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CA 02372511 2001-11-23
WO 00/77037 PCT/US00/14042
SECRETED AND TRANSMEMBRANE POLYPEPTIDES AND NUCLEIC ACIDS ENCODING
THE SAME
FIELD OF THE INVENTION
The present invention relates generally to the identification and isolation of
novel DNA and to the
recombinant production of novel polypeptides.
BACKGROUND OF THE INVENTION
Extracellular proteins play important roles in, among other things, 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.
Secreted proteins have various industrial applications, including as
pharmaceuticals, diagnostics,
biosensors and bioreactors. Most protein drugs available at present, such as
thrombolytic agents, interferons,
interleukins, erythropoietins, colony stimulating factors, and various other
cytokines, are secretory proteins.
Their receptors, which are membrane proteins, also have potential as
therapeutic or diagnostic agents. Efforts
are being undertaken by both industry and academia to identify new, native
secreted proteins. Many efforts
are focused on the screening of mammalian recombinant DNA libraries to
identify the coding sequences for
novel secreted 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); U.S.
Patent No. 5,536,637)].
Membrane-bound proteins and receptors can play important roles in, among other
things, 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. 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. For instance,
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
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CA 02372511 2001-11-23
WO 00/77037 PCT/US00/14042
fibroblast growth factor receptor and nerve growth factor receptor.
Membrane-bound proteins and receptor molecules have various industrial
applications, including as
pharmaceutical and diagnostic agents. Receptor immunoadhesins, for instance,
can be employed as therapeutic
agents to block receptor-ligand interactions. The membrane-bound proteins can
also be employed for screening
of potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction.
Efforts are being undertaken by both industry and academia to identify new,
native receptor or
membrane-bound proteins. Many efforts are focused on the screening of
mammalian recombinant DNA
libraries to identify the coding sequences for novel receptor or membrane-
bound proteins.
1. PRO196
The abbreviations "TIE" or "tie" are acronyms, which stand for "tyrosine
kinase containing Ig and
EGF homology domains" and were coined to designate a new family of receptor
tyrosine kinases which are
almost exclusively expressed in vascular endothelial cells and early
hemopoietic cells, and are characterized
by the presence of an EGF-like domain, and extracellular folding units
stabilized by intra-chain disulfide bonds,
generally referred to as "immunoglobulin (IG)-like" folds. A tyrosine kinase
homologous cDNA fragment
from human leukemia cells (tie) was described by Partanen et al., Proc. Natl.
Acad. Sci. USA 87, 8913-8917
(1990). The mRNA of this human "tie" receptor has been detected in all human
fetal and mouse embryonic
tissues, and has been reported to be localized in the cardiac and vascular
endothelial cells. Korhonen et at.,
Blood 80, 2548-2555 (1992); PCT Application Publication No. WO 93/14124
(published 22 July 1993). The
rat homolog of human tie, referred to as "tie-1", was identified by
Maisonpierre et al., Oncogene 8, 1631-1637
(1993)). Another tie receptor, designated "tie-2" was originally identified in
rats ( Dumont et al., Oncogene
8, 1293-1301 (1993)), while the human homolog of tie-2, referred to as "ork"
was described in U.S. Patent
No. 5,447,860 (Ziegler). The murine homolog of tie-2 was originally termed
"tek." The cloning of a mouse
tie-2 receptor from a brain capillary cDNA library is disclosed in PCT
Application Publication No. WO
95/13387 (published 18 May 1995). The TIE receptors are believed to be
actively involved in angiogenesis,
and may play a role in hemopoiesis as well.
The expression cloning of human TIE-2 ligands has been described in PCT
Application Publication
No. WO 96/11269 (published 18 April 1996) and in U.S. Patent No. 5,521,073
(published 28 May 1996).
