAGGGCGGG TGGG.
[0352] For screening several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification, as per Ausubel et al., Current Protocols in
Molecular Biology (Green Publishing Associates and Wiley
Interscience, N.Y., 1989), with the PCR primer pair identified
above. A positive library was then screened by colony hybridization
to isolate clones encoding PR0261 (human WISP-2) using the probe
oligonucleotide and one of the PCR primers.
[0353] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue. The cDNA libraries used to isolate the
cDNA clones were constructed by standard methods using commercially
available reagents such as those from Invitrogen, San Diego, Calif.
The cDNA was primed with oligo dT containing a NotI site, linked
with blunt-to-SalT-hemikinased adaptors, cleaved with NotI, sized
appropriately by gel electrophoresis, and cloned in a defined
orientation into a suitable cloning vector (such as pRK5B or pRK5D;
pRK5B is a precursor of pRK5D that does not contain the SfiI site;
see Holmes et al., Science, 253:1278-1280 (1991)) in the unique
XhoI and NotI sites.
[0354] DNA sequencing of the clones isolated as described above
gave the DNA sequence for PR0261 (herein designated as UNQ228
(DNA33473-seq min); SEQ ID NO:38), which begins at nucleotide 34 of
SEQ ID NO:13 (FIG. 4A-4B) and the derived amino acid sequence for
PR0261 (SEQ ID NO:16).
[0355] The entire nucleotide sequence encoding human WISP-2 is
shown in FIGS. 4A-4B (SEQ ID NO:13). This sequence contains a
single open reading frame with an apparent translational initiation
site at nucleotide positions 22-24 of SEQ ID NO:13 and ending at
the stop codon after nucleotide 770 of SEQ ID NO:13 (FIGS. 4A-4B).
The predicted polypeptide precursor is 250 amino acids long (FIGS.
4A-4B). The putative signal sequence spans from amino acid
positions 1 to 23 of SEQ ID NO:16. Clone UNQ228 (DNA33473-seq min)
has been deposited with ATCC and is assigned ATCC deposit no.
209391.
[0356] Analysis of the amino acid sequence of the full-length
PR0261 polypeptide suggests that portions of it possess significant
homology to CTGF, thereby indicating that PR0261 is a novel growth
factor.
[0357] The entire nucleotide sequence encoding human WISP-3 from
this clone is shown in FIGS. 6A-6C (SEQ ID NO:30) This sequence
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 46-48 of SEQ ID NO:30 and
ending at the stop codon after nucleotide 1161 of SEQ ID NO:30
(FIGS. 6A-6C). The predicted polypeptide precursor is 372 amino
acids long (FIGS. 6A-6C). The putative signal sequence is from
amino acid positions 1 to 33 of SEQ ID NO:33. Clone UNQ462
(DNA56350-1176-2) has been deposited with ATCC and is assigned ATCC
deposit no. 209706.
Example 5
Isolation of cDNA Clones Encoding Human WISP-3
[0358] In this example, the gene encoding WISP-3 was cloned twice
essentially in parallel. First, it was determined whether the
databases described above contained any new members of the WISP
family. Two EST homologies to the WISPs were found and both were
cloned. Full-length clones were isolated corresponding to each of
these EST homologies. The efforts resulted in two full-length
clones of the same gene (the original EST homologies had been from
distinct regions of the same gene). The first clone obtained was
designated as DNA56350 and the second as DNA58800.
DNA56350
[0359] Based on the sequence of INCYTE 3208053, a virtual DNA 48917
was obtained and oligonucleotides were synthesized for use as
probes to isolate a clone of the full-length coding sequence for
PRO956 (human WISP-3). A pair of PCR primers, forward and reverse,
were synthesized having the sequences: TABLE-US-00007 (SEQ ID
NO:118) 5'-GTCTTGTGCAAGCAACAAAATGGACTCC (SEQ ID NO:119)
3'-GACACAATGTAAGTCGGAACGCTGTCG
[0360] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the INCYTE sequence, which probe has the
sequence: TABLE-US-00008 (SEQ ID NO:120)
5'-GCTCCAGAACATGTGGGATGGGAATATCTAACAGGGTGACCAATGAA AAC
[0361] A human fetal kidney library primed with oligo dT containing
a XhoI-NotI size cut greater than 3700 kb was screened for a source
of a full-length clone by PCR amplification with the PCR primer
pair identified above. The positive library was then used to
isolate clones encoding PR0956 (human WISP-3) using the probe
oligonucleotide and one of the PCR primers.
[0362] DNA sequencing of the clone isolated as described above gave
the DNA sequence for PR0956 (herein designated as UNQ462 (SEQ ID
NO:30), and the derived amino acid sequence for PR0956 (SEQ ID
NO:33).
[0363] The entire nucleotide sequence encoding human WISP-3 from
this clone is shown in FIG. 6 (SEQ ID NO:30). This sequence
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 46-48 of SEQ ID NO:30 and
ending at the stop codon after nucleotide 1161 of SEQ ID NO:30
(FIG. 6). The predicted polypeptide precursor is 372 amino acids
long (FIG. 6). The putative signal sequence is from amino acid
positions 1 to 33 of SEQ ID NO:33. Clone UNQ462 (DNA56350-1176-2)
has been deposited with ATCC and is assigned ATCC deposit no.
209706.
[0364] Analysis of the amino acid sequence of the full-length
PR0956 polypeptide suggests that portions of it possess significant
homology to CTGF, thereby indicating that PRO956 is a novel growth
factor. This, clone has an additional methionine just 5' of the
first methionine in this clone. The amino acid sequence of this
clone is 42% homologous to that of human WISP-1, and 32% homologous
to that of human WISP-2.
[0365] In situ hybridization of human WISP-3 is shown below. Using
the mapping techniques set forth above, it was found that human
WISP-3 was localized to chromosome 6q22-6q23 and was linked to the
marker AFM211ze5 with a LOD score of 1000. WISP-3 is approximately
18 megabases proximal to CTGF and 23 megabases promimal to the
human cellular oncogene MYB, which are also located at 6q22-6q23.
Martinerie et al., Oncogene, 7: 2529-2534 (1992); Meese et al.,
Genes Chromosomes Cancer, 1: 88-94 (1989).
[0366] The clone was inserted into pRK5E, described above. Upon
transformation of JM109 cells, the plasmid rendered the cells
ampicillin resistant. Upon digestion with BamHI and NotI, a
fragment was obtained having a human insert from ATG to the stop
codon as indicated in FIG. 6.
DNA58800
[0367] Based on the sequence of HS142L7, a virtual DNA 56506 was
obtained and oligonucleotides were synthesized for use as probes to
isolate a clone of the full-length coding sequence for PR0790
(human WISP-3). To this end, a pair of PCR primers, forward and
reverse, were synthesized having the sequences: TABLE-US-00009 (SEQ
ID NO:121) 5'-CCTGGAGTGAGCCTGGTGAGAGA (SEQ ID NO:122)
3'-ACACTGGGTGTGTTTCCCGACATAACA
[0368] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the HS 142L7 sequence, which probe has
the sequence: TABLE-US-00010 (SEQ ID NO:123)
5'-TGGTTGCTTGGCACAGATTTTACAGCATCCACAGCCATCTCTCA
[0369] A human bone marrow library primed with oligo dT containing
a XhoI-NotI size cut of 1-3 kb was screened for a source of a
full-length clone by PCR amplification with the PCR primer pair
identified above. The positive library was then used to isolate
clones encoding PR0790 (human WISP-3) using the probe
oligonucleotide and one of the PCR primers.
[0370] DNA sequencing of the clone isolated as described above gave
the DNA sequence for PRO790 (SEQ ID NO:34), and the derived amino
acid sequence for PR0790 (SEQ ID NO:37).
[0371] The entire nucleotide sequence encoding human WISP-3 from
this clone is shown in FIGS. 7A-7C (SEQ ID NO:34). This sequence
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 16-18 of SEQ ID NO:34 and
ending at the stop codon after nucleotide 1077 of SEQ ID NO:34
(FIGS. 7A-7C). The predicted polypeptide precursor is 355 amino
acids long (FIGS. 7A-7C). The putative signal sequence spans from
amino acid positions 1 to 15 of SEQ ID NO:37. This clone
DNA58800-1176-2 has been deposited with ATCC and is assigned ATCC
deposit no. 209707.
[0372] Analysis of the amino acid sequence of the full-length
PRO790 polypeptide suggests that portions of it possess significant
homology to CTGF, thereby indicating that, like PRO956 which is a
splice variant thereof, PR0790 is a novel growth factor.
[0373] In situ hybridization of human WISP-3 is shown below.
[0374] The clone was inserted into pRK5E, described above. Upon
transformation of JM109 cells, the plasmid rendered the cells
ampicillin resistant. Upon digestion with BamHI and NotI, a
fragment was obtained having a human insert from ATG to the stop
codon as indicated in FIGS. 7A-7C.
Example 6
Use of WISP-Encoding DNA as a Hybridization Probe
[0375] The following method describes use of a nucleotide sequence
encoding a WISP polypeptide as a hybridization probe.
[0376] DNA comprising the coding sequence of full-length or mature
human WISP-1 (as shown in FIGS. 3A-3C, SEQ ID NOS:4 or 3,
respectively), or full-length or mature mouse WISP-1 (as shown in
FIGS. 1A-1B, SEQ ID NOS:12 or 11, respectively), or of full-length
or putative mature human WISP-2 (as shown in FIG. 4A-4B, SEQ ID
NOS:16 or 15, respectively), or full-length or putative mature
mouse WISP-2 (as shown in FIGS. 2A-2B, SEQ ID NOS:20 or 19,
respectively) is employed as a probe to screen for homologous DNAs
(such as those encoding naturally occurring variants of these
particular WISP proteins in human tissue cDNA libraries or human
tissue genomic libraries.
[0377] Hybridization and washing of filters containing either
library DNAs is performed under the following high-stringency
conditions. Hybridization of radiolabeled WIS P-polypeptide-derived
probe (such as UNQ228 (DNA33473-seq min)-derived probe) to the
filters is performed in a solution of 50% formamide, 5.times.SSC,
0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH
6.8, 2.times.Denhardt's solution, and 10% dextran sulfate at
42.degree. C. for 20 hours. Washing of the filters is performed in
an aqueous solution of 0.1.times.SSC and 0.1% SDS at 42.degree.
C.
[0378] DNAs having a desired sequence identity with the DNA
encoding a full-length, native-sequence WISP polypeptide can then
be identified using standard techniques known in the art.
Example 7
Expression of WISP Polypeptide in E. coli
[0379] This example illustrates preparation of an unglycosylated
form of WISP polypeptide by recombinant expression in E. coli.
[0380] The DNA sequence encoding WISP polypeptide is initially
amplified using selected PCR primers. The primers should contain
restriction enzyme sites which correspond to the restriction enzyme
sites on the selected expression vector. A variety of expression
vectors may be employed. An example of a suitable vector is pBR322
(derived from E. coli; see Bolivar et al., Gene, 2: 95 (1977))
which contains genes for ampicillin and tetracycline resistance.
The vector is digested with restriction enzyme and
dephosphorylated. The PCR-amplified sequences are then ligated into
the vector. The vector will preferably include sequences which
encode an antibiotic-resistance gene, a trp promoter, a polyhis
leader (including the first six STII codons, polyhis sequence, and
enterokinase cleavage site), the WISP-coding region, lambda
transcriptional terminator, and an argU gene.
[0381] The ligation mixture is then used to transform a selected E.
coli strain using the methods described in Sambrook et al., supra.
Transformants are identified by their ability to grow on LB plates,
and antibiotic-resistant colonies are then selected. Plasmid DNA
can be isolated and confirmed by restriction analysis and DNA
sequencing.
[0382] Selected clones can be grown overnight in liquid culture
medium such as LB broth supplemented with antibiotics. The
overnight culture may subsequently be used to inoculate a
larger-scale culture. The cells are then grown to a desired optical
density, during which the expression promoter is turned on.
[0383] After the cells are cultured for several more hours, the
cells can be harvested by centrifugation. The cell pellet obtained
by the centrifugation can be solubilized using various agents known
in the art, and the WISP polypeptide can then be purified using a
metal-chelating column under conditions that allow tight binding of
the protein.
Example 8
Expression of WISP Polypeptide in Mammalian Cells
[0384] This example illustrates preparation of a potentially
glycosylated form of WISI-polypeptide by recombinant expression in
mammalian cells.
[0385] The vector, pRK5E, was employed as the expression vector.
The appropriate DNA encoding WISP polypeptide was ligated into
pRK5E with selected restriction enzymes to allow insertion of the
DNA for WISP polypeptide using ligation methods as described in
Sambrook et al., supra. The resulting vectors were
pRK5E.h.WIG-1.568.38, pRK5E.m.WIG-1.568.6his, pRK5E-m.WIG-2.1367.3,
plasmid encoding human WISP-2, DNA56350-1176-2, and
DNA58800-1176-2, all deposited at the ATCC. These vectors are
conveniently referred to collectively as pRK5E.WISP in the general
description below.
[0386] In one embodiment, the selected host cells may be 293 cells.
Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue
culture plates in medium such as DMEM supplemented with fetal calf
serum and optionally, nutrient components and/or antibiotics. About
10 .alpha.g pRK5E.WISP DNA is mixed with about 1 .mu.g DNA encoding
the VA RNA gene (Thimmappaya et al., Cell, 31:543 (1982)) and
dissolved in 500 .mu.l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M
CaCl.sub.2. To this mixture is added, dropwise, 500 .mu.l of 50 mM
HEPES (pH 7.35), 280 mM NaCl, 1.5 MM NaPO.sub.4, and a precipitate
is allowed to form for 10 minutes at 25.degree. C. The precipitate
is suspended and added to the 293 cells and allowed to settle for
about four hours at 37.degree. C. The culture medium is aspirated
off and 2 ml of 20% glycerol in phosphate-buffered saline (PBS) is
added for 30 seconds. The 293 cells are then washed with serum-free
medium, fresh medium is added, and the cells are incubated for
about 5 days.
[0387] Approximately 24 hours after the transfections, the culture
medium is removed and replaced with culture medium (alone) or
culture medium containing 200 .mu.Ci/ml .sup.35S-cysteine and 200
.mu.Ci/Ml .sup.35S-methionine. After a 12-hour incubation, the
conditioned medium is collected, concentrated on a spin filter, and
loaded onto a 15% SDS gel. The processed gel may be dried and
exposed to film for a selected period of time to reveal the
presence of the WISP polypeptide. The cultures containing
transfected cells may undergo further incubation (in serum-free
medium) and the medium is tested in selected bioassays.
[0388] In an alternative technique, the WISP polypeptide may be
introduced into 293 cells transiently using the dextran sulfate
method described by Somparyrac et al., Proc. Natl. Acad. Sci.,
12:7575 (1981). 293 cells are grown to maximal density in a spinner
flask and 700 .mu.g pRK5E.WISP DNA is added. The cells are first
concentrated from the spinner flask by centrifugation and washed
with PBS. The DNA-dextran precipitate is incubated on the cell
pellet for four hours. The cells are treated with 20% glycerol for
90 seconds, washed with tissue culture medium, and re-introduced
into the spinner flask containing tissue culture medium, 5 .mu.g/ml
bovine insulin, and 0.1 .mu.g/ml bovine transferrin. After about
four days, the conditioned media are centrifuged and filtered to
remove cells and debris. The sample containing expressed WISP
polypeptide can then be concentrated and purified by any selected
method, such as dialysis and/or column chromatography.
[0389] In another embodiment, the WISP polypeptide can be expressed
in CHO cells. The pRK5E.WISP can be transfected into CHO cells
using known reagents such as CaPO.sub.4 or DEAE-dextran. As
described above, the cell cultures can be incubated, and the medium
replaced with culture medium (alone) or medium containing a
radiolabel such as .sup.35S-methionine. After determining the
presence of the WISP polypeptide, the culture medium may be
replaced with serum-free medium. Preferably, the cultures are
incubated for about 6 days, and then the conditioned medium is
harvested. The medium containing the expressed WISP polypeptide can
then be concentrated and purified by any selected method.
[0390] Epitope-tagged WISP polypeptide may also be expressed in
host CHO cells. The WISP polypeptide may be subcloned out of the
pRK5 vector. Suva et al., Science, 237: 893-896 (1987) EP 307,247
published Mar. 15, 1989. The subclone insert can undergo PCR to
fuse in-frame with a selected epitope tag such as a poly-his tag
into a baculovirus expression vector. The poly-his-tagged WISP
polypeptide insert can then be subcloned into a SV40-driven vector
containing a selection marker such as DHFR for selection of stable
clones. Finally, the CHO cells can be transfected (as described
above) with the SV40-driven vector. Labeling may be performed, as
described above, to verify expression. The culture medium
containing the expressed poly-His-tagged WISP can then be
concentrated and purified by any selected method, such as by
Ni.sup.2+-chelate affinity chromatography.
[0391] In particular, mouse WISP-1 cDNA for insertion into
mammalian expression vectors was created via PCR using clone
m568.19A (see above) pure phage DNA as template and using as
primers m.568.pcr.top4 (5'-TGACTTCCAGGCATGAGGTGGCTCCTG; SEQ ID
NO:124) and m.568.pcr.bot3 (5'-ATTGGCAATCTCTTCGAAGTCAGGGTAAGATTCC;
SEQ ID NO:125) for the 6-his version, or m.568.pcr.top4 (SEQ ID
NO:124) and 568.pcr.bot5, which has a 3'-terminal XbaI site
(5'-GGTACGTCTAGACTAATTGGCAATCTCTTCGAAGTCAGGG; SEQ ID NO:126) for
the non-his version. The insert integrity was confirmed by
sequencing and analyzed. The PCR was run using Pfu polymerase and
the conditions were: TABLE-US-00011 temp. time denaturation
94.degree. C. 1 min annealing 62.degree. C. 30 sec extension
72.degree. C. 1.5 min # of cycles: 25
[0392] For transient expression in 293 cells analyzed by Western
blot, the above inserts were ligated into the pRK5 vector referred
to above at the BamHI/XbaI site using the BOEHRINGER MANNHEIM.TM.
rapid ligation kit. The resulting plasmids were designated
pRK5.mu.WISP-1.6his and pRK5.mu.WISP-1.nohis for the His-tagged and
non-His-tagged inserts, respectively. Then the 293 cells were
plated and allowed to reach approximately 85% confluency overnight
(37.degree. C./5% CO.sub.2). The plated cells were transfected with
plasmid DNA pRK5.mu.WISP-1.6his or pRK5.mu.WISP-1.nohis by using
lipofectamine (Gibco BRL) at a 4.5:1 lipid:DNA ratio.
[0393] Transfection efficiency (>70% usually) was monitored
using a GFP expression plasmid (pGREEN LANTERN.TM. from Gibco BRL).
Approximately 5 hours post-transfection, the medium was changed to
fresh SF media (50:50 with 1.times.L-Glu and 1.times.P/S) for
protein production. The conditioned media was allowed to accumulate
for specified amounts of time (depending on the experiment) before
harvesting.
[0394] The medium was harvested and concentrated in the presence of
1 mM PMSF using the CENTRICON-10.TM. concentrator. The
concentrated, conditioned media (usually 1.5 ml) was then bound to
Ni-.sup.++ NTA agarose beads (Qiagen) for 2 hours (for the
His-tagged version only). Protein was eluted from the beads by
boiling for 10 minutes in 2.times.SDS loading buffer (Novex) with
or without beta-mercaptoethanol for reduced vs. non-reduced
protein, respectively.
[0395] The protein was visualized by SDS-PAGE using 4-20% gradient
TRIS-glycine gels, 10-wells, 1 mm thickness (Novex). Gels ran at
125 volts (constant) for 95 minutes. Western transfer was achieved
using a NOVEX.TM. transfer apparatus to PVDF membranes (Novex) at
200 mA (constant) for 45 minutes. The blots were blocked for a
minimum of 1 hour at room temperature in blocking buffer
(PBS+TWEEN-20.TM. (0.5%), 5% dry milk, and 3% goat serum). Blots
were incubated in primary antibody (for His-tagged protein:
INVITROGEN.TM. anti-his (C-terminal)-HRP-conjugated antibody or for
the non-His version: polyclonal anti-murine WISP-1 antibody
prepared as described below) at a 1:2000 dilution in fresh blocking
buffer for 1 hour at room temperature. The non-His-tagged protein
blots were incubated in secondary antibody (PIERCE.TM. goat
anti-rabbit IgG(H+L) HRP conjugated) diluted 1:2000 in fresh
blocking buffer. The blots were incubated in chemiluminescent
substrate (ECL.TM. from Amersham or SUPERSIGNAL.TM. from Pierce)
for 1 minute before exposing to film.
[0396] For transient expression analyzed by antibody staining, 293
cells were cultured, plated, and transfected as described above.
The cells were fixed to culture dishes for 2 minutes in 1:1
methanol:acetone solution. Fixed cells were then incubated in
primary antibody (for His-tagged protein: INVITROGEN.TM. anti-his
(C-term)-HRP-conjugated antibody or for the non-His version:
polyclonal anti-murine WISP-1 antibody prepared as described below)
diluted 1:1000 in PBS with 10% fetal bovine serum for 2 hours. The
non-His-tagged protein blots were then incubated in secondary
antibody (PIERCE.TM. goat anti-rabbit IgG(H+L) HRP conjugated)
diluted 1:150 in PBS with 10% fetal bovine serum for 1 hour. The P
incubation was in color reagent substrate for HRP for up to 1 hour
(1.0% O-dianisidine-saturated ETOH, 0.01% hydrogen peroxide in
PBS).
[0397] For stable expression of mouse WISP-1 in mammalian cells,
the starting vector employed was pRK5.CMV.puro-dhfR, the sequence
of which is shown in FIGS. 16A-16D. This vector has two SAR
sequences cloned into KpnI, SapI sites of the SVIDS, splice-donor
vector, and has the pSVI backbone with the pRK5 cloning linker
(pSV15) and the intron made from pSVI.WTSD.D by adding a
linearization linker ML) into the HpaI site. The sequence is edited
to include changes in vector puc 118 backbone derived from the
sequence of pRK5 and includes a four-base insertion after MCS
characteristic of the SVI vector.
[0398] The above inserts were ligated into pRK5.CMV.puro-dhfR at
the BamHI/XbaI site using the BOEHRINGER MANNHEIM.TM. rapid
ligation kit, producing pRK5.CMV.puro-dhfR.mu.WISP-1.6his or
pRK5.CMV.puro-dhfr.mu.WISP-1.nohis. This construct allows for
stable selection of expressing cells using either puromycin (2
.mu.g/ml in 293 cells or 10 .mu.g/ml in CHO-DP12 cells) or the DHFR
deletion in the CHO-DP12 line, which allows for subsequent
amplification in methotrexate. Isolated colonies representative of
stably transfected cells were picked, cultured under selective
pressure, and analyzed by antibody staining or Western blot as
described above.
Example 9
Expression of WISP Polypeptide in Yeast
[0399] The following method describes recombinant expression of a
WISP polypeptide in yeast.
[0400] First, yeast expression vectors are constructed for
intracellular production or secretion of a WISP polypeptide from
the ADH2/GAPDH promoter. DNA encoding a WISP polypeptide and the
promoter is inserted into suitable restriction enzyme sites in the
selected plasmid to direct intracellular expression. For secretion,
DNA encoding a WISP polypeptide can be cloned into the selected
plasmid, together with DNA encoding the ADH2/GAPDH promoter, a
native WISP signal peptide or other mammalian signal peptide or
yeast alpha-factor or invertase secretory signal/leader sequence,
and linker sequences (if needed) for expression.
[0401] Yeast cells, such as yeast strain AB110, can then be
transformed with the expression plasmids described above and
cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by precipitation with 10%
trichloroacetic acid and separation by SDS-PAGE, followed by
staining of the gels with Coomassie Blue stain.
[0402] Recombinant WISP polypeptide can subsequently be isolated
and purified by removing the yeast cells from the fermentation
medium by centrifugation and then concentrating the medium using
selected cartridge filters. The concentrate containing the WISP
polypeptide may further be purified using selected column
chromatography resins.
Example 10
Expression of WISP Polypeptide in Baculovirus-Infected Insect Cells
and Purification Thereof
[0403] The following method describes recombinant expression of a
WISP polypeptide in baculovirus-infected insect cells, and
purification thereof.
General
[0404] The sequence coding for WISP polypeptide is fused upstream
of an epitope tag contained within a baculovirus expression vector.
Such epitope tags include poly-His tags and immunoglobulin tags
(like Fc regions of IgG). A variety of plasmids may be employed,
including plasmids derived from commercially available plasmids
such as pVL1393 (Novagen). Briefly, the sequence encoding WISP
polypeptide or the desired portion of the coding sequence (such as
the sequence encoding the mature protein if the protein is
extracellular) is amplified by PCR with primers complementary to
the 5' and 3' regions. The 5' primer may incorporate flanking
(selected) restriction enzyme sites. The product is then digested
with those selected restriction enzymes and subcloned into the
expression vector.
[0405] Recombinant baculovirus is generated by co-transfecting the
above plasmid and BACULOGOLD.TM. virus DNA (Pharmingen) into
Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using
lipofectin (commercially available from Gibco-BRL). After 4-5 days
of incubation at 28.degree. C., the released viruses are harvested
and used for further amplifications, viral infection and protein
expression are performed as described by O'Reilley et al.,
Baculovirus Expression Vectors: A Laboratory Manual (oxford: Oxford
University Press, 1994).
[0406] Expressed poly-His-tagged WISP polypeptide can then be
purified, for example, by Ni.sup.2--chelate affinity chromatography
as follows. Extracts are prepared from recombinant virus-infected
Sf9 cells as described by Rupert et al., Nature, 362:175-179
(1993). Briefly, Sf9 cells are washed, resuspended in sonication
buffer (25 mL HEPES, pH 7.9; 12.5 mm MgCl.sub.2; 0.1 mM EDTA; 10%
glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20
seconds on ice. The sonicates are cleared by centrifugation, and
the supernatant is diluted 50-fold in loading buffer (50 mM
phosphate, 300 mM NaCl, 10% glycerol, pH 7.8), and filtered through
a 0.45 .mu.m filter. A Ni.sup.2--NTA agarose column (commercially
available from Qiagen) is prepared with a bed volume of 5 mL,
washed with 25 mL of water, and equilibrated with 25 mL of loading
buffer. The filtered cell extract is loaded onto the column at 0.5
mL per minute. The column is washed to baseline A.sub.280 with
loading buffer, at which point fraction collection is started.
Next, the column is washed with a secondary wash buffer (50 mM
phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes
non-specifically bound protein. After reaching A.sub.280 baseline
again, the column is developed with a 0 to 500 mM imidazole
gradient in the secondary wash buffer. One-mL fractions are
collected and analyzed by SDS-PAGE and silver staining or Western
blot with Ni.sup.2+-NTA-conjugated to alkaline phosphatase
(Qiagen). Fractions containing the eluted His.sub.10-tagged WISP
polypeptide are pooled and dialyzed against loading buffer.
[0407] Alternatively, purification of the IgG-tagged (or Fc-tagged)
WISP polypeptide can be performed using known chromatography
techniques, including, for instance, Protein A or protein G column
chromatography.
Specific
[0408] 1. Expression
[0409] In particular, mouse WISP-1 polypeptide was expressed in a
baculovirus expression system similar to that described above using
as the baculovirus transfer vector pb.PH.mu.568.9.IgG.baculo or
pbPH.mu.568.8his.baculo. FIGS. 17A-17D show the sequence (SEQ ID
NO:54) of plasmid pb.PH.IgG, which was used to prepare
pb.PH.mu.568.9.IgG.baculo. FIGS. 18A-18D show the sequence (SEQ ID
NO:55) of plasmid pbPH.His.c, which was used to prepare
pbPH.mu.568.8his.baculo.
[0410] Both of these baculovirus transfer vectors are based on
pVL1393 (Pharmingen), which has neither the His nor Fc tags. The
pb.PH.IgG vector (FIG. 17) allows the expression of foreign
proteins under control of the AcNPV polyhedrin promoter, which is
active in the very late phase of virus infection. The foreign
protein can be expressed as a C-terminal human IgG fusicn protein.
The His(b)-tag will not be translated as a result of the IgG stop
coc'-.on just 51 of the His(8)-tag. The sequence encoding the
foreign protein should Le inserted as a 3' blunt-ended fragment
into the unique StuI site preceeding the His-taq. In that case an
additional proline residue will be added. The 5' site can be either
BamHI, EcoRI, NotI, NcoI, and NheI.
[0411] The IgG vector was constructed by NdeI digestion of the
pVL1393.IgG plasmid followed by Klenow treatment to fill in the
sticky end site. This is followed by a NcoI digest and insertion
into the pbPH.His.c.times.NcoI/SmaI-digested vector.
[0412] The sequence of pbPH.His.c shown in FIGS. 18A-18D contains
the backbone sequence of the vector pVL1392, which contains
approximately the EcoRI/XmaIII fragment of AcMNPV C-6, from
position 0.0 to 5.7 mu. Possee et al., Virology, 185: 229-241
(1991). It allows the expression of foreign proteins under control
of the Autographa californica nuclear polyhedrosis virus (AcNPV)
polyhedrin gene promoter, which is active in the very late phase of
virus infection.
[0413] The foreign protein can be expressed as a C-terminally His-
or a IgG (Fc region only)-tagged protein. The sequence encoding the
foreign protein should be inserted as a 31-blunt-ended fragment
into the unique SmaI site preceding the His-tag or the SluI site
for IgG. In that case an additional glycine residue will be added
for His tags and a proline will be added for IgG tags. The 5' site
can be either BamHI, NotI, EcoRI, or NcoI. BamHI was used for
both.
[0414] The vectors were constructed by inserting a PCR insert into
BamHIISmaI for the His vector and BamHIIStuj for the IgG vector.
The PCR insert was made using 5'-phosphorylated primers as follows:
m.568.pcr.top6 (5'-TTTCCCTTTGGATCCTAAACCAACATGAGGTGGCTCCTGCCC; SEQ
ID NO:127) and m.568.pcr.bot3 (SEQ ID NO:125), 5' phosphorylated. A
twenty-cycle PCR reaction with Pfu polymerase enzyme was performed
using the following conditions: 1 min at 95.degree. C., 30 sec at
60.degree. C., 3.5 min at 72.degree. C. The PCR product was
purified with QIAQUICK.TM. and digested with BamHI at 37.degree. C.
for 1 hr. The digested PCR insert was ligated into the digested
vector using a 1:3 ratio of insert to vector with 1 pl T4 DNA
ligase (Bio Labs). ULTRA MAX.TM. DH5a FT competent cells, 100 pl,
(Gibco BRL Cat #10643-013) were added to the ligation product, and
the mixture was incubated on ice for 30 min, followed by a heat
shock at 42.degree. C. for 45 sec. Individual colonies were picked
and miniscreen DNA was prepared using QIA PREpTl (Qiagen).
Construct sequencing was performed using ABI Prism's dRHODAMINE
DYE.TM. terminator cycle sequencing.
[0415] The plasmid pb.PH.IgG has a polylinker
BamHI-NotI-EcoRI-NcfI-SrfI-StuI-(IgG Fc region
only)-Stop-XbaI-SpeI-PslI-BglII. The location of particular regions
in this plasmid is as follows: Insertion of polylinker/foreign
gene: 4129-4912 (BamHI-BglII), polh coding: 4913-5479, ORF 1629:
7134-4820; ORF 588 (PK1): 7133-7723; ColEI origin of replication:
-7973-8858, and ampicillin coding: 9779-8230. The plasmid
pbPH.His.c has a polylinker BamHI-NotI-EcoRI-NcoI-SrfI-SmaI-(His
8)-Stop-XbaI-SpeI-PstI-BglII. The NcoI site of pbPH.His.c resides
within a Kozak sequence. The location of particular regions in this
plasmid is as follows: ORF 504 (PTP): 76-582, ORF 984 (ORF2):
1600-614, ORF 453 (ORF3): 2323-1868, conotoxin: 1818-1657, ORF 327
(ORF4): 2352-2681, ORF 630 (lef-2): 2662-3294, ORF 603: 3937-3332,
ORF polh: 4093 (mutated codon ATG/'ATT), insertion of
polylinker/foreign gene: 4129-4218 (BamHI-BglII), polh coding:
4224-4790, ORF 1629: 6445-4820, ORF 588 (PK1): 6444-7034, ColEI
origin of replication: 7284-8169, and ampicillin
coding:9090-8230.
[0416] The mouse WISP-1 cDNA disclosed herein was inserted into the
vectors pbPH.His.c and pb.PH.IgG to produce the respective
expression plasmids by creating a 3' blunt-ended fragment for
cloning into the unique SmaI site preceding the His-tag or IgG-tag.
An additional glycine residue was added to the His protein
produced. An additional proline was added to the IgG protein. The
51 site of the cDNA insert was BamHI.
[0417] 2. Purification
[0418] For purification purposes, either a poly-His tag or the Fc
portion of human IgG was added to the C-terminal coding region of
the cDNA before expression. The conditioned media from the
transfected cells (0.5 to 2 L) was harvested by centrifugation to
remove the cells and filtered through 0.22 micron filters. For the
poly-His-tagged constructs, the protein was purified using a
Ni*2-NTA column (Qiagen). Before purification, imidazole was added
to the conditioned media to a concentration of 5 mM. The
conditioned media was pumped onto a 6-ml Ni*2-NTA column
equilibrated in 20 mM HEPES, pH 7.4, buffer containing 0.3 M NaCl
and 5 mM imidazole at a flow rate of 4-5 ml/min at 4.degree. C.
After loading, the column was washed with additional equilibration
buffer and the protein was eluted with equilibration buffer
containing 0.25 M imidazole. The highly purified protein was
subsequently desalted into a storage buffer containing 10 MM HEPES,
0.14 M NaCl, and 4% mannitol, pH 6.8, with a 25 ml G25
SUPERFINE.TM. (Pharmacia) column and stored at -80.degree. C.
[0419] Immunoadhesin (Fc-containing) constructs of WISP-1 protein
were purified from the conditioned media as follows. The
conditioned media was pumped onto a 5-ml Protein A column
(Pharmacia) which had been equilibrated in a 20 mm Na phosphate
buffer, pH 6.8. After loading, the column was washed extensively
with equilibration buffer before elution with 100 mM citric acid,
pH 3.5. The eluted protein was immediately neutralized by
collecting 1-ml fractions into tubes containing 275 uL of 1 M Tris,
pH 9, buffer. The highly purified protein was subsequently desalted
into storage buffer as described above for the poly-His-tagged
proteins. The homogeneity of the protein was assessed by SDS
polyacrylamide gels and by N-terminal amino acid sequencing by
Edman degradation.
Example 11
Axis Duplication Assay
[0420] Xenopus embryos were injected with human WISP-2 mRNA into
either a presumptive ventral or presumptive dorsal vegetal
blastomere at the 8- to 16-cell stage to overexpress locally the
encoded protein and assay for its developmental effects. The
methods used are described in Sokol et al., Cell, 67: 741-752
(1991).
[0421] More specifically, for synthesis of capped RNA, human WISP-2
and mouse Wnt-1 cDNAs were cloned into the pGEMHE vector (gift of
Dr. Todd Evans, AECOM) to prepare pGEMHE.hu.WISP-2.8H and
pGEMHE.mu.Wnt-1, respectively. The constructs were linearized at
the 3' end using the SphI restriction enzyme. Capped RNAs were
synthesized using AMBION's T7 MESSAGEMACHINE.TM. RNA synthesis
kit.
[0422] For obtaining mature oocytes, an adult female Xenopus laevis
was injected with 200 I.U pregnant mare serum 3 days before use.
The night before the experiment, the female frog was injected with
800 I.U of human chorionic gonadotropin. Fresh oocytes were
squeezed from female frogs the next morning. In vitro fertilization
of oocytes was performed by mixing oocytes with minced testes from
a sacrificed male frog. Fertilized eggs were dejellied with 2%
cysteine (pH 7.8) for 10 minutes. Dejellied eggs were washed once
with distilled water and transferred to 0.1.times.Modified Barth's
Solution (MBS) (Methods in Cell Biology, Volume 36, Xenopus laevis:
Practical uses in Cell and Molecular Biology, Kay and Peng, Eds
(New York: Academic Press, 1991)) with 5% Ficoll. Eggs were lined
on injection trays which contained 0.1.times.MBS with 5% Ficoll for
injection. After injection, embryos were kept in 0.1.times.MBS in
an 18.degree. C. incubator. Embryos were staged according to
Nieuwkoop and Faber, Normal Table of Xenopus laevis:
(Daudin)-(Amsterdam: North-Holland, 1967).
[0423] For animal cap assays, embryos were injected at the 2-cell
stage with 1 ng of capped RNA, and animal caps were isolated at
stage 8 and cultured in 1.times.MMR for another 24 hours for the
RT-PCR assay. Total RNA was isolated from harvested animal caps
using a RNEASy.TM. kit (Qiagen). RNA samples (approximately 1 Pg)
were reverse transcribed using random hexamer and GIBCO:BRL
SUPERSCRIPT JIT' reverse transcriptase. The annealing temperature
for the PCR reactions was 55.degree. C. unless noted otherwise.
[0424] For axis duplication assays, embryos at the 8-cell stage
were injected with 1 ng capped RNA at either the dorsal or ventral
vegetal blastomere and incubated in 0.1.times.MBS for 72 hours.
[0425] The sequences of PCR primers used in this experiment were:
TABLE-US-00012 (SEQ ID NO:128) EF-1a.U: 5'-CAGATTGGTGCTGGATATGC
(SEQ ID NO:129) EF-1a.D: 5'-ACTGCCTTGATTACTCCTAC (SEQ ID NO:130)
noggin.U: 5'-AGTTGCAGATGTGGCTCT (SEQ ID NO:131) noggin.D:
5'-AGTCCAAGAGTCTCAGCA (SEQ ID NO:132) goosecoid.U:
5'-ACAACTGGAAGCACTGGA (SEQ ID NO:133) goosecoid.D:
5'-TCTTATTCCAGAGGAACC (SEQ ID NO:134) cardiac-actin.U:
5'-TCCCTGTACGCTTCTGGTCGTA (SEQ ID NO:135) cardiac-actin.D:
5'-TCTCAAAGTCCAAAGCCACATA (SEQ ID NO:136) NCAM.U:
5'-CACAGTTCCAGCAAATAC (SEQ ID NO:137) NCAM.D:
5'-GGAATCAGGCGGTACAGT
[0426] It was found that human WISP-2 can partially induce axis
duplication in this assay.
Example 12
Thymidine Incorporation Assay
[0427] In a (.sup.3H)-thymidine incorporation assay, 19 different
cell lines, including RAG (renal adenocarcinoma, mouse) and NRK-49F
(normal kidney fibroblasts, rat) cells, identified in Table I
below, were plated in 96-well plates at 3.times.104 in HGDMEM with
10% serum. Twenty four hours after plating, the medium was changed
to HGDMEM with 0.2% serum before adding the test proteins. WISP
proteins were added to a final concentration of approximately 3.6
ng/ul. Serial dilutions were made in a total volume of 70
.mu.l/well of fresh media. After 18 hr incubation at 37.degree. C.,
5 .mu.Ci/ml (.sup.3H)thymidine was added for 5 hrs. Medium was
aspirated and cells were removed with 1.times. trypsin onto a GF/C
filter using Packard's.TM. 96-well FILTERMATE 196.TM.. The filters
were dried and 40 .mu.l of scintillation fluid was added for
counting on a top count, microplate scintillation counter
(Packard).
[0428] The results are shown in Table I: TABLE-US-00013 TABLE I
IH-Thymidine Incorporation Assay Results mWISP-1- hWISP-1- hWISP-2-
Cell line Type ATCC No. IgG IgG IgG HT-29 adenocarcinoma HTB-38 No
change No change (human moderately well- colon) differentiated
Wi-Dr adenocarcinoma CCL-218 No change No change (human colon)
Calu-1 epidermoid HTB-54 inhibits .about.1.1x inhibits .about.1.2X
(human carcinoma grade lung, III, metastasis to pleura Calu-6
anaplastic HTB-56 No change stimulates (human carcinoma,
.about.1.4X lung) probably lung SK-MES-1 squamous HTB-58 No change
No change (human carcinoma, pleural lung) effusion A549 carcinoma
CCL-185 inhibits-1.5x inhibits .about.1.7X (human lung) H460 large
cell HTB-177 inhibits .about.1.4X inhibits .about.1.3X (human
carcinoma lung) SW900 squamous cell HTB-59 no change no change
(human carcinoma lung) MRC5 normal diploid CCL-171 no change no
change (human lung) IMR-90 normal diploid CCL-186 stimulates
stimulates (human .about.1.1x .about.1.5X lung) Wnt-I
myo-epithelial inhibits .about.2x C57 mg (mouse mammary gland) MLg
lung stimulates .about.4X (mouse lung) LL/2 lung carcinoma inhibits
.about.2x (mouse lung) JC (mouse carcinoma inhibits .about.2x
inhibits .about.3x mammary gland) N MuMG normal stimulates
.about.2X stimulates (mouse .about.1.4X mammary gland) NRK-49F
normal fibroblast stimulates .about.3X stimulates (rat kidney)
.about.3.5x RAG adenocarcinoma stimulates stimulates .about.3X
stimulates .about.4X (mouse .about.4.5X kidney) NIH/3T3 fibroblast
stimulates .about.3X (mouse embryo) UCLA-P3 inhibits .about.1.5x
inhibits .about.2X (human lung)
[0429] It is seen that WISP-1 and WISP-2 exhibit both stimulatory
and inhibitory effects on proliferation of normal and tumor cells,
depending on the cell line employed.
Example 13
Preparation of Antibodies that Bind WISP Polypeptide
[0430] 1. Polyclonal Antibodies
[0431] Polyclonal antisera were generated in female New Zealand
White rabbits against murine WISP-1 and human WISP-2. The antigens
used were proteins fused with histidine for murine WISP-1 and
proteins fused with the Fc portion of IgG for human WISP-2. The
same protocol was used for both proteins. Each protein was
homogenized with Freund's complete adjuvant for the primary
injection and with Freund's incomplete adjuvant for all subsequent
boosts. For the primary immunization and the first boost, 3.3 .mu.g
per kg body weight was injected directly into the popliteal lymph
nodes as described in Bennett et al., J. Biol. Chem., 266:
23060-23067 (1991) and "Production of Antibodies by Inoculation
into Lymph Nodes" by Morton Sigel et al. in Methods in Enzymology,
Vol. 93 (New York: Academic Press, 1983). For all subsequent
boosts, 3.3 .mu.g per kg body weight was injected into subcutaneous
and intramuscular sites. Injections were done every 3 weeks with
bleeds taken on the following two weeks.
[0432] 2. Monoclonal Antibodies
[0433] Techniques for producing monoclonal antibodies that can
specifically bind a WISP polypeptides are known in the art and are
described, for instance, in Goding, supra. Immunogens that may be
employed include purified WISP polypeptide, fusion proteins
containing WISP polypeptide, and cells expressing recombinant WISP
polypeptide on the cell surface. Selection of the immunogen can be
made by the skilled artisan without undue experimentation.
[0434] Mice, such as Balb/c, are immunized with the WISP immunogen
emulsified in complete Freund's adjuvant and injected
subcutaneously or intraperitoneally in an amount from 1 to 100
micrograms. Alternatively, the immunogen is emulsified in MPL-TDM
adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and
injected into the animal's hind foot pads. The immunized mice are
then boosted 10 to 12 days later with additional immunogen
emulsified in the selected adjuvant. Thereafter, for several weeks,
the mice may also be boosted with additional immunization
injections. Serum samples may be periodically obtained from the
mice by retro-orbital bleeding for testing in ELISA assays to
detect antibodies to WISP polypeptide.
[0435] After a suitable antibody titer has been detected, the
animals "positive" for antibodies can be injected with a final
intravenous injection of a WISP polypeptide. Three to four days
later, the mice are sacrificed and the spleen cells are harvested.
The spleen cells are then fused (using 35% PEG) to a selected
murine myeloma cell line such as P3X63AgU.1, available from ATCC,
No. CRL 1597, or x63-AgB.653 (Kearney et al., J. Immunology, 123:
1548 (1979)). The fusions generate hybridoma cells which can then
be plated in 96-well tissue culture plates containing HAT
(hypoxanthine, aminopterin, and thymidine) medium to inhibit
proliferation of non-fused cells, myeloma hybrids, and spleen cell
hybrids.
[0436] The hybridoma cells will be screened in an ELISA for
reactivity against a WISP polypeptide. Determination of "positive"
hybridoma cells secreting the desired monoclonal antibodies against
a WISP polypeptide is within the skill in the art.
[0437] The positive hybridoma cells can be injected
intraperitoneally into syngeneic Balb/c mice to produce ascites
containing the anti-WISP polypeptide monoclonal antibodies.
Alternatively, the hybridoma cells can be grown in tissue culture
flasks or roller bottles. Purification of the monoclonal antibodies
produced in the ascites can be accomplished using ammonium sulfate
precipitation, followed by gel-exclusion chromatography.
Alternatively, affinity chromatography based upon binding of
antibody to protein A or protein G can be employed.
[0438] Specifically, for each of the human WISP-1 antibodies, five
female Balb-c mice were pre-bled and then injected via their hind
foot pads with purified human WISP-1, tagged with the Fc portion of
IgG and emulsified prior to injection in MPL-TDM adjuvant (Ribi
Immunochemical Research, Hamilton, Mont.) in a 1:1 ratio of WISP
antigen to adjuvant. The dosing schedule for the WISP-1 immunogen
was as follows: TABLE-US-00014 Injection Date- Dose/Site
Dose/Animal Concentration Day 16 of month 1 50 .mu.l site 100
.mu.l/animal 6 .mu.g/animal Day 12 of month 2 50 .mu.l/site 100
.mu.l/animal 6 .mu.g/animal Day 21 of month 2 50 .mu.l/site 100
.mu.l/animal 6 .mu.g/animal Day 28 of month 2 50 .mu.l/site 100
.mu.l/animal 2 .mu.g/animal Day 4 of month 3 50 .mu.l/site 100
.mu.l/animal 2 .mu.g/animal Day 11 of month 3 50 .mu.l/site 100
.mu.l/animal 2 .mu.g/animal Day 18 of month 3 50 .mu.l/site 100
.mu.l/animal 2 .mu.g/animal Day 25 of month 3 50 .mu.l/site 100
.mu.l/animal 2 .mu.g/animal
[0439] For WISP-1, the mice were bled on Day 10 of month 4. After
the mice were bled, the monoclonal antibodies were made by
harvesting their spleens and by fusion as indicated above, using as
the murine myeloma cell line X63.AgB.653.
[0440] The five monoclonal antibodies generated to human WISP-1
are: TABLE-US-00015 10F2.2A7 gamma2b/kappa 10A9.2B1 gamma2a/kappa
8F7.1B1 gamma1/kappa 1H1.ID5 gamma1/kappa 2G7.2H4 gamma1/kappa
[0441] For WISP-2 monoclonal antibodies the same regimen is
employed except that purified human WISP-2 is used as immunogen in
the above protocol rather than purified human WISP-1 and the dosing
schedule for the WISP-2 immunogen is as follows: TABLE-US-00016
Injection Date- Dose/Site Dose/Animal Concentration Day 16 of month
1 50 .mu.l/site 100 .mu.l/animal 6 .mu.g/animal Day 21 of month 2
50 .mu.l/site 100 .mu.l/animal 1 .mu.g/animal Day 28 of month 2 50
.mu.l/site 100 .mu.l/animal 1 .mu.g/animal Day 4 of month 3 50
.mu.l/site 100 .mu.l/animal 1 .mu.g/animal Day 11 of month 3 50
.mu.l/site 100 .mu.l/animal 1 .mu.g/animal Day 18 of month 3 50
.mu.l/site 100 .mu.l/animal 1 .mu.g/animal Day 25 of month 3 50
.mu.l/site 100 .mu.l/animal 1 .mu.g/animal
Example 14
Uses of Antibodies that Bind WISP Polypeptide
[0442] 1. Cell Lines
[0443] The established human breast tumor cells BT474 and
MDA-MB-231 (which are available from ATCC) are grown in minimum
essential medium (Gibco, Grand Island, N.Y.) supplemented with 10%
heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, Utah),
sodium pyruvate, L-glutamine (2 mM), non-essential amino acids, and
2.times. vitamin solution and maintained at 37.degree. C. in 5%
CO.sub.2. Zhang et al., Invas. & Metas., 11:204-215 (1991);
Price et al., Cancer Res., 50:717-721 (1990).
[0444] 2. Antibodies
[0445] Anti-WISP-1 or anti-WISP-2 monoclonal antibodies that may be
prepared as described above are harvested with PBS containing 25 mM
EDTA and used to immunize BALB/c mice. The mice are given
injections i.p. of 107 cells in 0.5 ml PBS on weeks 0, 2, 5 and 7.
The mice with antisera that immunoprecipitated 32p-labeled Wnt-1
are given i.p. injections of a wheatgerm agglutinin-SEPHAROSE.TM.
(WGA)-purified Wnt membrane extract on weeks 9 and 13. This is
followed by an i.v. injection of 0.1 ml of the Wnt-1 preparation,
and the splenocytes are fused with mouse myeloma line X63-Ag8.653.
Hybridoma supernatants are screened for Wnt-1 binding by ELISA and
radioimmunoprecipitation. MOPC-21 (IgG1) (Cappell, Durham, N.C.) is
used as an isotype-matched control.
[0446] Additionally, the anti-ErbB2 IgG.sub.1.kappa. murine
monoclonal antibodies 4D5 (ATCC CRL 10463 deposited May 24, 1990)
and 7C2, specific for the extracellular domain of ErbB2, may be
used with the above antibodies. They are produced as described in
Fendly et al., Cancer Research, 50:1550-1558 (1990) and
WO89/06692.
[0447] 3. Analysis of Cell Cycle Status and Viability
[0448] Cells are simultaneously examined for viability and cell
cycle status by flow cytometry on a FACSTAR PLUS.TM. (Becton
Dickinson Immunocytometry Systems USA, San Jose, Calif.). Breast
tumor cells are harvested by washing the monolayer with PBS,
incubating cells in 0.05% trypsin and 0.53 mM EDTA (Gibco), and
resuspending them in culture medium. The cells are washed twice
with PBS containing 1% FBS and the pellet is incubated for 30
minutes on ice with 50 .mu.l of 400 .mu.M 7-aminoactinomycin D
(7AAD) (Molecular Probes, Eugene, Oreg.), a vital dye which stains
all permeable cells. Cells are then fixed with 1.0 ml of 0.5%
paraformaldehyde in PBS and simultaneously permeabilized and
stained for 16 hours at 4.degree. C. with 220 .mu.l of 10 .mu.g/ml
HOECHST 33342.TM. dye (also a DNA binding dye) containing 5% TWEEN
20.TM..
[0449] The data from 1.times.10.sup.4 cells are collected and
stored using LYSYS II.TM. software and analyzed using
PAINT-A-GATE.TM. software (Becton Dickinson). Darzynkiewica et al.,
Cytometry, 13:795-808 (1992); Picker et al., J. Immunol.,
150:1105-1121 (1993). The viability and percentage of cells in each
stage of the cell cycle are determined on gated single cells using
7AAD and Hoechst staining, respectively. (Cell doublets are
excluded by pulse analysis of width vs. area of the Hoechst
signal.) Cell numbers are determined using a hemocytometer.
[0450] 4. DNA Synthesis ((.sup.3H)-Thymidine Incorporation
Assay)
[0451] The assay was performed exactly as described in Example 12,
except that the WISP polypeptides used as test proteins were
replaced by the polyclonal antibodies generated in New Zealand
White rabbits against murine WISP-1 and human WISP-2 described in
Example 13, and not all the cell lines in Example 12 were tested.
The results are shown in Table II: TABLE-US-00017 TABLE II
.sup.3H-Thymidine Incorporation Assay Results Cell line Type ATCC
No. pAB.mWISP-1 pAB.mWISP-2 HT-29 adenocarcinoma HTB-38 No change
No change (human moderately colon) well-differentiated Wi-Dr
adenocarcinoma CCL-218 No change No change (human colon) N MuMG
normal stimulates .about.3X (mouse mammary gland) NRK-49F normal
fibroblast stimulates .about.2X (rat kidney) RAG adenocarcinoma
stimulates .about.4.X (mouse kidney) NIH/3T3 fibroblast stimulates
.about.2X (mouse embryo)
[0452] It is seen that the polyclonal antibodies to mouse WISP-1
and to human WISP-2 exhibited both stimulatory and inhibitory
effects on proliferation of normal and tumor cells, depending on
the cell line employed.
[0453] 5. Affinity of Binding to Putative Receptor
[0454] Radioiodinated anti-WISP-1 and anti-WISP-2 antibodies are
prepared by the IODOGEN.TM. method. Fracker et al., Biochem.
Biophys. Res. Comm., 80:849-857 (1978). Binding assays are
performed using appropriate receptor-expressing cells (such as, for
mouse anti-WISP antibodies, MLG, a mouse lung cell line available
from the ATCC) cultured in 96-well tissue culture plates (Falcon,
Becton Dickinson Labware, Lincoln Park, N.J.). The cells are
trypsinized and seeded in wells of 96-well plates at a density of
10.sup.4 cells/well and allowed to adhere overnight. The monolayers
are washed with cold culture medium supplemented with 0.1% sodium
azide and then incubated in triplicate with 100 .mu.l of serial
dilutions of .sup.125-anti-WISP-1 or WISP-2 antibodies in cold
culture medium containing 0.1% sodium azide for 4 hours on ice.
Non-specific binding is estimated by the preincubation of each
sample with a 100-fold molar excess of nonradioactive antibodies in
a total volume of 100 .mu.l. Unbound radioactivity is removed by
two washes with cold medium containing 0.1% sodium azide. The
cell-associated radioactivity is detected in a gamma counter after
solubilization of the cells with 150 .mu.l of 0.1 M NaOH/well. The
WISP-1 and WISP-2 binding constants (Kd) and anti-WISP antibody
binding affinities are determined by Scatchard analysis.
[0455] It is expected that the antibodies against WISP-1 and WISP-2
will affect the growth of these cells.
Example 15
Further Uses of Antibodies that Bind WISP Polypeptide
[0456] 1. WISP-1 and WISP-2
[0457] This example shows that the WISP-1 and WISP-2 genes are
amplified in the genome of certain human lung, colon, and/or breast
malignant tumors and/or cell lines. Amplification is associated
with overexpression of the gene product, indicating that the WISP-1
and WISP-2 proteins are useful targets for therapeutic intervention
in certain cancers such as colon, lung, breast, and other cancers.
A therapeutic agent may take the form of antagonists of WISP
molecules, for example, murine-human, chimeric, humanized, or human
antibodies against WISP-1 and WISP-2, such as the antibodies
prepared as described above.
[0458] The starting material for the screen was genomic DNA
isolated from a variety of cancers. The DNA is quantitated
precisely, e.g., fluorometrically. As a negative control, DNA was
isolated from the cells of ten normal healthy individuals, pooled,
and used as an assay control for the gene copy in healthy
individuals.
[0459] The 5' nuclease assay (for example, TAQMAN.TM.) and
real-time quantitative PCR (for example, ABI PRIZM 7700.TM.
Sequence Detection System.TM. (Perkin Elmer, Applied Biosystems
Division, Foster City, Calif.)), were used to find series
potentially amplified in certain cancers. The results were used to
determine whether the DNAs encoding WISP-1 and WISP-2 are
over-represented in any of the primary lung or colon cancers or
cancer cell lines or breast cancer cell lines that were screened.
The primary lung cancers were obtained from indiviuals with tumors
of the type and stage as indicated in Table III. An explanation of
the abbreviations used for the designation of the primary tumors
listed in Table III and the primary tumors and cell lines referred
to throughout this example is given below:
[0460] Human lung carcinoma cell lines include A549 (SRCC768),
Calu-1 (SRCC769), Calu-6 (SRCC770), H157 (SRCC771), H441 (SRCC772),
H460 (SRCC773), SKMES-1 (SRCC774) and SW900 (SRCC775), all
available from ATCC. Primary human lung tumor cells usually derive
from adenocarcinomas, squamous cell carcinomas, large cell
carcinomas, non-small cell carcinomas, small cell carcinomas, and
broncho alveolar carcinomas, and include, for example, SRCC724
(squamous cell carcinoma abbreviated as "SqCCa"), SRCC725
(non-small cell carcinoma, abbreviated as "NSCCa"), SRCC726
(adenocarcinoma, abbreviated as "AdenoCa"), SRCC727
(adenocarcinoma), SRCC728 (squamous cell carcinoma), SRCC729
(adenocarcinoma), SRCC730 (adeno/squamous cell carcinoma), SRCC731
(adenocarcinoma), SRCC732 (squamous cell carcinoma), SRCC733
(adenocarcinoma), SRCC734 (adenocarcinoma), SRCC735 (broncho
alveolar carcinoma, abbreviated as "BAC"), SRCC736 (squamous cell
carcinoma), SRCC738 (squamous cell carcinoma), SRCC739 (squamous
cell carcinoma), SRCC740 (squamous cell carcinoma), and SRCC740
(lung cell carcinoma, abbreviated as "LCCa").
[0461] Colon cancer cell lines include, for example, ATCC cell
lines SW480 (adenocarcinoma, SRCC776), SW620 (lymph node metastasis
of colon adenocarcinoma, SRCC777), COL0320 (adenocarcinoma,
SRCC778), HT29 (adenocarcinoma, SRCC779), HM7 (carcinoma, SRCC780),
CaWiDr (adenocarcinoma, srcc781), HCT116 (carcinoma, SRCC782),
SKC01 (adenocarcinoma, SRCC-83), SW403 (adenocarcinoma, SRCC784),
LS174T (carcinoma, SRCC785), and HM7 (a high mucin producing
variant of ATCC colon adenocarcinoma cell line LS 174T, obtained
from Dr. Robert Warren, UCSF). Primary colon tumors include colon
adenoocarcinomas designated CT2 (SRCC742), CT3 (SRCC743), CT8
(SRCC744), CT10 (SRCC-45), CT12 (SRCC746), CT14 (SRCC747), CT15
(SRCC748), CT17 (SRCC750), CT1 (SRCC751), CT4 (SRCC752), CT5
(SRCC753), CT6 (SRCC754), CT7 (SRCC755), CT9 (SRCC756), CT11
(SRCC757), CT18 (SRCC758), and DcR3, BACrev, BACfwd, T160, and
T159.
[0462] Human breast carcinoma cell lines include, for example,
HBL100 (SRCC759), MB435s (SRCC760), T47D (SRCC761), MB468
(SRCC762), MB175 (SRCC763), MB361 (SRCC764), BT20 (SRCC765), MCF7
(SRCC766), and SKBR3 (SRCC767).
[0463] The results are reported in delta (.DELTA.) CT units. One
unit corresponds to one PCR cycle or approximately a 2-fold
amplification relative to normal, two units corresponds to 4-fold,
3 units to 8-fold amplification and so on. Quantitation was
obtained using primers derived from the 3'-untranslated regions of
the WISP-1 and WISP-2 cDNAs and a TAQMAN.TM. fluorescent probe
corresponding to the respective intervening sequences. Using the 3'
region tends to avoid crossing intron-exon boundaries in the
genomic DNA, an essential requirement for accurate assessment of
gene amplification using this method. The sequences for the primers
and probes (forward, reverse, and probe) used for the
WISP-1-encoding and WISP-2-encoding gene amplification were as
follows: TABLE-US-00018 WISP-1 Probe and primers: hu.WISP1.TMP
(probe) (SEQ ID NO:138) 5'-AGCCTTTCCAAGTCACTAGAAGTCCT, GCTGG
hu.WISP1.TMF (forward primer) (SEQ ID NO:139)
5'-CTGGACTACACCCAAGCCTGA hu.WISP1.TMR (reverse primer) (SEQ ID
NO:140) 5'-CATTTCTTGGGATTTAGGCAAGA WISP-2 probe and primers:
DNA33473.3utr-5 (forward primer) (SEQ ID NO:141)
5'-TCTAGCCCACTCCCTGCCT DNA33473.3utr-3 (reverse primer) (SEQ ID
NO:142) 5'-GAAGTCGGAGAGAAAGCTCGC DNA33473.3utr-probe (SEQ ID
NO:143) 5'-CACACACAGCCTATATCAAACAT'UCACACG
[0464] The 5' nuclease assay reaction is a fluorescent PCR-based
technique which makes use of the 5' exonuclease activity of Taq DNA
polymerase enzyme to monitor amplification in real time. Two
oligonucleotide primers are used to generate an amplicon typical of
a PCR reaction. A third oligonucleotide, or probe, is designed to
detect nucleotide sequence located between the two PCR primers. The
probe is non-extendible by Taq DNA polymerase enzyme, and is
labeled with a reporter fluorescent dye and a quencher fluorescent
dye. Any laser-induced emission from the reporter dye is quenched
by the quenching dye when the two dyes are located close together
as they are on the probe. During the amplification reaction, the
Taq DNA polymerase enzyme cleaves the probe in a template-dependent
manner. The resultant probe fragments disassociate in solution, and
signal from the released reporter dye is free from the quenching
effect of the second fluorophore. One molecule of reporter dye is
liberated for each new molecule synthesized, and detection of the
unquenched reporter dye provides the basis for quantitative
interpretation of the data.
[0465] The 5' nuclease procedure is run on a real-time quantitative
PCR device such as the ABI PRIM 7700.TM. Sequence Detection
System.TM.. The system consists of a thermocyler, laser,
charge-coupled device (CCD), camera and computer. The system
amplifies samples in a 96-well format on a thermocycler. During
amplification, laser-induced fluorescent signal is collected in
real-time through fiber optics cables for all 96 wells, and
detected at the CCD. The system includes software for running the
instrument and for analyzing the data.
[0466] 5'-Nuclease assay data are initially expressed as Ct, or the
threshold cycle. This is defined as the cycle at which the reporter
signal accumulates above the background level of fluorescence. The
.DELTA.Ct values are used as quantitative measurement of the
relative number of starting copies of a particular target sequence
in a nucleic acid sample when comparing cancer DNA results to
normal human DNA results.
[0467] The results of the first run performed are shown in FIGS.
19A-D and 20A-D for WISP-1 and WISP-2, respectively, and controls.
Note the pattern shown in FIG. 19B (marked huWISP-1). The standard
deviation for two samples of normal human DNA is shown in the
column marked Nor Hu. This was used as a quality control tool. If
the standard deviation was unacceptably large, the entire run was
repeated. The nine additional columns corresponded to the human
colon cancer cell lines noted above. The delta CT's for HT29 and
WIDr were >3, corresponding to an about 8-fold
over-representation of the WISP-1 gene in these samples compared to
the normal samples. Similarly, FIG. 19B suggests an about 4-fold
amplification of WISP-1 in the HCT116, SKCo-1, and SW403 cell
lines.
[0468] As a comparison, see FIG. 20B (marked huFASr). The generally
small delta CT values indicate that this gene was not significantly
amplified in any of the cell lines (the value of 1 for SW620
corresponding to 2-fold amplification is within the noise level for
the assay)
[0469] The WISP-1 result was confirmed in three replicate
reactions. See FIGS. 21A-D, 22A-D, and 23A-C. The pattern and delta
CT values obtained were very similar in FIGS. 21A-C (marked
huWISP-1c, huWISP-1b, and huWISP-1a, respectively). The result was
essentially identical to that obtained in the first run. HT29 and
WIDr showed the highest levels of WISP-1 amplification, while
HCT116, SKCo-1, and SW403 cell lines showed somewhat lower levels
of WISP-1 gene amplification. Two additional reactions from a third
run were confirmatory. See FIGS. 25A and 25B.
[0470] The WISP-1 gene is located on chromosome 8, in the general
vicinity of the myc gene, which is known to be amplified in some
colon cancer cell lines. The pattern obtained using primers and
probe for the myc gene, namely, TABLE-US-00019 hu.c-myc.tm.p (SEQ
ID NO:144) 5'-CTTGAGACTGAAAGATTTAGCCATAATGTAAACTGCCT hu.c-myc.tm.f
(SEQ ID NO:145) 5'-CAAATGCAACCTCACAACCTTG, and hu.c-myc.tm.r (SEQ
ID NO:146) 5'-TTCTTTTATGCCCAAAGTCCAATT,
is consistent with a published report (Cancer Research, 57:
1769-1775 (1997)), tending to validate the 5' nuclease assay
method, but is clearly different from that obtained for WISP-1.
These data prove that the myc gene is not the target of the
amplification detected using the primers and probes for WISP-1.
[0471] The data using primers and probes based on the WISP-2 DNA
sequence suggest that this gene may be the target of low-level gene
amplification in most of the cell 14 nes examined. See FIGS. 20C,
22A-D, and 25C and D. Hence, antibodies to both WISP-1 and WISP-2,
particularly humanized antibodies, are expected to be of benefit in
combating certain types of cancer such as colon cancer, similar to
the humanized anti-HER-2 antibody in clinical use.
[0472] 2. WISP-2
[0473] Description of Tumors and Cell Lines
[0474] Amplification using several different tumor types was
performed for human WISP-2 (PR0261), as described below. Table III
describes the stage, T stage, and N stage of various primary tumors
which were used to screen the WISP-2 compound of the invention.
TABLE-US-00020 TABLE III Primary Lung and Colon Tumor Profiles
Other Dukes T N Primary Tumor Stage Stage Stage Stage Stage Human
lung tumor SqCCA (SRCC724) IB -- -- T1 N1 [LT1] Human lung tumor
NSCCa (SRCC725) IA -- -- T3 NO [LT1a] Human lung tumor AdenoCa
(SRCC726) IB -- -- T2 NO (LT2) Human lung tumor AdenoCa (SRCC727)
IB -- -- T1 N2 (LT3) Human lung tumor SqCCq (SRCC728) IIB -- -- T2
NO [LT4] Human lung tumor AdenoCa (SRCC729) IV -- -- T1 NO [LT6]
Human lung tumor Aden/SqCCa IB -- -- T1 NO (SRCC730) [LT7] Human
lung tumor AdenoCa (SRCC731) IIB -- -- T2 NO [LT9] Human lung tumor
SqCCa (SRCC732) IA -- -- T2 N1 [LT10] Human lung tumor AdenoCa
(SRCC733) IB -- -- T1 N1 [LT11] Human lung tumor AdenoCa (SRCC734)
IIA -- -- T2 NO [LT12] Human lung tumor BAC (SRCC735) IB -- -- T2
NO [LT13] Human lung tumor SqCCa (SRCC736) IB -- -- T2 NO [LT15]
Human lung tumor SqCCa (SRCC737) IB -- -- T2 NO [LT16] Human lung
tumor SqCCa (SRCC738) IIB -- -- T2 N1 [LT17) Human lung tumor SqCCa
(SRCC739) IB -- -- T2 NO [LT18] Human lung tumor SqCCa (SRCC740) IB
-- -- T2 NO [LT19] Human lung tumor LCCa (SRCC741) IIB -- -- T3 N1
[LT21] Human colon AdenoCa (SRCC742) (CT2] -- M1 D pT4 NO Human
colon AdenoCa (SRCC743) [CT3] -- B pT3 NO Human colon AdenoCa (SRCC
744) [CT8] B T3 NO Human colon AdenoCa (SRCC745) A pT2 NO [CT10]
Human colon AdenoCa (SRCC746) MO, R1 B T3 NO [CT12] Human colon
AdenoCa (SRCC747) pMO, RO B pT3 pNO [CT14] Human colon AdenoCa
(,(;RCC748) M1, R2 D T4 N2 (CT15] Human colon AdenoCa (SRCC749) pMO
B pT3 pNO [CT16] Human colon AdenoCa (SRCC750) C1 pT3 pN1 [CT17]
Human colon AdenoCa (SRCC751) [CT1] MO, R1 B pT3 NO Human colon
AdenoCa (SRCC752) (CT4] B pT3 MO Human colon AdenoCa (SRCC753)
(CT5] G2 C1 pT3 pNO Human colon AdenoCa (SRCC754) [CT6] PMO, RO B
pT3 pNO Human colon AdenoCa (SRCC755) [CT7] G1 A pT2 pNO Human
colon AdenoCa (SRCC756) [CT9] G3 D pT4 pN2 Human colon AdenoCa
(SRCC757) B T3 NO [CT11] Human colon AdenoCa (SRCC758) MO, RO B pT3
pNO [CT18]
[0475] DNA Preparation:
[0476] DNA was prepared from cultured cell lines, primary tumors,
and normal human blood (controls and framework and epicenter
mapping). The isolation was performed using purification kit #13362
(which includes 10 purification tips with a capacity of 400 .mu.g
genomic DNA each), buffer set #1960 and protease #19155 and #19101,
all from Quiagen, according to the manufacturer's instructions and
the description below.
[0477] Cell Culture Lysis:
[0478] Cells were washed and trypsinized at a concentration of
7.5.times.10.sup.8 per tip and pelleted by centrifuging at 1000 rpm
for 5 minutes at 4.degree. C., followed by washing again with 112
volume of PBS recentrifugation. The pellets were washed a third
time, and the suspended cells collected and washed 2.times. with
PBS. The cells were then suspended into 10 mL PBS. Buffer C1 was
equilibrated at 4.degree. C. Protease #19155 (Quiagen) was diluted
into 6.25 ml cold ddH.sub.2O to a final concentration of 20 mg/ml
and equilibrated at 4.degree. C. 10 mL of G2 Buffer was prepared by
diluting RNAse A stock (Quiagen) (100 mg/ml) to a final
concentration of 200 .mu.g/ml.
[0479] Buffer C1 (10 mL, 4.degree. C.) and ddH2O (40 mL, 4.degree.
C.) were then added to the 10 mL of cell suspension, mixed by
inverting and incubated on ice for 10 minutes. The cell nuclei were
pelleted by centrifuging in a BECKMAN.TM. swinging bucket rotor at
2500 rpm at 4.degree. C. for 15 minutes. The supernatant was
discarded and the nuclei were suspended with a vortex into 2 mL
Buffer C1 (at 4.degree. C.) and 6 mL ddH.sub.2O, followed by a
second 4.degree. C. centrifugation at 2500 rpm for 15 minutes. The
nuclei were then resuspended into the residual buffer using 200
.mu.l per tip. G2 buffer (10 ml) was added to the suspended nuclei
while gentle vortexing was applied. Upon completion of buffer
addition, vigorous vortexing was applied for 30 seconds. Quiagen
protease (200 pl, prepared as indicated above) was added and
incubated at 50.degree. C. for 60 minutes. The incubation and
centrifugation were repeated until the lysates were clear (e.g.,
incubating an additional 30-60 minutes, pelleting at 3000.times.g
for 10 min., 4.degree. C.).
[0480] Solid Human Tumor Sample Preparation and Lysis:
[0481] Tumor samples were weighed and placed into 50-ml conical
tubes and held on ice. Processing was limited to no more than 250
mg tissue per preparation (1 tip/preparation). The protease
solution was freshly prepared by diluting into 6.25 ml cold
ddH.sub.2O to a final concentration of 20 mg/ml and stored at
4.degree. C. G2 buffer (20 ml) was prepared by diluting DNAse A to
a final concentration of 200 mg/ml (from 100 mg/ml stock). The
tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds
using the large tip of the polytron in a laminar-flow TC hood to
order to avoid inhalation of aerosols, and held at room
temperature. Between samples, the polytron was cleaned by spinning
at 2.times.30 seconds each in 2L ddH.sub.2O, followed by G2 buffer
(50 ml) If tissue was still present on the generator tip, the
apparatus was disassembled and cleaned.
[0482] Protease (Quiagen), prepared as indicated above, 1.0 ml, was
added, followed by vortexing and incubation at 50.degree. C. for 3
hours. The incubation and centrifugation were repeated until the
lysates were clear (e.g., incubating additional 30-60 minutes,
pelleting at 3000.times.g for 10 min., 4.degree. C.).
[0483] Human Blood Preparation and Lysis:
[0484] Blood was drawn from healthy volunteers using standard
infectious agent protocols and citrated into 10 ml samples per tip.
Protease (Quiagen) was freshly prepared by dilution into 6.25 ml
cold ddH.sub.2O to a final concentration of 20 mg/ml and stored at
4.degree. C. G2 buffer was prepared by diluting RNAse A to a final
concentration of 200 .mu.g/ml from 100 mg/ml stock. The blood (10
ml) was placed into a 50-ml conical tube, and 10 ml C1 buffer and
30 ml ddH.sub.2O (both previously equilibrated to 4.degree. C.)
were added, and the components mixed by inverting and held on ice
for 10 minutes. The nuclei were pelleted with a BECKMAN.TM.
swinging bucket rotor at 2500 rpm, 4.degree. C. for 15 minutes and
the supernatant was discarded. With a vortex, the nuclei were
suspended into 2 ml C1 buffer (4.degree. C.) and 6 ml ddH.sub.2O
(4.degree. C.). Vortexing was repeated until the pellet was white.
The nuclei were then suspended into the residual buffer Using a
200-.mu.l tip. G2 buffer (10 ml) was added to the suspended nuclei
while gently vortexing, followed by vigorous vortexing for 30
seconds. Protease (Quiagen) was added (200 pl) and incubated at
50.degree. C. for 60 minutes. The incubation and centrifugation
were repeated until the lysates were clear (e.g., incubating an
additional 30-60 minutes, pelleting at 3000.times.g for 10 min.,
4.degree. C.).
[0485] Purification of Cleared Lysates; Isolation of Genomic
DNA:
[0486] Genomic DNA was equilibrated (1 sample per maxi tip
preparation) with 10 ml QBT buffer. QF elution buffer was
equilibrated at 50.degree. C. The samples were vortexed for 30
seconds, then loaded onto equilibrated tips and drained by gravity.
The tips were washed with 2.times.15 ml QC buffer. The DNA was
eluted into 30-ml silanized, autoclaved 30-ml COREX.TM. tubes with
15-ml QF buffer (50.degree. C.). Isopropanol (10.5 ml) was added to
each sample, and the tubes were covered with paraffin and mixed by
repeat ed inversion until the DNA precipitated. Samples were
pelleted by centrifugation in the SS-34 rotor at 15,000 rpm for 10
minutes at 4.degree. C. The pellet location was marked, the
supernatant discarded, and 10 ml 70% ethanol (4.degree. C.) was
added. Samples were pelleted again by centrifugation on the SS-34
rotor at 10,000 rpm for 10 minutes at 4.degree. C. The pellet
location was marked and the supernatant discarded. The tubes were
then placed on their side in a drying rack and dried 10 minutes at
37.degree. C., taking care not to overdry the samples.
[0487] After drying, the pellets were dissolved into 1.0 ml TE (pH
8.5) and placed at 50.degree. C. for 1-2 hours. Samples were held
overnight at 4.degree. C. as dissolution continued. The DNA
solution was then transferred to 1.5-ml tubes with a 26-gauge
needle on a tuberculin syringe. The transfer was repeated 5.times.
in order to shear the DNA. Samples were then placed at 50.degree.
C. for 1-2 hours.
[0488] Quantitation of Genomic DNA and Preparation for Gene
Amplification Assay:
[0489] The DNA levels in each tube were quantified by standard
A260, A280 spectrophotometry on a 1:20 dilution (5 .mu.l DNA+95 pl
ddH.sub.2O) using the 0.1-ml quartz cuvettes in the BECKMAN
DU640.TM. spectrophotometer. A260/A280 ratios were in the range of
1.8-1.9. Each DNA sample was then diluted further to approximately
200 ng/ml in TE (pH 8.5). If the original material was highly
concentrated (about 700 ng/.mu.l), the material was placed at
50.degree. C. for several hours until resuspended.
[0490] Fluorometric DNA quantitation was then performed on the
diluted material (20-600 ng/ml) using the manufacturer's guidelines
as modified below. This was accomplished by allowing a HOEFFER DYNA
QUANT 200.TM. fluorometer to warm up for about 15 minutes. The
HOECHST.TM. dye working solution (#H33258, 10 .mu.l, prepared
within 12 hours of use) was diluted into 100 ml 1.times.TNE buffer.
A 2-ml cuvette was filled with the fluorometer solution, placed
into the machine, and the machine was zeroed. pGEM 3Zf(+) (2 .mu.l,
lot #360851026) was added to 2 ml of fluorometer solution and
calibrated at 200 units. A second 2 .mu.l of pGEM 3Zf(+) DNA was
then tested and the reading confirmed at 400+/-10 units. Each
sample was then read at least in triplicate. When 3 samples were
found to be within 10% of each other, their average was taken and
this value was used as the quantification value.
[0491] The fluorometrically-determined concentration was then used
to dilute each sample to 10 ng/.mu.l in ddH.sub.2O. This was done
simultaneously on all template samples for a single TAQMAN.TM.
plate assay, and with enough material to run 500-1000 assays. The
samples were tested in triplicate with both B-actin and GAPDH on a
single plate with normal human DNA and no-template controls. The
diluted samples were used, provided that the CT value of normal
human DNA subtracted from test DNA was +/-1 CT. The diluted,
lot-qualified genomic DNA was stored in 1.0-ml aliquots at
-80.degree. C. Aliquots which were subsequently to be used in the
gene amplification assay were stored at 4.degree. C. Each 1-ml
aliquot is enough for 8-9 plates or 64 tests.
[0492] Framework Mapping and Epicenter Marking:
[0493] Human WISP-1 was reexamined with both framework and
epicenter mapping. Selected tumors from the above initial screen
were reexamined with both framework and epicenter mapping. Table IV
indicates the chromosomal mapping of the framework markers that
were used in the present example. The framework markers are located
approximately every 20 megabases along Chromosome 8 and were used
to control for aneuploidy. TABLE-US-00021 TABLE IV Framework
Markers Map Position on Stanford Human Genome Chromosome 8 Center
Marker Name H9 EST-00040 H59 WI-961 H121 SHGC-11323 H200 SHGC-7433
H256 AFMa183zfl
[0494] Table V describes the epicenter markers that were employed
in association with WISP-1. These markers are located in close
proximity to the gene for WISP-1 and are used to assess the
amplification status of the region of chromosome 8 in which the
gene for WISP-1 is located. The distance between individual markers
is measured in centirays (cR), which is a radiation. breakage unit
approximately equal to a 1% chance of a breakage between two
markers. One cR is very roughly equivalent to 20 kilobases. The
marker SHGC-32958 is the marker found to be the closest to the
location on chromosome 8 to which the gene encoding WISP-1 most
closely maps. TABLE-US-00022 TABLE V Epicenter Markers Map Position
on Stanford Human Genome Distance to next Chromosome 8 Center
Marker Name Marker (cR) H257 AFMa248te1 103(gap) H259 SHGC-36664 33
H261 AFM259xc5 63 H266 SHGC-32958 41 H267 AFMa175xc1 19 H268
AFM337wg5 87 H273 SHGC-33759 71 H274 SHGC-32752 5 H275 WI-7711 21
H277 HGC-34940 --
[0495] The framework markers for human WISP-2 are located
approximately every 20 megabases along Chromosome 20, and are used
to control for aneuploidy. The markers are shown in Table VI.
TABLE-US-00023 TABLE VI Framework Markers Map Position on Stanford
Human Genome Chromosome 20 Center Marker Name T10 SHGC-2797 T48
UT759 T73 AFMa339xf5 T115 SHGC-33922 T159 HGC-36268
[0496] The marker SHGC-33922 is the marker to which human WISP-2
DNA most closely maps. This marker is between the framework
markers.
[0497] Framework analysis showed that all markers were up in
tumors; thus, chromosome 20 was aneuploid in many, tumors. Since
the markers were up due to aneuploidy, epicenter analysis was not
done for human WISP-2 gene.
[0498] The .DELTA.Ct values of the above described framework
markers along Chromosome 8 relative for WISP-1 are indicated for
selected tumors in Tables VII and VIII. TABLE-US-00024 TABLE VII
Amplification of framework markers relative to Human WISP-1 DNA
Framework Markers (.DELTA.ct) Probe/Delta CT C-myc WISP-1 WISP-2 H9
H59 H121 H200 H256 Template (SD) (SD) (SD) (SD) (SD) (SD) (SD) (SD)
Nor Hu 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 (0.91) (0.01) (0.20)
(0.13) (0.20) (0.14) (0.16) (0.04) SW480 1.86 0.84 1.92 -1.18 1.01
0.17 0.165 0.81 SW620 1.45 0.98 1.60 0.45 0.75 1.00 0.81 0.52
Colo320 3.73 0.65 1.88 0.69 0.70 0.89 0.60 0.40 HT29 0.83 2.67 2.20
-1.13 -0.40 -0.55 1.00 2.42 HM7 -2.03 0.07 -0.28 -0.28 0.24 -0.48
0.12 -0.26 WiDr -0.13 2.91 1.67 -0.20 0.95 0.0'7 1.43 2.55 HCT116
-0.57 1.82 1.04 1.24 1.56 0.84 1.76 1.53 SKCO-1 0.19 1.68 0.97
-0.30 0.32 0.12 1.39 1.63 SW403 -0.72 1.34 1.77 0.23 0.53 0.26 1.48
1.48 Nor Hu -- 0.00 0.00 0.00 0.00 0.00 0.00 0.00 (0.18) (1.02)
(0.08) (0.13) (0.01) (0.16) (0.37) CT-2 -- 0.65 0.44 -0.25 0.11
0.07 0.13 0.95 CT-3 -- 0.90 0.95 -0.27 0.05 -0.10 -0.11 0.32 CT-8
-- 0.47 -0.34 0.07 -0.20 0.00 -0.04 0.07 CT-10 -- 0.76 0.50 0.23
-0.36 -0.08 0.17 0.70 CT-12 -- 1.30 2.14 -0.70 -0.45 0.24 0.47 1.75
CT-14 -- 1.17 -0.48 0.05 0.18 0.31 0.23 1.5! CT-15 -- 0.22 -0.13
0.13 -0.48 0.29 0.11 0.59 CT-16 -- 0.26 0.10 0.00 -0.15 -0.23 -0.09
0.95 CT-17 -- 0.57 -0.33 0.73 -0.11 -0.05 -0.11 0.25 Nor Hu -- 0.00
0.00 0.00 0.00 0.00 0.00 0.00 (0.45) (1.07) (0.04) (0.21) (0.18)
(0.03) (0.18) CT-1 -- 0.64 -0.37 -0.36 0.19 0.68 0.01 0.66 CT-4 --
0.15 -0.23 -1.00 0.24 -0.11 0.30 0.14 CT-5 -- 0.86 -1.23 -0.60
-0.25 0.22 0.51 0.62 CT-6 -- 0.03 0.39 -0.24 0.61 0.70 0.01 0.19
CT-7 -- -0.20 -1.36 -0.76 0.00 -0.09 -0.13 -0.16 CT-9 -- 0.30 -0.54
-0.50 0.29 0.54 0.11 0.18 CT-11 -- 0.48 0.14 -0.89 0.34 0.82 0.17
-0.06 CT-18 -- -0.20 -1.37- -0.52 0.32 0.66 0.08 0.12
[0499] TABLE-US-00025 TABLE VIII Amplification of framework markers
relative to Human WISP-1 DNA Framework Markers (8ct) Probe/Delta CT
Template WISP-2 (SD) T10 (SD) T48 (SD) T73 (SD) T115 (SD) T159 (SD)
Nor Hu 0.00 0.00 0.00 0.00 0.00 0.00 (0.05) (0.16) (0.09) (0.21)
(3.22) (0.09) SW480 1.31 1.32 0.63 1.94 -5.66 1.61. SW620 1.32 2.02
1.42 1.06 -10.95 1.48 Colo320 0.43 1.35 1.37 0.61 0.30 1.37 HT29
1.76 1.09 -2.23 1.26 -5.47 1.87 HM7 -0.32 0.32 0.38 0.41 -6.3 0.48
WiDr 1.76 1.61 -1.38 1.04 -7.36 1.55 HCT116 1.18 1.24 1.15 1.46
-8.38 1.49 SKCO-1 1.40 1.17 1.19 1.13 -5.34 1.61 SW403 1.92 2.24
-17.23 1.38 -3.66 2.12
[0500] Gene Amplification Assay Results:
[0501] The human WISP-2 (PR0261) compound of the invention was
screened in the following primary tumors and the resulting
.DELTA.Ct values are reported in Table IX. TABLE-US-00026 TABLE IX
.DELTA.Ct values in lung and colon primary tumor models Primary
Tumor PR0261 LT1 0.41 LT1a 1.08 LT2 0.27 LT3 0.98 LT4 0.32 LT6 0.45
LT7 0.03 LT9 0.18 LT10 1.16 LT11 0.67, 1.59, 0.63, 0.19, LT12 0.80,
1.73, 1.08, 2.23 LT13 1.02, 1.13, 1.01, 0.29 LT15 0.97, 2.64, 0.56,
2.38 LT16 0.80, 0.75, 0.82, 2.05 LT17 1.67, 2.01, 1.43, 0.93 LT18
1.22, 0.46, 0.15, -0.17 LT19 0.76, 1.38, 1.39, 2.33 LT21 0.04,
1.14, 0.48, 3.40 CT2 1.66 CT3 2.14 CT8 0.55 CT10 1.00 CT12 0.34
CT14 1.03 CT15 0.67 CT16 0.87 CT17 -0.19 M -0.06 CT4 1.00 CT5 1.07
CT6 -0.08 CT7 0.15 CT9 0.68 CT11 0.59 CT18 0.73 A549 -- Calu-1
Calu-6 H157 H441 H460 SKMES1 SW900 -- SW480 0.62, 1.86, 1.90, 1.91,
1.20, 2.36, 1.57, 1.68, 1.68, 1.53, 1.36, 2.50 1.59, SW620 0.66,
1.98, 1.65, 1.57, 1.85, 1.83, 1.63, 1.41, 1.61, 1.42, 1.24, 1.59
1.52, Colo320 -0.33, 2.49, 0.66, 0.99, 0.48, 1.06, 0.91, 1.24,
0.72, 1.04, 0.33, 0.46, 0.2-- HT29 0.46, 2.00, 1.95, 2.59, 1.61,
2.59, 2.58, 1.39, 1.49, 1.32 1.38, 1.40, HM7 -0.70, 0.54, 0.74,
0.67, -0.29, 0.66, 0.27, 0.-08, 0.64, 0.34, 0.09, 0.29, 0.21 WiDr
0.19, 1.84, 1.64, 1.58, 1.00, 0.91, 1.71, 0.87 1.44, 1.57, 0.93,
HCT116 0.25, 1.08, 1.29, 2.05, 1.04, 1.81, 2.01, 1.56, 1.29, 1.05,
1.07, 1.09, 0.96 SKC01 0.73, 2.10, 1.99, 1.50, 1.33, 2.13, 1.00,
1.33, 1.33, 1.29 1.26, 1.19 SW403 0.26, 2.15, 1.98, 1.52, 1.42,
1.67, 2.20, 2.19, 2.40, 1.40, 1.50, 1.291.43, LS174T 1.48 HBL100
1.40 MB435s 1.43 T47D 0.38 MB468 -0.08 MB175 0.23 MB361 0.37 BT20
1.66 MCF7 0.53 SKBR3 1.73
[0502] The .DELTA.Ct values for DNA33473 (PR0261; human WISP-2) in
a variety of primary lung and colon tumors as well as lung tumor
cell lines are reported in Table IX. A .DELTA.Ct value of >1 was
typically used as the threshold value for amplification scoring, as
this represents a doubling of the gene copy. Table IX indicates
that significant amplification of DNA33474 occurred in: primary
lung tumors LT1a, LT10, LT12, LT15, LT17 and LT19; (2) primary
colon tumors CT2, CT3, CT14, and CT5; (3) colon tumor cell lines
SW480, SW620, HT29, WiDr, HCT116, SKC01, SW403, and LS174T and (4)
breast tumor cell lines HBL100, MB435s, BT20 and SKBR3.
[0503] The .DELTA.Ct and average .DELTA.Ct values for the primary
lung tumors were the following: 1.08, 1.16, 1.17, 1.64, 1.50 and
1.47, respectively; those for the primary colon tumors were 1.16,
2.14, 1.03 and 1.07, respectively; those for the colon tumor cell
lines were 1.67, 1.54, 1.73, 1.24, 1.32, 1.35, 1.65, and 1.48,
respectively; and those for the breast tumor cell lines were 1.40,
1.43, 1.66, and 1.73, respectively.
[0504] For the lung tumors, this represents approximately a 2.1-,
2.2-, 2.2-, 3.1-, 2.8-, and 2.8-, respectively, fold increase in
gene copy relative to normal tissue. For the colon tumors, this
represents a 2.2-, 4.4-, 2.0-, and 2.1-, respectively, fold
increase in gene copy relative to normal tissue. For the colon
tumor cell lines, this represents a 3.2-, 2.9-, 3.3-, 2.4-, 2.5-,
2.5-, 3.1-, and 2.8-, respectively, fold increase in gene copy
relative to normal tissue. For the breast tumor cell lines, this
represents a 2.6-, 2.7-, 3.2-, and 3.3-, respectively, fold
increase in gene copy relative to normal tissue. Because
amplification of DNA33473 (PR0261) occurs in various tumors, it is
likely associated with tumor formation or growth. As a result,
antagonists (e.g., antibodies) directed against the protein encoded
by DNA33473 (PR0261) would be expected to be useful in cancer
therapy.
Example 16
In Situ Hybridization
[0505] In situ hybridization is a powerful and versatile technique
for the detection and localization of nucleic acid sequences within
cell or tissue preparations. It may be useful, for example, in
identifying sites of gene expression, analyzing the tissue
distribution of transcription, identifying and localizing viral
infection, following changes in specific mRNA synthesis, and aiding
in chromosome mapping.
[0506] In situ hybridization was performed following an optimized
version of the protocol by Lu and Gillett, Cell Vision 1: 169-176
(1994), using PCR-generated .sup.33P-labeled riboprobes. Briefly,
formalin-fixed, paraffin-embedded human tissues were sectioned,
deparaffinized, deproteinated in proteinase K (20 g/ml) for 15
minutes at 37.degree. C., and further processed for in situ
hybridization as described by Lu and Gillett, supra. A (.sup.33-P)
UTP-labeled antisense riboprobe was generated from a PCR product
and hybridized at 55.degree. C. overnight. The slides were dipped
in KODAK NTB2.TM. nuclear track emulsion and exposed for 4
weeks.
[0507] .sup.33P-Riboprobe synthesis
[0508] 6.0 .mu.l (125 mCi) of .sup.33P-UTP (Amersham BF 1002,
SA<2000 Ci/mmol) were speed-vacuum dried. To each tube
containing dried .sup.33P-UTP, the following ingredients were
added:
[0509] 2.0 .mu.l 5.times. transcription buffer
[0510] 1.0 .mu.l DTT (100 mM)
[0511] 2.0 .mu.l NTP mix (2.5 mM: 10 .mu.l each of 10 mM GTP, CTP
& ATP+10 .mu.l H.sub.2O)
[0512] 1.0 .mu.l UTP (50 .mu.M)
[0513] 1.0 .mu.l RNAsin
[0514] 1.0 .mu.l DNA template (1 .mu.g)
[0515] 1.0 .mu.l H.sub.2
[0516] 1.0 .mu.l RNA polymerase (for PCR products T3=AS, T7=S,
usually)
[0517] The tubes were incubated at 37.degree. C. for one hour. A
total of 1.0 .mu.l RQ1 DNase was added, followed by incubation at
37.degree. C. for 15 minutes. A total of 90 .mu.l TE (10 mM Tris pH
7.6/1 mM EDTA, pH 8.0) was added, and the mixture was pipetted onto
DE81 paper. The remaining solution was loaded in a MICROCON-50.TM.
ultrafiltration unit, and spun using program 10 (6 minutes). The
filtration unit was inverted over a second tube and spun using
program 2 (3 minutes). After the final recovery spin, a total of
100 .mu.l TE was added. Then 1 .mu.l of the final product was
pipetted on DE81 paper and counted in 6 ml of BIOFLUOR II.TM..
[0518] The probe was run on a TBE/urea gel. A total of 1-3 .mu.l of
the probe or 5 .mu.l of RNA Mrk III was added to 3 .mu.l of loading
buffer. After heating on a 95.degree. C. heat block for three
minutes, the gel was immediately placed on ice. The wells of gel
were flushed, and the sample was loaded and run at 180-250 volts
for 45 minutes. The gel was wrapped in plastic wrap (SARAN.TM.
brand) and exposed to XAR film with an intensifying screen in a
-70.degree. C. freezer one hour to overnight.