A vector designated as Agt10 encoding a TIE-2 ligand named "htie-2 ligand 1"
or "hTLI" has been deposited
under ATCC Accession No. 75928. A plasmid encoding another TIE-2 ligand
designated "htie-2 2" or
"hTL2" is available under ATCC Accession No. 75928. This second ligand has
been described as an
antagonist of the TAI-2 receptor. The identification of secreted human and
mouse ligands for the TIE-2
receptor has been reported by Davis et al., Cell 87, 1161-1169 (1996). The
human ligand designated
"Angiopoietin-1 ", to reflect its role in angiogenesis and potential action
during hemopoiesis, is the same ligand
as the ligand variously designated as "htie-2 1" or "hTL-1" in WO 96/11269.
Angiopoietin-1 has been
described to play an angiogenic role later and distinct from that of VEGF
(Surf et al., Cell 87, 1171-1180
(1996)). Since TIE-2 is apparently upregulated during the pathologic
angiogenesis requisite for tumor growth
(Kaipainen et al., Cancer Res. 54, 6571-6577 (1994)) angiopoietin-1 has been
suggested to be additionally
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CA 02372511 2001-11-23
WO 00/77037 PCT/US00/14042
useful for specifically targeting tumor vasculature (Davis et al., supra).
2. PRO444
Efforts are being undertaken by both industry and academia to identify new,
native secreted proteins.
Many efforts are focused on the screening of mammalian recombinant DNA
libraries to identify the coding
sequences for novel secreted proteins. We herein describe the identification
and isolation of cDNA molecules
encoding novel secreted polypeptides, designated herein as PR0444
polypeptides.
3. PRO183
Efforts are being undertaken by both industry and academia to identify new,
native secreted proteins.
Many efforts are focused on the screening of mammalian recombinant DNA
libraries to identify the coding
sequences for novel secreted proteins. We herein describe the identification
and isolation of cDNA molecules
encoding novel polypeptides, designated herein as PRO 183 polypeptides.
4. PRO185
Efforts are being undertaken by both industry and academia to identify new,
native secreted proteins.
Many efforts are focused on the screening of mammalian recombinant DNA
libraries to identify the coding
sequences for novel secreted proteins. We herein describe the identification
and isolation of cDNA molecules
encoding novel polypeptides, designated herein as PRO 185 polypeptides.
5. PRO210 and PR0217
Epidermal growth factor (EGF) is a conventional mitogenic factor that
stimulates the proliferation of
various types of cells including epithelial cells and fibroblasts. EGF binds
to and activates the EGF receptor
(EGFR), which initiates intracellular signaling and subsequent effects. The
EGFR is expressed in neurons of
the cerebral cortex, cerebellum, and hippocampus in addition to other regions
of the central nervous system
(CNS). In addition, EGF is also expressed in various regions of the CNS.
Therefore, EGF acts not only on
mitotic cells, but also on postmitotic neurons. In fact, many studies have
indicated that EGF has neurotrophic
or neuromodulatory effects on various types of neurons in the CNS. For
example, EGF acts directly on
cultured cerebral cortical and cerebellar neurons, enhancing neurite outgrowth
and survival. On the other
hand, EGF also acts on other cell types, including septal cholinergic and
mesencephalic dopaminergic neurons,
indirectly through glial cells. Evidence of the effects of EGF on neurons in
the CNS is accumulating, but the
mechanisms of action remain essentially unknown. EGF-induced signaling in
mitotic cells is better understood
than in postmitotic neurons. Studies of cloned pheochromocytoma PC 12 cells
and cultured cerebral cortical
neurons have suggested that the EGF-induced neurotrophic actions are mediated
by sustained activation of the
EGFR and mitogen-activated protein kinase (MAPK) in response to EGF. The
sustained intracellular signaling
correlates with the decreased rate of EGFR down-regulation, which might
determine the response of neuronal
cells to EGF. It is likely that EGF is a multi-potent growth factor that acts
upon various types of cells
including mitotic cells and postmitotic neurons.
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CA 02372511 2001-11-23
WO 00/77037 PCT/US00/14042
EGF is produced by the salivary and Brunner's glands of the gastrointestinal
system, kidney, pancreas,
thyroid gland, pituitary gland, and the nervous system, and is found in body
fluids such as saliva, blood,
cerebrospinal fluid (CSF), urine, amniotic fluid, prostatic fluid, pancreatic
juice, and breast milk,
Plata-Salaman, CR Peptides 12: 653-663 (1991).