[0519] .sup.33P-Hybridization
[0520] A. Pretreatment of Frozen Sections
[0521] The slides were removed from the freezer, placed on aluminum
trays, and thawed at room temperature for 5 minutes. The trays were
placed in a 55.degree. C. incubator for five minutes to reduce
condensation. The slides were fixed for 10 minutes in 4%
paraformaldehyde on ice in the fume hood, and washed in
0.5.times.SSC for 5 minutes, at room temperature (25 ml
20.times.SSC+975 ml s.c. H.sub.2O). After deproteination in 0.5
.mu.g/ml proteinase K for 10 minutes at 37.degree. C. (12.5 .mu.l
of 10 mg/ml stock in 250 ml prewarmed RNAse-free RNAse buffer), the
sections were washed in 0.5.times.SSC for 10 minutes at room
temperature. The sections were dehydrated in 70%, 95%, and 100%
ethanol, 2 minutes each.
[0522] B. Pretreatment of Paraffin-Embedded Sections
[0523] The slides were deparaffinized, placed in s.c. H.sub.2O, and
rinsed twice in 2.times.SSC at room temperature, for 5 minutes each
time. The sections were deproteinated in 20 .mu.g/ml proteinase K
(500 .mu.l of 10 mg/ml in 250 ml RNAse-free RNAse buffer;
37.degree. C., 15 minutes) for human embryo tissue, or 8.times.
proteinase K (100 .mu.l in 250 ml RNAse buffer, 37.degree. C., 30
minutes) for formalin tissues. Subsequent rinsing in 0.5.times.SSC
and dehydration were performed as described above.
[0524] C. Prehybridization
[0525] The slides were laid out in a plastic box lined with Box
buffer (4.times.SSC, 50% formamide) The filter paper was saturated.
The tissue was covered with 50 .mu.l of hybridization buffer (3.75
g dextran sulfate+6 ml s.c. H.sub.2O), vortexed, and heated in the
microwave for 2 minutes with the cap loosened. After cooling on
ice, 18.75 ml formamide, 3.75 ml 20.times.SSC, and 9 ml s.c.
H.sub.2O were added, and the tissue was vortexed well and incubated
at 42.degree. C. for 1-4 hours.
[0526] D. Hybridization
[0527] 1.0.times.10.sup.6 cpm probe and 1.0 .mu.l tRNA (50 mg/ml
stock) per slide were heated at 95.degree. C. for 3 minutes. The
slides were cooled on ice, and 48 .mu.l hybridization buffer was
added per slide. After vortexing, 50 .mu.l .sup.33P mix was added
to 50 .mu.l prehybridization on the slide. The slides were
incubated overnight at 55.degree. C.
[0528] E. Washes
[0529] Washing was done for 2.times.10 minutes with 2.times.SSC,
EDTA at room temperature (400 ml 20.times.SSC+16 ml 0.25 M EDTA,
V.sub.f=4 L), followed by RNAseA treatment at 37.degree. C. for 30
minutes (500 .mu.l of 10 mg/ml in 250 ml RNAse buffer=20 .mu.g/ml).
The slides were washed 2.times.10 minutes with 2.times.SSC, EDTA at
room temperature. The stringency wash conditions were as follows: 2
hours at 55.degree. C., 0.1.times.SSC, EDTA (20 ml 20.times.SSC+16
ml EDTA, V.sub.f=4 L).
[0530] F. Oligonucleotides
[0531] In situ analysis was performed on DNA sequences disclosed
herein. The oligonucleotides employed for these analyses are as
follows. TABLE-US-00027 (1) Mouse WISP-1 (Clone 568) Notrim-p1:
(SEQ ID NO:147) 5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC GTC CCT GGC
CAG TGC TGT GAG-3' Notrim-p2: (SEQ ID NO:148) 5'-CTA TGA AAT TAA
CCC TCA CTA AAG GGA GGG CCA GGC TTT GCT TCC ATT-3' (2) Human WISP-1
hmWISP-1 p1: (SEQ ID NO:149) 5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC
TGG AGG CAT GGC ACA GGA AC-31 hmWISP-1 p2: (SEQ ID NO:150) 5'-CTA
TGA AAT TAA CCC TCA CTA AAG GGA TCC GGA TCA GGC TTG GGT GTA-3' (3)
Mouse WISP-2 (Clone 1367.3) 1367.p1: (SEQ ID NO:151) 5'-GGA TTC TAA
TAC GAC TCA CTA TAG GGC AGC TTG GGA TGG AGG TCT TTC-3' 1367.p2:
(SEQ ID NO:152) 5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA GGG CAC TGG
GGT GGT GT-3' (4) Human WISP-2 (DNA33473) DNA33473-p1: (SEQ ID
NO:153) 5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC GCG AGG ACG GCG GCT
TCA-3' DNA33473-p2: (SEQ ID NO:154) 5'-CTA TGA AAT TAA CCC TCA CTA
AAG GGA AGA GTC GCG GCC GCC CTT TTT-3' (5) Human WISP-3 WISP3-p1
(SEQ ID NO:155) 5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC GGG GCT CCT
CTT CTC CAC TCT-3' WISP3-p2 (SEQ ID NO:156) 5'-CTA TGA AAT TAA CCC
TCA CTA AAG GGA GCT GTC GCA AGG CTG AAT GTA-3'
[0532] G. Results
[0533] In situ analysis was performed on the above DNA sequences
disclosed herein. The results from these analyses are as
follows.
(1) Mouse WISP-1
Expression in Mouse Tissues
[0534] Mouse Fetal Tissues: In situ hybridization of mouse WISP-1
showed strong expression in embryonic mesenchymal tissues. At E10.5
expression was observed in tissues that would develop into skeletal
elements in the adult; this pattern was maintained at later stages
of embryonic development. In later stages (E12.5 and E15.5),
expression was highest in osteoblasts at the sites of bone
formation. Expression was also observed in the embryonic heart,
where the signal was particularly strong in the atria at E12.5
(atria were not included in sections at E15.5).
[0535] Mouse Adult Tissues: No expression was observed in any of
the adult tissues examined, including heart, lung, kidney, adrenal,
liver, pancreas, cerebrum, and cerebellum. These results do not
correlate with the Northern data.
[0536] Additional sites of expression in the fetus were the walls
of developing blood vessels and in fibroblast-like cells within the
hepatic portal tract mesenchyme.
Expression in Normal and Wnt-1 Transgenic Tumors
[0537] Expression with the antisense probe was observed over
fibroblast-like cells lying adjacent to the subcutaneous skeletal
muscle in P10 (post-natal day 10 pups) and in pregnant females.
Expression was not observed over breast epithelial cells at any of
the time points examined in the study.
[0538] Expression of mouse WISP-1 was high in all three of the
Wnt-1 transgenic tumors tested and appeared to be confined to the
supporting fibroblast-like cells within the delicate connective
tissue stroma. Some expression was seen over the tumor cells
themselves; however, this likely represents overspill from tumor
fibroblasts, rather than true expression by tumor cells.
[0539] In summary, mouse WISP-1 was expressed in embryonic skeletal
mesenchyme and at sites of bone formation. It was additionally
expressed in fibroblasts in the sub-cutus of growing pups and
pregnant females. It is likely to play a role in osteogenesis, and
may be involved in repair after injury. Expression was also
observed in the embryonic heart.
(2) Human WISP-1
Expression in Human Tissues
[0540] Human Fetal Tissue The fetal tissues examined (E12-E16
weeks) included: placenta, umbilical cord, liver, kidney, adrenals,
thyroid, lungs, heart, great vessels, oesophagus, stomach, small
intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body
wall, pelvis, and lower limb.
[0541] Human WISP-1 exhibited expression at sites of connective
tissue interfaces in the fetus, for example, developing portal
tracts, fascial planes in muscle, and connective tissue surrounding
developing skeletal elements and tendons. Expression also was seen
in the epithelium of the developing renal cortex and in
spindle-shaped fibroblast-like cells in the fetal adrenal. Human
WISP-1 was strongly expressed by osteoblasts at sites of bone
formation in the fetal limb.
[0542] Human Adult Tissue The adult tissues examined were: liver,
kidney, adrenal, myocardium, aorta, spleen, lung, skin,
chondrosarcoma, eye, stomach, gastric carcinoma, colon, colonic
carcinoma, renal cell carcinoma, prostate, bladder mucosa, and gall
bladder, as well as tissue with acetominophen-induced liver injury
and hepatic cirrhosis.
[0543] No expression was seen in normal or diseased adult tissues
in this study.
[0544] In summary, the overall pattern of expression of human
WISP-1 was broadly similar to that observed for the mouse gene as
noted above. The human WISP-1 probe did not cross react with the
mouse embryo section.
Expression in Human Breast Carcinoma and Normal Breast Tissue
[0545] Human WISP-1 was negative on benign and malignant epithelial
cells, but showed specific hybridization in mesenchmal cells,
particularly in areas of tissue repair, including dystrophic
ossification. Most positive cells had the morphology of
fibroblasts; smooth muscle cells appeared to be negative.
[0546] In summary, this study shows expression of human WISP-1 RNA
in mesenchymal cells involved in tissue repair and/or collagen
deposition. The signal was particularly strong in benign
fibroblast-like cells adjacent to either infiltrating breast
carcinoma cells or tissue destruction due to benign, inflammatory
conditions (duct rupture). Of note is the fact that deposition of
benign osteoid seemed to correlate with strong expression of the
RNA.
(3) Mouse WISP-2
Expression in Normal Mouse Tissues
[0547] Mouse Fetal Tissues: Expression of mouse WISP-2 was observed
in osteoblasts in an E15.5 mouse embryo, within the developing
mandible.
[0548] Mouse Adult Tissues: Expression of mouse WISP-2 was observed
in stromal cells around the origin, and within the cusps of the
mitral and tricuspid valves of the adult heart. Expression was also
observed in the adventitial cells of the renal artery; expression
was presumed to be present at this site in all arteries.
[0549] All other tissues were negative.
Expression in Wnt-1 Tumors
[0550] The results demonstrated specific expression of mouse WISP-2
in the stroma of all Wnt-1 tumors examined. There was a signal over
mononuclear cells with open vesicular nuclei, possibly macrophages.
No expression was observed in either the benign or the malignant
epithelium.
(4) Human WISP-2
Expression in Human Tissues
[0551] Strong expression of the WISP-2-encoding gene was observed
in dermal fibroblasts in normal adult skin. Additionally, strong
expression was seen in two cirrhotic livers, at sites of active
hepatic fibrosis. Moderate expression was found over fasiculata
cells of adrenal cortex. This localization supports a role for
human WISP-2 in extracellular matrix formation or turnover.
Expression in Human Breast Carcinoma and Normal Breast Tissue, and
in Lung Carcinoma
[0552] Human WISP-2 showed a similar hybridization pattern to human
WISP-1 (described above) in the two breast tumors examined. It was
negative on benign and malignant epithelial cells, but showed
specific hybridization in mesenchmal cells, particularly in areas
of tissue repair, including dystrophic ossification. The signal
appeared to localize to the same cell population for both probes
WISP-1 and WISP-2; however, in some areas (breast tumor 02), the
signal for WISP-2 was significantly stronger than that for human
WISP-1. Most positive cells had the morphology of fibroblasts;
smooth muscle cells appeared to be negative. The signal for human
WISP-2 was less intense in the lung tumor tissue; however, this
section also showed less tissue repair compared with the breast
tumor slides. Normal lung and kidney tissue were essentially
negative for human WISP-2, as for human WISP-1.
[0553] In summary, this study shows expression of human WISP-2 RNA
in mesenchymal cells involved in tissue repair and/or collagen
deposition. The signal was particularly strong in benign
fibroblast-like cells adjacent to either infiltrating breast
carcinoma cells or tissue destruction due to benign, inflammatory
conditions (duct rupture). Of note is the fact that deposition of
benign osteoid seemed to correlate with strong expression of the
RNA.
(5) Human WISP-3
[0554] Expression in Normal Adult and Fetal Tissues and in Human
Breast Carcinoma and Normal Breast Tissue and in Colon
Carcinoma
[0555] The analysis shows strong expression of human WISP-3 in
dermal fibroblasts in normal adult skin and in cirrhotic livers at
sites of active hepatic fibrosis. This localization pattern
supports a role for this growth factor in extracellular matrix
formation and turnover.
[0556] The probe for human WISP-3 was negative on most tissues
examined it showed a weak, diffuse positivity on sections of an
osteosarcoma; some of the positive cells do represent malignant
cells. WISP-3 was negative on all normal and fetal tissues
examined.
Example 17
Ability of WISP Polypeptides to Inhibit VEGF-Stimulated
Proliferation of Endothelial Cell Growth
[0557] The ability of mouse and human WISP-1 and human WISP-2
polypeptides to inhibit VEGF-stimulated proliferation of
endothelial cells was tested. Specifically, bovine adrenal cortical
capillary endothelial (ACE) cells (from primary culture, maximum
12-14 passages) were plated on 96-well microtiter plates (Amersham
Life Science) at a density of 500 cells/well per 100 .mu.L in
low-glucose DMEM, 10% calf serum, 2 mM glutamine, 1.times.
pen/strept, and flingizone, supplemented with 3 ng/mL VEGF.
Controls were plated the same way but some did not include VEGF. A
test sample of either mouse WISP-1, human WISP-1 conjugated to IgG,
or human WISP-2 (PR0261) conjugated to poly-His was added in a
100-.mu.l volume for a 200-.mu.L final volume. Cells were incubated
for 5-7 days at 37.degree. C. The media were aspirated and the
cells washed 1.times. with PBS. An acid phosphatase reaction
mixture (100 .mu.L, 0.1 M sodium acetate, pH 5.5, 0.1%
TRITON-100.TM., 10 mM p-nitrophenyl phosphate) was added. After
incubation for 2 hours at 37.degree. C., the reaction was stopped
by addition of 10 .mu.l, 1 N NaOH. OD was measured on a microtiter
plate reader at 405 mn. Controls were: no cells, cells alone,
cells+FGF (5 ng/mL), cells+VEGF (3 ng/mL), cells+VEGF (3
ng/ml)+TGF-.beta. (1 ng/ml), and cells+VEGF (3 ng/mL)+LIF (5 ng/mL)
(TGF-.beta. at a 1 ng/ml concentration is known to block 70-90% of
VEGF-stimulated cell proliferation.)
[0558] The results were assessed by calculating the percentage
inhibition of VEGF(3ng/ml)-stimulated cell proliferation,
determined by measuring acid phosphatase activity at OD405 nm (1)
relative to cells without stimulation, and (2) relative to the
reference TGF-.beta. inhibition of VEGF-stimulated activity. The
results, as shown in Table X below, are indicative of the utility
of the WISP polypeptides in cancer therapy and specifically in
inhibiting tumor angiogenesis. The numerical values (relative
inhibition) shown in Table x are determined by calculating the
percent inhibition of VEGF-stimulated proliferation by the mouse
WISP-1, human WISP-1-IgG, and human WISP-2-poly-His polypeptides
relative to cells without stimulation and then dividing that
percentage into the percent inhibition obtained by TGF-.beta. at 1
ng/ml, which is known to block 70-90% of VEGF-stimulated cell
proliferation. Human WISP-1 and human WISP-2 appear to be
particularly useful as angiostatic agents. TABLE-US-00028 TABLE X
Polypeptide Concentration (nM) Relative Inhibition Mouse WISP-1 0.1
113 '' 1.0 108 '' 10.0 109 Human WISP-1-IgG 1.1 1 '' 11.0 0.95 ''
110.0 0.9 Human WisP-2-poly-His 0.01 0.95 '' 0.01% 1.1 '' 0.1 0.62
'' 1.0 1.03 '' 1.0 0.5 '' 1.0 0.6
Deposit of Material
[0559] The following materials have been deposited with the
American Type Culture Collection, 10801 University Blvd., Manassas,
Va., USA (ATCC): TABLE-US-00029 Material ATCC Dep. No. Deposit Date
pRK5E.h.WIG-1.568.38 209533 Dec. 10, 1997 pRK5E.m.WIG-1.568.6his
209537 Dec. 10, 1997 Plasmid 209391 Oct. 17, 1997 (encoding human
WISP-2) pRKE.m.WIG-2.1367.3 209538 Dec. 10, 1997 DNA56350-1176-2
209706 Mar. 26, 1998 DNA58800-1176-2 209707 Mar. 26, 1998
[0560] These deposits were made under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of viable cultures of the deposits for 30 years from the date of
deposit. The deposits will be made available by ATCC under the
terms of the Budapest Treaty, and subject to an agreement between
Genentech, Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of the cultures of the deposits to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 USC .sctn.122 and the
Commissioner's rules pursuant thereto (including 37 CFR .sctn.1.14
with particular reference to 886 OG 638).
[0561] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited materials is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
[0562] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the constructs deposited, since the deposited embodiment is
intended as a single illustration of certain aspects of the
invention and any constructs that are functionally equivalent are
within the scope of this invention. The deposits of materials
herein do not constitute an admission that the written description
herein contained is inadequate to enable the practice of any aspect
of the invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Sequence CWU 1
1
156 1 2830 DNA Homo sapiens 1 cccacgcgtc cgctgggccc agctcccccg
agaggtggtc ggatcctctg 50 ggctgctcgg tcgatgcctg tgccactgac
gtccaggcat gaggtggttc 100 ctgccctgga cgctggcagc agtgacagca
gcagccgcca gcaccgtcct 150 ggccacggcc ctctctccag cccctacgac
catggacttt actccagctc 200 cactggagga cacctcctca cgcccccaat
tctgcaagtg gccatgtgag 250 tgcccgccat ccccaccccg ctgcccgctg
ggggtcagcc tcatcacaga 300 tggctgtgag tgctgtaaga tgtgcgctca
gcagcttggg gacaactgca 350 cggaggctgc catctgtgac ccccaccggg
gcctctactg tgactacagc 400 ggggaccgcc cgaggtacgc aataggagtg
tgtgcacagg tggtcggtgt 450 gggctgcgtc ctggatgggg tgcgctacaa
caacggccag tccttccagc 500 ctaactgcaa gtacaactgc acgtgcatcg
acggcgcggt gggctgcaca 550 ccactgtgcc tccgagtgcg ccccccgcgt
ctctggtgcc cccacccgcg 600 gcgcgtgagc atacctggcc actgctgtga
gcagtgggta tgtgaggacg 650 acgccaagag gccacgcaag accgcacccc
gtgacacagg agccttcgat 700 gctgtgggtg aggtggaggc atggcacagg
aactgcatag cctacacaag 750 cccctggagc ccttgctcca ccagctgcgg
cctgggggtc tccactcgga 800 tctccaatgt taacgcccag tgctggcctg
agcaagagag ccgcctctgc 850 aacttgcggc catgcgatgt ggacatccat
acactcatta aggcagggaa 900 gaagtgtctg gctgtgtacc agccagaggc
atccatgaac ttcacacttg 950 cgggctgcat cagcacacgc tcctatcaac
ccaagtactg tggagtttgc 1000 atggacaata ggtgctgcat cccctacaag
tctaagacta tcgacgtgtc 1050 cttccagtgt cctgatgggc ttggcttctc
ccgccaggtc ctatggatta 1100 atgcctgctt ctgtaacctg agctgtagga
atcccaatga catctttgct 1150 gacttggaat cctaccctga cttctcagaa
attgccaact aggcaggcac 1200 aaatcttggg tcttggggac taacccaatg
cctgtgaagc agtcagccct 1250 tatggccaat aacttttcac caatgagcct
tagttaccct gatctggacc 1300 cttggcctcc atttctgtct ctaaccattc
aaatgacgcc tgatggtgct 1350 gctcaggccc atgctatgag ttttctcctt
gatatcattc agcatctact 1400 ctaaagaaaa atgcctgtct ctagctgttc
tggactacac ccaagcctga 1450 tccagccttt ccaagtcact agaagtcctg
ctggatcttg cctaaatccc 1500 aagaaatgga atcaggtaga cttttaatat
cactaatttc ttctttagat 1550 gccaaaccac aagactcttt gggtccattc
agatgaatag atggaatttg 1600 gaacaataga ataatctatt atttggagcc
tgccaagagg tactgtaatg 1650 ggtaattctg acgtcagcgc accaaaacta
tcctgattcc aaatatgtat 1700 gcacctcaag gtcatcaaac atttgccaag
tgagttgaat agttgcttaa 1750 ttttgatttt taatggaaag ttgtatccat
taacctgggc attgttgagg 1800 ttaagtttct cttcacccct acactgtgaa
gggtacagat taggtttgtc 1850 ccagtcagaa ataaaatttg ataaacattc
ctgttgatgg gaaaagcccc 1900 cagttaatac tccagagaca gggaaaggtc
agcccatttc agaaggacca 1950 attgactctc acactgaatc agctgctgac
tggcagggct ttgggcagtt 2000 ggccaggctc ttccttgaat cttctccctt
gtcctgcttg ggttcatagg 2050 aattggtaag gcctctggac tggcctgtct
ggcccctgag agtggtgccc 2100 tggaacactc ctctactctt acagagcctt
gagagaccca gctgcagacc 2150 atgccagacc cactgaaatg accaagacag
gttcaggtag gggtgtgggt 2200 caaaccaaga agtgggtgcc cttggtagca
gcctggggtg acctctagag 2250 ctggaggctg tgggactcca ggggcccccg
tgttcaggac acatctattg 2300 cagagactca tttcacagcc tttcgttctg
ctgaccaaat ggccagtttt 2350 ctggtaggaa gatggaggtt taccagttgt
ttagaaacag aaatagactt 2400 aataaaggtt taaagctgaa gaggttgaag
ctaaaaggaa aaggttgttg 2450 ttaatgaata tcaggctatt atttattgta
ttaggaaaat ataatattta 2500 ctgttagaat tcttttattt agggcctttt
ctgtgccaga cattgctctc 2550 agtgctttgc atgtattagc tcactgaatc
ttcacgacaa tgttgagaag 2600 ttcccattat tatttctgtt cttacaaatg
tgaaacggaa gctcatagag 2650 gtgagaaaac tcaaccagag tcacccagtt
ggtgactggg aaagttagga 2700 ttcagatcga aattggactg tctttataac
ccatattttc cccctgtttt 2750 tagagcttcc aaatgtgtca gaataggaaa
acattgcaat aaatggcttg 2800 attttttaaa aaaaaaaaaa aaaaaaaaaa 2830 2
2830 DNA Homo sapiens 2 tttttttttt tttttttttt tttaaaaaat caagccattt
attgcaatgt 50 tttcctattc tgacacattt ggaagctcta aaaacagggg
gaaaatatgg 100 gttataaaga cagtccaatt tcgatctgaa tcctaacttt
cccagtcacc 150 aactgggtga ctctggttga gttttctcac ctctatgagc
ttccgtttca 200 catttgtaag aacagaaata ataatgggaa cttctcaaca
ttgtcgtgaa 250 gattcagtga gctaatacat gcaaagcact gagagcaatg
tctggcacag 300 aaaaggccct aaataaaaga attctaacag taaatattat
attttcctaa 350 tacaataaat aatagcctga tattcattaa caacaacctt
ttccttttag 400 cttcaacctc ttcagcttta aacctttatt aagtctattt
ctgtttctaa 450 acaactggta aacctccatc ttcctaccag aaaactggcc
atttggtcag 500 cagaacgaaa ggctgtgaaa tgagtctctg caatagatgt
gtcctgaaca 550 cgggggcccc tggagtccca cagcctccag ctctagaggt
caccccaggc 600 tgctaccaag ggcacccact tcttggtttg acccacaccc
ctacctgaac 650 ctgtcttggt catttcagtg ggtctggcat ggtctgcagc
tgggtctctc 700 aaggctctgt aagagtagag gagtgttcca gggcaccact
ctcaggggcc 750 agacaggcca gtccagaggc cttaccaatt cctatgaacc
caagcaggac 800 aagggagaag attcaaggaa gagcctggcc aactgcccaa
agccctgcca 850 gtcagcagct gattcagtgt gagagtcaat tggtccttct
gaaatgggct 900 gacctttccc tgtctctgga gtattaactg ggggcttttc
ccatcaacag 950 gaatgtttat caaattttat ttctgactgg gacaaaccta
atctgtaccc 1000 ttcacagtgt aggggtgaag agaaacttaa cctcaacaat
gcccaggtta 1050 atggatacaa ctttccatta aaaatcaaaa ttaagcaact
attcaactca 1100 cttggcaaat gtttgatgac cttgaggtgc atacatattt
ggaatcagga 1150 tagttttggt gcgctgacgt cagaattacc cattacagta
cctcttggca 1200 ggctccaaat aatagattat tctattgttc caaattccat
ctattcatct 1250 gaatggaccc aaagagtctt gtggtttggc atctaaagaa
gaaattagtg 1300 atattaaaag tctacctgat tccatttctt gggatttagg
caagatccag 1350 caggacttct agtgacttgg aaaggctgga tcaggcttgg
gtgtagtcca 1400 gaacagctag agacaggcat ttttctttag agtagatgct
gaatgatatc 1450 aaggagaaaa ctcatagcat gggcctgagc agcaccatca
ggcgtcattt 1500 gaatggttag agacagaaat ggaggccaag ggtccagatc
agggtaacta 1550 aggctcattg gtgaaaagtt attggccata agggctgact
gcttcacagg 1600 cattgggtta gtccccaaga cccaagattt gtgcctgcct
agttggcaat 1650 ttctgagaag tcagggtagg attccaagtc agcaaagatg
tcattgggat 1700 tcctacagct caggttacag aagcaggcat taatccatag
gacctggcgg 1750 gagaagccaa gcccatcagg acactggaag gacacgtcga
tagtcttaga 1800 cttgtagggg atgcagcacc tattgtccat gcaaactcca
cagtacttgg 1850 gttgatagga gcgtgtgctg atgcagcccg caagtgtgaa
gttcatggat 1900 gcctctggct ggtacacagc cagacacttc ttccctgcct
taatgagtgt 1950 atggatgtcc acatcgcatg gccgcaagtt gcagaggcgg
ctctcttgct 2000 caggccagca ctgggcgtta acattggaga tccgagtgga
gacccccagg 2050 ccgcagctgg tggagcaagg gctccagggg cttgtgtagg
ctatgcagtt 2100 cctgtgccat gcctccacct cacccacagc atcgaaggct
cctgtgtcac 2150 ggggtgcggt cttgcgtggc ctcttggcgt cgtcctcaca
tacccactgc 2200 tcacagcagt ggccaggtat gctcacgcgc cgcgggtggg
ggcaccagag 2250 acgcgggggg cgcactcgga ggcacagtgg tgtgcagccc
accgcgccgt 2300 cgatgcacgt gcagttgtac ttgcagttag gctggaagga
ctggccgttg 2350 ttgtagcgca ccccatccag gacgcagccc acaccgacca
cctgtgcaca 2400 cactcctatt gcgtacctcg ggcggtcccc gctgtagtca
cagtagaggc 2450 cccggtgggg gtcacagatg gcagcctccg tgcagttgtc
cccaagctgc 2500 tgagcgcaca tcttacagca ctcacagcca tctgtgatga
ggctgacccc 2550 cagcgggcag cggggtgggg atggcgggca ctcacatggc
cacttgcaga 2600 attgggggcg tgaggaggtg tcctccagtg gagctggagt
aaagtccatg 2650 gtcgtagggg ctggagagag ggccgtggcc aggacggtgc
tggcggctgc 2700 tgctgtcact gctgccagcg tccagggcag gaaccacctc
atgcctggac 2750 gtcagtggca caggcatcga ccgagcagcc cagaggatcc
gaccacctct 2800 cgggggagct gggcccagcg gacgcgtggg 2830 3 345 PRT
Homo sapiens 3 Thr Ala Leu Ser Pro Ala Pro Thr Thr Met Asp Phe Thr
Pro Ala 1 5 10 15 Pro Leu Glu Asp Thr Ser Ser Arg Pro Gln Phe Cys
Lys Trp Pro 20 25 30 Cys Glu Cys Pro Pro Ser Pro Pro Arg Cys Pro
Leu Gly Val Ser 35 40 45 Leu Ile Thr Asp Gly Cys Glu Cys Cys Lys
Met Cys Ala Gln Gln 50 55 60 Leu Gly Asp Asn Cys Thr Glu Ala Ala
Ile Cys Asp Pro His Arg 65 70 75 Gly Leu Tyr Cys Asp Tyr Ser Gly
Asp Arg Pro Arg Tyr Ala Ile 80 85 90 Gly Val Cys Ala Gln Val Val
Gly Val Gly Cys Val Leu Asp Gly 95 100 105 Val Arg Tyr Asn Asn Gly
Gln Ser Phe Gln Pro Asn Cys Lys Tyr 110 115 120 Asn Cys Thr Cys Ile
Asp Gly Ala Val Gly Cys Thr Pro Leu Cys 125 130 135 Leu Arg Val Arg
Pro Pro Arg Leu Trp Cys Pro His Pro Arg Arg 140 145 150 Val Ser Ile
Pro Gly His Cys Cys Glu Gln Trp Val Cys Glu Asp 155 160 165 Asp Ala
Lys Arg Pro Arg Lys Thr Ala Pro Arg Asp Thr Gly Ala 170 175 180 Phe
Asp Ala Val Gly Glu Val Glu Ala Trp His Arg Asn Cys Ile 185 190 195
Ala Tyr Thr Ser Pro Trp Ser Pro Cys Ser Thr Ser Cys Gly Leu 200 205
210 Gly Val Ser Thr Arg Ile Ser Asn Val Asn Ala Gln Cys Trp Pro 215
220 225 Glu Gln Glu Ser Arg Leu Cys Asn Leu Arg Pro Cys Asp Val Asp
230 235 240 Ile His Thr Leu Ile Lys Ala Gly Lys Lys Cys Leu Ala Val
Tyr 245 250 255 Gln Pro Glu Ala Ser Met Asn Phe Thr Leu Ala Gly Cys
Ile Ser 260 265 270 Thr Arg Ser Tyr Gln Pro Lys Tyr Cys Gly Val Cys
Met Asp Asn 275 280 285 Arg Cys Cys Ile Pro Tyr Lys Ser Lys Thr Ile
Asp Val Ser Phe 290 295 300 Gln Cys Pro Asp Gly Leu Gly Phe Ser Arg
Gln Val Leu Trp Ile 305 310 315 Asn Ala Cys Phe Cys Asn Leu Ser Cys
Arg Asn Pro Asn Asp Ile 320 325 330 Phe Ala Asp Leu Glu Ser Tyr Pro
Asp Phe Ser Glu Ile Ala Asn 335 340 345 4 367 PRT Homo sapiens 4
Met Arg Trp Phe Leu Pro Trp Thr Leu Ala Ala Val Thr Ala Ala 1 5 10
15 Ala Ala Ser Thr Val Leu Ala Thr Ala Leu Ser Pro Ala Pro Thr 20
25 30 Thr Met Asp Phe Thr Pro Ala Pro Leu Glu Asp Thr Ser Ser Arg
35 40 45 Pro Gln Phe Cys Lys Trp Pro Cys Glu Cys Pro Pro Ser Pro
Pro 50 55 60 Arg Cys Pro Leu Gly Val Ser Leu Ile Thr Asp Gly Cys
Glu Cys 65 70 75 Cys Lys Met Cys Ala Gln Gln Leu Gly Asp Asn Cys
Thr Glu Ala 80 85 90 Ala Ile Cys Asp Pro His Arg Gly Leu Tyr Cys
Asp Tyr Ser Gly 95 100 105 Asp Arg Pro Arg Tyr Ala Ile Gly Val Cys
Ala Gln Val Val Gly 110 115 120 Val Gly Cys Val Leu Asp Gly Val Arg
Tyr Asn Asn Gly Gln Ser 125 130 135 Phe Gln Pro Asn Cys Lys Tyr Asn
Cys Thr Cys Ile Asp Gly Ala 140 145 150 Val Gly Cys Thr Pro Leu Cys
Leu Arg Val Arg Pro Pro Arg Leu 155 160 165 Trp Cys Pro His Pro Arg
Arg Val Ser Ile Pro Gly His Cys Cys 170 175 180 Glu Gln Trp Val Cys
Glu Asp Asp Ala Lys Arg Pro Arg Lys Thr 185 190 195 Ala Pro Arg Asp
Thr Gly Ala Phe Asp Ala Val Gly Glu Val Glu 200 205 210 Ala Trp His
Arg Asn Cys Ile Ala Tyr Thr Ser Pro Trp Ser Pro 215 220 225 Cys Ser
Thr Ser Cys Gly Leu Gly Val Ser Thr Arg Ile Ser Asn 230 235 240 Val
Asn Ala Gln Cys Trp Pro Glu Gln Glu Ser Arg Leu Cys Asn 245 250 255
Leu Arg Pro Cys Asp Val Asp Ile His Thr Leu Ile Lys Ala Gly 260 265
270 Lys Lys Cys Leu Ala Val Tyr Gln Pro Glu Ala Ser Met Asn Phe 275
280 285 Thr Leu Ala Gly Cys Ile Ser Thr Arg Ser Tyr Gln Pro Lys Tyr
290 295 300 Cys Gly Val Cys Met Asp Asn Arg Cys Cys Ile Pro Tyr Lys
Ser 305 310 315 Lys Thr Ile Asp Val Ser Phe Gln Cys Pro Asp Gly Leu
Gly Phe 320 325 330 Ser Arg Gln Val Leu Trp Ile Asn Ala Cys Phe Cys
Asn Leu Ser 335 340 345 Cys Arg Asn Pro Asn Asp Ile Phe Ala Asp Leu
Glu Ser Tyr Pro 350 355 360 Asp Phe Ser Glu Ile Ala Asn 365 367 5
345 PRT Homo sapiens 5 Thr Ala Leu Ser Pro Ala Pro Thr Thr Met Asp
Phe Thr Pro Ala 1 5 10 15 Pro Leu Glu Asp Thr Ser Ser Arg Pro Gln
Phe Cys Lys Trp Pro 20 25 30 Cys Glu Cys Pro Pro Ser Pro Pro Arg
Cys Pro Leu Gly Val Ser 35 40 45 Leu Ile Thr Asp Gly Cys Glu Cys
Cys Lys Met Cys Ala Gln Gln 50 55 60 Leu Gly Asp Asn Cys Thr Glu
Ala Ala Ile Cys Asp Pro His Arg 65 70 75 Gly Leu Tyr Cys Asp Tyr
Ser Gly Asp Arg Pro Arg Tyr Ala Ile 80 85 90 Gly Val Cys Ala Gln
Val Val Gly Val Gly Cys Val Leu Asp Gly 95 100 105 Val Arg Tyr Asn
Asn Gly Gln Ser Phe Gln Pro Asn Cys Lys Tyr 110 115 120 Asn Cys Thr
Cys Ile Asp Gly Ala Val Gly Cys Thr Pro Leu Cys 125 130 135 Leu Arg
Val Arg Pro Pro Arg Leu Trp Cys Pro His Pro Arg Arg 140 145 150 Val
Ser Ile Pro Gly His Cys Cys Glu Gln Trp Ile Cys Glu Asp 155 160 165
Asp Ala Lys Arg Pro Arg Lys Thr Ala Pro Arg Asp Thr Gly Ala 170 175
180 Phe Asp Ala Val Gly Glu Val Glu Ala Trp His Arg Asn Cys Ile 185
190 195 Ala Tyr Thr Ser Pro Trp Ser Pro Cys Ser Thr Ser Cys Gly Leu
200 205 210 Gly Val Ser Thr Arg Ile Ser Asn Val Asn Ala Gln Cys Trp
Pro 215 220 225 Glu Gln Glu Ser Arg Leu Cys Asn Leu Arg Pro Cys Asp
Val Asp 230 235 240 Ile His Thr Leu Ile Lys Ala Gly Lys Lys Cys Leu
Ala Val Tyr 245 250 255 Gln Pro Glu Ala Ser Met Asn Phe Thr Leu Ala
Gly Cys Ile Ser 260 265 270 Thr Arg Ser Tyr Gln Pro Lys Tyr Cys Gly
Val Cys Met Asp Asn 275 280 285 Arg Cys Cys Ile Pro Tyr Lys Ser Lys
Thr Ile Asp Val Ser Phe 290 295 300 Gln Cys Pro Asp Gly Leu Gly Phe
Ser Arg Gln Val Leu Trp Ile 305 310 315 Asn Ala Cys Phe Cys Asn Leu
Ser Cys Arg Asn Pro Asn Asp Ile 320 325 330 Phe Ala Asp Leu Glu Ser
Tyr Pro Asp Phe Ser Glu Ile Ala Asn 335 340 345 6 345 PRT Homo
sapiens 6 Thr Ala Leu Ser Pro Ala Pro Thr Thr Met Asp Phe Thr Pro
Ala 1 5 10 15 Pro Leu Glu Asp Thr Ser Ser Arg Pro Gln Phe Cys Lys
Trp Pro 20 25 30 Cys Glu Cys Pro Pro Ser Pro Pro Arg Cys Pro Leu
Gly Val Ser 35 40 45 Leu Ile Thr Asp Gly Cys Glu Cys Cys Lys Met
Cys Ala Gln Gln 50 55 60 Leu Gly Asp Asn Cys Thr Glu Ala Ala Ile
Cys Asp Pro His Arg 65 70 75 Gly Leu Tyr Cys Asp Tyr Ser Gly Asp
Arg Pro Arg Tyr Ala Ile 80 85 90 Gly Val Cys Ala Gln Val Val Gly
Val Gly Cys Val Leu Asp Gly 95 100 105 Val Arg Tyr Asn Asn Gly Gln
Ser Phe Gln Pro Asn Cys Lys Tyr 110 115 120 Asn Cys Thr Cys Ile Asp
Gly Ala Val Gly Cys Thr Pro Leu Cys 125 130 135 Leu Arg Val Arg Pro
Pro Arg Leu Trp Cys Pro His Pro Arg Arg 140 145 150 Val Ser Ile Pro
Gly His Cys Cys Glu Gln Trp Val Cys Glu Asp 155 160 165 Asp Ala Lys
Arg Pro Arg Lys Thr Ala Pro Arg Asp Thr Gly Ser 170 175
180 Phe Asp Ala Val Gly Glu Val Glu Ala Trp His Arg Asn Cys Ile 185
190 195 Ala Tyr Thr Ser Pro Trp Ser Pro Cys Ser Thr Ser Cys Gly Leu
200 205 210 Gly Val Ser Thr Arg Ile Ser Asn Val Asn Ala Gln Cys Trp
Pro 215 220 225 Glu Gln Glu Ser Arg Leu Cys Asn Leu Arg Pro Cys Asp
Val Asp 230 235 240 Ile His Thr Leu Ile Lys Ala Gly Lys Lys Cys Leu
Ala Val Tyr 245 250 255 Gln Pro Glu Ala Ser Met Asn Phe Thr Leu Ala
Gly Cys Ile Ser 260 265 270 Thr Arg Ser Tyr Gln Pro Lys Tyr Cys Gly
Val Cys Met Asp Asn 275 280 285 Arg Cys Cys Ile Pro Tyr Lys Ser Lys
Thr Ile Asp Val Ser Phe 290 295 300 Gln Cys Pro Asp Gly Leu Gly Phe
Ser Arg Gln Val Leu Trp Ile 305 310 315 Asn Ala Cys Phe Cys Asn Leu
Ser Cys Arg Asn Pro Asn Asp Ile 320 325 330 Phe Ala Asp Leu Glu Ser
Tyr Pro Asp Phe Ser Glu Ile Ala Asn 335 340 345 7 367 PRT Homo
sapiens 7 Met Arg Trp Phe Leu Pro Trp Thr Leu Ala Ala Val Thr Ala
Ala 1 5 10 15 Ala Ala Ser Thr Val Leu Ala Thr Ala Leu Ser Pro Ala
Pro Thr 20 25 30 Thr Met Asp Phe Thr Pro Ala Pro Leu Glu Asp Thr
Ser Ser Arg 35 40 45 Pro Gln Phe Cys Lys Trp Pro Cys Glu Cys Pro
Pro Ser Pro Pro 50 55 60 Arg Cys Pro Leu Gly Val Ser Leu Ile Thr
Asp Gly Cys Glu Cys 65 70 75 Cys Lys Met Cys Ala Gln Gln Leu Gly
Asp Asn Cys Thr Glu Ala 80 85 90 Ala Ile Cys Asp Pro His Arg Gly
Leu Tyr Cys Asp Tyr Ser Gly 95 100 105 Asp Arg Pro Arg Tyr Ala Ile
Gly Val Cys Ala Gln Val Val Gly 110 115 120 Val Gly Cys Val Leu Asp
Gly Val Arg Tyr Asn Asn Gly Gln Ser 125 130 135 Phe Gln Pro Asn Cys
Lys Tyr Asn Cys Thr Cys Ile Asp Gly Ala 140 145 150 Val Gly Cys Thr
Pro Leu Cys Leu Arg Val Arg Pro Pro Arg Leu 155 160 165 Trp Cys Pro
His Pro Arg Arg Val Ser Ile Pro Gly His Cys Cys 170 175 180 Glu Gln
Trp Ile Cys Glu Asp Asp Ala Lys Arg Pro Arg Lys Thr 185 190 195 Ala
Pro Arg Asp Thr Gly Ala Phe Asp Ala Val Gly Glu Val Glu 200 205 210
Ala Trp His Arg Asn Cys Ile Ala Tyr Thr Ser Pro Trp Ser Pro 215 220
225 Cys Ser Thr Ser Cys Gly Leu Gly Val Ser Thr Arg Ile Ser Asn 230
235 240 Val Asn Ala Gln Cys Trp Pro Glu Gln Glu Ser Arg Leu Cys Asn
245 250 255 Leu Arg Pro Cys Asp Val Asp Ile His Thr Leu Ile Lys Ala
Gly 260 265 270 Lys Lys Cys Leu Ala Val Tyr Gln Pro Glu Ala Ser Met
Asn Phe 275 280 285 Thr Leu Ala Gly Cys Ile Ser Thr Arg Ser Tyr Gln
Pro Lys Tyr 290 295 300 Cys Gly Val Cys Met Asp Asn Arg Cys Cys Ile
Pro Tyr Lys Ser 305 310 315 Lys Thr Ile Asp Val Ser Phe Gln Cys Pro
Asp Gly Leu Gly Phe 320 325 330 Ser Arg Gln Val Leu Trp Ile Asn Ala
Cys Phe Cys Asn Leu Ser 335 340 345 Cys Arg Asn Pro Asn Asp Ile Phe
Ala Asp Leu Glu Ser Tyr Pro 350 355 360 Asp Phe Ser Glu Ile Ala Asn
365 367 8 367 PRT Homo sapiens 8 Met Arg Trp Phe Leu Pro Trp Thr
Leu Ala Ala Val Thr Ala Ala 1 5 10 15 Ala Ala Ser Thr Val Leu Ala
Thr Ala Leu Ser Pro Ala Pro Thr 20 25 30 Thr Met Asp Phe Thr Pro
Ala Pro Leu Glu Asp Thr Ser Ser Arg 35 40 45 Pro Gln Phe Cys Lys
Trp Pro Cys Glu Cys Pro Pro Ser Pro Pro 50 55 60 Arg Cys Pro Leu
Gly Val Ser Leu Ile Thr Asp Gly Cys Glu Cys 65 70 75 Cys Lys Met
Cys Ala Gln Gln Leu Gly Asp Asn Cys Thr Glu Ala 80 85 90 Ala Ile
Cys Asp Pro His Arg Gly Leu Tyr Cys Asp Tyr Ser Gly 95 100 105 Asp
Arg Pro Arg Tyr Ala Ile Gly Val Cys Ala Gln Val Val Gly 110 115 120
Val Gly Cys Val Leu Asp Gly Val Arg Tyr Asn Asn Gly Gln Ser 125 130
135 Phe Gln Pro Asn Cys Lys Tyr Asn Cys Thr Cys Ile Asp Gly Ala 140
145 150 Val Gly Cys Thr Pro Leu Cys Leu Arg Val Arg Pro Pro Arg Leu
155 160 165 Trp Cys Pro His Pro Arg Arg Val Ser Ile Pro Gly His Cys
Cys 170 175 180 Glu Gln Trp Val Cys Glu Asp Asp Ala Lys Arg Pro Arg
Lys Thr 185 190 195 Ala Pro Arg Asp Thr Gly Ser Phe Asp Ala Val Gly
Glu Val Glu 200 205 210 Ala Trp His Arg Asn Cys Ile Ala Tyr Thr Ser
Pro Trp Ser Pro 215 220 225 Cys Ser Thr Ser Cys Gly Leu Gly Val Ser
Thr Arg Ile Ser Asn 230 235 240 Val Asn Ala Gln Cys Trp Pro Glu Gln
Glu Ser Arg Leu Cys Asn 245 250 255 Leu Arg Pro Cys Asp Val Asp Ile
His Thr Leu Ile Lys Ala Gly 260 265 270 Lys Lys Cys Leu Ala Val Tyr
Gln Pro Glu Ala Ser Met Asn Phe 275 280 285 Thr Leu Ala Gly Cys Ile
Ser Thr Arg Ser Tyr Gln Pro Lys Tyr 290 295 300 Cys Gly Val Cys Met
Asp Asn Arg Cys Cys Ile Pro Tyr Lys Ser 305 310 315 Lys Thr Ile Asp
Val Ser Phe Gln Cys Pro Asp Gly Leu Gly Phe 320 325 330 Ser Arg Gln
Val Leu Trp Ile Asn Ala Cys Phe Cys Asn Leu Ser 335 340 345 Cys Arg
Asn Pro Asn Asp Ile Phe Ala Asp Leu Glu Ser Tyr Pro 350 355 360 Asp
Phe Ser Glu Ile Ala Asn 365 367 9 1766 DNA Mus musculus Unsure 10
Unknown base 9 taacaaggcn gtcctgcttg gagaggcatc cgcatcctct
gggctgagcc 50 gtagctcctg tgacgctgac ttccaggcat gaggtggctc
ctgccctgga 100 cgctggcagc cgtggcagtc ctgagggtgg gcaacatcct
ggccacggcc 150 ctctctccaa cccccacaac aatgaccttc accccagcac
cactagagga 200 aacgactaca cgccccgaat tctgcaagtg gccatgtgag
tgcccacaat 250 ccccacctcg ctgcccactg ggcgtcagcc taatcacaga
tggctgtgaa 300 tgctgtaaga tatgtgccca gcagcttggg gacaactgca
cagaggctgc 350 catctgtgac ccacaccggg gcctctactg cgattacagt
ggggatcgcc 400 cgaggtacgc aataggagtg tgtgcacagg tggtcggtgt
gggctgtgtc 450 ctggatggcg tacgctacac caatggcgag tccttccaac
ccaactgcag 500 gtacaactgt acctgcattg atggcacggt gggctgcaca
ccgctgtgcc 550 taagccccag gcccccacgc ctctggtgcc gccagccccg
gcacgtgaga 600 gtccctggcc agtgctgtga gcagtgggtg tgtgatgatg
acgcaaggag 650 accacgccag actgcactgt tggacaccag agcctttgca
gcgtcaggcg 700 ccgtggagca acggtatgag aactgcatag cctacactag
tccctggagc 750 ccctgctcta ccacctgtgg cctaggtatc tccactcgga
tctctaacgt 800 caatgcccgg tgctggccag agcaggaaag tcgcctctgc
aacctgcggc 850 catgtgatgt ggacatccaa ctacacatca aggcagggaa
gaaatgcctg 900 gctgtgtacc agccagagga ggccacgaac ttcactctcg
caggctgtgt 950 cagcacacgc acctaccgac ccaagtactg cggagtctgt
actgacaata 1000 ggtgttgcat cccctacaag tccaagacca tcagtgtgga
tttccagtgt 1050 ccagaggggc caggtttctc ccggcaggtc ctatggatta
atgcttgctt 1100 ctgcaacctg agctgcagga atcctaacga tatctttgct
gacttggaat 1150 cttaccctga cttcgaagag attgccaatt aggtgggtgt
gtggctcagg 1200 gtaaagttcc atgctgcaaa gcagccagcc ctttgtggtc
caggacttca 1250 caattgagcc ttatttcatc tacttcctac tcgattctga
attcccagtt 1300 tctgttcctg ttttgacaat cgtaatggcc caggagagtg
ctgctcaggc 1350 tcagacaatg ggttcctcct tggggacatt ctacatcatt
ccaaggaaaa 1400 cacatctctg actgttcaca atggaagcaa agcctggccc
agctagtctg 1450 gctccagcct gggcaagttg tcagaagttg tgatgggatt
gtccaaggaa 1500 aagcatcagc tgaagaacca gtatcatgaa gtccttcctc
agatgccaag 1550 cctagggatg ctgggatcct ttcagacaga tggatgggat
tggggacaca 1600 ggaataagct attattttac ccttgccaaa tgatactatc
ctgggtattt 1650 ctgcctaaaa acataccaaa agtgttcttg ttccactgat
ctgtatatca 1700 caagtcacca aacattttcc aggtgaggac ccatagttgt
gtcattctgt 1750 tttgccaatt gaaaaa 1766 10 1766 DNA Mus musculus
Unsure 1757 Unknown base. 10 tttttcaatt ggcaaaacag aatgacacaa
ctatgggtcc tcacctggaa 50 aatgtttggt gacttgtgat atacagatca
gtggaacaag aacacttttg 100 gtatgttttt aggcagaaat acccaggata
gtatcatttg gcaagggtaa 150 aataatagct tattcctgtg tccccaatcc
catccatctg tctgaaagga 200 tcccagcatc cctaggcttg gcatctgagg
aaggacttca tgatactggt 250 tcttcagctg atgcttttcc ttggacaatc
ccatcacaac ttctgacaac 300 ttgcccaggc tggagccaga ctagctgggc
caggctttgc ttccattgtg 350 aacagtcaga gatgtgtttt ccttggaatg
atgtagaatg tccccaagga 400 ggaacccatt gtctgagcct gagcagcact
ctcctgggcc attacgattg 450 tcaaaacagg aacagaaact gggaattcag
aatcgagtag gaagtagatg 500 aaataaggct caattgtgaa gtcctggacc
acaaagggct ggctgctttg 550 cagcatggaa ctttaccctg agccacacac
ccacctaatt ggcaatctct 600 tcgaagtcag ggtaagattc caagtcagca
aagatatcgt taggattcct 650 gcagctcagg ttgcagaagc aagcattaat
ccataggacc tgccgggaga 700 aacctggccc ctctggacac tggaaatcca
cactgatggt cttggacttg 750 taggggatgc aacacctatt gtcagtacag
actccgcagt acttgggtcg 800 gtaggtgcgt gtgctgacac agcctgcgag
agtgaagttc gtggcctcct 850 ctggctggta cacagccagg catttcttcc
ctgccttgat gtgtagttgg 900 atgtccacat cacatggccg caggttgcag
aggcgacttt cctgctctgg 950 ccagcaccgg gcattgacgt tagagatccg
agtggagata cctaggccac 1000 aggtggtaga gcaggggctc cagggactag
tgtaggctat gcagttctca 1050 taccgttgct ccacggcgcc tgacgctgca
aaggctctgg tgtccaacag 1100 tgcagtctgg cgtggtctcc ttgcgtcatc
atcacacacc cactgctcac 1150 agcactggcc agggactctc acgtgccggg
gctggcggca ccagaggcgt 1200 gggggcctgg ggcttaggca cagcggtgtg
cagcccaccg tgccatcaat 1250 gcaggtacag ttgtacctgc agttgggttg
gaaggactcg ccattggtgt 1300 agcgtacgcc atccaggaca cagcccacac
cgaccacctg tgcacacact 1350 cctattgcgt acctcgggcg atccccactg
taatcgcagt agaggccccg 1400 gtgtgggtca cagatggcag cctctgtgca
gttgtcccca agctgctggg 1450 cacatatctt acagcattca cagccatctg
tgattaggct gacgcccagt 1500 gggcagcgag gtggggattg tgggcactca
catggccact tgcagaattc 1550 ggggcgtgta gtcgtttcct ctagtggtgc
tggggtgaag gtcattgttg 1600 tgggggttgg agagagggcc gtggccagga
tgttgcccac cctcaggact 1650 gccacggctg ccagcgtcca gggcaggagc
cacctcatgc ctggaagtca 1700 gcgtcacagg agctacggct cagcccagag
gatgcggatg cctctccaag 1750 caggacngcc ttgtta 1766 11 345 PRT Mus
musculus 11 Thr Ala Leu Ser Pro Thr Pro Thr Thr Met Thr Phe Thr Pro
Ala 1 5 10 15 Pro Leu Glu Glu Thr Thr Thr Arg Pro Glu Phe Cys Lys
Trp Pro 20 25 30 Cys Glu Cys Pro Gln Ser Pro Pro Arg Cys Pro Leu
Gly Val Ser 35 40 45 Leu Ile Thr Asp Gly Cys Glu Cys Cys Lys Ile
Cys Ala Gln Gln 50 55 60 Leu Gly Asp Asn Cys Thr Glu Ala Ala Ile
Cys Asp Pro His Arg 65 70 75 Gly Leu Tyr Cys Asp Tyr Ser Gly Asp
Arg Pro Arg Tyr Ala Ile 80 85 90 Gly Val Cys Ala Gln Val Val Gly
Val Gly Cys Val Leu Asp Gly 95 100 105 Val Arg Tyr Thr Asn Gly Glu
Ser Phe Gln Pro Asn Cys Arg Tyr 110 115 120 Asn Cys Thr Cys Ile Asp
Gly Thr Val Gly Cys Thr Pro Leu Cys 125 130 135 Leu Ser Pro Arg Pro
Pro Arg Leu Trp Cys Arg Gln Pro Arg His 140 145 150 Val Arg Val Pro
Gly Gln Cys Cys Glu Gln Trp Val Cys Asp Asp 155 160 165 Asp Ala Arg
Arg Pro Arg Gln Thr Ala Leu Leu Asp Thr Arg Ala 170 175 180 Phe Ala
Ala Ser Gly Ala Val Glu Gln Arg Tyr Glu Asn Cys Ile 185 190 195 Ala
Tyr Thr Ser Pro Trp Ser Pro Cys Ser Thr Thr Cys Gly Leu 200 205 210
Gly Ile Ser Thr Arg Ile Ser Asn Val Asn Ala Arg Cys Trp Pro 215 220
225 Glu Gln Glu Ser Arg Leu Cys Asn Leu Arg Pro Cys Asp Val Asp 230
235 240 Ile Gln Leu His Ile Lys Ala Gly Lys Lys Cys Leu Ala Val Tyr
245 250 255 Gln Pro Glu Glu Ala Thr Asn Phe Thr Leu Ala Gly Cys Val
Ser 260 265 270 Thr Arg Thr Tyr Arg Pro Lys Tyr Cys Gly Val Cys Thr
Asp Asn 275 280 285 Arg Cys Cys Ile Pro Tyr Lys Ser Lys Thr Ile Ser
Val Asp Phe 290 295 300 Gln Cys Pro Glu Gly Pro Gly Phe Ser Arg Gln
Val Leu Trp Ile 305 310 315 Asn Ala Cys Phe Cys Asn Leu Ser Cys Arg
Asn Pro Asn Asp Ile 320 325 330 Phe Ala Asp Leu Glu Ser Tyr Pro Asp
Phe Glu Glu Ile Ala Asn 335 340 345 12 367 PRT Mus musculus 12 Met
Arg Trp Leu Leu Pro Trp Thr Leu Ala Ala Val Ala Val Leu 1 5 10 15
Arg Val Gly Asn Ile Leu Ala Thr Ala Leu Ser Pro Thr Pro Thr 20 25
30 Thr Met Thr Phe Thr Pro Ala Pro Leu Glu Glu Thr Thr Thr Arg 35
40 45 Pro Glu Phe Cys Lys Trp Pro Cys Glu Cys Pro Gln Ser Pro Pro
50 55 60 Arg Cys Pro Leu Gly Val Ser Leu Ile Thr Asp Gly Cys Glu
Cys 65 70 75 Cys Lys Ile Cys Ala Gln Gln Leu Gly Asp Asn Cys Thr
Glu Ala 80 85 90 Ala Ile Cys Asp Pro His Arg Gly Leu Tyr Cys Asp
Tyr Ser Gly 95 100 105 Asp Arg Pro Arg Tyr Ala Ile Gly Val Cys Ala
Gln Val Val Gly 110 115 120 Val Gly Cys Val Leu Asp Gly Val Arg Tyr
Thr Asn Gly Glu Ser 125 130 135 Phe Gln Pro Asn Cys Arg Tyr Asn Cys
Thr Cys Ile Asp Gly Thr 140 145 150 Val Gly Cys Thr Pro Leu Cys Leu
Ser Pro Arg Pro Pro Arg Leu 155 160 165 Trp Cys Arg Gln Pro Arg His
Val Arg Val Pro Gly Gln Cys Cys 170 175 180 Glu Gln Trp Val Cys Asp
Asp Asp Ala Arg Arg Pro Arg Gln Thr 185 190 195 Ala Leu Leu Asp Thr
Arg Ala Phe Ala Ala Ser Gly Ala Val Glu 200 205 210 Gln Arg Tyr Glu
Asn Cys Ile Ala Tyr Thr Ser Pro Trp Ser Pro 215 220 225 Cys Ser Thr
Thr Cys Gly Leu Gly Ile Ser Thr Arg Ile Ser Asn 230 235 240 Val Asn
Ala Arg Cys Trp Pro Glu Gln Glu Ser Arg Leu Cys Asn 245 250 255 Leu
Arg Pro Cys Asp Val Asp Ile Gln Leu His Ile Lys Ala Gly 260 265 270
Lys Lys Cys Leu Ala Val Tyr Gln Pro Glu Glu Ala Thr Asn Phe 275 280
285 Thr Leu Ala Gly Cys Val Ser Thr Arg Thr Tyr Arg Pro Lys Tyr 290
295 300 Cys Gly Val Cys Thr Asp Asn Arg Cys Cys Ile Pro Tyr Lys Ser
305 310 315 Lys Thr Ile Ser Val Asp Phe Gln Cys Pro Glu Gly Pro Gly
Phe 320 325 330 Ser Arg Gln Val Leu Trp Ile Asn Ala Cys Phe Cys Asn
Leu Ser 335 340 345 Cys Arg Asn Pro Asn Asp Ile Phe Ala Asp Leu Glu
Ser Tyr Pro 350 355 360 Asp Phe Glu Glu Ile Ala Asn 365 367 13
1293
DNA Homo sapiens 13 cccacgcgtc cggctgggga catgagaggc acaccgaaga
cccacctcct 50 ggccttctcc ctcctctgcc tcctctcaaa ggtgcgtacc
cagctgtgcc 100 cgacaccatg tacctgcccc tggccacctc cccgatgccc
gctgggagta 150 cccctggtgc tggatggctg tggctgctgc cgggtatgtg
cacggcggct 200 gggggagccc tgcgaccaac tccacgtctg cgacgccagc
cagggcctgg 250 tctgccagcc cggggcagga cccggtggcc ggggggccct
gtgcctcttg 300 gcagaggacg acagcagctg tgaggtgaac ggccgcctgt
atcgggaagg 350 ggagaccttc cagccccact gcagcatccg ctgccgctgc
gaggacggcg 400 gcttcacctg cgtgccgctg tgcagcgagg atgtgcggct
gcccagctgg 450 gactgccccc accccaggag ggtcgaggtc ctgggcaagt
gctgccctga 500 gtgggtgtgc ggccaaggag ggggactggg gacccagccc
cttccagccc 550 aaggacccca gttttctggc cttgtctctt ccctgccccc
tggtgtcccc 600 tgcccagaat ggagcacggc ctggggaccc tgctcgacca
cctgtgggct 650 gggcatggcc acccgggtgt ccaaccagaa ccgcttctgc
cgactggaga 700 cccagcgccg cctgtgcctg tccaggccct gcccaccctc
caggggtcgc 750 agtccacaaa acagtgcctt ctagagccgg gctgggaatg
gggacacggt 800 gtccaccatc cccagctggt ggccctgtgc ctgggccctg
ggctgatgga 850 agatggtccg tgcccaggcc cttggctgca ggcaacactt
tagcttgggt 900 ccaccatgca gaacaccaat attaacacgc tgcctggtct
gtctggatcc 950 cgaggtatgg cagaggtgca agacctagtc ccctttcctc
taactcactg 1000 cctaggaggc tggccaaggt gtccagggtc ctctagccca
ctccctgcct 1050 acacacacag cctatatcaa acatgcacac gggcgagctt
tctctccgac 1100 ttcccctggg caagagatgg gacaagcagt cccttaatat
tgaggctgca 1150 gcaggtgctg ggctggactg gccatttttc tgggggtagg
atgaagagaa 1200 ggcacacaga gattctggat ctcctgctgc cttttctgga
gtttgtaaaa 1250 ttgttcctga atacaagcct atgcgtgaaa aaaaaaaaaa aaa
1293 14 1293 DNA Homo sapiens 14 tttttttttt ttttttcacg cataggcttg
tattcaggaa caattttaca 50 aactccagaa aaggcagcag gagatccaga
atctctgtgt gccttctctt 100 catcctaccc ccagaaaaat ggccagtcca
gcccagcacc tgctgcagcc 150 tcaatattaa gggactgctt gtcccatctc
ttgcccaggg gaagtcggag 200 agaaagctcg cccgtgtgca tgtttgatat
aggctgtgtg tgtaggcagg 250 gagtgggcta gaggaccctg gacaccttgg
ccagcctcct aggcagtgag 300 ttagaggaaa ggggactagg tcttgcacct
ctgccatacc tcgggatcca 350 gacagaccag gcagcgtgtt aatattggtg
ttctgcatgg tggacccaag 400 ctaaagtgtt gcctgcagcc aagggcctgg
gcacggacca tcttccatca 450 gcccagggcc caggcacagg gccaccagct
ggggatggtg gacaccgtgt 500 ccccattccc agcccggctc tagaaggcac
tgttttgtgg actgcgaccc 550 ctggagggtg ggcagggcct ggacaggcac
aggcggcgct gggtctccag 600 tcggcagaag cggttctggt tggacacccg
ggtggccatg cccagcccac 650 aggtggtcga gcagggtccc caggccgtgc
tccattctgg gcaggggaca 700 ccagggggca gggaagagac aaggccagaa
aactggggtc cttgggctgg 750 aaggggctgg gtccccagtc cccctccttg
gccgcacacc cactcagggc 800 agcacttgcc caggacctcg accctcctgg
ggtgggggca gtcccagctg 850 ggcagccgca catcctcgct gcacagcggc
acgcaggtga agccgccgtc 900 ctcgcagcgg cagcggatgc tgcagtgggg
ctggaaggtc tccccttccc 950 gatacaggcg gccgttcacc tcacagctgc
tgtcgtcctc tgccaagagg 1000 cacagggccc cccggccacc gggtcctgcc
ccgggctggc agaccaggcc 1050 ctggctggcg tcgcagacgt ggagttggtc
gcagggctcc cccagccgcc 1100 gtgcacatac ccggcagcag ccacagccat
ccagcaccag gggtactccc 1150 agcgggcatc ggggaggtgg ccaggggcag
gtacatggtg tcgggcacag 1200 ctgggtacgc acctttgaga ggaggcagag
gagggagaag gccaggaggt 1250 gggtcttcgg tgtgcctctc atgtccccag
ccggacgcgt ggg 1293 15 227 PRT Homo sapiens 15 Gln Leu Cys Pro Thr
Pro Cys Thr Cys Pro Trp Pro Pro Pro Arg 1 5 10 15 Cys Pro Leu Gly
Val Pro Leu Val Leu Asp Gly Cys Gly Cys Cys 20 25 30 Arg Val Cys
Ala Arg Arg Leu Gly Glu Pro Cys Asp Gln Leu His 35 40 45 Val Cys
Asp Ala Ser Gln Gly Leu Val Cys Gln Pro Gly Ala Gly 50 55 60 Pro
Gly Gly Arg Gly Ala Leu Cys Leu Leu Ala Glu Asp Asp Ser 65 70 75
Ser Cys Glu Val Asn Gly Arg Leu Tyr Arg Glu Gly Glu Thr Phe 80 85
90 Gln Pro His Cys Ser Ile Arg Cys Arg Cys Glu Asp Gly Gly Phe 95
100 105 Thr Cys Val Pro Leu Cys Ser Glu Asp Val Arg Leu Pro Ser Trp
110 115 120 Asp Cys Pro His Pro Arg Arg Val Glu Val Leu Gly Lys Cys
Cys 125 130 135 Pro Glu Trp Val Cys Gly Gln Gly Gly Gly Leu Gly Thr
Gln Pro 140 145 150 Leu Pro Ala Gln Gly Pro Gln Phe Ser Gly Leu Val
Ser Ser Leu 155 160 165 Pro Pro Gly Val Pro Cys Pro Glu Trp Ser Thr
Ala Trp Gly Pro 170 175 180 Cys Ser Thr Thr Cys Gly Leu Gly Met Ala
Thr Arg Val Ser Asn 185 190 195 Gln Asn Arg Phe Cys Arg Leu Glu Thr
Gln Arg Arg Leu Cys Leu 200 205 210 Ser Arg Pro Cys Pro Pro Ser Arg
Gly Arg Ser Pro Gln Asn Ser 215 220 225 Ala Phe 227 16 250 PRT Homo
sapiens 16 Met Arg Gly Thr Pro Lys Thr His Leu Leu Ala Phe Ser Leu
Leu 1 5 10 15 Cys Leu Leu Ser Lys Val Arg Thr Gln Leu Cys Pro Thr
Pro Cys 20 25 30 Thr Cys Pro Trp Pro Pro Pro Arg Cys Pro Leu Gly
Val Pro Leu 35 40 45 Val Leu Asp Gly Cys Gly Cys Cys Arg Val Cys
Ala Arg Arg Leu 50 55 60 Gly Glu Pro Cys Asp Gln Leu His Val Cys
Asp Ala Ser Gln Gly 65 70 75 Leu Val Cys Gln Pro Gly Ala Gly Pro
Gly Gly Arg Gly Ala Leu 80 85 90 Cys Leu Leu Ala Glu Asp Asp Ser
Ser Cys Glu Val Asn Gly Arg 95 100 105 Leu Tyr Arg Glu Gly Glu Thr
Phe Gln Pro His Cys Ser Ile Arg 110 115 120 Cys Arg Cys Glu Asp Gly
Gly Phe Thr Cys Val Pro Leu Cys Ser 125 130 135 Glu Asp Val Arg Leu
Pro Ser Trp Asp Cys Pro His Pro Arg Arg 140 145 150 Val Glu Val Leu
Gly Lys Cys Cys Pro Glu Trp Val Cys Gly Gln 155 160 165 Gly Gly Gly
Leu Gly Thr Gln Pro Leu Pro Ala Gln Gly Pro Gln 170 175 180 Phe Ser
Gly Leu Val Ser Ser Leu Pro Pro Gly Val Pro Cys Pro 185 190 195 Glu
Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly Leu 200 205 210
Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu 215 220
225 Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro Ser 230
235 240 Arg Gly Arg Ser Pro Gln Asn Ser Ala Phe 245 250 17 1734 DNA
Mus musculus 17 cccacgcgtc cgcgctcctg atctccagag gaccccgggc
tgggacaggg 50 gccttggcga ggctgcagct gctgtggcag tagcttggga
tggaggtctt 100 tcttgctggg aactgaggag ctgagaggct cctgtcaggc
tcctgtccta 150 aactcttggc acttgcggtg gcttgggctt cacacactgt
cagacacctt 200 cttggtggcc tcctcggcct caggtttgaa gctggctcca
caagggacac 250 ggtgacatga ggggcaaccc actgatccat cttctggcca
tttccttcct 300 ctgcattctc tcaatggtgt attcccagct gtgcccagca
ccctgtgcct 350 gtccttggac accaccccag tgcccaccgg gggtacccct
ggtgctggat 400 ggctgtggct gctgtcgagt gtgtgcacgg aggctggggg
agtcctgcga 450 ccacctgcat gtctgcgacc ccagccaggg cctggtttgt
cagcctgggg 500 caggccccag tggccgtggt gctgtgtgcc tcttcgaaga
ggatgacggg 550 agctgtgagg tgaatggccg caggtacctg gatggggaga
cctttaaacc 600 caattgcagg gttttgtgcc gctgtgatga cggtggtttc
acctgcctgc 650 cgctgtgcag tgaggatgtg cggctgccca gctgggactg
cccacgcccc 700 aggagaatac aggtgccagg aaggtgctgc cccgagtggg
tgtgtgacca 750 ggcagtgatg cagccggcaa tccagccctc ctcagcccaa
ggacaccaac 800 tttctgccct tgtcactcct gcatctgccg atggcccctg
tccaaactgg 850 agcacagcct ggggcccctg ctcaaccacc tgtgggttgg
gcatagccac 900 ccgagtatcc aaccagaacc gattctgcca actggagatc
cagcgtcgcc 950 tgtgtctgtc cagaccctgc ctggcatcca ggagccacgg
ctcatggaac 1000 agtgccttct agagccattg cggggatgtg gatacagggc
ctgccattct 1050 cagcaaatgt ccctaggacc aggccctgga ctgatggtag
atgcccctct 1100 ccatgctctt ggctgcagtt aactgtcctg ggtggattca
gtgtccagag 1150 cctctgagcg atccctgctc tgtctgaggt gggggaagca
ggtgaccagc 1200 tccatttctc tggattctga cccaggcttc tgggttctcc
tggctagttc 1250 ctcaaaactt ccctgtatga aaaggacaac caaaaggacc
tttaaagcta 1300 agctgtactg ggcaagcctg gccaccatgc tggggatagt
gacagtaata 1350 ggtaccaggc agcagattgc ctgaaacatc caggtccctt
cttggacttc 1400 tatgtgcttg tcccaaagat tatgggtgac cttgtaagtg
tgcctttcct 1450 gatctgagaa caccctgccc ggctgggaag aattttctgg
gaacatgaag 1500 agatggaatc acactattct taagagcgtt tgccaagtcc
aggaacttga 1550 cctttgtatt tgtaaaaata cacatctctt aaatgctcac
aaagcaagag 1600 gctccacact tctggcaggc cagggccttt ctcttcagca
tgagagagac 1650 aaggaacagt agagtaccct cctctggagg actggcccgg
tctggaataa 1700 acacccaaat caagtgtgga aaaaaaaaaa aaaa 1734 18 1734
DNA Mus musculus 18 tttttttttt tttttccaca cttgatttgg gtgtttattc
cagaccgggc 50 cagtcctcca gaggagggta ctctactgtt ccttgtctct
ctcatgctga 100 agagaaaggc cctggcctgc cagaagtgtg gagcctcttg
ctttgtgagc 150 atttaagaga tgtgtatttt tacaaataca aaggtcaagt
tcctggactt 200 ggcaaacgct cttaagaata gtgtgattcc atctcttcat
gttcccagaa 250 aattcttccc agccgggcag ggtgttctca gatcaggaaa
ggcacactta 300 caaggtcacc cataatcttt gggacaagca catagaagtc
caagaaggga 350 cctggatgtt tcaggcaatc tgctgcctgg tacctattac
tgtcactatc 400 cccagcatgg tggccaggct tgcccagtac agcttagctt
taaaggtcct 450 tttggttgtc cttttcatac agggaagttt tgaggaacta
gccaggagaa 500 cccagaagcc tgggtcagaa tccagagaaa tggagctggt
cacctgcttc 550 ccccacctca gacagagcag ggatcgctca gaggctctgg
acactgaatc 600 cacccaggac agttaactgc agccaagagc atggagaggg
gcatctacca 650 tcagtccagg gcctggtcct agggacattt gctgagaatg
gcaggccctg 700 tatccacatc cccgcaatgg ctctagaagg cactgttcca
tgagccgtgg 750 ctcctggatg ccaggcaggg tctggacaga cacaggcgac
gctggatctc 800 cagttggcag aatcggttct ggttggatac tcgggtggct
atgcccaacc 850 cacaggtggt tgagcagggg ccccaggctg tgctccagtt
tggacagggg 900 ccatcggcag atgcaggagt gacaagggca gaaagttggt
gtccttgggc 950 tgaggagggc tggattgccg gctgcatcac tgcctggtca
cacacccact 1000 cggggcagca ccttcctggc acctgtattc tcctggggcg
tgggcagtcc 1050 cagctgggca gccgcacatc ctcactgcac agcggcaggc
aggtgaaacc 1100 accgtcatca cagcggcaca aaaccctgca attgggttta
aaggtctccc 1150 catccaggta cctgcggcca ttcacctcac agctcccgtc
atcctcttcg 1200 aagaggcaca cagcaccacg gccactgggg cctgccccag
gctgacaaac 1250 caggccctgg ctggggtcgc agacatgcag gtggtcgcag
gactccccca 1300 gcctccgtgc acacactcga cagcagccac agccatccag
caccaggggt 1350 acccccggtg ggcactgggg tggtgtccaa ggacaggcac
agggtgctgg 1400 gcacagctgg gaatacacca ttgagagaat gcagaggaag
gaaatggcca 1450 gaagatggat cagtgggttg cccctcatgt caccgtgtcc
cttgtggagc 1500 cagcttcaaa cctgaggccg aggaggccac caagaaggtg
tctgacagtg 1550 tgtgaagccc aagccaccgc aagtgccaag agtttaggac
aggagcctga 1600 caggagcctc tcagctcctc agttcccagc aagaaagacc
tccatcccaa 1650 gctactgcca cagcagctgc agcctcgcca aggcccctgt
cccagcccgg 1700 ggtcctctgg agatcaggag cgcggacgcg tggg 1734 19 228
PRT Mus musculus 19 Gln Leu Cys Pro Ala Pro Cys Ala Cys Pro Trp Thr
Pro Pro Gln 1 5 10 15 Cys Pro Pro Gly Val Pro Leu Val Leu Asp Gly
Cys Gly Cys Cys 20 25 30 Arg Val Cys Ala Arg Arg Leu Gly Glu Ser
Cys Asp His Leu His 35 40 45 Val Cys Asp Pro Ser Gln Gly Leu Val
Cys Gln Pro Gly Ala Gly 50 55 60 Pro Ser Gly Arg Gly Ala Val Cys
Leu Phe Glu Glu Asp Asp Gly 65 70 75 Ser Cys Glu Val Asn Gly Arg
Arg Tyr Leu Asp Gly Glu Thr Phe 80 85 90 Lys Pro Asn Cys Arg Val
Leu Cys Arg Cys Asp Asp Gly Gly Phe 95 100 105 Thr Cys Leu Pro Leu
Cys Ser Glu Asp Val Arg Leu Pro Ser Trp 110 115 120 Asp Cys Pro Arg
Pro Arg Arg Ile Gln Val Pro Gly Arg Cys Cys 125 130 135 Pro Glu Trp
Val Cys Asp Gln Ala Val Met Gln Pro Ala Ile Gln 140 145 150 Pro Ser
Ser Ala Gln Gly His Gln Leu Ser Ala Leu Val Thr Pro 155 160 165 Ala
Ser Ala Asp Gly Pro Cys Pro Asn Trp Ser Thr Ala Trp Gly 170 175 180
Pro Cys Ser Thr Thr Cys Gly Leu Gly Ile Ala Thr Arg Val Ser 185 190
195 Asn Gln Asn Arg Phe Cys Gln Leu Glu Ile Gln Arg Arg Leu Cys 200
205 210 Leu Ser Arg Pro Cys Leu Ala Ser Arg Ser His Gly Ser Trp Asn
215 220 225 Ser Ala Phe 228 20 251 PRT Mus musculus 20 Met Arg Gly
Asn Pro Leu Ile His Leu Leu Ala Ile Ser Phe Leu 1 5 10 15 Cys Ile
Leu Ser Met Val Tyr Ser Gln Leu Cys Pro Ala Pro Cys 20 25 30 Ala
Cys Pro Trp Thr Pro Pro Gln Cys Pro Pro Gly Val Pro Leu 35 40 45
Val Leu Asp Gly Cys Gly Cys Cys Arg Val Cys Ala Arg Arg Leu 50 55
60 Gly Glu Ser Cys Asp His Leu His Val Cys Asp Pro Ser Gln Gly 65
70 75 Leu Val Cys Gln Pro Gly Ala Gly Pro Ser Gly Arg Gly Ala Val
80 85 90 Cys Leu Phe Glu Glu Asp Asp Gly Ser Cys Glu Val Asn Gly
Arg 95 100 105 Arg Tyr Leu Asp Gly Glu Thr Phe Lys Pro Asn Cys Arg
Val Leu 110 115 120 Cys Arg Cys Asp Asp Gly Gly Phe Thr Cys Leu Pro
Leu Cys Ser 125 130 135 Glu Asp Val Arg Leu Pro Ser Trp Asp Cys Pro
Arg Pro Arg Arg 140 145 150 Ile Gln Val Pro Gly Arg Cys Cys Pro Glu
Trp Val Cys Asp Gln 155 160 165 Ala Val Met Gln Pro Ala Ile Gln Pro
Ser Ser Ala Gln Gly His 170 175 180 Gln Leu Ser Ala Leu Val Thr Pro
Ala Ser Ala Asp Gly Pro Cys 185 190 195 Pro Asn Trp Ser Thr Ala Trp
Gly Pro Cys Ser Thr Thr Cys Gly 200 205 210 Leu Gly Ile Ala Thr Arg
Val Ser Asn Gln Asn Arg Phe Cys Gln 215 220 225 Leu Glu Ile Gln Arg
Arg Leu Cys Leu Ser Arg Pro Cys Leu Ala 230 235 240 Ser Arg Ser His
Gly Ser Trp Asn Ser Ala Phe 245 250 251 21 345 PRT Homo sapiens 21
Thr Ala Leu Ser Pro Ala Pro Thr Thr Met Asp Phe Thr Pro Ala 1 5 10
15 Pro Leu Glu Asp Thr Ser Ser Arg Pro Gln Phe Cys Lys Trp Pro 20
25 30 Cys Glu Cys Pro Pro Ser Pro Pro Arg Cys Pro Leu Gly Val Ser
35 40 45 Leu Ile Thr Asp Gly Cys Glu Cys Cys Lys Met Cys Ala Gln
Gln 50 55 60 Leu Gly Asp Asn Cys Thr Glu Ala Ala Ile Cys Asp Pro
His Arg 65 70 75 Gly Leu Tyr Cys Asp Tyr Ser Gly Asp Arg Pro Arg
Tyr Ala Ile 80 85 90 Gly Val Cys Ala Gln Val Val Gly Val Gly Cys
Val Leu Asp Gly 95 100 105 Val Arg Tyr Asn Asn Gly Gln Ser Phe Gln
Pro Asn Cys Lys Tyr 110 115 120 Asn Cys Thr Cys Ile Asp Gly Ala Val
Gly Cys Thr Pro Leu Cys 125 130 135 Leu Arg Val Arg Pro Pro Arg Leu
Trp Cys Pro His Pro Arg Arg 140 145 150 Val Ser Ile Pro Gly His Cys
Cys Glu Gln Trp Ile Cys Glu Asp 155 160 165 Asp Ala Lys Arg Pro Arg
Lys Thr Ala Pro Arg Asp Thr Gly Ser 170 175 180 Phe Asp Ala Val Gly
Glu Val Glu Ala Trp His Arg Asn Cys Ile 185 190
195 Ala Tyr Thr Ser Pro Trp Ser Pro Cys Ser Thr Ser Cys Gly Leu 200
205 210 Gly Val Ser Thr Arg Ile Ser Asn Val Asn Ala Gln Cys Trp Pro
215 220 225 Glu Gln Glu Ser Arg Leu Cys Asn Leu Arg Pro Cys Asp Val
Asp 230 235 240 Ile His Thr Leu Ile Lys Ala Gly Lys Lys Cys Leu Ala
Val Tyr 245 250 255 Gln Pro Glu Ala Ser Met Asn Phe Thr Leu Ala Gly
Cys Ile Ser 260 265 270 Thr Arg Ser Tyr Gln Pro Lys Tyr Cys Gly Val
Cys Met Asp Asn 275 280 285 Arg Cys Cys Ile Pro Tyr Lys Ser Lys Thr
Ile Asp Val Ser Phe 290 295 300 Gln Cys Pro Asp Gly Leu Gly Phe Ser
Arg Gln Val Leu Trp Ile 305 310 315 Asn Ala Cys Phe Cys Asn Leu Ser
Cys Arg Asn Pro Asn Asp Ile 320 325 330 Phe Ala Asp Leu Glu Ser Tyr
Pro Asp Phe Ser Glu Ile Ala Asn 335 340 345 22 367 PRT Homo sapiens
22 Met Arg Trp Phe Leu Pro Trp Thr Leu Ala Ala Val Thr Ala Ala 1 5
10 15 Ala Ala Ser Thr Val Leu Ala Thr Ala Leu Ser Pro Ala Pro Thr
20 25 30 Thr Met Asp Phe Thr Pro Ala Pro Leu Glu Asp Thr Ser Ser
Arg 35 40 45 Pro Gln Phe Cys Lys Trp Pro Cys Glu Cys Pro Pro Ser
Pro Pro 50 55 60 Arg Cys Pro Leu Gly Val Ser Leu Ile Thr Asp Gly
Cys Glu Cys 65 70 75 Cys Lys Met Cys Ala Gln Gln Leu Gly Asp Asn
Cys Thr Glu Ala 80 85 90 Ala Ile Cys Asp Pro His Arg Gly Leu Tyr
Cys Asp Tyr Ser Gly 95 100 105 Asp Arg Pro Arg Tyr Ala Ile Gly Val
Cys Ala Gln Val Val Gly 110 115 120 Val Gly Cys Val Leu Asp Gly Val
Arg Tyr Asn Asn Gly Gln Ser 125 130 135 Phe Gln Pro Asn Cys Lys Tyr
Asn Cys Thr Cys Ile Asp Gly Ala 140 145 150 Val Gly Cys Thr Pro Leu
Cys Leu Arg Val Arg Pro Pro Arg Leu 155 160 165 Trp Cys Pro His Pro
Arg Arg Val Ser Ile Pro Gly His Cys Cys 170 175 180 Glu Gln Trp Ile
Cys Glu Asp Asp Ala Lys Arg Pro Arg Lys Thr 185 190 195 Ala Pro Arg
Asp Thr Gly Ser Phe Asp Ala Val Gly Glu Val Glu 200 205 210 Ala Trp
His Arg Asn Cys Ile Ala Tyr Thr Ser Pro Trp Ser Pro 215 220 225 Cys
Ser Thr Ser Cys Gly Leu Gly Val Ser Thr Arg Ile Ser Asn 230 235 240
Val Asn Ala Gln Cys Trp Pro Glu Gln Glu Ser Arg Leu Cys Asn 245 250
255 Leu Arg Pro Cys Asp Val Asp Ile His Thr Leu Ile Lys Ala Gly 260
265 270 Lys Lys Cys Leu Ala Val Tyr Gln Pro Glu Ala Ser Met Asn Phe
275 280 285 Thr Leu Ala Gly Cys Ile Ser Thr Arg Ser Tyr Gln Pro Lys
Tyr 290 295 300 Cys Gly Val Cys Met Asp Asn Arg Cys Cys Ile Pro Tyr
Lys Ser 305 310 315 Lys Thr Ile Asp Val Ser Phe Gln Cys Pro Asp Gly
Leu Gly Phe 320 325 330 Ser Arg Gln Val Leu Trp Ile Asn Ala Cys Phe
Cys Asn Leu Ser 335 340 345 Cys Arg Asn Pro Asn Asp Ile Phe Ala Asp
Leu Glu Ser Tyr Pro 350 355 360 Asp Phe Ser Glu Ile Ala Asn 365 367
23 1403 DNA Homo sapiens 23 gccagtctgg gcccagctcc cccgagaggt
ggtcggatcc tctgggctgc 50 tcggtcgatg cctgtgccac tgacgtccag
gcatgaggtg gttcctgccc 100 tggacgctgg cagcagtgac agcagcagcc
gccagcaccg tcctggccac 150 ggccctctct ccagccccta cgaccatgga
ctttacccca gctccactgg 200 aggacacctc ctcacgcccc caattctgca
agtggccatg tgagtgcccg 250 ccatccccac cccgctgccc gctgggggtc
agcctcatca cagatggctg 300 tgagtgctgt aagatgtgcg ctcagcagct
tggggacaac tgcacggagg 350 ctgccatctg tgacccccac cggggcctct
actgtgacta cagcggggac 400 cgcccgagag gtggtcggtg tgggctgcgt
cctggatggg gtgcgctaca 450 acaacggcca gtccttccag cctaactgca
agtacaactg cacgtgcatc 500 gacggcgcgg tgggctgcac accactgtgc
ctccgagtgc gccccccgcg 550 tctctggtgc ccccacccgc ggcgcgtgag
catacctggc cactgctgtg 600 agcagtgggt atgtgaggac gacgccaaga
ggccacgcaa gaccgcaccc 650 cgtgacacag gagccttcga tgctgtgggt
gaggtggagg catggcacag 700 gaactgcata gcctacacaa gcccctggag
cccttgctcc accagctgcg 750 gcctgggggt ctccactcgg atctccaatg
ttaacgccca gtgctggcct 800 gagcaagaga gccgcctctg caacttgcgg
ccatgcgatg tggacatcca 850 tacactcatt aaggcaggga agaagtgtct
ggctgtgtac cagccagagg 900 catccatgaa cttcacactt gcgggctgca
tcagcacacg ctcctatcaa 950 cccaagtact gtggagtttg catggacaat
aggtgctgca tcccctacaa 1000 gtctaagact atcgacgtgt ccttccagtg
tcctgatggg cttggcttct 1050 cccgccaggt cctatggatt aatgcctgct
tctgtaacct gagctgtagg 1100 aatcccaatg acatctttgc tgacttggaa
tcctaccctg acttctcaga 1150 aattgccaac taggcaggca caaatcttgg
gtcttgggga ctaacccaat 1200 gcctgtgaag cagtcagccc ttatggccaa
taacttttca ccaatgagcc 1250 ttagttaccc tgatctggac ccttggcctc
catttctgtc tctaaccatt 1300 caaatgacgc ctgatggtgc tgctcaggcc
catgctatga gttttctcct 1350 tgatatcatt cagcatctac tctaaagaaa
aatgcctgtc tctagctgtt 1400 ctg 1403 24 693 DNA Homo sapiens 24
tttaattaaa cccccaaggg ctgcggaagg agcatatctg gtgctcctga 50
tgggccggcc agtctgggcc cagctccccc gagaggtggt cggatcctct 100
gggctgctcg gtcgatgcct gtgccactga cgtccaggca tgaggtggtt 150
cctgccctgg acgctggcag cagtgacagc agcagccgcc agcaccgtcc 200
tggccacggc cctctctcca gcccctacga ccatggactt taccccagct 250