EGF is mediated by its membrane specific receptor, which contains an intrinsic
tyrosine kinase.
Stoscheck CM et al., J. Cell Biochem. 31: 135-152 (1986). EGF is believed to
function by binding to the
extracellular portion of its receptor which induces a transmembrane signal
that activates the intrinsic tyrosine
kinase.
Purification and sequence analysis of the EGF-like domain has revealed the
presence of six conserved
cysteine residues which cross-bind to create three peptide loops, Savage CR et
al., J. Biol. Chem. 248:
7669-7672 (1979). It is now generally known that several other peptides can
react with the EGF receptor
which share the same generalized motif XnCX7CX4/5CX10CXCX5GX2CXn, where X
represents any
non-cysteine amino acid, and n is a variable repeat number. Non isolated
peptides having this motif include
TGF-a, amphiregulin, schwannoma-derived growth factor (SDGF), heparin-binding
EGF-like growth factors
and certain virally encoded peptides (e.g., Vaccinia virus, Reisner AH, Nature
313: 801-803 (1985), Shope
fibroma virus, Chang W., et al., Mol Cell Biol. 7: 535-540 (1987), Molluscum
contagiosum, Porter CD &
Archard LC, J. Gen. Virol. 68: 673-682 (1987), and Myxoma virus, Upton C et
al., J. Virol. 61: 1271-1275
(1987). Prigent SA & Lemoine N.R., Prog. Growth Factor Res. 4: 1-24 (1992).
EGF-like domains are not confined to growth factors but have been observed in
a variety of
cell-surface and extracellular proteins which have interesting properties in
cell adhesion, protein-protein
interaction and development, Laurence DJR & Gusterson BA, Tumor Biol. 11: 229-
261 (1990). These
proteins include blood coagulation factors (factors VI, IX, X, XII, protein C,
protein S, protein Z, tissue
plasminogen activator, urokinase), extracellular matrix components (laminin,
cytotactin, entactin), cell surface
receptors (LDL receptor, thrombomodulin receptor) and immunity-related
proteins (complement Clr,
uromodulin).
Even more interesting, the general structure pattern of EGF-like precursors is
preserved through lower
organisms as well as in mammalian cells. A number of genes with developmental
significance have been
identified in invertebrates with EGF-like repeats. For example, the notch gene
of Drosophila encodes 36
tandemly arranged 40 amino acid repeats which show homology to EGF, Wharton W
et al., Cell 43: 557-581
(1985). Hydropathy plots indicate a putative membrane spanning domain, with
the EGF-related sequences
being located on the extracellular side of the membrane. Other homeotic genes
with EGF-like repeats include
Delta, 95F and 5ZD which were identified using probes based on Notch, and the
nematode gene Lin-12 which
encodes a putative receptor for a developmental signal transmitted between two
specified cells.
Specifically, EGF has been shown to have potential in the preservation and
maintenance of
gastrointestinal mucosa and the repair of acute and chronic mucosal lesions,
Konturek, PC et al., Eur. J.
Gastroenterol Hepatol. 7 (10), 933-37 (1995), including the treatment of
necrotizing enterocolitis,
Zollinger-Ellison syndrome, gastrointestinal ulceration gastrointestinal
ulcerations and congenital microvillus
atrophy, A. Guglietta & PB Sullivan, Eur. J. Gastroenterol Hepatol, 7(10), 945-
50 (1995). Additionally, EGF
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CA 02372511 2001-11-23
WO 00/77037 PCT/US00/14042
has been implicated in hair follicle differentiation; C.L. du Cros, J. Invest.
Dermatol. 101 (1 Suppl.),
106S-113S (1993), SG Hillier, Clin. Endocrinol. 33(4), 427-28 (1990); kidney
function, L.L. Hamm et al.,
Semin. Nephrol. 13 (1): 109-15 (1993), RC Harris, Am. J. Kidney Dis. 17(6):
627-30 (1991); tear fluid, GB
van Setten et al., Int. Ophthalmol 15(6); 359-62 (1991); vitamin K mediated
blood coagulation, J. Stenflo et
al., Blood 78(7): 1637-51 (1991). EGF is also implicated various skin disease
characterized by abnormal
keratinocyte differentiation, e.g., psoriasis, epithelial cancers such as
squamous cell carcinomas of the lung,
epidermoid carcinoma of the vulva and gliomas. King, LE et al., Am. J. Med.