ccactggagg acacctcctc acgcccccaa ttctgcaagt ggccatgtga 300
gtgcccgcca tccccacccc gctgcccgct gggggtcagc ctcatcacag 350
atggctgtga gtgctgtaag atgtgcgctc agcagcttgg ggacaactgc 400
acggaggctg ccatctgtga cccccaccgg ggcctctact gtgactacag 450
cggggaccgc ccgaggtacg caataggagt gtgtgcacgc agggaagaag 500
tgtctggctg tgtaccagcc agaggcatcc atgaacttca cacttgcggg 550
ctgcatcagc acacgctcct atcaacccaa gtactgtgga gtttgcatgg 600
acaacaggtg ctgcatcccc tacaagtcta agactatcga cgtgtccttc 650
cagtgtcctg atgggcttgg cttctcccgc caggtcctat gga 693 25 683 DNA Homo
sapiens 25 cagaatttga actgggatcc acctgtctct aaagatgggt ttcctcccat
50 gcttccacac tgcctctctt gatcagaaac atacaaggag ctgagaacat 100
gtcctccact ccctgggtac ctttgctggt tagaagccaa cttgctgtcc 150
tgtggggagg tacagccaat ttctgtgttc ctctgagttc tggggaccgc 200
agaccttagt gtggtgaaag tgagcgttgg gggctggtgg gagctgtaga 250
ttcatgcaga ttctgttccc cacacacaga tgctgtgggt gaggtggagg 300
catggcacag gaactgcata gcctacacaa gcccctggag cccttgctcc 350
accagctgcg gcctgggggt ctccactcgg atctccaatg ttaacgccca 400
gtgctggcct gagcaagaga gccgcctctg caacttgcgg ccatgcgatg 450
tggacatcca tacactcatt aaggcaggga agaagtgtct ggctgtgtac 500
cagccagagg catccatgaa cttcacactt gcgggctgca tcagcacacg 550
ctcctatcaa cccaagtact gtggagtttg catggacaat aggtgctgca 600
tcccctacaa gtctaagact atcgacgtgt ccttccagtg tcctgatggg 650
cttggcttct cccgccaggt cgtatggatt aat 683 26 1202 DNA Homo sapiens
26 gtctgggccc agctcccccg agaggtggtc ggatcctctg ggctgctcgg 50
tcgatgcctg tgccactgac gtccaggcat gaggtggttc ctgccctgga 100
cgctggcagc agtgacagca gcagccgcca gcaccgtcct ggccacggcc 150
ctctctccag cccctacgac catggacttt accccagctc cactggagga 200
cacctcctca cgcccccaat tctgcaagtg gccatgtgag tgcccgccat 250
ccccaccccg ctgcccgctg ggggtcagcc tcatcacaga tggctgtgag 300
tgctgtaaga tgtgcgctca gcagcttggg gacaactgca cggaggctgc 350
catctgtgac ccccaccggg gcctctactg tgactacagc ggggaccgcc 400
cgaggtacgc aataggagtg tgtgcacgca gggaagaagt gtctggctgt 450
gtaccagcca gaggcatcca tgaacttcac acttgcgggc tgcatcagca 500
cacgctccta tcaacccaag tactgtggag tttgcatgga caacaggtgc 550
tgcatcccct acaagtctaa gactatcgac gtgtccttcc agtgtcctga 600
tgggcttggc ttctcccgcc aggtcctatg gattaatgcc tgcttctgta 650
acctgagctg taggaatccc aatgacatct ttgctgactt ggaatcctac 700
cctgacttct cagaaattgc caactaggca ggcacaaatc ttgggtcttg 750
gggactaacc caatgcctgt gaagcagtca gcccttatgg ccaataactt 800
ttcaccaatg agccttagtt accctgatct ggacccttgg cctccatttc 850
tgtctctaac cattcaaatg acgcctgatg gtgctgctca ggcccatgct 900
atgagttttc tccttgatat cattcagcat ctactctaaa gaaaaatgcc 950
tgtctctagc tgttctggac tacacccaag cctgatccag cctttccaag 1000
tcactagaag tcctgctgga tcttgcctaa atcccaagaa atggaatcag 1050
gtagactttt aatatcacta atttcttctt tagatgccaa accacaagac 1100
tctttgggtc cattcagatg aatagatgga atttggaaca atagaataat 1150
ctattatttg gagcctgcca agaggtactg taatgggtaa ttctgacgtc 1200 ag 1202
27 1183 DNA Homo sapiens 27 cagaacagct agagacaggc atttttcttt
agagtagatg ctgaatgata 50 tcaaggagaa aactcatagc atgggcctga
gcagcaccat caggcgtcat 100 ttgaatggtt agagacagaa atggaggcca
agggtccaga tcagggtaac 150 taaggctcat tggtgaaaag ttattggcca
taagggctga ctgcttcaca 200 ggcattgggt tagtccccaa gacccaagat
ttgtgcctgc ctagttggca 250 atttctgaga agtcagggta ggattccaag
tcagcaaaga tgtcattggg 300 attcctacag ctcaggttac agaagcaggc
attaatccat aggacctggc 350 gggagaagcc aagcccatca ggacactgga
aggacacgtc gatagtctta 400 gacttgtagg ggatgcagca cctattgtcc
atgcaaactc cacagtactt 450 gggttgatag gagcgtgtgc tgatgcagcc
cgcaagtgtg aagttcatgg 500 atgcctctgg ctggtacaca gccagacact
tcttccctgc cttaatgagt 550 gtatggatgt ccacatcgca tggccgcaag
ttgcagaggc ggctctcttg 600 ctcaggccag cactgggcgt taacattgga
gatccgagtg gagaccccca 650 ggccgcagct ggtggagcaa gggctccagg
ggcttgtgta ggctatgcag 700 ttcctgtgcc atgcctccac ctcacccaca
gcatctgtgt gtggggaaca 750 gaatctgcat gaatctacag ctcccaccag
cccccaacgc tcactttcac 800 cacactaagg tctgcggtcc ccagaactca
gaggaacaca gaaattggct 850 gtacctcccc acaggacagc aagttggctt
ctaaccagca aaggtaccca 900 gggagtggag gacatgttct cagctccttg
tatgtttctg atcaagagag 950 gcagtgtgga agcatgggag gaaacccatc
tttagagaca ggtggatccc 1000 agttcaaatt ctgctctacc acctacaagc
tgtgtgatct tagataaccc 1050 accctgggcc tgtctcccca ttagaacaat
aacacctgcc tgtgcggctg 1100 gcaacacaat aataagggcc tagattttta
ctgagtatgc atcaatcatc 1150 cttgctaagt gctgggaatg ggactttttt ttt
1183 28 546 DNA Homo sapiens 28 cctgatctgg acccttggcc tccaattctg
tctgtaacca ttcaaatgac 50 gcctggtggt gctgctcagg cccatagcaa
ggttcagcct ggttaagtcc 100 aagctgaatt agcggccgcg tcgacagtag
gagtgtgtgc acatgctgtg 150 ggtgaggtgg aggcatggca caggaactgc
atagcctaca caagcccctg 200 gagcccttgc tccaccagct gcggcctggg
ggtctccact cggatctcca 250 atgttaacgc ccagtgctgg cctgagcaag
agagccgcct ctgcaacttg 300 cggccatgcg atgtggacat ccatacactc
attaaggcag ggaagaagtg 350 tctggctgtg taccagccag aggcatccat
gaacttcaca cttgcgggct 400 gcatcagcac acgctcctat caacccaagt
actgtggagt ttgcatggac 450 aataggtgct gcatccccta caagtctaag
actatcgacg tgtccttcca 500 gtgtcctgat gggcttggct tctcccgcca
ggtcctatgg attaat 546 29 1101 DNA Homo sapiens 29 ggcccagctc
ccccgagagg tggtcggatc ctctgggctg ctcggtcgat 50 gcctgtgcca
ctgacgtcca ggcatgaggt ggttcctgcc ctggacgctg 100 gcagcagtga
cagcagcagc cgccagcacc gtcctggcca cggccctctc 150 tccagcccct
acgaccatgg actttacccc agctccactg gaggacacct 200 cctcacgccc
ccaattctgc aagtggccat gtgagtgccc gccatcccca 250 ccccgctgcc
cgctgggggt cagcctcatc acagatggct gtgagtgctg 300 taagatgtgc
gctcagcagc ttggggacaa ctgcacggag gctgccatct 350 gtgaccccca
ccggggcctc tactgtgact acagcgggga ccgcccgaga 400 ggtggtcggt
gtgggctgcg tcctggatgg ggtgcgctac aacaacggcc 450 agtccttcca
gcctaactgc aagtacaact gcacgtgcat cgacggcgcg 500 gtgggctgca
caccactgtg cctccgagtg cgccccccgc gtctctggtg 550 cccccacccg
cggcgcgtga gcatacctgg ccactgctgt gagcagtgga 600 tatgtgagga
cgacgccaag aggccacgca agaccgcacc ccgtgacaca 650 ggagccttcg
atgccagaag cgcccgctcc ctcagagatg tgacaaccaa 700 aatcatctcc
agacctttcc aaatacaccc taggagacaa aattgctcgg 750 tggagaagca
gtcctgtgag gacaggagga ggcgtggagg aaagctttgt 800 ccccagcagc
cccagggaag caaggcagct ctcccaccac cacctcccca 850 ggagggccac
acgagggtca cggggggagc agggaggcgg aagctgtctg 900 ccattgtgtc
tggcccagtg accctgttct gaccgagcac aagcggagcc 950 cctgcctagc
cgagatgctg tgggtgaggt ggaggcatgg cacaggaact 1000 gcatagccta
cacaagcccc tggagccctt gctccaccag ctgcggcctg 1050 ggggtctcca
ctcggatctc caatgttaac gcccagtgct ggcctgagca 1100 a 1101 30 1335 DNA
Homo sapiens Unsure 1205, 1318 Unknown base. 30 gtggggtttg
cagaggagac aggggagctt tgtgtacccg gagcaatgaa 50 caagcggcga
cttctctacc cctcagggtg gctccacggt cccagcgaca 100 tgcaggggct
cctcttctcc actcttctgc ttgctggcct ggcacagttc 150 tgctgcaggg
tacagggcac tggaccatta gatacaacac ctgaaggaag 200 gcctggagaa
gtgtcagatg cacctcagcg taaacagttt tgtcactggc 250 cctgcaaatg
ccctcagcag aagccccgtt gccctcctgg agtgagcctg 300 gtgagagatg
gctgtggatg ctgtaaaatc tgtgccaagc aaccagggga 350 aatctgcaat
gaagctgacc tctgtgaccc acacaaaggg ctgtattgtg 400 actactcagt
agacaggcct aggtacgaga ctggagtgtg tgcatacctt 450 gtagctgttg
ggtgcgagtt caaccaggta cattatcata atggccaagt 500 gtttcagccc
aaccccttgt tcagctgcct ctgtgtgagt ggggccattg 550 gatgcacacc
tctgttcata ccaaagctgg ctggcagtca ctgctctgga 600 gctaaaggtg
gaaagaagtc tgatcagtca aactgtagcc tggaaccatt 650 actacagcag
ctttcaacaa gctacaaaac aatgccagct tatagagatc 700 tcccacttat
ttggaaaaaa aaatgtcttg tgcaagcaac aaaatggact 750 ccctgctcca
gaacatgtgg gatgggaata tctaacaggg tgaccaatga 800 aaacagcaac
tgtgaaatga gaaaagagaa aagactgtgt tacattcagc 850 cttgcgacag
caatatatta aagacaataa agattcccaa aggaaaaaca 900 tgccaaccta
ctttccaact ctccaaagct gaaaaatttg tcttttctgg 950 atgctcaagt
actcagagtt acaaacccac tttttgtgga atatgcttgg 1000 ataagagatg
ctgtatccct aataagtcta aaatgattac tattcaattt 1050 gattgcccaa
atgaggggtc atttaaatgg aagatgctgt ggattacatc 1100 ttgtgtgtgt
cagagaaact gcagagaacc tggagatata ttttctgagc 1150 tcaagattct
gtaaaaccaa gcaaatgggg gaaaagttag tcaatcctgt 1200 catanaataa
aaaaattagt gagtataaaa tggtggcaaa tctactttgt 1250 ttaaaacagt
atgaatgcct attctcagat cactacattt aaggcattag 1300 aaacttttaa
aaagttanct taaaaatata cataa 1335 31 1335 DNA Homo sapiens Unsure
18, 131 Unknown base. 31 ttatgtatat ttttaagnta actttttaaa
agtttctaat gccttaaatg 50 tagtgatctg agaataggca ttcatactgt
tttaaacaaa gtagatttgc 100 caccatttta tactcactaa tttttttatt
ntatgacagg attgactaac 150 ttttccccca tttgcttggt tttacagaat
cttgagctca gaaaatatat 200 ctccaggttc tctgcagttt ctctgacaca
cacaagatgt aatccacagc 250 atcttccatt taaatgaccc ctcatttggg
caatcaaatt gaatagtaat 300 cattttagac ttattaggga tacagcatct
cttatccaag catattccac 350 aaaaagtggg tttgtaactc tgagtacttg
agcatccaga aaagacaaat 400 ttttcagctt tggagagttg gaaagtaggt
tggcatgttt ttcctttggg 450 aatctttatt gtctttaata tattgctgtc
gcaaggctga atgtaacaca 500 gtcttttctc ttttctcatt tcacagttgc
tgttttcatt ggtcaccctg 550 ttagatattc ccatcccaca tgttctggag
cagggagtcc attttgttgc 600 ttgcacaaga catttttttt tccaaataag
tgggagatct ctataagctg 650 gcattgtttt gtagcttgtt gaaagctgct
gtagtaatgg ttccaggcta 700 cagtttgact gatcagactt ctttccacct
ttagctccag agcagtgact 750 gccagccagc tttggtatga acagaggtgt
gcatccaatg gccccactca 800 cacagaggca gctgaacaag gggttgggct
gaaacacttg gccattatga 850 taatgtacct ggttgaactc gcacccaaca
gctacaaggt atgcacacac 900 tccagtctcg tacctaggcc tgtctactga
gtagtcacaa tacagccctt 950 tgtgtgggtc acagaggtca gcttcattgc
agatttcccc tggttgcttg 1000 gcacagattt tacagcatcc acagccatct
ctcaccaggc tcactccagg 1050 agggcaacgg ggcttctgct gagggcattt
gcagggccag tgacaaaact 1100 gtttacgctg aggtgcatct gacacttctc
caggccttcc ttcaggtgtt 1150 gtatctaatg gtccagtgcc ctgtaccctg
cagcagaact gtgccaggcc 1200 agcaagcaga agagtggaga agaggagccc
ctgcatgtcg ctgggaccgt 1250 ggagccaccc tgaggggtag agaagtcgcc
gcttgttcat tgctccgggt 1300 acacaaagct cccctgtctc ctctgcaaac cccac
1335 32 339 PRT Homo sapiens 32 Gln Phe Cys Cys Arg Val Gln Gly Thr
Gly Pro Leu Asp Thr Thr 1 5 10 15 Pro Glu Gly Arg Pro Gly Glu Val
Ser Asp Ala Pro Gln Arg Lys 20 25 30 Gln Phe Cys His Trp Pro Cys
Lys Cys Pro Gln Gln Lys Pro Arg 35 40 45 Cys Pro Pro Gly Val Ser
Leu Val Arg Asp Gly Cys Gly Cys Cys 50 55 60 Lys Ile Cys Ala Lys
Gln Pro Gly Glu Ile Cys Asn Glu Ala Asp 65 70 75 Leu Cys Asp Pro
His Lys Gly Leu Tyr Cys Asp Tyr Ser Val Asp 80 85 90 Arg Pro Arg
Tyr Glu Thr Gly Val Cys Ala Tyr Leu Val Ala Val 95 100 105 Gly Cys
Glu Phe Asn Gln Val His Tyr His Asn Gly Gln Val Phe 110 115 120 Gln
Pro Asn Pro Leu Phe Ser Cys Leu Cys Val Ser Gly Ala Ile 125 130 135
Gly Cys Thr Pro Leu Phe Ile Pro Lys Leu Ala Gly Ser His Cys 140 145
150 Ser Gly Ala Lys Gly Gly Lys Lys Ser Asp Gln Ser Asn Cys Ser 155
160 165 Leu Glu Pro Leu Leu Gln Gln Leu Ser Thr Ser Tyr Lys Thr Met
170 175 180 Pro Ala Tyr Arg Asp Leu Pro Leu Ile Trp Lys Lys Lys Cys
Leu 185 190 195 Val Gln Ala Thr Lys Trp Thr Pro Cys Ser Arg Thr Cys
Gly Met 200 205 210 Gly Ile Ser Asn Arg Val Thr Asn Glu Asn Ser Asn
Cys Glu Met 215 220 225 Arg Lys Glu Lys Arg Leu Cys Tyr Ile Gln Pro
Cys Asp Ser Asn 230 235 240 Ile Leu Lys Thr Ile Lys Ile Pro Lys Gly
Lys Thr Cys Gln Pro 245 250 255 Thr Phe Gln Leu Ser Lys Ala Glu Lys
Phe Val Phe Ser Gly Cys 260 265 270 Ser Ser Thr Gln Ser Tyr Lys Pro
Thr Phe Cys Gly Ile Cys Leu 275 280 285 Asp Lys Arg Cys Cys Ile Pro
Asn Lys Ser Lys Met Ile Thr Ile 290 295 300 Gln Phe Asp Cys Pro Asn
Glu Gly Ser Phe Lys Trp Lys Met Leu 305 310 315 Trp Ile Thr Ser Cys
Val Cys Gln Arg Asn Cys Arg Glu Pro Gly 320 325 330 Asp Ile Phe Ser
Glu Leu Lys Ile Leu 335 339 33 372 PRT Homo sapiens 33 Met Asn Lys
Arg Arg Leu Leu Tyr Pro Ser Gly Trp Leu His Gly 1 5 10 15 Pro Ser
Asp Met Gln Gly Leu Leu Phe Ser Thr Leu Leu Leu Ala 20 25 30 Gly
Leu Ala Gln Phe Cys Cys Arg Val Gln Gly Thr Gly Pro Leu 35 40 45
Asp Thr Thr Pro Glu Gly Arg Pro Gly Glu Val Ser Asp Ala Pro 50 55
60 Gln Arg Lys Gln Phe Cys His Trp Pro Cys Lys Cys Pro Gln Gln 65
70 75 Lys Pro Arg Cys Pro Pro Gly Val Ser Leu Val Arg Asp Gly Cys
80 85 90 Gly Cys Cys Lys Ile Cys Ala Lys Gln Pro Gly Glu Ile Cys
Asn 95 100 105 Glu Ala Asp Leu Cys Asp Pro His Lys Gly Leu Tyr Cys
Asp Tyr 110 115 120 Ser Val Asp Arg Pro Arg Tyr Glu Thr Gly Val Cys
Ala Tyr Leu 125 130 135 Val Ala Val Gly Cys Glu Phe Asn Gln Val His
Tyr His Asn Gly 140 145 150 Gln Val Phe Gln Pro Asn Pro Leu Phe Ser
Cys Leu Cys Val Ser 155 160 165 Gly Ala Ile Gly Cys Thr Pro Leu Phe
Ile Pro Lys Leu Ala Gly 170 175 180 Ser His Cys Ser Gly Ala Lys Gly
Gly Lys Lys Ser Asp Gln Ser 185 190 195 Asn Cys Ser Leu Glu Pro Leu
Leu Gln Gln Leu Ser Thr Ser Tyr 200 205 210 Lys Thr Met Pro Ala Tyr
Arg Asp Leu Pro Leu Ile Trp Lys Lys 215 220 225 Lys Cys Leu Val Gln
Ala Thr Lys Trp Thr Pro Cys Ser Arg Thr 230 235 240 Cys Gly Met Gly
Ile Ser Asn Arg Val Thr Asn Glu Asn Ser Asn 245 250 255 Cys Glu Met
Arg Lys Glu Lys Arg Leu Cys Tyr Ile Gln Pro Cys 260 265 270 Asp Ser
Asn Ile Leu Lys Thr Ile Lys Ile Pro Lys Gly Lys Thr 275 280 285 Cys
Gln Pro Thr Phe Gln Leu Ser Lys Ala Glu Lys Phe Val Phe 290 295 300
Ser Gly Cys Ser Ser Thr Gln Ser Tyr Lys Pro Thr Phe Cys Gly 305 310
315 Ile Cys Leu Asp Lys Arg Cys Cys Ile Pro Asn Lys Ser Lys Met 320
325 330 Ile Thr Ile Gln Phe Asp Cys Pro Asn Glu Gly Ser Phe Lys Trp
335 340 345 Lys Met Leu Trp Ile Thr Ser Cys Val Cys Gln Arg Asn Cys
Arg 350 355 360 Glu Pro Gly Asp Ile Phe Ser Glu Leu Lys Ile Leu 365
370 372 34 1212 DNA Homo sapiens 34 cacggtccca gcgacatgca
ggggctcctc ttctccactc ttctgcttgc 50 tggcctggca cagttctgct
gcagggtaca gggcactgga ccattagata 100 caacacctga aggaaggcct
ggagaagtgt cagatgcacc tcagcgtaaa 150 cagttttgtc actggccctg
caaatgccct cagcagaagc cccgttgccc 200 tcctggagtg agcctggtga
gagatggctg tggatgctgt aaaatctgtg 250 ccaagcaacc aggggaaatc
tgcaatgaag ctgacctctg tgacccacac 300 aaagggctgt attgtgacta
ctcagtagac aggcctaggt acgagactgg 350 agtgtgtgca taccttgtag
ctgttgggtg cgagttcaac caggtacatt 400 atcataatgg ccaagtgttt
cagcccaacc ccttgttcag ctgcctctgt 450 gtgagtgggg ccattggatg
cacacctctg ttcataccaa agctggctgg 500 cagtcactgc tctggagcta
aaggtggaaa gaagtctgat cagtcaaact 550 gtagcctgga accattacta
cagcagcttt caacaagcta caaaacaatg 600 ccagcttata gaaatctccc
acttatttgg aaaaaaaaat gtcttgtgca 650 agcaacaaaa tggactccct
gctccagaac atgtgggatg ggaatatcta 700 acagggtgac caatgaaaac
agcaactgtg aaatgagaaa agagaaaaga 750 ctgtgttaca ttcagccttg
cgacagcaat atattaaaga caataaagat 800 tcccaaagga aaaacatgcc
aacctacttt ccaactctcc aaagctgaaa 850 aatttgtctt ttctggatgc
tcaagtactc agagttacaa acccactttt 900 tgtggaatat gcttggataa
gagatgctgt atccctaata agtctaaaat 950 gattactatt caatttgatt
gcccaaatga ggggtcattt aaatggaaga 1000 tgctgtggat tacatcttgt
gtgtgtcaga gaaactgcag agaacctgga 1050 gatatatttt ctgagctcaa
gattctgtaa aaccaagcaa atgggggaaa 1100 agttagtcaa tcctgtcata
taataaaaaa attagtgagt aaaaaaaaaa 1150 aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa agaaaaaaaa 1200 aaaaaaaaaa aa 1212 35 1212
DNA Homo sapiens 35 tttttttttt tttttttttt cttttttttt tttttttttt
tttttttttt 50 tttttttttt tttttttttt ttactcacta atttttttat
tatatgacag 100 gattgactaa cttttccccc atttgcttgg ttttacagaa
tcttgagctc 150 agaaaatata tctccaggtt ctctgcagtt tctctgacac
acacaagatg 200 taatccacag catcttccat ttaaatgacc cctcatttgg
gcaatcaaat 250 tgaatagtaa tcattttaga cttattaggg atacagcatc
tcttatccaa 300 gcatattcca caaaaagtgg gtttgtaact ctgagtactt
gagcatccag 350 aaaagacaaa tttttcagct ttggagagtt ggaaagtagg
ttggcatgtt 400 tttcctttgg gaatctttat tgtctttaat atattgctgt
cgcaaggctg 450 aatgtaacac agtcttttct cttttctcat ttcacagttg
ctgttttcat 500 tggtcaccct gttagatatt cccatcccac atgttctgga
gcagggagtc 550 cattttgttg cttgcacaag acattttttt ttccaaataa
gtgggagatt 600 tctataagct ggcattgttt tgtagcttgt tgaaagctgc
tgtagtaatg 650 gttccaggct acagtttgac tgatcagact tctttccacc
tttagctcca 700 gagcagtgac tgccagccag ctttggtatg aacagaggtg
tgcatccaat 750 ggccccactc acacagaggc agctgaacaa ggggttgggc
tgaaacactt 800 ggccattatg ataatgtacc tggttgaact cgcacccaac
agctacaagg 850 tatgcacaca ctccagtctc gtacctaggc ctgtctactg
agtagtcaca 900 atacagccct ttgtgtgggt cacagaggtc agcttcattg
cagatttccc 950 ctggttgctt ggcacagatt ttacagcatc cacagccatc
tctcaccagg 1000 ctcactccag gagggcaacg gggcttctgc tgagggcatt
tgcagggcca 1050 gtgacaaaac tgtttacgct gaggtgcatc tgacacttct
ccaggccttc 1100 cttcaggtgt tgtatctaat ggtccagtgc cctgtaccct
gcagcagaac 1150 tgtgccaggc cagcaagcag aagagtggag aagaggagcc
cctgcatgtc 1200 gctgggaccg tg 1212 36 339 PRT Homo sapiens 36 Gln
Phe Cys Cys Arg Val Gln Gly Thr Gly Pro Leu Asp Thr Thr 1 5 10 15
Pro Glu Gly Arg Pro Gly Glu Val Ser Asp Ala Pro Gln Arg Lys 20 25
30 Gln Phe Cys His Trp Pro Cys Lys Cys Pro Gln Gln Lys Pro Arg 35
40 45 Cys Pro Pro Gly Val Ser Leu Val Arg Asp Gly Cys Gly Cys Cys
50 55 60 Lys Ile Cys Ala Lys Gln Pro Gly Glu Ile Cys Asn Glu Ala
Asp 65 70 75 Leu Cys Asp Pro His Lys Gly Leu Tyr Cys Asp Tyr Ser
Val Asp 80 85 90 Arg Pro Arg Tyr Glu Thr Gly Val Cys Ala Tyr Leu
Val Ala Val 95 100 105 Gly Cys Glu Phe Asn Gln Val His Tyr His Asn
Gly Gln Val Phe 110 115 120 Gln Pro Asn Pro Leu Phe Ser Cys Leu Cys
Val Ser Gly Ala Ile 125 130 135 Gly Cys Thr Pro Leu Phe Ile Pro Lys
Leu Ala Gly Ser His Cys 140 145 150 Ser Gly Ala Lys Gly Gly Lys Lys
Ser Asp Gln Ser Asn Cys Ser 155 160 165 Leu Glu Pro Leu Leu Gln Gln
Leu Ser Thr Ser Tyr Lys Thr Met 170 175 180 Pro Ala Tyr Arg Asn Leu
Pro Leu Ile Trp Lys Lys Lys Cys Leu 185 190 195 Val Gln Ala Thr Lys
Trp Thr Pro Cys Ser Arg Thr Cys Gly Met 200 205 210 Gly Ile Ser Asn
Arg Val Thr Asn Glu Asn Ser Asn Cys Glu Met 215 220 225 Arg Lys Glu
Lys Arg Leu Cys Tyr Ile Gln Pro Cys Asp Ser Asn 230 235 240 Ile Leu
Lys Thr Ile Lys Ile Pro Lys Gly Lys Thr Cys Gln Pro 245 250 255 Thr
Phe Gln Leu Ser Lys Ala Glu Lys Phe Val Phe Ser Gly Cys 260 265 270
Ser Ser Thr Gln Ser Tyr Lys Pro Thr Phe Cys Gly Ile Cys Leu 275 280
285 Asp Lys Arg Cys Cys Ile Pro Asn Lys Ser Lys Met Ile Thr Ile 290
295 300 Gln Phe Asp Cys Pro Asn Glu Gly Ser Phe Lys Trp Lys Met Leu
305 310 315 Trp Ile Thr Ser Cys Val Cys Gln Arg Asn Cys Arg Glu Pro
Gly 320 325 330 Asp Ile Phe Ser Glu Leu Lys Ile Leu 335 339 37 354
PRT Homo sapiens 37 Met Gln Gly Leu Leu Phe Ser Thr Leu Leu Leu Ala
Gly Leu Ala 1 5 10 15 Gln Phe Cys Cys Arg Val Gln Gly Thr Gly Pro
Leu Asp Thr Thr 20 25 30 Pro Glu Gly Arg Pro Gly Glu Val Ser Asp
Ala Pro Gln Arg Lys 35 40 45 Gln Phe Cys His Trp Pro Cys Lys Cys
Pro Gln Gln Lys Pro Arg 50 55 60 Cys Pro Pro Gly Val Ser Leu Val
Arg Asp Gly Cys Gly Cys Cys 65 70 75 Lys Ile Cys Ala Lys Gln Pro
Gly Glu Ile Cys Asn Glu Ala Asp 80 85 90 Leu Cys Asp Pro His Lys
Gly Leu Tyr Cys Asp Tyr Ser Val Asp 95 100 105 Arg Pro Arg Tyr Glu
Thr Gly Val Cys Ala Tyr Leu Val Ala Val 110 115 120 Gly Cys Glu Phe
Asn Gln Val His Tyr His Asn Gly Gln Val Phe 125 130 135 Gln Pro Asn
Pro Leu Phe Ser Cys Leu Cys Val Ser Gly Ala Ile 140 145 150 Gly Cys
Thr Pro Leu Phe Ile Pro Lys Leu Ala Gly Ser His Cys 155 160 165 Ser
Gly Ala Lys Gly Gly Lys Lys Ser Asp Gln Ser Asn Cys Ser 170 175 180
Leu Glu Pro Leu Leu Gln Gln Leu Ser Thr Ser Tyr Lys Thr Met 185 190
195 Pro Ala Tyr Arg Asn Leu Pro Leu Ile Trp Lys Lys Lys Cys Leu 200
205 210 Val Gln Ala Thr Lys Trp Thr Pro Cys Ser Arg Thr Cys Gly Met
215 220 225 Gly Ile Ser Asn Arg Val Thr Asn Glu Asn Ser Asn Cys Glu
Met 230 235 240 Arg Lys Glu Lys Arg Leu Cys Tyr Ile Gln Pro Cys Asp
Ser Asn 245 250 255 Ile Leu Lys Thr Ile Lys Ile Pro Lys Gly Lys Thr
Cys Gln Pro 260 265 270 Thr Phe Gln Leu Ser Lys Ala Glu Lys Phe Val
Phe Ser Gly Cys 275 280 285 Ser Ser Thr Gln Ser Tyr Lys Pro Thr Phe
Cys Gly Ile Cys Leu 290 295 300 Asp Lys Arg Cys Cys Ile Pro Asn Lys
Ser Lys Met Ile Thr Ile 305 310 315 Gln Phe Asp Cys Pro Asn Glu Gly
Ser Phe Lys Trp Lys Met Leu 320 325 330 Trp Ile Thr Ser Cys Val Cys
Gln Arg Asn Cys Arg Glu Pro Gly 335 340 345 Asp Ile Phe Ser Glu Leu
Lys Ile Leu 350 354 38 738 DNA Homo sapiens 38 ccgaagaccc
acctcctggc cttctccctc ctctgcctcc tctcaaaggt 50 gcgtacccag
ctgtgcccga caccatgtac ctgcccctgg ccacctcccc 100 gatgcccgct
gggagtaccc ctggtgctgg atggctgtgg ctgctgccgg 150 gtatgtgcac
ggcggctggg ggagccctgc gaccaactcc acgtctgcga 200 cgccagccag
ggcctggtct gccagcccgg ggcaggaccc ggtggccggg 250 gggccctgtg
cctcttggca gaggacgaca gcagctgtga ggtgaacggc 300 cgcctgtatc
gggaagggga gaccttccag ccccactgca gcatccgctg 350 ccgctgcgag
gacggcggct tcacctgcgt gccgctgtgc agcgaggatg 400 tgcggctgcc
cagctgggac tgcccccacc ccaggagggt cgaggtcctg 450 ggcaagtgct
gccctgagtg ggtgtgcggc caaggagggg gactggggac 500 ccagcccctt
ccagcccaag gaccccagtt ttctggcctt gtctcttccc 550 tgccccctgg
tgtcccctgc ccagaatgga gcacggcctg gggaccctgc 600 tcgaccacct
gtgggctggg catggccacc cgggtgtcca accagaaccg 650 cttctgccga
ctggagaccc agcgccgcct gtgcctgtcc aggccctgcc 700 caccctccag
gggtcgcagt ccacaaaaca gtgccttc 738 39 841 DNA Artificial sequence
misc_feature 1-841 Sequence is synthesized. 39 ctgcagggga
catgagaggc acaccgaaga cccacctcct ggccttctcc 50 ctcctctgcc
tcctctcaaa ggtgcgtacc cagctgtgcc cgacaccatg 100 tacctgcccc
tggccacctc cccgatgccc gctgggagta cccctggtgg 150 tggatggctg
tggctgctgc cgggtatgtg cacggcggct gggggagccc 200 tgcgaccaac
tccacgtctg cgacgccagc cagggcctgg tctgccagcc 250 cggggcagga
cccggtggcc ggggggccct gtgcctcttg gcagaggacg 300 acagcagctg
tgaggtgaac ggccgcctgt atcgggaagg ggagaccttc 350 cagccccact
gcagcatccg ctgccgctgc gaggacggcg gcttcacctg 400 cgtgccgctg
tgcagcgagg atgtgcggct gcccagctgg gactgccccc 450 accccaggag
ggtcgaggtc ctgggcaagt gctgccctga gtgggtgtgc 500 ggccaaggag
ggggactggg gaccagccct tccagcccaa ggaccccagt 550 tttctggcct
tgtctcttcc ctgccccctg gtgtcccctg cccagaatgg 600 agcacggcct
ggggaccctg ctcgaccacc tgtgggctgg gcatggccac 650 ccgggtgtcc
aaccagaacc gcttctgccg actggagacc cagcgccgcc 700 tgtgcctgtc
caggccctgc ccaccctcca ggggtcgcag tccacaaaac 750 agtgccttct
agagccgggc tgggaatggg gacacggtgt ccaccatccc 800 cagctggtgg
ccctgtgcct gggccctggg ctgatggaag a 841 40 14 DNA Artificial
sequence misc_feature 1-14 Sequence is synthesized. 40 ttttgtacaa
gctt 14 41 44 DNA Artificial sequence misc_feature 1-44 Sequence is
synthesized. 41 ctaatacgac tcactatagg gctcgagcgg ccgcccgggc aggt 44
42 43 DNA Artificial sequence misc_feature 1-43 Sequence is
synthesized 42 tgtagcgtga agacgacaga aagggcgtgg tgcggagggc ggt 43
43 10 DNA Artificial Sequence misc_feature 1-10 Sequence is
synthesized 43 acctgcccgg 10 44 11 DNA Artificial sequence
misc_feature 1-11 Sequence is synthesized 44 accgccctcc g 11 45 22
DNA Artificial sequence misc_feature 1-22 Sequence is synthesized
45 ctaatacgac tcactatagg gc 22 46 21 DNA Artificial sequence
misc_feature 1-21 Sequence is synthesized 46 tgtagcgtga agacgacaga
a 21 47 22 DNA Artificial sequence misc_feature 1-22 Sequence is
synthesized 47 tcgagcggcc gcccgggcag gt 22 48 22 DNA Artificial
sequence misc_feature 1-22 Sequence is synthesized 48 agggcgtggt
gcggagggcg gt 22 49 20 DNA Artificial sequence misc_feature 1-20
Sequence is synthesized 49 accacagtcc atgccatcac 20 50 20 DNA
Artificial sequence misc_feature 1-20 Sequence is synthesized 50
tccaccaccc tgttgctgta 20 51 163 DNA Artificial sequence
misc_feature 1-163 Sequence is synthesized 51 tgtaatacga ctcactatag
ggcgaattgg gcccgacgtc gcatgctccc 50 ggccgccatg gccgcgggat
tatcactagt gcggccgcct gcaggtcgac 100 catatgggag agctcccaac
gcgttggatg catagcttga gtattctata 150 gtgtcaccta aat 163 52 163 DNA
Artificial sequence misc_feature 1-163 Sequence is synthesized 52
atttaggtga cactatagaa tactcaagct atgcatccaa cgcgttggga 50
gctctcccat atggtcgacc tgcaggcggc cgcactagtg attatcccgc 100
ggccatggcg gccgggagca tgcgacgtcg ggcccaattc gccctatagt 150
gagtcgtatt aca 163 53 10325 DNA Artificial sequence misc_feature
1-10325 Sequence is synthesized. 53 ttcgagctcg cccgacattg
attattgact agagtcgatc accggttatt 50 aatagtaatc aattacgggg
tcatagttca tagcccatat atggagttcc 100 gcgttacata acttacggta
aatggcccgc ctggctgacc gcccaacgac 150 ccccgcccat tgacgtcaat
aatgacgtat gttcccatag taacgccaat 200 agggactttc cattgacgtc
aatgggtgga gtatttacgg taaactgccc 250 acttggcagt acatcaagtg
tatcatatgc caagtacgcc ccctattgac 300 gtcaatgacg gtaaatggcc
cgcctggcat tatgcccagt acatgacctt 350 atgggacttt cctacttggc
agtacatcta cgtattagtc atcgctatta 400 ccatggtgat gcggttttgg
cagtacatca atgggcgtgg atagcggttt 450 gactcacggg gatttccaag
tctccacccc attgacgtca atgggagttt 500 gttttggcac caaaatcaac
gggactttcc aaaatgtcgt aacaactccg 550 ccccattgac gcaaatgggc
ggtaggcgtg tacggtggga ggtctatata 600 agcagagctc gtttagtgaa
ccgtcagatc gcctggagac gccatccacg 650 ctgttttgac ctgggcccgg
ccgaggccgc ctcggcctct gagctattcc 700 agaagtagtg aggaggcttt
tttggaggcc taggcttttg caaaaagcta 750 gcttatccgg ccgggaacgg
tgcattggaa cgcggattcc ccgtgccaag 800 agtgacgtaa gtaccgccta
tagagcgact agtccaccat gaccgagtac 850 aagcccacgg tgcgcctcgc
cacccgcgac gacgtcccgc gggccgtacg 900 caccctcgcc gccgcgttcg
ccgactaccc cgccacgcgc cacaccgtag 950 acccggaccg ccacatcgag
cgggtcaccg agctgcaaga actcttcctc 1000 acgcgcgtcg ggctcgacat
cggcaaggtg tgggtcgcgg acgacggcgc 1050 cgcggtggcg gtctggacca
cgccggagag cgtcgaagcg ggggcggtgt 1100 tcgccgagat cggcccgcgc
atggccgagt tgagcggttc ccggctggcc 1150 gcgcagcaac agatggaagg
cctcctggcg ccgcaccggc ccaaggagcc 1200 cgcgtggttc ctggccaccg
tcggcgtctc gcccgaccac cagggcaagg 1250 gtctgggcag cgccgtcgtg
ctccccggag tggaggcggc cgagcgcgcc 1300 ggggtgcccg ccttcctgga
gacctccgcg ccccgcaacc tccccttcta 1350 cgagcggctc ggcttcaccg
tcaccgccga cgtcgagtgc ccgaaggacc 1400 gcgcgacctg gtgcatgacc
cgcaagcccg gtgccaacat ggttcgacca 1450 ttgaactgca tcgtcgccgt
gtcccaaaat atggggattg gcaagaacgg 1500 agacctaccc tgccctccgc
tcaggaacgc gttcaagtac ttccaaagaa 1550 tgaccacaac ctcttcagtg
gaaggtaaac agaatctggt gattatgggt 1600 aggaaaacct ggttctccat
tcctgagaag aatcgacctt taaaggacag 1650 aattaatata gttctcagta
gagaactcaa agaaccacca cgaggagctc 1700 attttcttgc caaaagtttg
gatgatgcct taagacttat tgaacaaccg 1750 gaattggcaa gtaaagtaga
catggtttgg atagtcggag gcagttctgt 1800 ttaccaggaa gccatgaatc
aaccaggcca ccttagactc tttgtgacaa 1850 ggatcatgca ggaatttgaa
agtgacacgt ttttcccaga aattgatttg 1900 gggaaatata aacctctccc
agaataccca ggcgtcctct ctgaggtcca 1950 ggaggaaaaa ggcatcaagt
ataagtttga agtctacgag aagaaagact 2000 aacgttaact gctcccctcc
taaagctatg catttttata agaccatggg 2050 acttttgctg gctttagatc
cccttggctt cgttagaacg cagctacaat 2100 taatacataa ccttatgtat
catacacata cgatttaggt gacactatag 2150 ataacatcca ctttgccttt
ctctccacag gtgtccactc ccaggtccaa 2200 ctgcacctcg gttctatcga
ttgaattccc cggggatcct ctagagtcga 2250 cctgcagaag cttcgatggc
cgccatggcc caacttgttt attgcagctt 2300 ataatggtta caaataaagc
aatagcatca caaatttcac aaataaagca 2350 tttttttcac tgcattctag
ttgtggtttg tccaaactca tcaatgtatc 2400 ttatcatgtc tggatcgatc
gggaattaat tcggcgcagc accatggcct 2450 gaaataacct ctgaaagagg
aacttggtta ggtaccgact agtcgcgtta 2500 cataacttac ggtaaatggc
ccgcctggct gaccgcccaa cgacccccgc 2550 ccattgacgt caataatgac
gtatgttccc atagtaacgc caatagggac 2600 tttccattga cgtcaatggg
tggagtattt acggtaaact gcccacttgg 2650 cagtacatca agtgtatcat
atgccaagta cgccccctat tgacgtcaat 2700 gacggtaaat ggcccgcctg
gcattatgcc cagtacatga ccttatggga 2750 ctttcctact tggcagtaca
tctacgtatt agtcatcgct attaccatgg 2800 tgatgcggtt ttggcagtac
atcaatgggc gtggatagcg gtttgactca 2850 cggggatttc caagtctcca
ccccattgac gtcaatggga gtttgttttg 2900 actagtagca aggtcgccac
gcacaagatc aatattaaca atcagtcatc 2950 tctctttagc aataaaaagg
tgaaaaatta cattttaaaa atgacaccat 3000 agacgatgta tgaaaataat
ctacttggaa ataaatctag gcaaagaagt 3050 gcaagactgt tacccagaaa
acttacaaat tgtaaatgag aggttagtga 3100 agatttaaat gaatgaagat
ctaaataaac ttataaattg tgagagaaat 3150 taatgaatgt ctaagttaat
gcagaaacgg agagacatac tatattcatg 3200 aactaaaaga cttaatattg
tgaaggtata ctttcttttc acataaattt 3250 gtagtcaata tgttcacccc
aaaaaagctg tttgttaact tgtcaacctc 3300 atttcaaaat gtatatagaa
agcccaaaga caataacaaa aatattcttg 3350 tagaacaaaa tgggaaagaa
tgttccacta aatatcaaga tttagagcaa 3400 agcatgagat gtgtggggat
agacagtgag gctgataaaa tagagtagag 3450 ctcagaaaca gacccattga
tatatgtaag tgacctatga aaaaaatatg 3500 gcattttaca atgggaaaat
gatgatcttt ttctttttta gaaaaacagg 3550 gaaatatatt tatatgtaaa
aaataaaagg gaacccatat gtcataccat 3600 acacacaaaa aaattccagt
gaattataag tctaaatgga gaaggcaaaa 3650 ctttaaatct tttagaaaat
aatatagaag catgccatca tgacttcagt 3700 gtagagaaaa atttcttatg
actcaaagtc ctaaccacaa agaaaagatt 3750 gttaattaga ttgcatgaat
attaagactt atttttaaaa ttaaaaaacc 3800 attaagaaaa gtcaggccat
agaatgacag aaaatatttg caacacccca 3850 gtaaagagaa ttgtaatatg
cagattataa aaagaagtct tacaaatcag 3900 taaaaaataa aactagacaa
aaatttgaac agatgaaaga gaaactctaa 3950 ataatcatta cacatgagaa
actcaatctc agaaatcaga gaactatcat 4000 tgcatataca ctaaattaga
gaaatattaa aaggctaagt aacatctgtg 4050 gcaatattga tggtatataa
ccttgatatg atgtgatgag aacagtactt 4100 taccccatgg gcttcctccc
caaaccctta ccccagtata aatcatgaca 4150 aatatacttt aaaaaccatt
accctatatc taaccagtac tcctcaaaac 4200 tgtcaaggtc atcaaaaata
agaaaagtct gaggaactgt caaaactaag 4250 aggaacccaa ggagacatga
gaattatatg taatgtggca ttctgaatga 4300 gatcccagaa cagaaaaaga
acagtagcta aaaaactaat gaaatataaa 4350 taaagtttga actttagttt
tttttaaaaa agagtagcat taacacggca 4400 aagtcatttt catatttttc
ttgaacatta agtacaagtc tataattaaa 4450 aattttttaa atgtagtctg
gaacattgcc agaaacagaa gtacagcagc 4500 tatctgtgct gtcgcctaac
tatccatagc tgattggtct aaaatgagat 4550 acatcaacgc tcctccatgt
tttttgtttt ctttttaaat gaaaaacttt 4600 attttttaag aggagtttca
ggttcatagc aaaattgaga ggaaggtaca 4650 ttcaagctga ggaagttttc
ctctattcct agtttactga gagattgcat 4700 catgaatggg tgttaaattt
tgtcaaatgc tttttctgtg tctatcaata 4750 tgaccatgtg attttcttct
ttaacctgtt gatgggacaa attacgttaa 4800 ttgattttca aacgttgaac
cacccttaca tatctggaat aaattctact 4850 tggttgtggt gtatattttt
tgatacattc ttggattctt tttgctaata 4900 ttttgttgaa aatgtttgta
tctttgttca tgagagatat tggtctgttg 4950 ttttcttttc ttgtaatgtc
attttctagt tccggtatta aggtaatgct 5000 ggcctagttg aatgatttag
gaagtattcc ctctgcttct gtcttctgag 5050 gtaccgcggc cgcccgtcgt
tttacaacgt cgtgactggg aaaaccctgg 5100 cgttacccaa cttaatcgcc
ttgcagcaca tccccctttc gccagctggc 5150 gtaatagcga agaggcccgc
accgatcgcc cttcccaaca gttgcgcagc 5200 ctgaatggcg aatggcgcct
gatgcggtat tttctcctta cgcatctgtg 5250 cggtatttca caccgcatac
gtcaaagcaa ccatagtacg cgccctgtag 5300 cggcgcatta agcgcggcgg
gtgtggtggt tacgcgcagc gtgaccgcta 5350 cacttgccag cgccctagcg