Sci. 296: 154-158 (1988).
Of great interest is mounting evidence that genetic alterations in growth
factors signaling pathways
are closely linked to developmental abnormalities and to chronic diseases
including cancer. Aaronson SA,
Science 254: 1146-1153 (1991). For example, c-erb-2 (also known as HER-2), a
proto-oncogene with close
structural similarity to EGF receptor protein, is overexpressed in human
breast cancer. King et al., Science
229: 974-976 (1985); Gullick, WJ, Hormones and their actions, Cooke BA et al.,
eds, Amsterdam, Elsevier,
pp 349-360 (1986).
6. PR0215
Protein-protein interactions include receptor and antigen complexes and
signaling mechanisms. As
more is known about the structural and functional mechanisms underlying
protein-protein interactions, protein-
protein interactions can be more easily manipulated to regulate the particular
result of the protein-protein
interaction. Thus, the underlying mechanisms of protein-protein interactions
are of interest to the scientific
and medical community.
All proteins containing leucine-rich repeats are thought to be involved in
protein-protein interactions.
Leucine-rich repeats are short sequence motifs present in a number of proteins
with diverse functions and
cellular locations. The crystal structure of ribonuclease inhibitor protein
has revealed that leucine-rich repeats
correspond to beta-alpha structural units. These units are arranged so that
they form a parallel beta-sheet with
one surface exposed to solvent, so that the protein acquires an unusual,
nonglubular shape. These two features
have been indicated as responsible for the protein-binding functions of
proteins containing leucine-rich repeats.
See, Kobe and Deisenhofer, Trends Biochem. Sci., 19(10):415-421 (Oct. 1994).
A study has been reported on leucine-rich proteoglycans which serve as tissue
organizers, orienting
and ordering collagen fibrils during ontogeny and are involved in pathological
processes such as wound
healing, tissue repair, and tumor stroma formation. lozzo, R. V., Crit. Rev.
Biochem. Mol. Biol., 32(2):141-
174 (1997). Others studies implicating leucine rich proteins in wound healing
and tissue repair are De La
Salle, C., et al., Vouv. Rev. Fr. Hematol. (Germany), 37(4):215-222 (1995),
reporting mutations in the
leucine rich motif in a complex associated with the bleeding disorder Bernard-
Soulier syndrome and
Chlemetson, K. J., Thromb. Haemost. (Germany), 74(1):111-116 (July 1995),
reporting that platelets have
leucine rich repeats. Another protein of particular interest which has been
reported to have leucine-rich repeats
is the SLIT protein which has been reported to be useful in treating neuro-
degenerative diseases such as
Alzheimer's disease, nerve damage such as in Parkinson's disease, and for
diagnosis of cancer, see,
Artavanistsakonas, S. and Rothberg, J. M., W09210518-A1 by Yale University.
Other studies reporting on
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WO 00/77037 PCT/US00/14042
the biological functions of proteins having leucine-rich repeats include:
Tayar, N., et al., Mol. Cell
Endocrinol., (Ireland), 125(1-2):65-70 (Dec. 1996) (gonadotropin receptor
involvement); Miura, Y., et al.,
Nippon Rinsho (Japan), 54(7):1784-1789 (July 1996) (apoptosis involvement);
Harris, P. C., et al., J. Am.
Soc. Nephrol., 6(4):1125-1133 (Oct. 1995) (kidney disease involvement); and
Ruoslahti, E. I., et al.,
WO9110727-A by La Jolla Cancer Research Foundation (decorin binding to
transforming growth factorp
involvement for treatment for cancer, wound healing and scarring).
7. PRO242. PRO1318 and PRO1600
Leukocytes include monocytes, macrophages, basophils, and eosinophils and play
an important role
in the immune response. These cells are important in the mechanisms initiated
by T and/or B lymphocytes and
secrete a range of cytokines which recruit and activate other inflammatory
cells and contribute to tissue
destruction.
Thus, investigation of the regulatory processes by which leukocytes move to
their appropriate
destination and interact with other cells is critical. Currently, leukocytes
are thought to move from the blood
to injured or inflamed tissues by rolling along the endothelial cells of the
blood vessel wall. This movement
is mediated by transient interactions between selectins and their ligands.
Next, the leukocyte must move
through the vessel wall and into the tissues. This diapedesis and
extravasation step involves cell activation
which promotes a more stable leukocyte-endothelial cell interaction, again
mediated by integrins and their
ligands.
Chemokines are a large family of structurally related polypeptide cytokines.
These molecules
stimulate leukocyte movement and may explain leukocyte trafficking in
different inflammatory situations.
Chemokines mediate the expression of particular adhesion molecules on
endothelial cells, and they produce
chemoattractants which activate specific cell types. In addition, the
chemokines stimulate proliferation and
regulate activation of specific cell types. In both of these activities,
chemokines demonstrate a high degree of
target cell specificity.
The chemokine family is divided into two subfamilies based on whether two
amino terminal cysteine
residues are immediately adjacent (C-C) or separated by one amino acid (C-X-
C). Chemokines of the C-X-C
family generally activate neutrophils and fibroblasts while the C-C chemokines
act on a more diverse group
of target cells including monocytes/macrophages, basophils, eosinophils and T
lymphocytes. The known
chemokines of both subfamilies are synthesized by many diverse cell types as
reviewed in Thomson A. (1994)
The Cytokine Handbook, 2 d Ed. Academic Press, N.Y.
Known chemokines include macrophage inflammatory proteins alpha and beta (MIP-
1 alpha and beta
), 1-309, RANTES, and monocyte chemotactic protein (MCP-1).
MIP-1 alpha and MIP-1 beta were first purified from a stimulated mouse
macrophage cell line and
elicited an inflammatory response when injected into normal tissues. MIP-1
alpha and MIP-1 beta consist of
68-69 amino acids and share approximately 70% identity in their mature
secreted forms. Both are expressed
in T cells, B cells and monocytes which are stimulated by mitogens, anti-CD3
and endotoxin, and both
polypeptides bind heparin and stimulate monocytes. MIP-1 alpha acts as a
chemoattractant for the CD-8 subset
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WO 00/77037 PCT/US00/14042
of T lymphocytes and eosinophils, while MIP-1 beta chemoattracts the CD-4
subset of T lymphocytes. In
addition, these proteins are known to stimulate myelopoiesis in mice.
RANTES is regulated by interleukins-1 and -4, transforming nerve factor and
interferon- gamma and
is expressed in T cells, platelets, stimulated rheumatoid synovial
fibroblasts, and in some tumor cell lines.
RANTES affects lymphocytes, monocytes, basophils and eosinophils. RANTES
expression is substantially
reduced upon T cell stimulation.
Monocyte chemotactic protein (MCP-1) is a 76 amino acid protein which appears
to be expressed in
almost all cells and tissues upon stimulation by a variety of agents. However,
the targets of MCP-1 are limited
to monocytes and basophils. In these cells, MCP-1 induces a MCP-l receptor.
Two related proteins, MCP-2
and MCP-3, have 62% and 73% identity, respectively, with MCP-1 and share its
chemoattractant specificity
or monocytes.
Current techniques for diagnosis of abnormalities in inflamed or diseased
issues mainly rely on
observation of clinical symptoms or serological analyses of body tissues or
fluids for hormones, polypeptides
or various metabolites. Problems exist with these diagnostic techniques.
First, patients may not manifest
clinical symptoms at early stages of disease. Second, serological tests do not
always differentiate between
invasive diseases and genetic syndromes. Thus, the identification of expressed
chemokines is important to the
development of new diagnostic techniques, effective therapies, and to aid in
the understanding of molecular
pathogenesis.
The chemokine molecules were reviewed in Schall TJ (1994) Chemotactic
Cytokines: Targets for
Therapeutic Development. International Business Communications, Southborough
Mass. pp 180-270; and in
Paul WE (1993) Fundamental Immunology, 3rd Ed. Raven Press, N.Y. pp 822-826.