cccgctcctt tcgctttctt cccttccttt 5400 ctcgccacgt tcgccggctt
tccccgtcaa gctctaaatc gggggctccc 5450 tttagggttc cgatttagtg
ctttacggca cctcgacccc aaaaaacttg 5500 atttgggtga tggttcacgt
agtgggccat cgccctgata gacggttttt 5550 cgccctttga cgttggagtc
cacgttcttt aatagtggac tcttgttcca 5600 aactggaaca acactcaacc
ctatctcggg ctattctttt gatttataag 5650 ggattttgcc gatttcggcc
tattggttaa aaaatgagct gatttaacaa 5700 aaatttaacg cgaattttaa
caaaatatta acgtttacaa ttttatggtg 5750 cactctcagt acaatctgct
ctgatgccgc atagttaagc cagccccgac 5800 acccgccaac acccgctgac
gcgccctgac gggcttgtct gctcccggca 5850 tccgcttaca gacaagctgt
gaccgtctcc gggagctgca tgtgtcagag 5900 gttttcaccg tcatcaccga
aacgcgcgag acgaaagggc ctcgtgatac 5950 gcctattttt ataggttaat
gtcatgataa taatggtttc ttagacgtca 6000 ggtggcactt ttcggggaaa
tgtgcgcgga acccctattt gtttattttt 6050 ctaaatacat tcaaatatgt
atccgctcat gagacaataa ccctgataaa 6100 tgcttcaata atattgaaaa
aggaagagta tgagtattca acatttccgt 6150 gtcgccctta ttcccttttt
tgcggcattt tgccttcctg tttttgctca 6200 cccagaaacg ctggtgaaag
taaaagatgc tgaagatcag ttgggtgcac 6250 gagtgggtta catcgaactg
gatctcaaca gcggtaagat ccttgagagt 6300 tttcgccccg aagaacgttt
tccaatgatg agcactttta aagttctgct 6350 atgtggcgcg gtattatccc
gtattgacgc cgggcaagag caactcggtc 6400 gccgcataca ctattctcag
aatgacttgg ttgagtactc accagtcaca 6450 gaaaagcatc ttacggatgg
catgacagta agagaattat gcagtgctgc 6500 cataaccatg agtgataaca
ctgcggccaa cttacttctg acaacgatcg 6550 gaggaccgaa ggagctaacc
gcttttttgc acaacatggg ggatcatgta 6600 actcgccttg atcgttggga
accggagctg aatgaagcca taccaaacga 6650 cgagcgtgac accacgatgc
ctgtagcaat ggcaacaacg ttgcgcaaac 6700 tattaactgg cgaactactt
actctagctt cccggcaaca attaatagac 6750 tggatggagg cggataaagt
tgcaggacca cttctgcgct cggcccttcc 6800 ggctggctgg tttattgctg
ataaatctgg agccggtgag cgtgggtctc 6850 gcggtatcat tgcagcactg
gggccagatg gtaagccctc ccgtatcgta 6900 gttatctaca cgacggggag
tcaggcaact atggatgaac gaaatagaca 6950 gatcgctgag ataggtgcct
cactgattaa gcattggtaa ctgtcagacc 7000 aagtttactc atatatactt
tagattgatt taaaacttca tttttaattt 7050 aaaaggatct aggtgaagat
cctttttgat aatctcatga ccaaaatccc 7100 ttaacgtgag ttttcgttcc
actgagcgtc agaccccgta gaaaagatca 7150 aaggatcttc ttgagatcct
ttttttctgc gcgtaatctg ctgcttgcaa 7200 acaaaaaaac caccgctacc
agcggtggtt tgtttgccgg atcaagagct 7250 accaactctt tttccgaagg
taactggctt cagcagagcg cagataccaa 7300 atactgtcct tctagtgtag
ccgtagttag gccaccactt caagaactct 7350 gtagcaccgc ctacatacct
cgctctgcta atcctgttac cagtggctgc 7400 tgccagtggc gataagtcgt
gtcttaccgg gttggactca agacgatagt 7450 taccggataa ggcgcagcgg
tcgggctgaa cggggggttc gtgcacacag 7500 cccagcttgg agcgaacgac
ctacaccgaa ctgagatacc tacagcgtga 7550 gctatgagaa agcgccacgc
ttcccgaagg gagaaaggcg gacaggtatc 7600 cggtaagcgg cagggtcgga
acaggagagc gcacgaggga gcttccaggg 7650 ggaaacgcct ggtatcttta
tagtcctgtc gggtttcgcc acctctgact 7700 tgagcgtcga tttttgtgat
gctcgtcagg ggggcggagc ctatggaaaa 7750 acgccagcaa cgcggccttt
ttacggttcc tggccttttg ctggcctttt 7800 gctcacatgt tctttcctgc
gttatcccct gattctgtgg ataaccgtat 7850 taccgccttt gagtgagctg
ataccgctcg ccgcagccga acgaccgagc 7900 gcagcgagtc agtgagcgag
gaagcggaag agcccgcggg caaggtcgcc 7950 acgcacaaga tcaatattaa
caatcagtca tctctcttta gcaataaaaa 8000 ggtgaaaaat tacattttaa
aaatgacacc atagacgatg tatgaaaata 8050 atctacttgg aaataaatct
aggcaaagaa gtgcaagact gttacccaga 8100 aaacttacaa attgtaaatg
agaggttagt gaagatttaa atgaatgaag 8150 atctaaataa acttataaat
tgtgagagaa attaatgaat gtctaagtta 8200 atgcagaaac ggagagacat
actatattca tgaactaaaa gacttaatat 8250 tgtgaaggta tactttcttt
tcacataaat ttgtagtcaa tatgttcacc 8300 ccaaaaaagc tgtttgttaa
cttgtcaacc tcatttcaaa atgtatatag 8350 aaagcccaaa gacaataaca
aaaatattct tgtagaacaa aatgggaaag 8400 aatgttccac taaatatcaa
gatttagagc aaagcatgag atgtgtgggg 8450 atagacagtg aggctgataa
aatagagtag agctcagaaa cagacccatt 8500 gatatatgta agtgacctat
gaaaaaaata tggcatttta caatgggaaa 8550 atgatgatct ttttcttttt
tagaaaaaca gggaaatata tttatatgta 8600 aaaaataaaa gggaacccat
atgtcatacc atacacacaa aaaaattcca 8650 gtgaattata agtctaaatg
gagaaggcaa aactttaaat cttttagaaa 8700 ataatataga agcatgccat
catgacttca gtgtagagaa aaatttctta 8750 tgactcaaag tcctaaccac
aaagaaaaga ttgttaatta gattgcatga 8800 atattaagac ttatttttaa
aattaaaaaa ccattaagaa aagtcaggcc 8850 atagaatgac agaaaatatt
tgcaacaccc cagtaaagag aattgtaata 8900 tgcagattat aaaaagaagt
cttacaaatc agtaaaaaat aaaactagac 8950 aaaaatttga acagatgaaa
gagaaactct aaataatcat tacacatgag 9000 aaactcaatc tcagaaatca
gagaactatc attgcatata cactaaatta 9050 gagaaatatt aaaaggctaa
gtaacatctg tggcaatatt gatggtatat 9100 aaccttgata tgatgtgatg
agaacagtac tttaccccat gggcttcctc 9150 cccaaaccct taccccagta
taaatcatga caaatatact ttaaaaacca 9200 ttaccctata tctaaccagt
actcctcaaa actgtcaagg tcatcaaaaa 9250 taagaaaagt ctgaggaact
gtcaaaacta agaggaaccc aaggagacat 9300 gagaattata tgtaatgtgg
cattctgaat gagatcccag aacagaaaaa 9350 gaacagtagc taaaaaacta
atgaaatata aataaagttt gaactttagt 9400 tttttttaaa aaagagtagc
attaacacgg caaagtcatt ttcatatttt 9450 tcttgaacat taagtacaag
tctataatta aaaatttttt aaatgtagtc 9500 tggaacattg ccagaaacag
aagtacagca gctatctgtg ctgtcgccta 9550 actatccata gctgattggt
ctaaaatgag atacatcaac gctcctccat 9600 gttttttgtt ttctttttaa
atgaaaaact ttatttttta agaggagttt 9650 caggttcata gcaaaattga
gaggaaggta cattcaagct gaggaagttt 9700 tcctctattc ctagtttact
gagagattgc atcatgaatg ggtgttaaat 9750 tttgtcaaat gctttttctg
tgtctatcaa tatgaccatg tgattttctt 9800 ctttaacctg ttgatgggac
aaattacgtt aattgatttt caaacgttga 9850 accaccctta catatctgga
ataaattcta cttggttgtg gtgtatattt 9900 tttgatacat tcttggattc
tttttgctaa tattttgttg aaaatgtttg 9950 tatctttgtt catgagagat
attggtctgt tgttttcttt tcttgtaatg 10000 tcattttcta gttccggtat
taaggtaatg ctggcctagt tgaatgattt 10050 aggaagtatt ccctctgctt
ctgtcttctg aagcggaaga gcgcccaata 10100 cgcaaaccgc ctctccccgc
gcgttggccg attcattaat gcagctggca 10150 cgacaggttt cccgactgga
aagcgggcag tgagcgcaac gcaattaatg 10200 tgagttagct cactcattag
gcaccccagg ctttacactt tatgcttccg 10250 gctcgtatgt tgtgtggaat
tgtgagcgga taacaatttc acacaggaaa 10300 cagctatgac atgattacga attaa
10325 54 10379 DNA Artificial sequence misc-feature 1-10379
Sequence is synthesized. 54 aagctttact cgtaaagcga gttgaaggat
catatttagt tgcgtttatg 50 agataagatt gaaagcacgt gtaaaatgtt
tcccgcgcgt tggcacaact 100 atttacaatg cggccaagtt ataaaagatt
ctaatctgat atgttttaaa 150 acacctttgc ggcccgagtt gtttgcgtac
gtgactagcg aagaagatgt 200 gtggaccgca gaacagatag taaaacaaaa
ccctagtatt ggagcaataa 250 tcgatttaac caacacgtct aaatattatg
atggtgtgca ttttttgcgg 300 gcgggcctgt tatacaaaaa aattcaagta
cctggccaga ctttgccgcc 350 tgaaagcata gttcaagaat ttattgacac
ggtaaaagaa tttacagaaa 400 agtgtcccgg catgttggtg ggcgtgcact
gcacacacgg tattaatcgc 450 accggttaca tggtgtgcag atatttaatg
cacaccctgg gtattgcgcc 500 gcaggaagcc atagatagat tcgaaaaagc
cagaggtcac aaaattgaaa 550 gacaaaatta cgttcaagat ttattaattt
aattaatatt atttgcattc 600 tttaacaaat actttatcct attttcaaat
tgttgcgctt cttccagcga 650 accaaaacta tgcttcgctt gctccgttta
gcttgtagcc gatcagtggc 700 gttgttccaa tcgacggtag gattaggccg
gatattctcc accacaatgt 750 tggcaacgtt gatgttacgt ttatgctttt
ggttttccac gtacgtcttt 800 tggccggtaa tagccgtaaa cgtagtgccg
tcgcgcgtca cgcacaacac 850 cggatgtttg cgcttgtccg cggggtattg
aaccgcgcga tccgacaaat 900 ccaccacttt ggcaactaaa tcggtgacct
gcgcgtcttt tttctgcatt 950 atttcgtctt tcttttgcat ggtttcctgg
aagccggtgt acatgcggtt 1000 tagatcagtc atgacgcgcg tgacctgcaa
atctttggcc tcgatctgct 1050 tgtccttgat ggcaacgatg cgttcaataa
actcttgttt tttaacaagt 1100 tcctcggttt tttgcgccac caccgcttgc
agcgcgtttg tgtgctcggt 1150 gaatgtcgca atcagcttag tcaccaactg
tttgctctcc tcctcccgtt 1200 gtttgatcgc gggatcgtac ttgccggtgc
agagcacttg aggaattact 1250 tcttctaaaa gccattcttg taattctatg
gcgtaaggca atttggactt 1300 cataatcagc tgaatcacgc cggatttagt
aatgagcact gtatgcggct 1350 gcaaatacag cgggtcgccc cttttcacga
cgctgttaga ggtagggccc 1400 ccattttgga tggtctgctc aaataacgat
ttgtatttat tgtctacatg 1450 aacacgtata gctttatcac aaactgtata
ttttaaactg ttagcgacgt 1500 ccttggccac gaaccggacc tgttggtcgc
gctctagcac gtaccgcagg 1550 ttgaacgtat cttctccaaa tttaaattct
ccaattttaa cgcgagccat 1600 tttgatacac gtgtgtcgat tttgcaacaa
ctattgtttt ttaacgcaaa 1650 ctaaacttat tgtggtaagc aataattaaa
tatgggggaa catgcgccgc 1700 tacaacactc gtcgttatga acgcagacgg
cgccggtctc ggcgcaagcg 1750 gctaaaacgt gttgcgcgtt caacgcggca
aacatcgcaa aagccaatag 1800 tacagttttg atttgcatat taacggcgat
tttttaaatt atcttattta 1850 ataaatagtt atgacgccta caactccccg
cccgcgttga ctcgctgcac 1900 ctcgagcagt tcgttgacgc cttcctccgt
gtggccgaac acgtcgagcg 1950 ggtggtcgat gaccagcggc gtgccgcacg
cgacgcacaa gtatctgtac 2000 accgaatgat cgtcgggcga aggcacgtcg
gcctccaagt ggcaatattg 2050 gcaaattcga aaatatatac agttgggttg
tttgcgcata tctatcgtgg 2100 cgttgggcat gtacgtccga acgttgattt
gcatgcaagc cgaaattaaa 2150 tcattgcgat tagtgcgatt aaaacgttgt
acatcctcgc ttttaatcat 2200 gccgtcgatt aaatcgcgca atcgagtcaa
gtgatcaaag tgtggaataa 2250 tgttttcttt gtattcccga gtcaagcgca
gcgcgtattt taacaaacta 2300 gccatcttgt aagttagttt catttaatgc
aactttatcc aataatatat 2350 tatgtatcgc acgtcaagaa ttaacaatgc
gcccgttgtc gcatctcaac 2400 acgactatga tagagatcaa ataaagcgcg
aattaaatag cttgcgacgc 2450 aacgtgcacg atctgtgcac gcgttccggc
acgagctttg attgtaataa 2500 gtttttacga agcgatgaca tgacccccgt
agtgacaacg atcacgccca 2550 aaagaactgc cgactacaaa attaccgagt
atgtcggtga cgttaaaact 2600 attaagccat ccaatcgacc gttagtcgaa
tcaggaccgc tggtgcgaga 2650 agccgcgaag tatggcgaat gcatcgtata
acgtgtggag tccgctcatt 2700 agagcgtcat gtttagacaa gaaagctaca
tatttaattg atcccgatga 2750 ttttattgat aaattgaccc taactccata
cacggtattc tacaatggcg 2800 gggttttggt caaaatttcc ggactgcgat
tgtacatgct gttaacggct 2850 ccgcccacta ttaatgaaat taaaaattcc
aattttaaaa aacgcagcaa 2900 gagaaacatt tgtatgaaag aatgcgtaga
aggaaagaaa aatgtcgtcg 2950 acatgctgaa caacaagatt aatatgcctc
cgtgtataaa aaaaatattg 3000 aacgatttga aagaaaacaa tgtaccgcgc
ggcggtatgt acaggaagag 3050 gtttatacta aactgttaca ttgcaaacgt
ggtttcgtgt gccaagtgtg 3100 aaaaccgatg tttaatcaag gctctgacgc
atttctacaa ccacgactcc 3150 aagtgtgtgg gtgaagtcat gcatctttta
atcaaatccc aagatgtgta 3200 taaaccacca aactgccaaa aaatgaaaac
tgtcgacaag ctctgtccgt 3250 ttgctggcaa ctgcaagggt ctcaatccta
tttgtaatta ttgaataata 3300 aaacaattat aaatgctaaa tttgtttttt
attaacgata caaaccaaac 3350 gcaacaagaa catttgtagt attatctata
attgaaaacg cgtagttata 3400 atcgctgagg taatatttaa aatcattttc
aaatgattca cagttaattt 3450 gcgacaatat aattttattt tcacataaac
tagacgcctt gtcgtcttct 3500 tcttcgtatt ccttctcttt ttcatttttc
tcctcataaa aattaacata 3550 gttattatcg tatccatata tgtatctatc
gtatagagta aattttttgt 3600 tgtcataaat atatatgtct tttttaatgg
ggtgtatagt accgctgcgc 3650 atagtttttc tgtaatttac aacagtgcta
ttttctggta gttcttcgga 3700 gtgtgttgct ttaattatta aatttatata
atcaatgaat ttgggatcgt 3750 cggttttgta caatatgttg ccggcatagt
acgcagcttc ttctagttca 3800 attacaccat tttttagcag caccggatta
acataacttt ccaaaatgtt 3850 gtacgaaccg ttaaacaaaa acagttcacc
tcccttttct atactattgt 3900 ctgcgagcag ttgtttgttg ttaaaaataa
cagccattgt aatgagacgc 3950 acaaactaat atcacaaact ggaaatgtct
atcaatatat agttgctgat 4000 atcatggaga taattaaaat gataaccatc
tcgcaaataa ataagtattt 4050 tactgttttc gtaacagttt tgtaataaaa
aaacctataa atattccgga 4100 ttattcatac cgtcccacca tcgggcgcgg
atccgcggcc gcgaattcta 4150 aaccaccatg gctagcaggc ctgacaaaac
tcacacatgc ccaccgtgcc 4200 cagcacctga actcctgggg ggaccgtcag
tcttcctctt ccccccaaaa 4250 cccaaggaca ccctcatgat ctcccggacc
cctgaggtca catgcgtggt 4300 ggtggacgtg agccacgaag accctgaggt
caagttcaac tggtacgtgg 4350 acggcgtgga ggtgcataat gccaagacaa
agccgcggga ggagcagtac 4400 aacagcacgt accgtgtggt cagcgtcctc
accgtcctgc accaggactg 4450 gctgaatggc aaggagtaca agtgcaaggt
ctccaacaaa gccctcccag 4500 cccccatcga gaaaaccatc tccaaagcca
aagggcagcc ccgagaacca 4550 caggtgtaca ccctgccccc atcccgggaa
gagatgacca agaaccaggt 4600 cagcctgacc tgcctggtca aaggcttcta
tcccagcgac atcgccgtgg 4650 agtgggagag caatgggcag ccggagaaca
actacaagac cacgcctccc 4700 gtgctggact ccgacggctc cttcttcctc
tacagcaagc tcaccgtgga 4750 caagagcagg tggcagcagg ggaacgtctt
ctcatgctcc gtgatgcatg 4800 aggctctgca caaccactac acgcagaaga
gcctctccct gtctccgggt 4850 aaatgacata gggcatcatc atcatcatca
tcatcattaa ttctagacta 4900 gtctgcagat ctgatccttt cctgggaccc
ggcaagaacc aaaaactcac 4950 tctcttcaag gaaatccgta atgttaaacc
cgacacgatg aagcttgtcg 5000 ttggatggaa aggaaaagag ttctacaggg
aaacttggac ccgcttcatg 5050 gaagacagct tccccattgt taacgaccaa
gaagtgatgg atgttttcct 5100 tgttgtcaac atgcgtccca ctagacccaa
ccgttgttac aaattcctgg 5150 cccaacacgc tctgcgttgc gaccccgact
atgtacctca tgacgtgatt 5200 aggatcgtcg agccttcatg ggtgggcagc
aacaacgagt accgcatcag 5250 cctggctaag aagggcggcg gctgcccaat
aatgaacctt cactctgagt 5300 acaccaactc gttcgaacag ttcatcgatc
gtgtcatctg ggagaacttc 5350 tacaagccca tcgtttacat cggtaccgac
tctgctgaag aggaggaaat 5400 tctccttgaa gtttccctgg tgttcaaagt
aaaggagttt gcaccagacg 5450 cacctctgtt cactggtccg gcgtattaaa
acacgataca ttgttattag 5500 tacatttatt aagcgctaga ttctgtgcgt
tgttgattta cagacaattg 5550 ttgtacgtat tttaataatt cattaaattt
ataatcttta gggtggtatg 5600 ttagagcgaa aatcaaatga ttttcagcgt
ctttatatct gaatttaaat 5650 attaaatcct caatagattt gtaaaatagg
tttcgattag tttcaaacaa 5700 gggttgtttt tccgaaccga tggctggact
atctaatgga ttttcgctca 5750 acgccacaaa acttgccaaa tcttgtagca
gcaatctagc tttgtcgata 5800 ttcgtttgtg ttttgttttg taataaaggt
tcgacgtcgt tcaaaatatt 5850 atgcgctttt gtatttcttt catcactgtc
gttagtgtac aattgactcg 5900 acgtaaacac gttaaataaa gcttggacat
atttaacatc gggcgtgtta 5950 gctttattag gccgattatc gtcgtcgtcc
caaccctcgt cgttagaagt 6000 tgcttccgaa gacgattttg ccatagccac
acgacgccta ttaattgtgt 6050 cggctaacac gtccgcgatc aaatttgtag
ttgagctttt tggaattatt 6100 tctgattgcg ggcgtttttg ggcgggtttc
aatctaactg tgcccgattt 6150 taattcagac aacacgttag aaagcgatgg
tgcaggcggt ggtaacattt 6200 cagacggcaa atctactaat ggcggcggtg
gtggagctga tgataaatct 6250 accatcggtg gaggcgcagg cggggctggc
ggcggaggcg gaggcggagg 6300 tggtggcggt gatgcagacg gcggtttagg
ctcaaatgtc tctttaggca 6350 acacagtcgg cacctcaact attgtactgg
tttcgggcgc cgtttttggt 6400 ttgaccggtc tgagacgagt gcgatttttt
tcgtttctaa tagcttccaa 6450 caattgttgt ctgtcgtcta aaggtgcagc
gggttgaggt tccgtcggca 6500 ttggtggagc gggcggcaat tcagacatcg
atggtggtgg tggtggtgga 6550 ggcgctggaa tgttaggcac gggagaaggt
ggtggcggcg gtgccgccgg 6600 tataatttgt tctggtttag tttgttcgcg
cacgattgtg ggcaccggcg 6650 caggcgccgc tggctgcaca acggaaggtc
gtctgcttcg aggcagcgct 6700 tggggtggtg gcaattcaat attataattg
gaatacaaat cgtaaaaatc 6750 tgctataagc attgtaattt cgctatcgtt
taccgtgccg atatttaaca 6800 accgctcaat gtaagcaatt gtattgtaaa
gagattgtct caagctccgc 6850 acgccgataa caagcctttt catttttact
acagcattgt agtggcgaga 6900 cacttcgctg tcgtcgacgt acatgtatgc
tttgttgtca aaaacgtcgt 6950 tggcaagctt taaaatattt aaaagaacat
ctctgttcag caccactgtg 7000 ttgtcgtaaa tgttgttttt gataatttgc
gcttccgcag tatcgacacg 7050 ttcaaaaaat tgatgcgcat caattttgtt
gttcctatta ttgaataaat 7100 aagattgtac agattcatat ctacgattcg
tcatggccac cacaaatgct 7150 acgctgcaaa cgctggtaca attttacgaa
aactgcaaaa acgtcaaaac 7200 tcggtataaa ataatcaacg ggcgctttgg
caaaatatct attttatcgc 7250 acaagcccac tagcaaattg tatttgcaga
aaacaatttc ggcgcacaat 7300 tttaacgctg acgaaataaa agttcaccag
ttaatgagcg accacccaaa 7350 ttttataaaa atctatttta atcacggttc
catcaacaac caagtgatcg 7400 tgatggacta cattgactgt cccgatttat
ttgaaacact acaaattaaa 7450 ggcgagcttt cgtaccaact tgttagcaat
attattagac agctgtgtga 7500 agcgctcaac gatttgcaca agcacaattt
catacacaac gacataaaac 7550 tcgaaaatgt cttatatttc gaagcacttg
atcgcgtgta tgtttgcgat 7600 tacggattgt gcaaacacga aaactcactt
agcgtgcacg acggcacgtt 7650 ggagtatttt agtccggaaa aaattcgaca
cacaactatg cacgtttcgt 7700 ttgactggta cgcggcgtgt taacatacaa
gttgctaacc ggcggttcgt 7750 aatcatggtc atagctgttt cctgtgtgaa
attgttatcc gctcacaatt 7800 ccacacaaca tacgagccgg aagcataaag
tgtaaagcct ggggtgccta 7850 atgagtgagc taactcacat taattgcgtt
gcgctcactg cccgctttcc 7900 agtcgggaaa cctgtcgtgc cagctgcatt
aatgaatcgg ccaacgcgcg 7950 gggagaggcg gtttgcgtat tgggcgctct
tccgcttcct cgctcactga 8000 ctcgctgcgc tcggtcgttc ggctgcggcg
agcggtatca gctcactcaa 8050 aggcggtaat acggttatcc acagaatcag
gggataacgc aggaaagaac 8100 atgtgagcaa aaggccagca aaaggccagg
aaccgtaaaa aggccgcgtt 8150 gctggcgttt ttccataggc tccgcccccc
tgacgagcat cacaaaaatc 8200 gacgctcaag tcagaggtgg cgaaacccga
caggactata aagataccag 8250 gcgtttcccc ctggaagctc cctcgtgcgc
tctcctgttc cgaccctgcc 8300 gcttaccgga tacctgtccg cctttctccc
ttcgggaagc gtggcgcttt 8350 ctcatagctc acgctgtagg tatctcagtt
cggtgtaggt cgttcgctcc 8400 aagctgggct gtgtgcacga accccccgtt
cagcccgacc gctgcgcctt 8450 atccggtaac tatcgtcttg agtccaaccc
ggtaagacac gacttatcgc 8500 cactggcagc agccactggt aacaggatta
gcagagcgag gtatgtaggc 8550 ggtgctacag agttcttgaa gtggtggcct
aactacggct acactagaag 8600 gacagtattt ggtatctgcg ctctgctgaa
gccagttacc ttcggaaaaa 8650 gagttggtag ctcttgatcc ggcaaacaaa
ccaccgctgg tagcggtggt 8700 ttttttgttt gcaagcagca gattacgcgc
agaaaaaaag gatctcaaga 8750 agatcctttg atcttttcta cggggtctga
cgctcagtgg aacgaaaact 8800 cacgttaagg gattttggtc atgagattat
caaaaaggat cttcacctag 8850 atccttttaa attaaaaatg aagttttaaa
tcaatctaaa gtatatatga 8900 gtaaacttgg tctgacagtt accaatgctt
aatcagtgag gcacctatct 8950 cagcgatctg tctatttcgt tcatccatag
ttgcctgact ccccgtcgtg 9000 tagataacta cgatacggga gggcttacca
tctggcccca gtgctgcaat 9050 gataccgcga gacccacgct caccggctcc
agatttatca gcaataaacc 9100 agccagccgg aagggccgag cgcagaagtg
gtcctgcaac tttatccgcc 9150 tccatccagt ctattaattg ttgccgggaa
gctagagtaa gtagttcgcc 9200 agttaatagt ttgcgcaacg ttgttgccat
tgctacaggc atcgtggtgt 9250 cacgctcgtc gtttggtatg gcttcattca
gctccggttc ccaacgatca 9300 aggcgagtta catgatcccc catgttgtgc
aaaaaagcgg ttagctcctt 9350 cggtcctccg atcgttgtca gaagtaagtt
ggccgcagtg ttatcactca 9400 tggttatggc agcactgcat aattctctta
ctgtcatgcc atccgtaaga 9450 tgcttttctg tgactggtga gtactcaacc
aagtcattct gagaatagtg 9500 tatgcggcga ccgagttgct cttgcccggc
gtcaatacgg gataataccg 9550 cgccacatag cagaacttta aaagtgctca
tcattggaaa acgttcttcg 9600 gggcgaaaac tctcaaggat cttaccgctg
ttgagatcca gttcgatgta 9650 acccactcgt gcacccaact gatcttcagc
atcttttact ttcaccagcg 9700 tttctgggtg agcaaaaaca ggaaggcaaa
atgccgcaaa aaagggaata 9750 agggcgacac ggaaatgttg aatactcata
ctcttccttt ttcaatatta 9800 ttgaagcatt tatcagggtt attgtctcat
gagcggatac atatttgaat 9850 gtatttagaa aaataaacaa ataggggttc
cgcgcacatt tccccgaaaa 9900 gtgccacctg acgtctaaga aaccattatt
atcatgacat taacctataa 9950 aaataggcgt atcacgaggc cctttcgtct
cgcgcgtttc ggtgatgacg 10000 gtgaaaacct ctgacacatg cagctcccgg
agacggtcac agcttgtctg 10050 taagcggatg ccgggagcag acaagcccgt
cagggcgcgt cagcgggtgt 10100 tggcgggtgt cggggctggc ttaactatgc
ggcatcagag cagattgtac 10150 tgagagtgca ccatatatgc ggtgtgaaat
accgcacaga tgcgtaagga 10200 gaaaataccg catcaggcgc cattcgccat
tcaggctgcg caactgttgg 10250 gaagggcgat cggtgcgggc ctcttcgcta
ttacgccagc tggcgaaagg 10300 gggatgtgct gcaaggcgat taagttgggt
aacgccaggg ttttcccagt 10350 cacgacgttg taaaacgacg gccagtgcc 10379
55 9690 DNA Artificial sequence misc_feature 1-9690 Sequence is
synthesized. 55 aagctttact cgtaaagcga gttgaaggat catatttagt
tgcgtttatg 50 agataagatt gaaagcacgt gtaaaatgtt tcccgcgcgt
tggcacaact 100 atttacaatg cggccaagtt ataaaagatt ctaatctgat
atgttttaaa 150 acacctttgc ggcccgagtt gtttgcgtac gtgactagcg
aagaagatgt 200 gtggaccgca gaacagatag taaaacaaaa ccctagtatt
ggagcaataa 250 tcgatttaac caacacgtct aaatattatg atggtgtgca
ttttttgcgg 300 gcgggcctgt tatacaaaaa aattcaagta cctggccaga
ctttgccgcc 350 tgaaagcata gttcaagaat ttattgacac ggtaaaagaa
tttacagaaa 400 agtgtcccgg catgttggtg ggcgtgcact gcacacacgg
tattaatcgc 450 accggttaca tggtgtgcag atatttaatg cacaccctgg
gtattgcgcc 500 gcaggaagcc atagatagat tcgaaaaagc cagaggtcac
aaaattgaaa 550 gacaaaatta cgttcaagat ttattaattt aattaatatt
atttgcattc 600 tttaacaaat actttatcct attttcaaat tgttgcgctt
cttccagcga 650 accaaaacta tgcttcgctt gctccgttta gcttgtagcc
gatcagtggc 700 gttgttccaa tcgacggtag gattaggccg gatattctcc
accacaatgt 750 tggcaacgtt gatgttacgt ttatgctttt ggttttccac
gtacgtcttt 800 tggccggtaa tagccgtaaa cgtagtgccg tcgcgcgtca
cgcacaacac 850 cggatgtttg cgcttgtccg cggggtattg aaccgcgcga
tccgacaaat 900 ccaccacttt ggcaactaaa tcggtgacct gcgcgtcttt
tttctgcatt 950 atttcgtctt tcttttgcat ggtttcctgg aagccggtgt
acatgcggtt 1000 tagatcagtc atgacgcgcg tgacctgcaa atctttggcc
tcgatctgct 1050 tgtccttgat ggcaacgatg cgttcaataa actcttgttt
tttaacaagt 1100 tcctcggttt tttgcgccac caccgcttgc agcgcgtttg
tgtgctcggt 1150 gaatgtcgca atcagcttag tcaccaactg tttgctctcc
tcctcccgtt 1200 gtttgatcgc gggatcgtac ttgccggtgc agagcacttg
aggaattact 1250 tcttctaaaa gccattcttg taattctatg gcgtaaggca
atttggactt 1300 cataatcagc tgaatcacgc cggatttagt aatgagcact
gtatgcggct 1350 gcaaatacag cgggtcgccc cttttcacga cgctgttaga
ggtagggccc 1400 ccattttgga tggtctgctc aaataacgat ttgtatttat
tgtctacatg 1450 aacacgtata gctttatcac aaactgtata ttttaaactg
ttagcgacgt 1500 ccttggccac gaaccggacc tgttggtcgc gctctagcac
gtaccgcagg 1550 ttgaacgtat cttctccaaa tttaaattct ccaattttaa
cgcgagccat 1600 tttgatacac gtgtgtcgat tttgcaacaa ctattgtttt
ttaacgcaaa 1650 ctaaacttat tgtggtaagc aataattaaa tatgggggaa
catgcgccgc 1700 tacaacactc gtcgttatga acgcagacgg cgccggtctc
ggcgcaagcg 1750 gctaaaacgt gttgcgcgtt caacgcggca aacatcgcaa
aagccaatag 1800 tacagttttg atttgcatat taacggcgat tttttaaatt
atcttattta 1850 ataaatagtt atgacgccta caactccccg cccgcgttga
ctcgctgcac 1900 ctcgagcagt tcgttgacgc cttcctccgt gtggccgaac
acgtcgagcg 1950 ggtggtcgat gaccagcggc gtgccgcacg cgacgcacaa
gtatctgtac 2000 accgaatgat cgtcgggcga aggcacgtcg gcctccaagt
ggcaatattg 2050 gcaaattcga aaatatatac agttgggttg tttgcgcata
tctatcgtgg 2100 cgttgggcat gtacgtccga acgttgattt gcatgcaagc
cgaaattaaa 2150 tcattgcgat tagtgcgatt aaaacgttgt acatcctcgc
ttttaatcat 2200 gccgtcgatt aaatcgcgca atcgagtcaa gtgatcaaag
tgtggaataa 2250 tgttttcttt gtattcccga gtcaagcgca gcgcgtattt
taacaaacta 2300 gccatcttgt aagttagttt catttaatgc aactttatcc
aataatatat 2350 tatgtatcgc acgtcaagaa ttaacaatgc gcccgttgtc
gcatctcaac 2400 acgactatga tagagatcaa ataaagcgcg aattaaatag
cttgcgacgc 2450 aacgtgcacg atctgtgcac gcgttccggc acgagctttg
attgtaataa 2500 gtttttacga agcgatgaca tgacccccgt agtgacaacg
atcacgccca 2550 aaagaactgc cgactacaaa attaccgagt atgtcggtga
cgttaaaact 2600 attaagccat ccaatcgacc gttagtcgaa tcaggaccgc
tggtgcgaga 2650 agccgcgaag tatggcgaat gcatcgtata acgtgtggag
tccgctcatt 2700 agagcgtcat gtttagacaa gaaagctaca tatttaattg
atcccgatga 2750 ttttattgat aaattgaccc taactccata cacggtattc
tacaatggcg 2800 gggttttggt caaaatttcc ggactgcgat tgtacatgct
gttaacggct 2850 ccgcccacta ttaatgaaat
taaaaattcc aattttaaaa aacgcagcaa 2900 gagaaacatt tgtatgaaag
aatgcgtaga aggaaagaaa aatgtcgtcg 2950 acatgctgaa caacaagatt
aatatgcctc cgtgtataaa aaaaatattg 3000 aacgatttga aagaaaacaa
tgtaccgcgc ggcggtatgt acaggaagag 3050 gtttatacta aactgttaca
ttgcaaacgt ggtttcgtgt gccaagtgtg 3100 aaaaccgatg tttaatcaag
gctctgacgc atttctacaa ccacgactcc 3150 aagtgtgtgg gtgaagtcat
gcatctttta atcaaatccc aagatgtgta 3200 taaaccacca aactgccaaa
aaatgaaaac tgtcgacaag ctctgtccgt 3250 ttgctggcaa ctgcaagggt
ctcaatccta tttgtaatta ttgaataata 3300 aaacaattat aaatgctaaa
tttgtttttt attaacgata caaaccaaac 3350 gcaacaagaa catttgtagt
attatctata attgaaaacg cgtagttata 3400 atcgctgagg taatatttaa
aatcattttc aaatgattca cagttaattt 3450 gcgacaatat aattttattt
tcacataaac tagacgcctt gtcgtcttct 3500 tcttcgtatt ccttctcttt
ttcatttttc tcctcataaa aattaacata 3550 gttattatcg tatccatata
tgtatctatc gtatagagta aattttttgt 3600 tgtcataaat atatatgtct
tttttaatgg ggtgtatagt accgctgcgc 3650 atagtttttc tgtaatttac
aacagtgcta ttttctggta gttcttcgga 3700 gtgtgttgct ttaattatta
aatttatata atcaatgaat ttgggatcgt 3750 cggttttgta caatatgttg
ccggcatagt acgcagcttc ttctagttca 3800 attacaccat tttttagcag
caccggatta acataacttt ccaaaatgtt 3850 gtacgaaccg ttaaacaaaa
acagttcacc tcccttttct atactattgt 3900 ctgcgagcag ttgtttgttg
ttaaaaataa cagccattgt aatgagacgc 3950 acaaactaat atcacaaact
ggaaatgtct atcaatatat agttgctgat 4000 atcatggaga taattaaaat
gataaccatc tcgcaaataa ataagtattt 4050 tactgttttc gtaacagttt
tgtaataaaa aaacctataa atattccgga 4100 ttattcatac cgtcccacca
tcgggcgcgg atccgcggcc gcgaattcta 4150 aaccaccatg ggcagctgcc
cgggcatcat catcatcatc atcatcatta 4200 attctagact agtctgcaga
tctgatcctt tcctgggacc cggcaagaac 4250 caaaaactca ctctcttcaa
ggaaatccgt aatgttaaac ccgacacgat 4300 gaagcttgtc gttggatgga
aaggaaaaga gttctacagg gaaacttgga 4350 cccgcttcat ggaagacagc
ttccccattg ttaacgacca agaagtgatg 4400 gatgttttcc ttgttgtcaa
catgcgtccc actagaccca accgttgtta 4450 caaattcctg gcccaacacg
ctctgcgttg cgaccccgac tatgtacctc 4500 atgacgtgat taggatcgtc
gagccttcat gggtgggcag caacaacgag 4550 taccgcatca gcctggctaa
gaagggcggc ggctgcccaa taatgaacct 4600 tcactctgag tacaccaact
cgttcgaaca gttcatcgat cgtgtcatct 4650 gggagaactt ctacaagccc
atcgtttaca tcggtaccga ctctgctgaa 4700 gaggaggaaa ttctccttga
agtttccctg gtgttcaaag taaaggagtt 4750 tgcaccagac gcacctctgt
tcactggtcc ggcgtattaa aacacgatac 4800 attgttatta gtacatttat
taagcgctag attctgtgcg ttgttgattt 4850 acagacaatt gttgtacgta
ttttaataat tcattaaatt tataatcttt 4900 agggtggtat gttagagcga
aaatcaaatg attttcagcg tctttatatc 4950 tgaatttaaa tattaaatcc
tcaatagatt tgtaaaatag gtttcgatta 5000 gtttcaaaca agggttgttt
ttccgaaccg atggctggac tatctaatgg 5050 attttcgctc aacgccacaa
aacttgccaa atcttgtagc agcaatctag 5100 ctttgtcgat attcgtttgt
gttttgtttt gtaataaagg ttcgacgtcg 5150 ttcaaaatat tatgcgcttt
tgtatttctt tcatcactgt cgttagtgta 5200 caattgactc gacgtaaaca
cgttaaataa agcttggaca tatttaacat 5250 cgggcgtgtt agctttatta
ggccgattat cgtcgtcgtc ccaaccctcg 5300 tcgttagaag ttgcttccga
agacgatttt gccatagcca cacgacgcct 5350 attaattgtg tcggctaaca
cgtccgcgat caaatttgta gttgagcttt 5400 ttggaattat ttctgattgc
gggcgttttt gggcgggttt caatctaact 5450 gtgcccgatt ttaattcaga
caacacgtta gaaagcgatg gtgcaggcgg 5500 tggtaacatt tcagacggca
aatctactaa tggcggcggt ggtggagctg 5550 atgataaatc taccatcggt
ggaggcgcag gcggggctgg cggcggaggc 5600 ggaggcggag gtggtggcgg
tgatgcagac ggcggtttag gctcaaatgt 5650 ctctttaggc aacacagtcg
gcacctcaac tattgtactg gtttcgggcg 5700 ccgtttttgg tttgaccggt
ctgagacgag tgcgattttt ttcgtttcta 5750 atagcttcca acaattgttg
tctgtcgtct aaaggtgcag cgggttgagg 5800 ttccgtcggc attggtggag
cgggcggcaa ttcagacatc gatggtggtg 5850 gtggtggtgg aggcgctgga
atgttaggca cgggagaagg tggtggcggc 5900 ggtgccgccg gtataatttg
ttctggttta gtttgttcgc gcacgattgt 5950 gggcaccggc gcaggcgccg
ctggctgcac aacggaaggt cgtctgcttc 6000 gaggcagcgc ttggggtggt
ggcaattcaa tattataatt ggaatacaaa 6050 tcgtaaaaat ctgctataag
cattgtaatt tcgctatcgt ttaccgtgcc 6100 gatatttaac aaccgctcaa
tgtaagcaat tgtattgtaa agagattgtc 6150 tcaagctccg cacgccgata
acaagccttt tcatttttac tacagcattg 6200 tagtggcgag acacttcgct
gtcgtcgacg tacatgtatg ctttgttgtc 6250 aaaaacgtcg ttggcaagct
ttaaaatatt taaaagaaca tctctgttca 6300 gcaccactgt gttgtcgtaa
atgttgtttt tgataatttg cgcttccgca 6350 gtatcgacac gttcaaaaaa
ttgatgcgca tcaattttgt tgttcctatt 6400 attgaataaa taagattgta
cagattcata tctacgattc gtcatggcca 6450 ccacaaatgc tacgctgcaa
acgctggtac aattttacga aaactgcaaa 6500 aacgtcaaaa ctcggtataa
aataatcaac gggcgctttg gcaaaatatc 6550 tattttatcg cacaagccca
ctagcaaatt gtatttgcag aaaacaattt 6600 cggcgcacaa ttttaacgct
gacgaaataa aagttcacca gttaatgagc 6650 gaccacccaa attttataaa
aatctatttt aatcacggtt ccatcaacaa 6700 ccaagtgatc gtgatggact
acattgactg tcccgattta tttgaaacac 6750 tacaaattaa aggcgagctt
tcgtaccaac ttgttagcaa tattattaga 6800 cagctgtgtg aagcgctcaa
cgatttgcac aagcacaatt tcatacacaa 6850 cgacataaaa ctcgaaaatg
tcttatattt cgaagcactt gatcgcgtgt 6900 atgtttgcga ttacggattg
tgcaaacacg aaaactcact tagcgtgcac 6950 gacggcacgt tggagtattt
tagtccggaa aaaattcgac acacaactat 7000 gcacgtttcg tttgactggt
acgcggcgtg ttaacataca agttgctaac 7050 cggcggttcg taatcatggt
catagctgtt tcctgtgtga aattgttatc 7100 cgctcacaat tccacacaac
atacgagccg gaagcataaa gtgtaaagcc 7150 tggggtgcct aatgagtgag
ctaactcaca ttaattgcgt tgcgctcact 7200 gcccgctttc cagtcgggaa
acctgtcgtg ccagctgcat taatgaatcg 7250 gccaacgcgc ggggagaggc
ggtttgcgta ttgggcgctc ttccgcttcc 7300 tcgctcactg actcgctgcg
ctcggtcgtt cggctgcggc gagcggtatc 7350 agctcactca aaggcggtaa
tacggttatc cacagaatca ggggataacg 7400 caggaaagaa catgtgagca
aaaggccagc aaaaggccag gaaccgtaaa 7450 aaggccgcgt tgctggcgtt
tttccatagg ctccgccccc ctgacgagca 7500 tcacaaaaat cgacgctcaa
gtcagaggtg gcgaaacccg acaggactat 7550 aaagatacca ggcgtttccc
cctggaagct ccctcgtgcg ctctcctgtt 7600 ccgaccctgc cgcttaccgg
atacctgtcc gcctttctcc cttcgggaag 7650 cgtggcgctt tctcatagct
cacgctgtag gtatctcagt tcggtgtagg 7700 tcgttcgctc caagctgggc
tgtgtgcacg aaccccccgt tcagcccgac 7750 cgctgcgcct tatccggtaa
ctatcgtctt gagtccaacc cggtaagaca 7800 cgacttatcg ccactggcag
cagccactgg taacaggatt agcagagcga 7850 ggtatgtagg cggtgctaca
gagttcttga agtggtggcc taactacggc 7900 tacactagaa ggacagtatt
tggtatctgc gctctgctga agccagttac 7950 cttcggaaaa agagttggta
gctcttgatc cggcaaacaa accaccgctg 8000 gtagcggtgg tttttttgtt
tgcaagcagc agattacgcg cagaaaaaaa 8050 ggatctcaag aagatccttt
gatcttttct acggggtctg acgctcagtg 8100 gaacgaaaac tcacgttaag
ggattttggt catgagatta tcaaaaagga 8150 tcttcaccta gatcctttta
aattaaaaat gaagttttaa atcaatctaa 8200 agtatatatg agtaaacttg
gtctgacagt taccaatgct taatcagtga 8250 ggcacctatc tcagcgatct
gtctatttcg ttcatccata gttgcctgac 8300 tccccgtcgt gtagataact
acgatacggg agggcttacc atctggcccc 8350 agtgctgcaa tgataccgcg
agacccacgc tcaccggctc cagatttatc 8400 agcaataaac cagccagccg
gaagggccga gcgcagaagt ggtcctgcaa 8450 ctttatccgc ctccatccag
tctattaatt gttgccggga agctagagta 8500 agtagttcgc cagttaatag
tttgcgcaac gttgttgcca ttgctacagg 8550 catcgtggtg tcacgctcgt
cgtttggtat ggcttcattc agctccggtt 8600 cccaacgatc aaggcgagtt
acatgatccc ccatgttgtg caaaaaagcg 8650 gttagctcct tcggtcctcc
gatcgttgtc agaagtaagt tggccgcagt 8700 gttatcactc atggttatgg
cagcactgca taattctctt actgtcatgc 8750 catccgtaag atgcttttct
gtgactggtg agtactcaac caagtcattc 8800 tgagaatagt gtatgcggcg
accgagttgc tcttgcccgg cgtcaatacg 8850 ggataatacc gcgccacata
gcagaacttt aaaagtgctc atcattggaa 8900 aacgttcttc ggggcgaaaa
ctctcaagga tcttaccgct gttgagatcc 8950 agttcgatgt aacccactcg
tgcacccaac tgatcttcag catcttttac 9000 tttcaccagc gtttctgggt