8. PRO288
Control of cell numbers in mammals is believed to be determined, in part, by a
balance between cell
proliferation and cell death. One form of cell death, sometimes referred to as
necrotic cell death, is typically
characterized as a pathologic form of cell death resulting from some trauma or
cellular injury. In contrast,
there is another, "physiologic" form of cell death which usually proceeds in
an orderly or controlled manner.
This orderly or controlled form of cell death is often referred to as
"apoptosis" [see, e.g., Barr et al.,
Bio/Technology, 12:487-493 (1994); Steller et al., Science, 267:1445-1449
(1995)]. Apoptotic cell death
naturally occurs in many physiological processes, including embryonic
development and clonal selection in the
immune system [Itoh et al., Cell, 66:233-243 (1991)]. Decreased levels of
apoptotic cell death have been
associated with a variety of pathological conditions, including cancer, lupus,
and herpes virus infection
[Thompson, Science, 267:1456-1462 (1995)]. Increased levels of apoptotic cell
death may be associated with
a variety of other pathological conditions, including AIDS, Alzheimer's
disease, Parkinson's disease,
amyotrophic lateral sclerosis, multiple sclerosis, retinitis pigmentosa,
cerebellar degeneration, aplastic anemia,
myocardial infarction, stroke, reperfusion injury, and toxin-induced liver
disease [see, Thompson, 00].
Apoptotic cell death is typically accompanied by one or more characteristic
morphological and
biochemical changes in cells, such as condensation of cytoplasm, loss of
plasma membrane microvilli,
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segmentation of the nucleus, degradation of chromosomal DNA or loss of
mitochondrial function. A variety
of extrinsic and intrinsic signals are believed to trigger or induce such
morphological and biochemical cellular
changes [Raff, Nature, 356:397-400 (1992); Steller, sera; Sachs et al., Blood,
82:15 (1993)]. For instance,
they can be triggered by hormonal stimuli, such as glucocorticoid hormones for
immature thymocytes, as well
as withdrawal of certain growth factors [Watanabe-Fukunaga et al., Nature,
356:314-317 (1992)]. Also, some
identified oncogenes such as myc, rel, and E1A, and tumor suppressors, like
p53, have been reported to have
a role in inducing apoptosis. Certain chemotherapy drugs and some forms of
radiation have likewise been
observed to have apoptosis-inducing activity [Thompson, supra .
Various molecules, such as tumor necrosis factor-a ("TNF-a"), tumor necrosis
factor-R ("TNF-R"
or "lymphotoxin"), CD30ligand, CD27 ligand, CD40ligand, OX-40 ligand, 4-1BB
ligand, Apo-1 ligand (also
referred to as Fas ligand or CD95 ligand), and Apo-2 ligand (also referred to
as TRAIL) have been identified
as members of the tumor necrosis factor ("TNF") family of cytokines [See,
e.g., Gruss and Dower, Blood,
85:3378-3404 (1995); Wiley et al., Immuni , 2:673-682 (1995); Pitti et al., J.
Biol. Chem., 271:12687-12690
(1996)]. Among these molecules, TNF-a, TNF-p, CD30 ligand, 4-1BB ligand, Apo-1
ligand, and Apo-2
ligand (TRAIL) have been reported to be involved in apoptotic cell death. Both
TNF-a and TNF-R have been
reported to induce apoptotic death in susceptible tumor cells [Schmid et al.,
Proc. Natl. Acad. Sci., 83:1881
(1986); Dealtry et al., Eur. J. Immunol., 17:689 (1987)]. Zheng et al. have
reported that TNF-a is involved
in post-stimulation apoptosis of CD8-positive T cells [Zheng et al., Nature,
377:348-351 (1995)]. Other
investigators have reported that CD30 ligand may be involved in deletion of
self-reactive T cells in the thymus
[Amakawa et al., Cold Spring Harbor Laboratory Symposium on Programmed Cell
Death, Abstr. No. 10,
(1995)].
Mutations in the mouse Fas/Apo-1 receptor or ligand genes (called lpr and gld,
respectively) have
been associated with some autoimmune disorders, indicating that Apo-1 ligand
may play a role in regulating
the clonal deletion of self-reactive lymphocytes in the periphery [Krammer et
al., Curr. Op. Immunol., 6:279-
289 (1994); Nagata et al., Science, 267:1449-1456 (1995)]. Apo-1 ligand is
also reported to induce post-
stimulation apoptosis in CD4-positive T lymphocytes and in B lymphocytes, and
may be involved in the
elimination of activated lymphocytes when their function is no longer needed
[Krammer et al., supra; Nagata
et al., su ra . Agonist mouse monoclonal antibodies specifically binding to
the Apo-1 receptor have been
reported to exhibit cell killing activity that is comparable to or similar to
that of TNF-a [Yonehara et al., J.
Exp. Med., 169:1747-1756 (1989)].
Induction of various cellular responses mediated by such TNF family cytokines
is believed to be
initiated by their binding to specific cell receptors. Two distinct TNF
receptors of approximately 55-kDa
(TNFR1) and 75-kDa (TNFR2) have been identified [Hohman et al., J. Biol.
Chem., 264:14927-14934 (1989);
Brockhaus et al., Proc. Natl. Acad. Sci., 87:3127-3131 (1990); EP 417,563,
published March 20, 1991] and
human and mouse cDNAs corresponding to both receptor types have been isolated
and characterized [Loetscher
et al., Cell, 61:351 (1990); Schall et al., Cell, 61:361 (1990); Smith et al.,
Science, 248:1019-1023 (1990);
Lewis et al., Proc. Natl. Acad. Sci., 88:2830-2834 (1991); Goodwin et al.,
Mol. Cell. Biol., 11:3020-3026
(1991)]. Extensive polymorphisms have been associated with both TNF receptor
genes [see, e.g., Takao et
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CA 02372511 2001-11-23
WO 00/77037 PCT/US00/14042
al., Immunogenetics, 37:199-203 (1993)]. Both TNFRs share the typical
structure of cell surface receptors
including extracellular, transmembrane and intracellular regions. The
extracellular portions of both receptors
are found naturally also as soluble TNF-binding proteins [Nophar, Y. et al.,
EMBO J., 9:3269 (1990); and
Kohno, T. et al., Proc. Natl. Acad. Sci. U.S.A., 87:8331(1990)]. More
recently, the cloning of recombinant
soluble TNF receptors was reported by Hale et al. [J. Cell. Biochem.
Supplement 15F, 1991, p. 113 (P424)].
The extracellular portion of type 1 and type 2 TNFRs (TNFRI and TNFR2)
contains a repetitive
amino acid sequence pattern of four cysteine-rich domains (CRDs) designated 1
through 4, starting from the
NH2-terminus. Each CRD is about 40 amino acids long and contains 4 to 6
cysteine residues at positions
which are well conserved [Schall et al., supr; Loetscher et al., ser; Smith et
al., su r; Nophar et al.,
supr; Kohno et al., su r]. In TNFR1, the approximate boundaries of the four
CRDs are as follows: CRD1-
amino acids 14 to about 53; CRD2- amino acids from about 54 to about 97; CRD3-
amino acids from about
98 to about 138; CRD4- amino acids from about 139 to about 167. In TNFR2, CRDI
includes amino acids
17 to about 54; CRD2- amino acids from about 55 to about 97; CRD3- amino acids
from about 98 to about
140; and CRD4- amino acids from about 141 to about 179 [Banner et al., Cell,
73:431-435 (1993)]. The
potential role of the CRDs in ligand binding is also described by Banner et
al., suWr.
A similar repetitive pattern of CRDs exists in several other cell-surface
proteins, including the p75
nerve growth factor receptor (NGFR) [Johnson et al., Cell, 47:545 (1986);
Radeke et al., Nature, 325:593
(1987)], the B cell antigen CD40 [Stamenkovic et al., EMBO J., 8:1403 (1989)],
the T cell antigen OX40
[Mallet et al., EMBO J., 9:1063 (1990)] and the Fas antigen [Yonehara et al.,
sgWra and Itoh et al., supra].