gagcaaaaac aggaaggcaa aatgccgcaa 9050 aaaagggaat aagggcgaca
cggaaatgtt gaatactcat actcttcctt 9100 tttcaatatt attgaagcat
ttatcagggt tattgtctca tgagcggata 9150 catatttgaa tgtatttaga
aaaataaaca aataggggtt ccgcgcacat 9200 ttccccgaaa agtgccacct
gacgtctaag aaaccattat tatcatgaca 9250 ttaacctata aaaataggcg
tatcacgagg ccctttcgtc tcgcgcgttt 9300 cggtgatgac ggtgaaaacc
tctgacacat gcagctcccg gagacggtca 9350 cagcttgtct gtaagcggat
gccgggagca gacaagcccg tcagggcgcg 9400 tcagcgggtg ttggcgggtg
tcggggctgg cttaactatg cggcatcaga 9450 gcagattgta ctgagagtgc
accatatatg cggtgtgaaa taccgcacag 9500 atgcgtaagg agaaaatacc
gcatcaggcg ccattcgcca ttcaggctgc 9550 gcaactgttg ggaagggcga
tcggtgcggg cctcttcgct attacgccag 9600 ctggcgaaag ggggatgtgc
tgcaaggcga ttaagttggg taacgccagg 9650 gttttcccag tcacgacgtt
gtaaaacgac ggccagtgcc 9690 56 249 PRT Homo sapiens 56 Arg Gly Thr
Pro Lys Thr His Leu Leu Ala Phe Ser Leu Leu Cys 1 5 10 15 Leu Leu
Ser Lys Val Arg Thr Gln Leu Cys Pro Thr Pro Cys Thr 20 25 30 Cys
Pro Trp Pro Pro Pro Arg Cys Pro Leu Gly Val Pro Leu Val 35 40 45
Leu Asp Gly Cys Gly Cys Cys Arg Val Cys Ala Arg Arg Leu Gly 50 55
60 Glu Pro Cys Asp Gln Leu His Val Cys Asp Ala Ser Gln Gly Leu 65
70 75 Val Cys Gln Pro Gly Ala Gly Pro Gly Gly Arg Gly Ala Leu Cys
80 85 90 Leu Leu Ala Glu Asp Asp Ser Ser Cys Glu Val Asn Gly Arg
Leu 95 100 105 Tyr Arg Glu Gly Glu Thr Phe Gln Pro His Cys Ser Ile
Arg Cys 110 115 120 Arg Cys Glu Asp Gly Gly Phe Thr Cys Val Pro Leu
Cys Ser Glu 125 130 135 Asp Val Arg Leu Pro Ser Trp Asp Cys Pro His
Pro Arg Arg Val 140 145 150 Glu Val Leu Gly Lys Cys Cys Pro Glu Trp
Val Cys Gly Gln Gly 155 160 165 Gly Gly Leu Gly Thr Gln Pro Leu Pro
Ala Gln Gly Pro Gln Phe 170 175 180 Ser Gly Leu Val Ser Ser Leu Pro
Pro Gly Val Pro Cys Pro Glu 185 190 195 Trp Ser Thr Ala Trp Gly Pro
Cys Ser Thr Thr Cys Gly Leu Gly 200 205 210 Met Ala Thr Arg Val Ser
Asn Gln Asn Arg Phe Cys Arg Leu Glu 215 220 225 Thr Gln Arg Arg Leu
Cys Leu Ser Arg Pro Cys Pro Pro Ser Arg 230 235 240 Gly Arg Ser Pro
Gln Asn Ser Ala Phe 245 249 57 248 PRT Homo sapiens 57 Gly Thr Pro
Lys Thr His Leu Leu Ala Phe Ser Leu Leu Cys Leu 1 5 10 15 Leu Ser
Lys Val Arg Thr Gln Leu Cys Pro Thr Pro Cys Thr Cys 20 25 30 Pro
Trp Pro Pro Pro Arg Cys Pro Leu Gly Val Pro Leu Val Leu 35 40 45
Asp Gly Cys Gly Cys Cys Arg Val Cys Ala Arg Arg Leu Gly Glu 50 55
60 Pro Cys Asp Gln Leu His Val Cys Asp Ala Ser Gln Gly Leu Val 65
70 75 Cys Gln Pro Gly Ala Gly Pro Gly Gly Arg Gly Ala Leu Cys Leu
80 85 90 Leu Ala Glu Asp Asp Ser Ser Cys Glu Val Asn Gly Arg Leu
Tyr 95 100 105 Arg Glu Gly Glu Thr Phe Gln Pro His Cys Ser Ile Arg
Cys Arg 110 115 120 Cys Glu Asp Gly Gly Phe Thr Cys Val Pro Leu Cys
Ser Glu Asp 125 130 135 Val Arg Leu Pro Ser Trp Asp Cys Pro His Pro
Arg Arg Val Glu 140 145 150 Val Leu Gly Lys Cys Cys Pro Glu Trp Val
Cys Gly Gln Gly Gly 155 160 165 Gly Leu Gly Thr Gln Pro Leu Pro Ala
Gln Gly Pro Gln Phe Ser 170 175 180 Gly Leu Val Ser Ser Leu Pro Pro
Gly Val Pro Cys Pro Glu Trp 185 190 195 Ser Thr Ala Trp Gly Pro Cys
Ser Thr Thr Cys Gly Leu Gly Met 200 205 210 Ala Thr Arg Val Ser Asn
Gln Asn Arg Phe Cys Arg Leu Glu Thr 215 220 225 Gln Arg Arg Leu Cys
Leu Ser Arg Pro Cys Pro Pro Ser Arg Gly 230 235 240 Arg Ser Pro Gln
Asn Ser Ala Phe 245 248 58 247 PRT Homo sapiens 58 Thr Pro Lys Thr
His Leu Leu Ala Phe Ser Leu Leu Cys Leu Leu 1 5 10 15 Ser Lys Val
Arg Thr Gln Leu Cys Pro Thr Pro Cys Thr Cys Pro 20 25 30 Trp Pro
Pro Pro Arg Cys Pro Leu Gly Val Pro Leu Val Leu Asp 35 40 45 Gly
Cys Gly Cys Cys Arg Val Cys Ala Arg Arg Leu Gly Glu Pro 50 55 60
Cys Asp Gln Leu His Val Cys Asp Ala Ser Gln Gly Leu Val Cys 65 70
75 Gln Pro Gly Ala Gly Pro Gly Gly Arg Gly Ala Leu Cys Leu Leu 80
85 90 Ala Glu Asp Asp Ser Ser Cys Glu Val Asn Gly Arg Leu Tyr Arg
95 100 105 Glu Gly Glu Thr Phe Gln Pro His Cys Ser Ile Arg Cys Arg
Cys 110 115 120 Glu Asp Gly Gly Phe Thr Cys Val Pro Leu Cys Ser Glu
Asp Val 125 130 135 Arg Leu Pro Ser Trp Asp Cys Pro His Pro Arg Arg
Val Glu Val 140 145 150 Leu Gly Lys Cys Cys Pro Glu Trp Val Cys Gly
Gln Gly Gly Gly 155 160 165 Leu Gly Thr Gln Pro Leu Pro Ala Gln Gly
Pro Gln Phe Ser Gly 170 175 180 Leu Val Ser Ser Leu Pro Pro Gly Val
Pro Cys Pro Glu Trp Ser 185 190 195 Thr Ala Trp Gly Pro Cys Ser Thr
Thr Cys Gly Leu Gly Met Ala 200 205 210 Thr Arg Val Ser Asn Gln Asn
Arg Phe Cys Arg Leu Glu Thr Gln 215 220 225 Arg Arg Leu Cys Leu Ser
Arg Pro Cys Pro Pro Ser Arg Gly Arg 230 235 240 Ser Pro Gln Asn Ser
Ala Phe 245 247 59 246 PRT Homo sapiens 59 Pro Lys Thr His Leu Leu
Ala Phe Ser Leu Leu Cys Leu Leu Ser 1 5 10 15 Lys Val Arg Thr Gln
Leu Cys Pro Thr Pro Cys Thr Cys Pro Trp 20 25 30 Pro Pro Pro Arg
Cys Pro Leu Gly Val Pro Leu Val Leu Asp Gly 35 40 45 Cys Gly Cys
Cys Arg Val Cys Ala Arg Arg Leu Gly Glu Pro Cys 50 55 60 Asp Gln
Leu His Val Cys Asp Ala Ser Gln Gly Leu Val Cys Gln 65 70 75 Pro
Gly Ala Gly Pro Gly Gly Arg Gly Ala Leu Cys Leu Leu Ala 80 85 90
Glu Asp Asp Ser Ser Cys Glu Val Asn Gly Arg Leu Tyr Arg Glu 95 100
105 Gly Glu Thr Phe Gln Pro His Cys Ser Ile Arg Cys Arg Cys Glu 110
115 120 Asp Gly Gly Phe Thr Cys Val Pro Leu Cys Ser Glu Asp Val Arg
125 130 135 Leu Pro Ser Trp Asp Cys Pro His Pro Arg Arg Val Glu Val
Leu 140 145 150 Gly Lys Cys Cys Pro Glu Trp Val Cys Gly Gln Gly Gly
Gly Leu 155 160 165 Gly Thr Gln Pro Leu Pro Ala Gln Gly Pro Gln Phe
Ser Gly Leu 170 175 180 Val Ser Ser Leu Pro Pro Gly Val Pro Cys Pro
Glu Trp Ser Thr 185 190 195 Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly
Leu Gly Met Ala Thr 200 205 210 Arg Val Ser Asn Gln Asn Arg Phe Cys
Arg Leu Glu Thr Gln Arg 215 220 225 Arg Leu Cys Leu Ser Arg Pro Cys
Pro Pro Ser Arg Gly Arg Ser 230 235 240 Pro Gln Asn Ser Ala Phe 245
246 60 245 PRT Homo sapiens 60 Lys Thr His Leu Leu Ala Phe Ser Leu
Leu Cys Leu Leu Ser Lys 1 5 10 15 Val Arg Thr Gln Leu Cys Pro Thr
Pro Cys Thr Cys Pro Trp Pro 20 25 30 Pro Pro Arg Cys Pro Leu Gly
Val Pro Leu Val Leu Asp Gly Cys
35 40 45 Gly Cys Cys Arg Val Cys Ala Arg Arg Leu Gly Glu Pro Cys
Asp 50 55 60 Gln Leu His Val Cys Asp Ala Ser Gln Gly Leu Val Cys
Gln Pro 65 70 75 Gly Ala Gly Pro Gly Gly Arg Gly Ala Leu Cys Leu
Leu Ala Glu 80 85 90 Asp Asp Ser Ser Cys Glu Val Asn Gly Arg Leu
Tyr Arg Glu Gly 95 100 105 Glu Thr Phe Gln Pro His Cys Ser Ile Arg
Cys Arg Cys Glu Asp 110 115 120 Gly Gly Phe Thr Cys Val Pro Leu Cys
Ser Glu Asp Val Arg Leu 125 130 135 Pro Ser Trp Asp Cys Pro His Pro
Arg Arg Val Glu Val Leu Gly 140 145 150 Lys Cys Cys Pro Glu Trp Val
Cys Gly Gln Gly Gly Gly Leu Gly 155 160 165 Thr Gln Pro Leu Pro Ala
Gln Gly Pro Gln Phe Ser Gly Leu Val 170 175 180 Ser Ser Leu Pro Pro
Gly Val Pro Cys Pro Glu Trp Ser Thr Ala 185 190 195 Trp Gly Pro Cys
Ser Thr Thr Cys Gly Leu Gly Met Ala Thr Arg 200 205 210 Val Ser Asn
Gln Asn Arg Phe Cys Arg Leu Glu Thr Gln Arg Arg 215 220 225 Leu Cys
Leu Ser Arg Pro Cys Pro Pro Ser Arg Gly Arg Ser Pro 230 235 240 Gln
Asn Ser Ala Phe 245 61 244 PRT Homo sapiens 61 Thr His Leu Leu Ala
Phe Ser Leu Leu Cys Leu Leu Ser Lys Val 1 5 10 15 Arg Thr Gln Leu
Cys Pro Thr Pro Cys Thr Cys Pro Trp Pro Pro 20 25 30 Pro Arg Cys
Pro Leu Gly Val Pro Leu Val Leu Asp Gly Cys Gly 35 40 45 Cys Cys
Arg Val Cys Ala Arg Arg Leu Gly Glu Pro Cys Asp Gln 50 55 60 Leu
His Val Cys Asp Ala Ser Gln Gly Leu Val Cys Gln Pro Gly 65 70 75
Ala Gly Pro Gly Gly Arg Gly Ala Leu Cys Leu Leu Ala Glu Asp 80 85
90 Asp Ser Ser Cys Glu Val Asn Gly Arg Leu Tyr Arg Glu Gly Glu 95
100 105 Thr Phe Gln Pro His Cys Ser Ile Arg Cys Arg Cys Glu Asp Gly
110 115 120 Gly Phe Thr Cys Val Pro Leu Cys Ser Glu Asp Val Arg Leu
Pro 125 130 135 Ser Trp Asp Cys Pro His Pro Arg Arg Val Glu Val Leu
Gly Lys 140 145 150 Cys Cys Pro Glu Trp Val Cys Gly Gln Gly Gly Gly
Leu Gly Thr 155 160 165 Gln Pro Leu Pro Ala Gln Gly Pro Gln Phe Ser
Gly Leu Val Ser 170 175 180 Ser Leu Pro Pro Gly Val Pro Cys Pro Glu
Trp Ser Thr Ala Trp 185 190 195 Gly Pro Cys Ser Thr Thr Cys Gly Leu
Gly Met Ala Thr Arg Val 200 205 210 Ser Asn Gln Asn Arg Phe Cys Arg
Leu Glu Thr Gln Arg Arg Leu 215 220 225 Cys Leu Ser Arg Pro Cys Pro
Pro Ser Arg Gly Arg Ser Pro Gln 230 235 240 Asn Ser Ala Phe 244 62
243 PRT Homo sapiens 62 His Leu Leu Ala Phe Ser Leu Leu Cys Leu Leu
Ser Lys Val Arg 1 5 10 15 Thr Gln Leu Cys Pro Thr Pro Cys Thr Cys
Pro Trp Pro Pro Pro 20 25 30 Arg Cys Pro Leu Gly Val Pro Leu Val
Leu Asp Gly Cys Gly Cys 35 40 45 Cys Arg Val Cys Ala Arg Arg Leu
Gly Glu Pro Cys Asp Gln Leu 50 55 60 His Val Cys Asp Ala Ser Gln
Gly Leu Val Cys Gln Pro Gly Ala 65 70 75 Gly Pro Gly Gly Arg Gly
Ala Leu Cys Leu Leu Ala Glu Asp Asp 80 85 90 Ser Ser Cys Glu Val
Asn Gly Arg Leu Tyr Arg Glu Gly Glu Thr 95 100 105 Phe Gln Pro His
Cys Ser Ile Arg Cys Arg Cys Glu Asp Gly Gly 110 115 120 Phe Thr Cys
Val Pro Leu Cys Ser Glu Asp Val Arg Leu Pro Ser 125 130 135 Trp Asp
Cys Pro His Pro Arg Arg Val Glu Val Leu Gly Lys Cys 140 145 150 Cys
Pro Glu Trp Val Cys Gly Gln Gly Gly Gly Leu Gly Thr Gln 155 160 165
Pro Leu Pro Ala Gln Gly Pro Gln Phe Ser Gly Leu Val Ser Ser 170 175
180 Leu Pro Pro Gly Val Pro Cys Pro Glu Trp Ser Thr Ala Trp Gly 185
190 195 Pro Cys Ser Thr Thr Cys Gly Leu Gly Met Ala Thr Arg Val Ser
200 205 210 Asn Gln Asn Arg Phe Cys Arg Leu Glu Thr Gln Arg Arg Leu
Cys 215 220 225 Leu Ser Arg Pro Cys Pro Pro Ser Arg Gly Arg Ser Pro
Gln Asn 230 235 240 Ser Ala Phe 243 63 242 PRT Homo sapiens 63 Leu
Leu Ala Phe Ser Leu Leu Cys Leu Leu Ser Lys Val Arg Thr 1 5 10 15
Gln Leu Cys Pro Thr Pro Cys Thr Cys Pro Trp Pro Pro Pro Arg 20 25
30 Cys Pro Leu Gly Val Pro Leu Val Leu Asp Gly Cys Gly Cys Cys 35
40 45 Arg Val Cys Ala Arg Arg Leu Gly Glu Pro Cys Asp Gln Leu His
50 55 60 Val Cys Asp Ala Ser Gln Gly Leu Val Cys Gln Pro Gly Ala
Gly 65 70 75 Pro Gly Gly Arg Gly Ala Leu Cys Leu Leu Ala Glu Asp
Asp Ser 80 85 90 Ser Cys Glu Val Asn Gly Arg Leu Tyr Arg Glu Gly
Glu Thr Phe 95 100 105 Gln Pro His Cys Ser Ile Arg Cys Arg Cys Glu
Asp Gly Gly Phe 110 115 120 Thr Cys Val Pro Leu Cys Ser Glu Asp Val
Arg Leu Pro Ser Trp 125 130 135 Asp Cys Pro His Pro Arg Arg Val Glu
Val Leu Gly Lys Cys Cys 140 145 150 Pro Glu Trp Val Cys Gly Gln Gly
Gly Gly Leu Gly Thr Gln Pro 155 160 165 Leu Pro Ala Gln Gly Pro Gln
Phe Ser Gly Leu Val Ser Ser Leu 170 175 180 Pro Pro Gly Val Pro Cys
Pro Glu Trp Ser Thr Ala Trp Gly Pro 185 190 195 Cys Ser Thr Thr Cys
Gly Leu Gly Met Ala Thr Arg Val Ser Asn 200 205 210 Gln Asn Arg Phe
Cys Arg Leu Glu Thr Gln Arg Arg Leu Cys Leu 215 220 225 Ser Arg Pro
Cys Pro Pro Ser Arg Gly Arg Ser Pro Gln Asn Ser 230 235 240 Ala Phe
242 64 241 PRT Homo sapiens 64 Leu Ala Phe Ser Leu Leu Cys Leu Leu
Ser Lys Val Arg Thr Gln 1 5 10 15 Leu Cys Pro Thr Pro Cys Thr Cys
Pro Trp Pro Pro Pro Arg Cys 20 25 30 Pro Leu Gly Val Pro Leu Val
Leu Asp Gly Cys Gly Cys Cys Arg 35 40 45 Val Cys Ala Arg Arg Leu
Gly Glu Pro Cys Asp Gln Leu His Val 50 55 60 Cys Asp Ala Ser Gln
Gly Leu Val Cys Gln Pro Gly Ala Gly Pro 65 70 75 Gly Gly Arg Gly
Ala Leu Cys Leu Leu Ala Glu Asp Asp Ser Ser 80 85 90 Cys Glu Val
Asn Gly Arg Leu Tyr Arg Glu Gly Glu Thr Phe Gln 95 100 105 Pro His
Cys Ser Ile Arg Cys Arg Cys Glu Asp Gly Gly Phe Thr 110 115 120 Cys
Val Pro Leu Cys Ser Glu Asp Val Arg Leu Pro Ser Trp Asp 125 130 135
Cys Pro His Pro Arg Arg Val Glu Val Leu Gly Lys Cys Cys Pro 140 145
150 Glu Trp Val Cys Gly Gln Gly Gly Gly Leu Gly Thr Gln Pro Leu 155
160 165 Pro Ala Gln Gly Pro Gln Phe Ser Gly Leu Val Ser Ser Leu Pro
170 175 180 Pro Gly Val Pro Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro
Cys 185 190 195 Ser Thr Thr Cys Gly Leu Gly Met Ala Thr Arg Val Ser
Asn Gln 200 205 210 Asn Arg Phe Cys Arg Leu Glu Thr Gln Arg Arg Leu
Cys Leu Ser 215 220 225 Arg Pro Cys Pro Pro Ser Arg Gly Arg Ser Pro
Gln Asn Ser Ala 230 235 240 Phe 241 65 240 PRT Homo sapiens 65 Ala
Phe Ser Leu Leu Cys Leu Leu Ser Lys Val Arg Thr Gln Leu 1 5 10 15
Cys Pro Thr Pro Cys Thr Cys Pro Trp Pro Pro Pro Arg Cys Pro 20 25
30 Leu Gly Val Pro Leu Val Leu Asp Gly Cys Gly Cys Cys Arg Val 35
40 45 Cys Ala Arg Arg Leu Gly Glu Pro Cys Asp Gln Leu His Val Cys
50 55 60 Asp Ala Ser Gln Gly Leu Val Cys Gln Pro Gly Ala Gly Pro
Gly 65 70 75 Gly Arg Gly Ala Leu Cys Leu Leu Ala Glu Asp Asp Ser
Ser Cys 80 85 90 Glu Val Asn Gly Arg Leu Tyr Arg Glu Gly Glu Thr
Phe Gln Pro 95 100 105 His Cys Ser Ile Arg Cys Arg Cys Glu Asp Gly
Gly Phe Thr Cys 110 115 120 Val Pro Leu Cys Ser Glu Asp Val Arg Leu
Pro Ser Trp Asp Cys 125 130 135 Pro His Pro Arg Arg Val Glu Val Leu
Gly Lys Cys Cys Pro Glu 140 145 150 Trp Val Cys Gly Gln Gly Gly Gly
Leu Gly Thr Gln Pro Leu Pro 155 160 165 Ala Gln Gly Pro Gln Phe Ser
Gly Leu Val Ser Ser Leu Pro Pro 170 175 180 Gly Val Pro Cys Pro Glu
Trp Ser Thr Ala Trp Gly Pro Cys Ser 185 190 195 Thr Thr Cys Gly Leu
Gly Met Ala Thr Arg Val Ser Asn Gln Asn 200 205 210 Arg Phe Cys Arg
Leu Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg 215 220 225 Pro Cys Pro
Pro Ser Arg Gly Arg Ser Pro Gln Asn Ser Ala Phe 230 235 240 66 239
PRT Homo sapiens 66 Phe Ser Leu Leu Cys Leu Leu Ser Lys Val Arg Thr
Gln Leu Cys 1 5 10 15 Pro Thr Pro Cys Thr Cys Pro Trp Pro Pro Pro
Arg Cys Pro Leu 20 25 30 Gly Val Pro Leu Val Leu Asp Gly Cys Gly
Cys Cys Arg Val Cys 35 40 45 Ala Arg Arg Leu Gly Glu Pro Cys Asp
Gln Leu His Val Cys Asp 50 55 60 Ala Ser Gln Gly Leu Val Cys Gln
Pro Gly Ala Gly Pro Gly Gly 65 70 75 Arg Gly Ala Leu Cys Leu Leu
Ala Glu Asp Asp Ser Ser Cys Glu 80 85 90 Val Asn Gly Arg Leu Tyr
Arg Glu Gly Glu Thr Phe Gln Pro His 95 100 105 Cys Ser Ile Arg Cys
Arg Cys Glu Asp Gly Gly Phe Thr Cys Val 110 115 120 Pro Leu Cys Ser
Glu Asp Val Arg Leu Pro Ser Trp Asp Cys Pro 125 130 135 His Pro Arg
Arg Val Glu Val Leu Gly Lys Cys Cys Pro Glu Trp 140 145 150 Val Cys
Gly Gln Gly Gly Gly Leu Gly Thr Gln Pro Leu Pro Ala 155 160 165 Gln
Gly Pro Gln Phe Ser Gly Leu Val Ser Ser Leu Pro Pro Gly 170 175 180
Val Pro Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr 185 190
195 Thr Cys Gly Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg 200
205 210 Phe Cys Arg Leu Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro
215 220 225 Cys Pro Pro Ser Arg Gly Arg Ser Pro Gln Asn Ser Ala Phe
230 235 239 67 238 PRT Homo sapiens 67 Ser Leu Leu Cys Leu Leu Ser
Lys Val Arg Thr Gln Leu Cys Pro 1 5 10 15 Thr Pro Cys Thr Cys Pro
Trp Pro Pro Pro Arg Cys Pro Leu Gly 20 25 30 Val Pro Leu Val Leu
Asp Gly Cys Gly Cys Cys Arg Val Cys Ala 35 40 45 Arg Arg Leu Gly
Glu Pro Cys Asp Gln Leu His Val Cys Asp Ala 50 55 60 Ser Gln Gly
Leu Val Cys Gln Pro Gly Ala Gly Pro Gly Gly Arg 65 70 75 Gly Ala
Leu Cys Leu Leu Ala Glu Asp Asp Ser Ser Cys Glu Val 80 85 90 Asn
Gly Arg Leu Tyr Arg Glu Gly Glu Thr Phe Gln Pro His Cys 95 100 105
Ser Ile Arg Cys Arg Cys Glu Asp Gly Gly Phe Thr Cys Val Pro 110 115
120 Leu Cys Ser Glu Asp Val Arg Leu Pro Ser Trp Asp Cys Pro His 125
130 135 Pro Arg Arg Val Glu Val Leu Gly Lys Cys Cys Pro Glu Trp Val
140 145 150 Cys Gly Gln Gly Gly Gly Leu Gly Thr Gln Pro Leu Pro Ala
Gln 155 160 165 Gly Pro Gln Phe Ser Gly Leu Val Ser Ser Leu Pro Pro
Gly Val 170 175 180 Pro Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys
Ser Thr Thr 185 190 195 Cys Gly Leu Gly Met Ala Thr Arg Val Ser Asn
Gln Asn Arg Phe 200 205 210 Cys Arg Leu Glu Thr Gln Arg Arg Leu Cys
Leu Ser Arg Pro Cys 215 220 225 Pro Pro Ser Arg Gly Arg Ser Pro Gln
Asn Ser Ala Phe 230 235 238 68 237 PRT Homo sapiens 68 Leu Leu Cys
Leu Leu Ser Lys Val Arg Thr Gln Leu Cys Pro Thr 1 5 10 15 Pro Cys
Thr Cys Pro Trp Pro Pro Pro Arg Cys Pro Leu Gly Val 20 25 30 Pro
Leu Val Leu Asp Gly Cys Gly Cys Cys Arg Val Cys Ala Arg 35 40 45
Arg Leu Gly Glu Pro Cys Asp Gln Leu His Val Cys Asp Ala Ser 50 55
60 Gln Gly Leu Val Cys Gln Pro Gly Ala Gly Pro Gly Gly Arg Gly 65
70 75 Ala Leu Cys Leu Leu Ala Glu Asp Asp Ser Ser Cys Glu Val Asn
80 85 90 Gly Arg Leu Tyr Arg Glu Gly Glu Thr Phe Gln Pro His Cys
Ser 95 100 105 Ile Arg Cys Arg Cys Glu Asp Gly Gly Phe Thr Cys Val
Pro Leu 110 115 120 Cys Ser Glu Asp Val Arg Leu Pro Ser Trp Asp Cys
Pro His Pro 125 130 135 Arg Arg Val Glu Val Leu Gly Lys Cys Cys Pro
Glu Trp Val Cys 140 145 150 Gly Gln Gly Gly Gly Leu Gly Thr Gln Pro
Leu Pro Ala Gln Gly 155 160 165 Pro Gln Phe Ser Gly Leu Val Ser Ser
Leu Pro Pro Gly Val Pro 170 175 180 Cys Pro Glu Trp Ser Thr Ala Trp
Gly Pro Cys Ser Thr Thr Cys 185 190 195 Gly Leu Gly Met Ala Thr Arg
Val Ser Asn Gln Asn Arg Phe Cys 200 205 210 Arg Leu Glu Thr Gln Arg
Arg Leu Cys Leu Ser Arg Pro Cys Pro 215 220 225 Pro Ser Arg Gly Arg
Ser Pro Gln Asn Ser Ala Phe 230 235 237 69 236 PRT Homo sapiens 69
Leu Cys Leu Leu Ser Lys Val Arg Thr Gln Leu Cys Pro Thr Pro 1 5 10
15 Cys Thr Cys Pro Trp Pro Pro Pro Arg Cys Pro Leu Gly Val Pro 20
25 30 Leu Val Leu Asp Gly Cys Gly Cys Cys Arg Val Cys Ala Arg Arg
35 40 45 Leu Gly Glu Pro Cys Asp Gln Leu His Val Cys Asp Ala Ser
Gln 50 55 60 Gly Leu Val Cys Gln Pro Gly Ala Gly Pro Gly Gly Arg
Gly Ala 65 70 75 Leu Cys Leu Leu Ala Glu Asp Asp Ser Ser Cys Glu
Val Asn Gly 80 85 90 Arg Leu Tyr Arg Glu Gly Glu Thr Phe Gln Pro
His Cys Ser Ile 95 100 105 Arg Cys Arg Cys Glu Asp Gly Gly Phe Thr
Cys Val Pro Leu Cys 110 115 120 Ser Glu Asp Val Arg Leu Pro Ser Trp
Asp Cys Pro His Pro Arg 125 130 135 Arg Val Glu Val Leu Gly Lys Cys
Cys Pro Glu
Trp Val Cys Gly 140 145 150 Gln Gly Gly Gly Leu Gly Thr Gln Pro Leu
Pro Ala Gln Gly Pro 155 160 165 Gln Phe Ser Gly Leu Val Ser Ser Leu
Pro Pro Gly Val Pro Cys 170 175 180 Pro Glu Trp Ser Thr Ala Trp Gly
Pro Cys Ser Thr Thr Cys Gly 185 190 195 Leu Gly Met Ala Thr Arg Val
Ser Asn Gln Asn Arg Phe Cys Arg 200 205 210 Leu Glu Thr Gln Arg Arg
Leu Cys Leu Ser Arg Pro Cys Pro Pro 215 220 225 Ser Arg Gly Arg Ser
Pro Gln Asn Ser Ala Phe 230 235 236 70 235 PRT Homo sapiens 70 Cys
Leu Leu Ser Lys Val Arg Thr Gln Leu Cys Pro Thr Pro Cys 1 5 10 15
Thr Cys Pro Trp Pro Pro Pro Arg Cys Pro Leu Gly Val Pro Leu 20 25
30 Val Leu Asp Gly Cys Gly Cys Cys Arg Val Cys Ala Arg Arg Leu 35
40 45 Gly Glu Pro Cys Asp Gln Leu His Val Cys Asp Ala Ser Gln Gly
50 55 60 Leu Val Cys Gln Pro Gly Ala Gly Pro Gly Gly Arg Gly Ala
Leu 65 70 75 Cys Leu Leu Ala Glu Asp Asp Ser Ser Cys Glu Val Asn
Gly Arg 80 85 90 Leu Tyr Arg Glu Gly Glu Thr Phe Gln Pro His Cys
Ser Ile Arg 95 100 105 Cys Arg Cys Glu Asp Gly Gly Phe Thr Cys Val
Pro Leu Cys Ser 110 115 120 Glu Asp Val Arg Leu Pro Ser Trp Asp Cys
Pro His Pro Arg Arg 125 130 135 Val Glu Val Leu Gly Lys Cys Cys Pro
Glu Trp Val Cys Gly Gln 140 145 150 Gly Gly Gly Leu Gly Thr Gln Pro
Leu Pro Ala Gln Gly Pro Gln 155 160 165 Phe Ser Gly Leu Val Ser Ser
Leu Pro Pro Gly Val Pro Cys Pro 170 175 180 Glu Trp Ser Thr Ala Trp
Gly Pro Cys Ser Thr Thr Cys Gly Leu 185 190 195 Gly Met Ala Thr Arg
Val Ser Asn Gln Asn Arg Phe Cys Arg Leu 200 205 210 Glu Thr Gln Arg
Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro Ser 215 220 225 Arg Gly Arg
Ser Pro Gln Asn Ser Ala Phe 230 235 71 234 PRT Homo sapiens 71 Leu
Leu Ser Lys Val Arg Thr Gln Leu Cys Pro Thr Pro Cys Thr 1 5 10 15
Cys Pro Trp Pro Pro Pro Arg Cys Pro Leu Gly Val Pro Leu Val 20 25
30 Leu Asp Gly Cys Gly Cys Cys Arg Val Cys Ala Arg Arg Leu Gly 35
40 45 Glu Pro Cys Asp Gln Leu His Val Cys Asp Ala Ser Gln Gly Leu
50 55 60 Val Cys Gln Pro Gly Ala Gly Pro Gly Gly Arg Gly Ala Leu
Cys 65 70 75 Leu Leu Ala Glu Asp Asp Ser Ser Cys Glu Val Asn Gly
Arg Leu 80 85 90 Tyr Arg Glu Gly Glu Thr Phe Gln Pro His Cys Ser
Ile Arg Cys 95 100 105 Arg Cys Glu Asp Gly Gly Phe Thr Cys Val Pro
Leu Cys Ser Glu 110 115 120 Asp Val Arg Leu Pro Ser Trp Asp Cys Pro
His Pro Arg Arg Val 125 130 135 Glu Val Leu Gly Lys Cys Cys Pro Glu
Trp Val Cys Gly Gln Gly 140 145 150 Gly Gly Leu Gly Thr Gln Pro Leu
Pro Ala Gln Gly Pro Gln Phe 155 160 165 Ser Gly Leu Val Ser Ser Leu
Pro Pro Gly Val Pro Cys Pro Glu 170 175 180 Trp Ser Thr Ala Trp Gly
Pro Cys Ser Thr Thr Cys Gly Leu Gly 185 190 195 Met Ala Thr Arg Val
Ser Asn Gln Asn Arg Phe Cys Arg Leu Glu 200 205 210 Thr Gln Arg Arg
Leu Cys Leu Ser Arg Pro Cys Pro Pro Ser Arg 215 220 225 Gly Arg Ser
Pro Gln Asn Ser Ala Phe 230 234 72 233 PRT Homo sapiens 72 Leu Ser
Lys Val Arg Thr Gln Leu Cys Pro Thr Pro Cys Thr Cys 1 5 10 15 Pro
Trp Pro Pro Pro Arg Cys Pro Leu Gly Val Pro Leu Val Leu 20 25 30
Asp Gly Cys Gly Cys Cys Arg Val Cys Ala Arg Arg Leu Gly Glu 35 40
45 Pro Cys Asp Gln Leu His Val Cys Asp Ala Ser Gln Gly Leu Val 50
55 60 Cys Gln Pro Gly Ala Gly Pro Gly Gly Arg Gly Ala Leu Cys Leu
65 70 75 Leu Ala Glu Asp Asp Ser Ser Cys Glu Val Asn Gly Arg Leu
Tyr 80 85 90 Arg Glu Gly Glu Thr Phe Gln Pro His Cys Ser Ile Arg
Cys Arg 95 100 105 Cys Glu Asp Gly Gly Phe Thr Cys Val Pro Leu Cys
Ser Glu Asp 110 115 120 Val Arg Leu Pro Ser Trp Asp Cys Pro His Pro
Arg Arg Val Glu 125 130 135 Val Leu Gly Lys Cys Cys Pro Glu Trp Val
Cys Gly Gln Gly Gly 140 145 150 Gly Leu Gly Thr Gln Pro Leu Pro Ala
Gln Gly Pro Gln Phe Ser 155 160 165 Gly Leu Val Ser Ser Leu Pro Pro
Gly Val Pro Cys Pro Glu Trp 170 175 180 Ser Thr Ala Trp Gly Pro Cys
Ser Thr Thr Cys Gly Leu Gly Met 185 190 195 Ala Thr Arg Val Ser Asn
Gln Asn Arg Phe Cys Arg Leu Glu Thr 200 205 210 Gln Arg Arg Leu Cys
Leu Ser Arg Pro Cys Pro Pro Ser Arg Gly 215 220 225 Arg Ser Pro Gln
Asn Ser Ala Phe 230 233 73 232 PRT Homo sapiens 73 Ser Lys Val Arg
Thr Gln Leu Cys Pro Thr Pro Cys Thr Cys Pro 1 5 10 15 Trp Pro Pro
Pro Arg Cys Pro Leu Gly Val Pro Leu Val Leu Asp 20 25 30 Gly Cys
Gly Cys Cys Arg Val Cys Ala Arg Arg Leu Gly Glu Pro 35 40 45 Cys
Asp Gln Leu His Val Cys Asp Ala Ser Gln Gly Leu Val Cys 50 55 60
Gln Pro Gly Ala Gly Pro Gly Gly Arg Gly Ala Leu Cys Leu Leu 65 70
75 Ala Glu Asp Asp Ser Ser Cys Glu Val Asn Gly Arg Leu Tyr Arg 80
85 90 Glu Gly Glu Thr Phe Gln Pro His Cys Ser Ile Arg Cys Arg Cys
95 100 105 Glu Asp Gly Gly Phe Thr Cys Val Pro Leu Cys Ser Glu Asp
Val 110 115 120 Arg Leu Pro Ser Trp Asp Cys Pro His Pro Arg Arg Val
Glu Val 125 130 135 Leu Gly Lys Cys Cys Pro Glu Trp Val Cys Gly Gln
Gly Gly Gly 140 145 150 Leu Gly Thr Gln Pro Leu Pro Ala Gln Gly Pro
Gln Phe Ser Gly 155 160 165 Leu Val Ser Ser Leu Pro Pro Gly Val Pro
Cys Pro Glu Trp Ser 170 175 180 Thr Ala Trp Gly Pro Cys Ser Thr Thr
Cys Gly Leu Gly Met Ala 185 190 195 Thr Arg Val Ser Asn Gln Asn Arg
Phe Cys Arg Leu Glu Thr Gln 200 205 210 Arg Arg Leu Cys Leu Ser Arg
Pro Cys Pro Pro Ser Arg Gly Arg 215 220 225 Ser Pro Gln Asn Ser Ala
Phe 230 232 74 231 PRT Homo sapiens 74 Lys Val Arg Thr Gln Leu Cys
Pro Thr Pro Cys Thr Cys Pro Trp 1 5 10 15 Pro Pro Pro Arg Cys Pro
Leu Gly Val Pro Leu Val Leu Asp Gly 20 25 30 Cys Gly Cys Cys Arg
Val Cys Ala Arg Arg Leu Gly Glu Pro Cys 35 40 45 Asp Gln Leu His
Val Cys Asp Ala Ser Gln Gly Leu Val Cys Gln 50 55 60 Pro Gly Ala
Gly Pro Gly Gly Arg Gly Ala Leu Cys Leu Leu Ala 65 70 75 Glu Asp
Asp Ser Ser Cys Glu Val Asn Gly Arg Leu Tyr Arg Glu 80 85 90 Gly
Glu Thr Phe Gln Pro His Cys Ser Ile Arg Cys Arg Cys Glu 95 100 105
Asp Gly Gly Phe Thr Cys Val Pro Leu Cys Ser Glu Asp Val Arg 110 115
120 Leu Pro Ser Trp Asp Cys Pro His Pro Arg Arg Val Glu Val Leu 125
130 135 Gly Lys Cys Cys Pro Glu Trp Val Cys Gly Gln Gly Gly Gly Leu
140 145 150 Gly Thr Gln Pro Leu Pro Ala Gln Gly Pro Gln Phe Ser Gly
Leu 155 160 165 Val Ser Ser Leu Pro Pro Gly Val Pro Cys Pro Glu Trp
Ser Thr 170 175 180 Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly Leu Gly
Met Ala Thr 185 190 195 Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu
Glu Thr Gln Arg 200 205 210 Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro
Ser Arg Gly Arg Ser 215 220 225 Pro Gln Asn Ser Ala Phe 230 231 75
230 PRT Homo sapiens 75 Val Arg Thr Gln Leu Cys Pro Thr Pro Cys Thr
Cys Pro Trp Pro 1 5 10 15 Pro Pro Arg Cys Pro Leu Gly Val Pro Leu
Val Leu Asp Gly Cys 20 25 30 Gly Cys Cys Arg Val Cys Ala Arg Arg
Leu Gly Glu Pro Cys Asp 35 40 45 Gln Leu His Val Cys Asp Ala Ser
Gln Gly Leu Val Cys Gln Pro 50 55 60 Gly Ala Gly Pro Gly Gly Arg
Gly Ala Leu Cys Leu Leu Ala Glu 65 70 75 Asp Asp Ser Ser Cys Glu
Val Asn Gly Arg Leu Tyr Arg Glu Gly 80 85 90 Glu Thr Phe Gln Pro
His Cys Ser Ile Arg Cys Arg Cys Glu Asp 95 100 105 Gly Gly Phe Thr
Cys Val Pro Leu Cys Ser Glu Asp Val Arg Leu 110 115 120 Pro Ser Trp
Asp Cys Pro His Pro Arg Arg Val Glu Val Leu Gly 125 130 135 Lys Cys
Cys Pro Glu Trp Val Cys Gly Gln Gly Gly Gly Leu Gly 140 145 150 Thr
Gln Pro Leu Pro Ala Gln Gly Pro Gln Phe Ser Gly Leu Val 155 160 165
Ser Ser Leu Pro Pro Gly Val Pro Cys Pro Glu Trp Ser Thr Ala 170 175
180 Trp Gly Pro Cys Ser Thr Thr Cys Gly Leu Gly Met Ala Thr Arg 185
190 195 Val Ser Asn Gln Asn Arg Phe Cys Arg Leu Glu Thr Gln Arg Arg
200 205 210 Leu Cys Leu Ser Arg Pro Cys Pro Pro Ser Arg Gly Arg Ser
Pro 215 220 225 Gln Asn Ser Ala Phe 230 76 229 PRT Homo sapiens 76
Arg Thr Gln Leu Cys Pro Thr Pro Cys Thr Cys Pro Trp Pro Pro 1 5 10
15 Pro Arg Cys Pro Leu Gly Val Pro Leu Val Leu Asp Gly Cys Gly 20
25 30 Cys Cys Arg Val Cys Ala Arg Arg Leu Gly Glu Pro Cys Asp Gln
35 40 45 Leu His Val Cys Asp Ala Ser Gln Gly Leu Val Cys Gln Pro
Gly 50 55 60 Ala Gly Pro Gly Gly Arg Gly Ala Leu Cys Leu Leu Ala
Glu Asp 65 70 75 Asp Ser Ser Cys Glu Val Asn Gly Arg Leu Tyr Arg
Glu Gly Glu 80 85 90 Thr Phe Gln Pro His Cys Ser Ile Arg Cys Arg
Cys Glu Asp Gly 95 100 105 Gly Phe Thr Cys Val Pro Leu Cys Ser Glu
Asp Val Arg Leu Pro 110 115 120 Ser Trp Asp Cys Pro His Pro Arg Arg
Val Glu Val Leu Gly Lys 125 130 135 Cys Cys Pro Glu Trp Val Cys Gly
Gln Gly Gly Gly Leu Gly Thr 140 145 150 Gln Pro Leu Pro Ala Gln Gly
Pro Gln Phe Ser Gly Leu Val Ser 155 160 165 Ser Leu Pro Pro Gly Val
Pro Cys Pro Glu Trp Ser Thr Ala Trp 170 175 180 Gly Pro Cys Ser Thr
Thr Cys Gly Leu Gly Met Ala Thr Arg Val 185 190 195 Ser Asn Gln Asn
Arg Phe Cys Arg Leu Glu Thr Gln Arg Arg Leu 200 205 210 Cys Leu Ser
Arg Pro Cys Pro Pro Ser Arg Gly Arg Ser Pro Gln 215 220 225 Asn Ser
Ala Phe 229 77 228 PRT Homo sapiens 77 Thr Gln Leu Cys Pro Thr Pro
Cys Thr Cys Pro Trp Pro Pro Pro 1 5 10 15 Arg Cys Pro Leu Gly Val
Pro Leu Val Leu Asp Gly Cys Gly Cys 20 25 30 Cys Arg Val Cys Ala
Arg Arg Leu Gly Glu Pro Cys Asp Gln Leu 35 40 45 His Val Cys Asp
Ala Ser Gln Gly Leu Val Cys Gln Pro Gly Ala 50 55 60 Gly Pro Gly
Gly Arg Gly Ala Leu Cys Leu Leu Ala Glu Asp Asp 65 70 75 Ser Ser
Cys Glu Val Asn Gly Arg Leu Tyr Arg Glu Gly Glu Thr 80 85 90 Phe
Gln Pro His Cys Ser Ile Arg Cys Arg Cys Glu Asp Gly Gly 95 100 105
Phe Thr Cys Val Pro Leu Cys Ser Glu Asp Val Arg Leu Pro Ser 110 115
120 Trp Asp Cys Pro His Pro Arg Arg Val Glu Val Leu Gly Lys Cys 125
130 135 Cys Pro Glu Trp Val Cys Gly Gln Gly Gly Gly Leu Gly Thr Gln
140 145 150 Pro Leu Pro Ala Gln Gly Pro Gln Phe Ser Gly Leu Val Ser
Ser 155 160 165 Leu Pro Pro Gly Val Pro Cys Pro Glu Trp Ser Thr Ala
Trp Gly 170 175 180 Pro Cys Ser Thr Thr Cys Gly Leu Gly Met Ala Thr
Arg Val Ser 185 190 195 Asn Gln Asn Arg Phe Cys Arg Leu Glu Thr Gln
Arg Arg Leu Cys 200 205 210 Leu Ser Arg Pro Cys Pro Pro Ser Arg Gly
Arg Ser Pro Gln Asn 215 220 225 Ser Ala Phe 228 78 250 PRT Homo
sapiens 78 Arg Gly Asn Pro Leu Ile His Leu Leu Ala Ile Ser Phe Leu
Cys 1 5 10 15 Ile Leu Ser Met Val Tyr Ser Gln Leu Cys Pro Ala Pro
Cys Ala 20 25 30 Cys Pro Trp Thr Pro Pro Gln Cys Pro Pro Gly Val
Pro Leu Val 35 40 45 Leu Asp Gly Cys Gly Cys Cys Arg Val Cys Ala
Arg Arg Leu Gly 50 55 60 Glu Ser Cys Asp His Leu His Val Cys Asp
Pro Ser Gln Gly Leu 65 70 75 Val Cys Gln Pro Gly Ala Gly Pro Ser
Gly Arg Gly Ala Val Cys 80 85 90 Leu Phe Glu Glu Asp Asp Gly Ser
Cys Glu Val Asn Gly Arg Arg 95 100 105 Tyr Leu Asp Gly Glu Thr Phe
Lys Pro Asn Cys Arg Val Leu Cys 110 115 120 Arg Cys Asp Asp Gly Gly
Phe Thr Cys Leu Pro Leu Cys Ser Glu 125 130 135 Asp Val Arg Leu Pro
Ser Trp Asp Cys Pro Arg Pro Arg Arg Ile 140 145 150 Gln Val Pro Gly
Arg Cys Cys Pro Glu Trp Val Cys Asp Gln Ala 155 160 165 Val Met Gln
Pro Ala Ile Gln Pro Ser Ser Ala Gln Gly His Gln 170 175 180 Leu Ser
Ala Leu Val Thr Pro Ala Ser Ala Asp Gly Pro Cys Pro 185 190 195 Asn
Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly Leu 200 205 210
Gly Ile Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Gln Leu 215 220
225 Glu Ile Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Leu Ala Ser 230
235 240 Arg Ser His Gly Ser Trp Asn Ser Ala Phe 245 250 79 249 PRT
Homo sapiens 79 Gly Asn Pro Leu Ile His Leu Leu Ala Ile Ser Phe Leu
Cys Ile 1 5 10 15 Leu Ser Met Val Tyr Ser Gln Leu Cys Pro Ala Pro
Cys Ala Cys 20 25 30 Pro Trp Thr Pro Pro Gln Cys Pro Pro Gly Val
Pro Leu Val Leu 35 40 45 Asp Gly Cys Gly Cys Cys Arg Val Cys Ala
Arg Arg Leu Gly Glu 50 55 60 Ser Cys Asp His Leu His Val Cys Asp
Pro Ser Gln Gly Leu Val
65 70 75 Cys Gln Pro Gly Ala Gly Pro Ser Gly Arg Gly Ala Val Cys
Leu 80 85 90 Phe Glu Glu Asp Asp Gly Ser Cys Glu Val Asn Gly Arg
Arg Tyr 95 100 105 Leu Asp Gly Glu Thr Phe Lys Pro Asn Cys Arg Val
Leu Cys Arg 110 115 120 Cys Asp Asp Gly Gly Phe Thr Cys Leu Pro Leu
Cys Ser Glu Asp 125 130 135 Val Arg Leu Pro Ser Trp Asp Cys Pro Arg
Pro Arg Arg Ile Gln 140 145 150 Val Pro Gly Arg Cys Cys Pro Glu Trp
Val Cys Asp Gln Ala Val 155 160 165 Met Gln Pro Ala Ile Gln Pro Ser
Ser Ala Gln Gly His Gln Leu 170 175 180 Ser Ala Leu Val Thr Pro Ala
Ser Ala Asp Gly Pro Cys Pro Asn 185 190 195 Trp Ser Thr Ala Trp Gly
Pro Cys Ser Thr Thr Cys Gly Leu Gly 200 205 210 Ile Ala Thr Arg Val
Ser Asn Gln Asn Arg Phe Cys Gln Leu Glu 215 220 225 Ile Gln Arg Arg
Leu Cys Leu Ser Arg Pro Cys Leu Ala Ser Arg 230 235 240 Ser His Gly
Ser Trp Asn Ser Ala Phe 245 249 80 248 PRT Homo sapiens 80 Asn Pro
Leu Ile His Leu Leu Ala Ile Ser Phe Leu Cys Ile Leu 1 5 10 15 Ser
Met Val Tyr Ser Gln Leu Cys Pro Ala Pro Cys Ala Cys Pro 20 25 30
Trp Thr Pro Pro Gln Cys Pro Pro Gly Val Pro Leu Val Leu Asp 35 40
45 Gly Cys Gly Cys Cys Arg Val Cys Ala Arg Arg Leu Gly Glu Ser 50
55 60 Cys Asp His Leu His Val Cys Asp Pro Ser Gln Gly Leu Val Cys
65 70 75 Gln Pro Gly Ala Gly Pro Ser Gly Arg Gly Ala Val Cys Leu
Phe 80 85 90 Glu Glu Asp Asp Gly Ser Cys Glu Val Asn Gly Arg Arg
Tyr Leu 95 100 105 Asp Gly Glu Thr Phe Lys Pro Asn Cys Arg Val Leu
Cys Arg Cys 110 115 120 Asp Asp Gly Gly Phe Thr Cys Leu Pro Leu Cys
Ser Glu Asp Val 125 130 135 Arg Leu Pro Ser Trp Asp Cys Pro Arg Pro
Arg Arg Ile Gln Val 140 145 150 Pro Gly Arg Cys Cys Pro Glu Trp Val
Cys Asp Gln Ala Val Met 155 160 165 Gln Pro Ala Ile Gln Pro Ser Ser
Ala Gln Gly His Gln Leu Ser 170 175 180 Ala Leu Val Thr Pro Ala Ser
Ala Asp Gly Pro Cys Pro Asn Trp 185 190 195 Ser Thr Ala Trp Gly Pro
Cys Ser Thr Thr Cys Gly Leu Gly Ile 200 205 210 Ala Thr Arg Val Ser
Asn Gln Asn Arg Phe Cys Gln Leu Glu Ile 215 220 225 Gln Arg Arg Leu
Cys Leu Ser Arg Pro Cys Leu Ala Ser Arg Ser 230 235 240 His Gly Ser
Trp Asn Ser Ala Phe 245 248 81 247 PRT Homo sapiens 81 Pro Leu Ile
His Leu Leu Ala Ile Ser Phe Leu Cys Ile Leu Ser 1 5 10 15 Met Val
Tyr Ser Gln Leu Cys Pro Ala Pro Cys Ala Cys Pro Trp 20 25 30 Thr
Pro Pro Gln Cys Pro Pro Gly Val Pro Leu Val Leu Asp Gly 35 40 45
Cys Gly Cys Cys Arg Val Cys Ala Arg Arg Leu Gly Glu Ser Cys 50 55
60 Asp His Leu His Val Cys Asp Pro Ser Gln Gly Leu Val Cys Gln 65
70 75 Pro Gly Ala Gly Pro Ser Gly Arg Gly Ala Val Cys Leu Phe Glu
80 85 90 Glu Asp Asp Gly Ser Cys Glu Val Asn Gly Arg Arg Tyr Leu
Asp 95 100 105 Gly Glu Thr Phe Lys Pro Asn Cys Arg Val Leu Cys Arg
Cys Asp 110 115 120 Asp Gly Gly Phe Thr Cys Leu Pro Leu Cys Ser Glu
Asp Val Arg 125 130 135 Leu Pro Ser Trp Asp Cys Pro Arg Pro Arg Arg
Ile Gln Val Pro 140 145 150 Gly Arg Cys Cys Pro Glu Trp Val Cys Asp
Gln Ala Val Met Gln 155 160 165 Pro Ala Ile Gln Pro Ser Ser Ala Gln
Gly His Gln Leu Ser Ala 170 175 180 Leu Val Thr Pro Ala Ser Ala Asp
Gly Pro Cys Pro Asn Trp Ser 185 190 195 Thr Ala Trp Gly Pro Cys Ser
Thr Thr Cys Gly Leu Gly Ile Ala 200 205 210 Thr Arg Val Ser Asn Gln
Asn Arg Phe Cys Gln Leu Glu Ile Gln 215 220 225 Arg Arg Leu Cys Leu
Ser Arg Pro Cys Leu Ala Ser Arg Ser His 230 235 240 Gly Ser Trp Asn
Ser Ala Phe 245 247 82 246 PRT Homo sapiens 82 Leu Ile His Leu Leu
Ala Ile Ser Phe Leu Cys Ile Leu Ser Met 1 5 10 15 Val Tyr Ser Gln
Leu Cys Pro Ala Pro Cys Ala Cys Pro Trp Thr 20 25 30 Pro Pro Gln
Cys Pro Pro Gly Val Pro Leu Val Leu Asp Gly Cys 35 40 45 Gly Cys
Cys Arg Val Cys Ala Arg Arg Leu Gly Glu Ser Cys Asp 50 55 60 His
Leu His Val Cys Asp Pro Ser Gln Gly Leu Val Cys Gln Pro 65 70 75
Gly Ala Gly Pro Ser Gly Arg Gly Ala Val Cys Leu Phe Glu Glu 80 85
90 Asp Asp Gly Ser Cys Glu Val Asn Gly Arg Arg Tyr Leu Asp Gly 95
100 105 Glu Thr Phe Lys Pro Asn Cys Arg Val Leu Cys Arg Cys Asp Asp
110 115 120 Gly Gly Phe Thr Cys Leu Pro Leu Cys Ser Glu Asp Val Arg
Leu 125 130 135 Pro Ser Trp Asp Cys Pro Arg Pro Arg Arg Ile Gln Val
Pro Gly 140 145 150 Arg Cys Cys Pro Glu Trp Val Cys Asp Gln Ala Val
Met Gln Pro 155 160 165 Ala Ile Gln Pro Ser Ser Ala Gln Gly His Gln
Leu Ser Ala Leu 170 175 180 Val Thr Pro Ala Ser Ala Asp Gly Pro Cys
Pro Asn Trp Ser Thr 185 190 195 Ala Trp Gly Pro Cys Ser Thr Thr Cys
Gly Leu Gly Ile Ala Thr 200 205 210 Arg Val Ser Asn Gln Asn Arg Phe
Cys Gln Leu Glu Ile Gln Arg 215 220 225 Arg Leu Cys Leu Ser Arg Pro
Cys Leu Ala Ser Arg Ser His Gly 230 235 240 Ser Trp Asn Ser Ala Phe
245 246 83 245 PRT Homo sapiens 83 Ile His Leu Leu Ala Ile Ser Phe
Leu Cys Ile Leu Ser Met Val 1 5 10 15 Tyr Ser Gln Leu Cys Pro Ala
Pro Cys Ala Cys Pro Trp Thr Pro 20 25 30 Pro Gln Cys Pro Pro Gly
Val Pro Leu Val Leu Asp Gly Cys Gly 35 40 45 Cys Cys Arg Val Cys
Ala Arg Arg Leu Gly Glu Ser Cys Asp His 50 55 60 Leu His Val Cys
Asp Pro Ser Gln Gly Leu Val Cys Gln Pro Gly 65 70 75 Ala Gly Pro
Ser Gly Arg Gly Ala Val Cys Leu Phe Glu Glu Asp 80 85 90 Asp Gly
Ser Cys Glu Val Asn Gly Arg Arg Tyr Leu Asp Gly Glu 95 100 105 Thr
Phe Lys Pro Asn Cys Arg Val Leu Cys Arg Cys Asp Asp Gly 110 115 120
Gly Phe Thr Cys Leu Pro Leu Cys Ser Glu Asp Val Arg Leu Pro 125 130
135 Ser Trp Asp Cys Pro Arg Pro Arg Arg Ile Gln Val Pro Gly Arg 140
145 150 Cys Cys Pro Glu Trp Val Cys Asp Gln Ala Val Met Gln Pro Ala
155 160 165 Ile Gln Pro Ser Ser Ala Gln Gly His Gln Leu Ser Ala Leu
Val 170 175 180 Thr Pro Ala Ser Ala Asp Gly Pro Cys Pro Asn Trp Ser
Thr Ala 185 190 195 Trp Gly Pro Cys Ser Thr Thr Cys Gly Leu Gly Ile
Ala Thr Arg 200 205 210 Val Ser Asn Gln Asn Arg Phe Cys Gln Leu Glu
Ile Gln Arg Arg 215 220 225 Leu Cys Leu Ser Arg Pro Cys Leu Ala Ser
Arg Ser His Gly Ser 230 235 240 Trp Asn Ser Ala Phe 245 84 244 PRT
Homo sapiens 84 His Leu Leu Ala Ile Ser Phe Leu Cys Ile Leu Ser Met
Val Tyr 1 5 10 15 Ser Gln Leu Cys Pro Ala Pro Cys Ala Cys Pro Trp
Thr Pro Pro 20 25 30 Gln Cys Pro Pro Gly Val Pro Leu Val Leu Asp
Gly Cys Gly Cys 35 40 45 Cys Arg Val Cys Ala Arg Arg Leu Gly Glu
Ser Cys Asp His Leu 50 55 60 His Val Cys Asp Pro Ser Gln Gly Leu
Val Cys Gln Pro Gly Ala 65 70 75 Gly Pro Ser Gly Arg Gly Ala Val
Cys Leu Phe Glu Glu Asp Asp 80 85 90 Gly Ser Cys Glu Val Asn Gly
Arg Arg Tyr Leu Asp Gly Glu Thr 95 100 105 Phe Lys Pro Asn Cys Arg
Val Leu Cys Arg Cys Asp Asp Gly Gly 110 115 120 Phe Thr Cys Leu Pro
Leu Cys Ser Glu Asp Val Arg Leu Pro Ser 125 130 135 Trp Asp Cys Pro
Arg Pro Arg Arg Ile Gln Val Pro Gly Arg Cys 140 145 150 Cys Pro Glu
Trp Val Cys Asp Gln Ala Val Met Gln Pro Ala Ile 155 160 165 Gln Pro
Ser Ser Ala Gln Gly His Gln Leu Ser Ala Leu Val Thr 170 175 180 Pro
Ala Ser Ala Asp Gly Pro Cys Pro Asn Trp Ser Thr Ala Trp 185 190 195
Gly Pro Cys Ser Thr Thr Cys Gly Leu Gly Ile Ala Thr Arg Val 200 205
210 Ser Asn Gln Asn Arg Phe Cys Gln Leu Glu Ile Gln Arg Arg Leu 215
220 225 Cys Leu Ser Arg Pro Cys Leu Ala Ser Arg Ser His Gly Ser Trp
230 235 240 Asn Ser Ala Phe 244 85 243 PRT Homo sapiens 85 Leu Leu
Ala Ile Ser Phe Leu Cys Ile Leu Ser Met Val Tyr Ser 1 5 10 15 Gln
Leu Cys Pro Ala Pro Cys Ala Cys Pro Trp Thr Pro Pro Gln 20 25 30
Cys Pro Pro Gly Val Pro Leu Val Leu Asp Gly Cys Gly Cys Cys 35 40
45 Arg Val Cys Ala Arg Arg Leu Gly Glu Ser Cys Asp His Leu His 50
55 60 Val Cys Asp Pro Ser Gln Gly Leu Val Cys Gln Pro Gly Ala Gly
65 70 75 Pro Ser Gly Arg Gly Ala Val Cys Leu Phe Glu Glu Asp Asp
Gly 80 85 90 Ser Cys Glu Val Asn Gly Arg Arg Tyr Leu Asp Gly Glu
Thr Phe 95 100 105 Lys Pro Asn Cys Arg Val Leu Cys Arg Cys Asp Asp
Gly Gly Phe 110 115 120 Thr Cys Leu Pro Leu Cys Ser Glu Asp Val Arg
Leu Pro Ser Trp 125 130 135 Asp Cys Pro Arg Pro Arg Arg Ile Gln Val
Pro Gly Arg Cys Cys 140 145 150 Pro Glu Trp Val Cys Asp Gln Ala Val
Met Gln Pro Ala Ile Gln 155 160 165 Pro Ser Ser Ala Gln Gly His Gln
Leu Ser Ala Leu Val Thr Pro 170 175 180 Ala Ser Ala Asp Gly Pro Cys
Pro Asn Trp Ser Thr Ala Trp Gly 185 190 195 Pro Cys Ser Thr Thr Cys
Gly Leu Gly Ile Ala Thr Arg Val Ser 200 205 210 Asn Gln Asn Arg Phe
Cys Gln Leu Glu Ile Gln Arg Arg Leu Cys 215 220 225 Leu Ser Arg Pro
Cys Leu Ala Ser Arg Ser His Gly Ser Trp Asn 230 235 240 Ser Ala Phe
243 86 242 PRT Homo sapiens 86 Leu Ala Ile Ser Phe Leu Cys Ile Leu
Ser Met Val Tyr Ser Gln 1 5 10 15 Leu Cys Pro Ala Pro Cys Ala Cys
Pro Trp Thr Pro Pro Gln Cys 20 25 30 Pro Pro Gly Val Pro Leu Val
Leu Asp Gly Cys Gly Cys Cys Arg 35 40 45 Val Cys Ala Arg Arg Leu
Gly Glu Ser Cys Asp His Leu His Val 50 55 60 Cys Asp Pro Ser Gln
Gly Leu Val Cys Gln Pro Gly Ala Gly Pro 65 70 75 Ser Gly Arg Gly
Ala Val Cys Leu Phe Glu Glu Asp Asp Gly Ser 80 85 90 Cys Glu Val
Asn Gly Arg Arg Tyr Leu Asp Gly Glu Thr Phe Lys 95 100 105 Pro Asn
Cys Arg Val Leu Cys Arg Cys Asp Asp Gly Gly Phe Thr 110 115 120 Cys
Leu Pro Leu Cys Ser Glu Asp Val Arg Leu Pro Ser Trp Asp 125 130 135
Cys Pro Arg Pro Arg Arg Ile Gln Val Pro Gly Arg Cys Cys Pro 140 145
150 Glu Trp Val Cys Asp Gln Ala Val Met Gln Pro Ala Ile Gln Pro 155
160 165 Ser Ser Ala Gln Gly His Gln Leu Ser Ala Leu Val Thr Pro Ala
170 175 180 Ser Ala Asp Gly Pro Cys Pro Asn Trp Ser Thr Ala Trp Gly
Pro 185 190 195 Cys Ser Thr Thr Cys Gly Leu Gly Ile Ala Thr Arg Val
Ser Asn 200 205 210 Gln Asn Arg Phe Cys Gln Leu Glu Ile Gln Arg Arg
Leu Cys Leu 215 220 225 Ser Arg Pro Cys Leu Ala Ser Arg Ser His Gly
Ser Trp Asn Ser 230 235 240 Ala Phe 242 87 241 PRT Homo sapiens 87
Ala Ile Ser Phe Leu Cys Ile Leu Ser Met Val Tyr Ser Gln Leu 1 5 10
15 Cys Pro Ala Pro Cys Ala Cys Pro Trp Thr Pro Pro Gln Cys Pro 20
25 30 Pro Gly Val Pro Leu Val Leu Asp Gly Cys Gly Cys Cys Arg Val
35 40 45 Cys Ala Arg Arg Leu Gly Glu Ser Cys Asp His Leu His Val
Cys 50 55 60 Asp Pro Ser Gln Gly Leu Val Cys Gln Pro Gly Ala Gly
Pro Ser 65 70 75 Gly Arg Gly Ala Val Cys Leu Phe Glu Glu Asp Asp
Gly Ser Cys 80 85 90 Glu Val Asn Gly Arg Arg Tyr Leu Asp Gly Glu
Thr Phe Lys Pro 95 100 105 Asn Cys Arg Val Leu Cys Arg Cys Asp Asp
Gly Gly Phe Thr Cys 110 115 120 Leu Pro Leu Cys Ser Glu Asp Val Arg
Leu Pro Ser Trp Asp Cys 125 130 135 Pro Arg Pro Arg Arg Ile Gln Val
Pro Gly Arg Cys Cys Pro Glu 140 145 150 Trp Val Cys Asp Gln Ala Val
Met Gln Pro Ala Ile Gln Pro Ser 155 160 165 Ser Ala Gln Gly His Gln
Leu Ser Ala Leu Val Thr Pro Ala Ser 170 175 180 Ala Asp Gly Pro Cys
Pro Asn Trp Ser Thr Ala Trp Gly Pro Cys 185 190 195 Ser Thr Thr Cys
Gly Leu Gly Ile Ala Thr Arg Val Ser Asn Gln 200 205 210 Asn Arg Phe
Cys Gln Leu Glu Ile Gln Arg Arg Leu Cys Leu Ser 215 220 225 Arg Pro
Cys Leu Ala Ser Arg Ser His Gly Ser Trp Asn Ser Ala 230 235 240 Phe
241 88 240 PRT Homo sapiens 88 Ile Ser Phe Leu Cys Ile Leu Ser Met
Val Tyr Ser Gln Leu Cys 1 5 10 15 Pro Ala Pro Cys Ala Cys Pro Trp
Thr Pro Pro Gln Cys Pro Pro 20 25 30 Gly Val Pro Leu Val Leu Asp
Gly Cys Gly Cys Cys Arg Val Cys 35 40 45 Ala Arg Arg Leu Gly Glu
Ser Cys Asp His Leu His Val Cys Asp 50 55 60 Pro Ser Gln Gly Leu
Val Cys Gln Pro Gly Ala Gly Pro Ser Gly 65 70 75 Arg Gly Ala Val
Cys Leu Phe Glu Glu Asp Asp Gly Ser Cys Glu 80 85 90 Val Asn Gly
Arg Arg Tyr Leu Asp Gly Glu Thr Phe Lys Pro Asn 95 100 105 Cys Arg
Val Leu Cys Arg Cys Asp Asp Gly Gly Phe Thr Cys Leu 110 115 120 Pro
Leu Cys Ser Glu Asp Val Arg Leu Pro Ser Trp Asp Cys Pro 125 130
135 Arg Pro Arg Arg Ile Gln Val Pro Gly Arg Cys Cys Pro Glu Trp 140
145 150 Val Cys Asp Gln Ala Val Met Gln Pro Ala Ile Gln Pro Ser Ser
155 160 165 Ala Gln Gly His Gln Leu Ser Ala Leu Val Thr Pro Ala Ser
Ala 170 175 180 Asp Gly Pro Cys Pro Asn Trp Ser Thr Ala Trp Gly Pro
Cys Ser 185 190 195 Thr Thr Cys Gly Leu Gly Ile Ala Thr Arg Val Ser
Asn Gln Asn 200 205 210 Arg Phe Cys Gln Leu Glu Ile Gln Arg Arg Leu
Cys Leu Ser Arg 215 220 225 Pro Cys Leu Ala Ser Arg Ser His Gly Ser
Trp Asn Ser Ala Phe 230 235 240 89 239 PRT Homo sapiens 89 Ser Phe
Leu Cys Ile Leu Ser Met Val Tyr Ser Gln Leu Cys Pro 1 5 10 15 Ala
Pro Cys Ala Cys Pro Trp Thr Pro Pro Gln Cys Pro Pro Gly 20 25 30
Val Pro Leu Val Leu Asp Gly Cys Gly Cys Cys Arg Val Cys Ala 35 40
45 Arg Arg Leu Gly Glu Ser Cys Asp His Leu His Val Cys Asp Pro 50
55 60 Ser Gln Gly Leu Val Cys Gln Pro Gly Ala Gly Pro Ser Gly Arg
65 70 75 Gly Ala Val Cys Leu Phe Glu Glu Asp Asp Gly Ser Cys Glu
Val 80 85 90 Asn Gly Arg Arg Tyr Leu Asp Gly Glu Thr Phe Lys Pro
Asn Cys 95 100 105 Arg Val Leu Cys Arg Cys Asp Asp Gly Gly Phe Thr
Cys Leu Pro 110 115 120 Leu Cys Ser Glu Asp Val Arg Leu Pro Ser Trp
Asp Cys Pro Arg 125 130 135 Pro Arg Arg Ile Gln Val Pro Gly Arg Cys
Cys Pro Glu Trp Val 140 145 150 Cys Asp Gln Ala Val Met Gln Pro Ala
Ile Gln Pro Ser Ser Ala 155 160 165 Gln Gly His Gln Leu Ser Ala Leu
Val Thr Pro Ala Ser Ala Asp 170 175 180 Gly Pro Cys Pro Asn Trp Ser
Thr Ala Trp Gly Pro Cys Ser Thr 185 190 195 Thr Cys Gly Leu Gly Ile
Ala Thr Arg Val Ser Asn Gln Asn Arg 200 205 210 Phe Cys Gln Leu Glu
Ile Gln Arg Arg Leu Cys Leu Ser Arg Pro 215 220 225 Cys Leu Ala Ser
Arg Ser His Gly Ser Trp Asn Ser Ala Phe 230 235 239 90 238 PRT Homo
sapiens 90 Phe Leu Cys Ile Leu Ser Met Val Tyr Ser Gln Leu Cys Pro
Ala 1 5 10 15 Pro Cys Ala Cys Pro Trp Thr Pro Pro Gln Cys Pro Pro
Gly Val 20 25 30 Pro Leu Val Leu Asp Gly Cys Gly Cys Cys Arg Val
Cys Ala Arg 35 40 45 Arg Leu Gly Glu Ser Cys Asp His Leu His Val
Cys Asp Pro Ser 50 55 60 Gln Gly Leu Val Cys Gln Pro Gly Ala Gly
Pro Ser Gly Arg Gly 65 70 75 Ala Val Cys Leu Phe Glu Glu Asp Asp
Gly Ser Cys Glu Val Asn 80 85 90 Gly Arg Arg Tyr Leu Asp Gly Glu
Thr Phe Lys Pro Asn Cys Arg 95 100 105 Val Leu Cys Arg Cys Asp Asp
Gly Gly Phe Thr Cys Leu Pro Leu 110 115 120 Cys Ser Glu Asp Val Arg
Leu Pro Ser Trp Asp Cys Pro Arg Pro 125 130 135 Arg Arg Ile Gln Val
Pro Gly Arg Cys Cys Pro Glu Trp Val Cys 140 145 150 Asp Gln Ala Val
Met Gln Pro Ala Ile Gln Pro Ser Ser Ala Gln 155 160 165 Gly His Gln
Leu Ser Ala Leu Val Thr Pro Ala Ser Ala Asp Gly 170 175 180 Pro Cys
Pro Asn Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr 185 190 195 Cys
Gly Leu Gly Ile Ala Thr Arg Val Ser Asn Gln Asn Arg Phe 200 205 210
Cys Gln Leu Glu Ile Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys 215 220
225 Leu Ala Ser Arg Ser His Gly Ser Trp Asn Ser Ala Phe 230 235 238
91 237 PRT Homo sapiens 91 Leu Cys Ile Leu Ser Met Val Tyr Ser Gln
Leu Cys Pro Ala Pro 1 5 10 15 Cys Ala Cys Pro Trp Thr Pro Pro Gln
Cys Pro Pro Gly Val Pro 20 25 30 Leu Val Leu Asp Gly Cys Gly Cys
Cys Arg Val Cys Ala Arg Arg 35 40 45 Leu Gly Glu Ser Cys Asp His
Leu His Val Cys Asp Pro Ser Gln 50 55 60 Gly Leu Val Cys Gln Pro
Gly Ala Gly Pro Ser Gly Arg Gly Ala 65 70 75 Val Cys Leu Phe Glu
Glu Asp Asp Gly Ser Cys Glu Val Asn Gly 80 85 90 Arg Arg Tyr Leu
Asp Gly Glu Thr Phe Lys Pro Asn Cys Arg Val 95 100 105 Leu Cys Arg
Cys Asp Asp Gly Gly Phe Thr Cys Leu Pro Leu Cys 110 115 120 Ser Glu
Asp Val Arg Leu Pro Ser Trp Asp Cys Pro Arg Pro Arg 125 130 135 Arg
Ile Gln Val Pro Gly Arg Cys Cys Pro Glu Trp Val Cys Asp 140 145 150
Gln Ala Val Met Gln Pro Ala Ile Gln Pro Ser Ser Ala Gln Gly 155 160
165 His Gln Leu Ser Ala Leu Val Thr Pro Ala Ser Ala Asp Gly Pro 170
175 180 Cys Pro Asn Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys
185 190 195 Gly Leu Gly Ile Ala Thr Arg Val Ser Asn Gln Asn Arg Phe
Cys 200 205 210 Gln Leu Glu Ile Gln Arg Arg Leu Cys Leu Ser Arg Pro
Cys Leu 215 220 225 Ala Ser Arg Ser His Gly Ser Trp Asn Ser Ala Phe
230 235 237 92 236 PRT Homo sapiens 92 Cys Ile Leu Ser Met Val Tyr
Ser Gln Leu Cys Pro Ala Pro Cys 1 5 10 15 Ala Cys Pro Trp Thr Pro
Pro Gln Cys Pro Pro Gly Val Pro Leu 20 25 30 Val Leu Asp Gly Cys
Gly Cys Cys Arg Val Cys Ala Arg Arg Leu 35 40 45 Gly Glu Ser Cys
Asp His Leu His Val Cys Asp Pro Ser Gln Gly 50 55 60 Leu Val Cys
Gln Pro Gly Ala Gly Pro Ser Gly Arg Gly Ala Val 65 70 75 Cys Leu
Phe Glu Glu Asp Asp Gly Ser Cys Glu Val Asn Gly Arg 80 85 90 Arg
Tyr Leu Asp Gly Glu Thr Phe Lys Pro Asn Cys Arg Val Leu 95 100 105
Cys Arg Cys Asp Asp Gly Gly Phe Thr Cys Leu Pro Leu Cys Ser 110 115
120 Glu Asp Val Arg Leu Pro Ser Trp Asp Cys Pro Arg Pro Arg Arg 125
130 135 Ile Gln Val Pro Gly Arg Cys Cys Pro Glu Trp Val Cys Asp Gln
140 145 150 Ala Val Met Gln Pro Ala Ile Gln Pro Ser Ser Ala Gln Gly
His 155 160 165 Gln Leu Ser Ala Leu Val Thr Pro Ala Ser Ala Asp Gly
Pro Cys 170 175 180 Pro Asn Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr
Thr Cys Gly 185 190 195 Leu Gly Ile Ala Thr Arg Val Ser Asn Gln Asn
Arg Phe Cys Gln 200 205 210 Leu Glu Ile Gln Arg Arg Leu Cys Leu Ser
Arg Pro Cys Leu Ala 215 220 225 Ser Arg Ser His Gly Ser Trp Asn Ser
Ala Phe 230 235 236 93 235 PRT Homo sapiens 93 Ile Leu Ser Met Val
Tyr Ser Gln Leu Cys Pro Ala Pro Cys Ala 1 5 10 15 Cys Pro Trp Thr
Pro Pro Gln Cys Pro Pro Gly Val Pro Leu Val 20 25 30 Leu Asp Gly
Cys Gly Cys Cys Arg Val Cys Ala Arg Arg Leu Gly 35 40 45 Glu Ser
Cys Asp His Leu His Val Cys Asp Pro Ser Gln Gly Leu 50 55 60 Val
Cys Gln Pro Gly Ala Gly Pro Ser Gly Arg Gly Ala Val Cys 65 70 75
Leu Phe Glu Glu Asp Asp Gly Ser Cys Glu Val Asn Gly Arg Arg 80 85
90 Tyr Leu Asp Gly Glu Thr Phe Lys Pro Asn Cys Arg Val Leu Cys 95
100 105 Arg Cys Asp Asp Gly Gly Phe Thr Cys Leu Pro Leu Cys Ser Glu
110 115 120 Asp Val Arg Leu Pro Ser Trp Asp Cys Pro Arg Pro Arg Arg
Ile 125 130 135 Gln Val Pro Gly Arg Cys Cys Pro Glu Trp Val Cys Asp
Gln Ala 140 145 150 Val Met Gln Pro Ala Ile Gln Pro Ser Ser Ala Gln
Gly His Gln 155 160 165 Leu Ser Ala Leu Val Thr Pro Ala Ser Ala Asp
Gly Pro Cys Pro 170 175 180 Asn Trp Ser Thr Ala Trp Gly Pro Cys Ser
Thr Thr Cys Gly Leu 185 190 195 Gly Ile Ala Thr Arg Val Ser Asn Gln
Asn Arg Phe Cys Gln Leu 200 205 210 Glu Ile Gln Arg Arg Leu Cys Leu
Ser Arg Pro Cys Leu Ala Ser 215 220 225 Arg Ser His Gly Ser Trp Asn
Ser Ala Phe 230 235 94 234 PRT Homo sapiens 94 Leu Ser Met Val Tyr
Ser Gln Leu Cys Pro Ala Pro Cys Ala Cys 1 5 10 15 Pro Trp Thr Pro
Pro Gln Cys Pro Pro Gly Val Pro Leu Val Leu 20 25 30 Asp Gly Cys
Gly Cys Cys Arg Val Cys Ala Arg Arg Leu Gly Glu 35 40 45 Ser Cys
Asp His Leu His Val Cys Asp Pro Ser Gln Gly Leu Val 50 55 60 Cys
Gln Pro Gly Ala Gly Pro Ser Gly Arg Gly Ala Val Cys Leu 65 70 75
Phe Glu Glu Asp Asp Gly Ser Cys Glu Val Asn Gly Arg Arg Tyr 80 85
90 Leu Asp Gly Glu Thr Phe Lys Pro Asn Cys Arg Val Leu Cys Arg 95
100 105 Cys Asp Asp Gly Gly Phe Thr Cys Leu Pro Leu Cys Ser Glu Asp
110 115 120 Val Arg Leu Pro Ser Trp Asp Cys Pro Arg Pro Arg Arg Ile
Gln 125 130 135 Val Pro Gly Arg Cys Cys Pro Glu Trp Val Cys Asp Gln
Ala Val 140 145 150 Met Gln Pro Ala Ile Gln Pro Ser Ser Ala Gln Gly
His Gln Leu 155 160 165 Ser Ala Leu Val Thr Pro Ala Ser Ala Asp Gly
Pro Cys Pro Asn 170 175 180 Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr
Thr Cys Gly Leu Gly 185 190 195 Ile Ala Thr Arg Val Ser Asn Gln Asn
Arg Phe Cys Gln Leu Glu 200 205 210 Ile Gln Arg Arg Leu Cys Leu Ser
Arg Pro Cys Leu Ala Ser Arg 215 220 225 Ser His Gly Ser Trp Asn Ser
Ala Phe 230 234 95 233 PRT Homo sapiens 95 Ser Met Val Tyr Ser Gln
Leu Cys Pro Ala Pro Cys Ala Cys Pro 1 5 10 15 Trp Thr Pro Pro Gln
Cys Pro Pro Gly Val Pro Leu Val Leu Asp 20 25 30 Gly Cys Gly Cys
Cys Arg Val Cys Ala Arg Arg Leu Gly Glu Ser 35 40 45 Cys Asp His
Leu His Val Cys Asp Pro Ser Gln Gly Leu Val Cys 50 55 60 Gln Pro
Gly Ala Gly Pro Ser Gly Arg Gly Ala Val Cys Leu Phe 65 70 75 Glu
Glu Asp Asp Gly Ser Cys Glu Val Asn Gly Arg Arg Tyr Leu 80 85 90
Asp Gly Glu Thr Phe Lys Pro Asn Cys Arg Val Leu Cys Arg Cys 95 100
105 Asp Asp Gly Gly Phe Thr Cys Leu Pro Leu Cys Ser Glu Asp Val 110
115 120 Arg Leu Pro Ser Trp Asp Cys Pro Arg Pro Arg Arg Ile Gln Val
125 130 135 Pro Gly Arg Cys Cys Pro Glu Trp Val Cys Asp Gln Ala Val
Met 140 145 150 Gln Pro Ala Ile Gln Pro Ser Ser Ala Gln Gly His Gln
Leu Ser 155 160 165 Ala Leu Val Thr Pro Ala Ser Ala Asp Gly Pro Cys
Pro Asn Trp 170 175 180 Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys
Gly Leu Gly Ile 185 190 195 Ala Thr Arg Val Ser Asn Gln Asn Arg Phe
Cys Gln Leu Glu Ile 200 205 210 Gln Arg Arg Leu Cys Leu Ser Arg Pro
Cys Leu Ala Ser Arg Ser 215 220 225 His Gly Ser Trp Asn Ser Ala Phe
230 233 96 232 PRT Homo sapiens 96 Met Val Tyr Ser Gln Leu Cys Pro
Ala Pro Cys Ala Cys Pro Trp 1 5 10 15 Thr Pro Pro Gln Cys Pro Pro
Gly Val Pro Leu Val Leu Asp Gly 20 25 30 Cys Gly Cys Cys Arg Val
Cys Ala Arg Arg Leu Gly Glu Ser Cys 35 40 45 Asp His Leu His Val
Cys Asp Pro Ser Gln Gly Leu Val Cys Gln 50 55 60 Pro Gly Ala Gly
Pro Ser Gly Arg Gly Ala Val Cys Leu Phe Glu 65 70 75 Glu Asp Asp
Gly Ser Cys Glu Val Asn Gly Arg Arg Tyr Leu Asp 80 85 90 Gly Glu
Thr Phe Lys Pro Asn Cys Arg Val Leu Cys Arg Cys Asp 95 100 105 Asp
Gly Gly Phe Thr Cys Leu Pro Leu Cys Ser Glu Asp Val Arg 110 115 120
Leu Pro Ser Trp Asp Cys Pro Arg Pro Arg Arg Ile Gln Val Pro 125 130
135 Gly Arg Cys Cys Pro Glu Trp Val Cys Asp Gln Ala Val Met Gln 140
145 150 Pro Ala Ile Gln Pro Ser Ser Ala Gln Gly His Gln Leu Ser Ala
155 160 165 Leu Val Thr Pro Ala Ser Ala Asp Gly Pro Cys Pro Asn Trp
Ser 170 175 180 Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly Leu Gly
Ile Ala 185 190 195 Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Gln Leu
Glu Ile Gln 200 205 210 Arg Arg Leu Cys Leu Ser Arg Pro Cys Leu Ala
Ser Arg Ser His 215 220 225 Gly Ser Trp Asn Ser Ala Phe 230 232 97
231 PRT Homo sapiens 97 Val Tyr Ser Gln Leu Cys Pro Ala Pro Cys Ala
Cys Pro Trp Thr 1 5 10 15 Pro Pro Gln Cys Pro Pro Gly Val Pro Leu
Val Leu Asp Gly Cys 20 25 30 Gly Cys Cys Arg Val Cys Ala Arg Arg
Leu Gly Glu Ser Cys Asp 35 40 45 His Leu His Val Cys Asp Pro Ser
Gln Gly Leu Val Cys Gln Pro 50 55 60 Gly Ala Gly Pro Ser Gly Arg
Gly Ala Val Cys Leu Phe Glu Glu 65 70 75 Asp Asp Gly Ser Cys Glu
Val Asn Gly Arg Arg Tyr Leu Asp Gly 80 85 90 Glu Thr Phe Lys Pro
Asn Cys Arg Val Leu Cys Arg Cys Asp Asp 95 100 105 Gly Gly Phe Thr
Cys Leu Pro Leu Cys Ser Glu Asp Val Arg Leu 110 115 120 Pro Ser Trp
Asp Cys Pro Arg Pro Arg Arg Ile Gln Val Pro Gly 125 130 135 Arg Cys
Cys Pro Glu Trp Val Cys Asp Gln Ala Val Met Gln Pro 140 145 150 Ala
Ile Gln Pro Ser Ser Ala Gln Gly His Gln Leu Ser Ala Leu 155 160 165
Val Thr Pro Ala Ser Ala Asp Gly Pro Cys Pro Asn Trp Ser Thr 170 175
180 Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly Leu Gly Ile Ala Thr 185
190 195 Arg Val Ser Asn Gln Asn Arg Phe Cys Gln Leu Glu Ile Gln Arg
200 205 210 Arg Leu Cys Leu Ser Arg Pro Cys Leu Ala Ser Arg Ser His
Gly 215 220 225 Ser Trp Asn Ser Ala Phe 230 231 98 230 PRT Homo
sapiens 98 Tyr Ser Gln Leu Cys Pro Ala Pro Cys Ala Cys Pro Trp Thr
Pro 1 5 10 15 Pro Gln Cys Pro Pro Gly Val Pro Leu Val Leu Asp Gly
Cys Gly 20 25 30 Cys Cys Arg Val Cys Ala Arg Arg Leu Gly Glu Ser
Cys Asp His
35 40 45 Leu His Val Cys Asp Pro Ser Gln Gly Leu Val Cys Gln Pro
Gly 50 55 60 Ala Gly Pro Ser Gly Arg Gly Ala Val Cys Leu Phe Glu
Glu Asp 65 70 75 Asp Gly Ser Cys Glu Val Asn Gly Arg Arg Tyr Leu
Asp Gly Glu 80 85 90 Thr Phe Lys Pro Asn Cys Arg Val Leu Cys Arg
Cys Asp Asp Gly 95 100 105 Gly Phe Thr Cys Leu Pro Leu Cys Ser Glu
Asp Val Arg Leu Pro 110 115 120 Ser Trp Asp Cys Pro Arg Pro Arg Arg
Ile Gln Val Pro Gly Arg 125 130 135 Cys Cys Pro Glu Trp Val Cys Asp
Gln Ala Val Met Gln Pro Ala 140 145 150 Ile Gln Pro Ser Ser Ala Gln
Gly His Gln Leu Ser Ala Leu Val 155 160 165 Thr Pro Ala Ser Ala Asp
Gly Pro Cys Pro Asn Trp Ser Thr Ala 170 175 180 Trp Gly Pro Cys Ser
Thr Thr Cys Gly Leu Gly Ile Ala Thr Arg 185 190 195 Val Ser Asn Gln
Asn Arg Phe Cys Gln Leu Glu Ile Gln Arg Arg 200 205 210 Leu Cys Leu
Ser Arg Pro Cys Leu Ala Ser Arg Ser His Gly Ser 215 220 225 Trp Asn
Ser Ala Phe 230 99 229 PRT Homo sapiens 99 Ser Gln Leu Cys Pro Ala
Pro Cys Ala Cys Pro Trp Thr Pro Pro 1 5 10 15 Gln Cys Pro Pro Gly
Val Pro Leu Val Leu Asp Gly Cys Gly Cys 20 25 30 Cys Arg Val Cys
Ala Arg Arg Leu Gly Glu Ser Cys Asp His Leu 35 40 45 His Val Cys
Asp Pro Ser Gln Gly Leu Val Cys Gln Pro Gly Ala 50 55 60 Gly Pro
Ser Gly Arg Gly Ala Val Cys Leu Phe Glu Glu Asp Asp 65 70 75 Gly
Ser Cys Glu Val Asn Gly Arg Arg Tyr Leu Asp Gly Glu Thr 80 85 90
Phe Lys Pro Asn Cys Arg Val Leu Cys Arg Cys Asp Asp Gly Gly 95 100
105 Phe Thr Cys Leu Pro Leu Cys Ser Glu Asp Val Arg Leu Pro Ser 110
115 120 Trp Asp Cys Pro Arg Pro Arg Arg Ile Gln Val Pro Gly Arg Cys
125 130 135 Cys Pro Glu Trp Val Cys Asp Gln Ala Val Met Gln Pro Ala
Ile 140 145 150 Gln Pro Ser Ser Ala Gln Gly His Gln Leu Ser Ala Leu
Val Thr 155 160 165 Pro Ala Ser Ala Asp Gly Pro Cys Pro Asn Trp Ser
Thr Ala Trp 170 175 180 Gly Pro Cys Ser Thr Thr Cys Gly Leu Gly Ile
Ala Thr Arg Val 185 190 195 Ser Asn Gln Asn Arg Phe Cys Gln Leu Glu
Ile Gln Arg Arg Leu 200 205 210 Cys Leu Ser Arg Pro Cys Leu Ala Ser
Arg Ser His Gly Ser Trp 215 220 225 Asn Ser Ala Phe 229 100 22 DNA
Artificial sequence misc_feature 1-22 Sequence is synthesized. 100
ccagccagag gaggccacga ac 22 101 24 DNA Artificial sequence
misc_feature 1-24 Sequence is synthesized. 101 gtacttgggt
cggtaggtgc gtgt 24 102 23 DNA Artificial sequence misc_feature 1-23
Sequence is synthesized. 102 gtggcccatg ctctggcaga ggg 23 103 24
DNA Artificial sequence misc_feature 1-24 Sequence is synthesized.
103 gactggagca aggtcgtcct cgcc 24 104 24 DNA Artificial sequence
misc_feature 1-24 Sequence is synthesized. 104 gcaccaccca
caaggaagcc atcc 24 105 24 DNA Artificial sequence misc_feature 1-24
Sequence is synthesized. 105 gacgaaaggg aagccggcat cacc 24 106 24
DNA Artificial sequence misc_feature 1-24 Sequence is synthesized.
106 gagaaggtcg tgttcgagca aacc 24 107 24 DNA Artificial sequence
misc_feature 1-24 Sequence is synthesized. 107 cttctcgtgt
acttcctgtg cctg 24 108 24 DNA Artificial sequence misc_feature 1-24
Sequence is synthesized. 108 cacgtcagct ggcgttgcca gctc 24 109 23
PRT Artificial sequence misc_feature 1-23 Sequence is synthesized.
109 Gln Pro Glu Glu Ala Thr Asn Phe Thr Leu Ala Gly Cys Val Ser 1 5
10 15 Thr Arg Thr Tyr Arg Pro Lys Tyr 20 23 110 24 DNA Artificial
sequence misc_feature 1-24 Sequence is synthesized. 110 ggccctggcc
tgccagaagt gtgg 24 111 24 DNA Artificial sequence misc_feature 1-24
Sequence is synthesized. 111 gtgtgccttt cctgatctga gaac 24 112 50
DNA Artificial sequence misc_feature 1-50 Sequence is synthesized.
112 gtgattccat ctcttcatgt tcccagaaaa ttcttcccag ccgggcaggg 50 113
70 DNA Artificial sequence misc_feature 1-70 Sequence is
synthesized. 113 ccagccagag gaggccacga acttcactct cgcaggctgt
gtcagcacac 50 gcacctaccg acccaagtac 70 114 50 DNA Artificial
sequence misc_feature 1-50 Sequence is synthesized. 114 gcccctggag
cccttgctcc accagctgcg gcctgggggt ctccactcgg 50 115 23 DNA
Artificial sequence misc_feature 1-23 Sequence is synthesized. 115
aaaggtgcgt acccagctgt gcc 23 116 24 DNA Artificial sequence
misc_feature 1-24 Sequence is synthesized. 116 ggtcttggcg
aagacggctg acct 24 117 51 DNA Artificial sequence misc_feature 1-51
Sequence is synthesized. 117 cctggtgctg gatggctgtg gctgctgccg
ggtatgtgca cggcggctgg 50 g 51 118 28 DNA Artificial sequence
misc_feature 1-28 Sequence is synthesized. 118 gtcttgtgca
agcaacaaaa tggactcc 28 119 27 DNA Artificial sequence misc_feature
1-27 Sequence is synthesized. 119 gctgtcgcaa ggctgaatgt aacacag 27
120 50 DNA Artificial sequence misc_feature 1-50 Sequence is
synthesized. 120 gctccagaac atgtgggatg ggaatatcta acagggtgac
caatgaaaac 50 121 23 DNA Artificial sequence misc_feature 1-23
Sequence is synthesized. 121 cctggagtga gcctggtgag aga 23 122 27
DNA Artificial sequence misc_feature 1-27 Sequence is synthesized.
122 acaatacagc cctttgtgtg ggtcaca 27 123 44 DNA Artificial sequence
misc_feature 1-44 Sequence is synthesized. 123 tggttgcttg
gcacagattt tacagcatcc acagccatct ctca 44 124 27 DNA Artificial
sequence misc_feature 1-27 Sequence is synthesized. 124 tgacttccag
gcatgaggtg gctcctg 27 125 34 DNA Artificial sequence misc_feature
1-34 Sequence is synthesized. 125 attggcaatc tcttcgaagt cagggtaaga
ttcc 34 126 40 DNA Artificial sequence misc_feature 1-40 Sequence
is synthesized. 126 ggtacgtcta gactaattgg caatctcttc gaagtcaggg 40
127 42 DNA Artificial sequence misc_feature 1-42 Sequence is
synthesized. 127 tttccctttg gatcctaaac caacatgagg tggctcctgc cc 42
128 20 DNA Artificial sequence misc_feature 1-20 Sequence is
synthesized. 128 cagattggtg ctggatatgc 20 129 20 DNA Artificial
sequence misc_feature 1-20 Sequence is synthesized. 129 actgccttga
ttactcctac 20 130 18 DNA Artificial sequence misc_feature 1-18
Sequence is synthesized. 130 agttgcagat gtggctct 18 131 18 DNA
Artificial sequence misc_feature 1-18 Sequence is synthesized. 131
agtccaagag tctcagca 18 132 18 DNA Artificial sequence misc_feature
1-18 Sequence is synthesized. 132 acaactggaa gcactgga 18 133 18 DNA
Artificial sequence misc_feature 1-18 Sequence is synthesized. 133
tcttattcca gaggaacc 18 134 22 DNA Artificial sequence misc_feature
1-22 Sequence is synthesized. 134 tccctgtacg cttctggtcg ta 22 135
22 DNA Artificial sequence misc_feature 1-22 Sequence is
synthesized. 135 tctcaaagtc caaagccaca ta 22 136 18 DNA Artificial
sequence misc_feature 1-18 Sequence is synthesized. 136 cacagttcca
gcaaatac 18 137 18 DNA Artificial sequence misc_feature 1-18
Sequence is synthesized. 137 ggaatcaggc ggtacagt 18 138 31 DNA
Artificial sequence misc_feature 1-31 Sequence is synthesized. 138
agcctttcca agtcactaga agtcctgctg g 31 139 21 DNA Artificial
sequence misc_feature 1-21 Sequence is synthesized. 139 ctggactaca
cccaagcctg a 21 140 23 DNA Artificial sequence misc_feature 1-23
Sequence is synthesized. 140 catttcttgg gatttaggca aga 23 141 19
DNA Artificial sequence misc_feature 1-19 Sequence is synthesized.
141 tctagcccac tccctgcct 19 142 21 DNA Artificial sequence
misc_feature 1-21 Sequence is synthesized. 142 gaagtcggag
agaaagctcg c 21 143 30 DNA Artificial sequence misc_feature 1-30
Sequence is synthesized. 143 cacacacagc ctatatcaaa catgcacacg 30
144 38 DNA Artificial sequence misc_feature 1-38 Sequence is
synthesized. 144 cttgagactg aaagatttag ccataatgta aactgcct 38 145
22 DNA Artificial sequence misc_feature 1-22 Sequence is
synthesized. 145 caaatgcaac ctcacaacct tg 22 146 24 DNA Artificial
sequence misc_feature 1-24 Sequence is synthesized. 146 ttcttttatg
cccaaagtcc aatt 24 147 48 DNA Artificial sequence misc_feature 1-48
Sequence is synthesized. 147 ggattctaat acgactcact atagggcgtc
cctggccagt gctgtgag 48 148 48 DNA Artificial sequence misc_feature
1-48 Sequence is synthesized. 148 ctatgaaatt aaccctcact aaagggaggg
ccaggctttg cttccatt 48 149 47 DNA Artificial sequence misc_feature
1-47 Sequence is synthesized. 149 ggattctaat acgactcact atagggctgg
aggcatggca caggaac 47 150 48 DNA Artificial sequence misc_feature
1-48 Sequence is synthesized. 150 ctatgaaatt aaccctcact aaagggatcc
ggatcaggct tgggtgta 48 151 48 DNA Artificial sequence misc_feature
1-48 Sequence is synthesized. 151 ggattctaat acgactcact atagggcagc
ttgggatgga ggtctttc 48 152 44 DNA Artificial sequence misc_feature
1-44 Sequence is synthesized. 152 ctatgaaatt aaccctcact aaagggaggg
cactggggtg gtgt 44 153 45 DNA Artificial sequence misc_feature 1-45
Sequence is synthesized. 153 ggattctaat acgactcact atagggcgcg
aggacggcgg cttca 45 154 48 DNA Artificial sequence misc_feature
1-48 Sequence is synthesized. 154 ctatgaaatt aaccctcact aaagggaaga
gtcgcggccg cccttttt 48 155 48 DNA Artificial sequence misc_feature
1-48 Sequence is synthesized. 155 ggattctaat acgactcact atagggcggg
gctcctcttc tccactct 48 156 48 DNA Artificial sequence misc_feature
1-48 Sequence is synthesized. 156 ctatgaaatt aaccctcact aaagggagct
gtcgcaaggc tgaatgta 48
* * * * *
References