CRDs are also found in the soluble TNFR (sTNFR)-like T2 proteins of the Shope
and myxoma poxviruses
[Upton et al., Virology, 160:20-29 (1987); Smith et al., Biochem. Biophys.
Res. Commun., 176:335 (1991);
Upton et al., Virology, 184:370 (1991)]. Optimal alignment of these sequences
indicates that the positions of
the cysteine residues are well conserved. These receptors are sometimes
collectively referred to as members
of the TNF/NGF receptor superfamily. Recent studies on p75NGFR showed that the
deletion of CRD1
[Welcher, A.A. et al., Proc. Natl. Acad. Sci. USA, 88:159-163 (1991)] or a 5-
amino acid insertion in this
domain [Yan, H. and Chao, M.V., J. Biol. Chem., 266:12099-12104 (1991)] had
little or no effect on NGF
ra . p75 NGFR contains a proline-rich stretch of about 60 amino acids,
binding [Yan, H. and Chao, M.V., su
p
between its CRD4 and transmembrane region, which is not involved in NGF
binding [Peetre, C. et al., Eur.
J. Hematol., 41:414-419 (1988); Seckinger, P. et al., J. Biol. Chem.,
264:11966-11973 (1989); Yan, H. and
Chao, M.V., su ra . A similar proline-rich region is found in TNFR2 but not in
TNFRI.
Itoh et al. disclose that the Apo-1 receptor can signal an apoptotic cell
death similar to that signaled
by the 55-kDa TNFR1 [Itoh et al., supraa J. Expression of the Apo-1 antigen
has also been reported to be
down-regulated along with that of TNFRI when cells are treated with either TNF-
a or anti-Apo-1 mouse
monoclonal antibody [Krammer et al., supra; Nagata et al., supra .
Accordingly, some investigators have
hypothesized that cell lines that co-express both Apo-1 and TNFR1 receptors
may mediate cell killing through
common signaling pathways I[ d.].
The TNF family ligands identified to date, with the exception of lymphotoxin-
a, are type II
transmembrane proteins, whose C-terminus is extracellular. In contrast, the
receptors in the TNF receptor
9
CA 02372511 2001-11-23
WO 00/77037 PCT/US00/14042
(TNFR) family identified to date are type I transmembrane proteins. In both
the TNF ligand and receptor
families, however, homology identified between family members has been found
mainly in the extracellular
domain ("ECD"). Several of the TNF family cytokines, including TNF-a, Apo-1
ligand and CD40 ligand,
are cleaved proteolytically at the cell surface; the resulting protein in each
case typically forms a homotrimeric
molecule that functions as a soluble cytokine. TNF receptor family proteins
are also usually cleaved
proteolytically to release soluble receptor ECDs that can function as
inhibitors of the cognate cytokines.
Recently, other members of the TNFR family have been identified. In Marsters
et al., Curr. Biol.,
6:750 (1996), investigators describe a full length native sequence human
polypeptide, called Apo-3, which
exhibits similarity to the TNFR family in its extracellular cysteine-rich
repeats and resembles TNFR1 and
CD95 in that it contains a cytoplasmic death domain sequence [see also
Marsters et al., Curr. Biol., 6:1669
(1996)]. Apo-3 has also been referred to by other investigators as DR3, wsl-1
and TRAMP [Chinnaiyan et
al., Science, 274:990 (1996); Kitson et al., Nature, 384:372 (1996); Bodmer et
al., Immuni , 6:79 (1997)].
Pan et al. have disclosed another TNF receptor family member referred to as
"DR4" [Pan et al.,
Science, 276:111-113 (1997)]. The DR4 was reported to contain a cytoplasmic
death domain capable of
engaging the cell suicide apparatus. Pan et al. disclose that DR4 is believed
to be a receptor for the ligand
known as Apo-2 ligand or TRAIL.
In Sheridan et al., Science, 277:818-821 (1997) and Pan et al., Science,
277:815-818 (1997), another
molecule believed to be a receptor for the Apo-2 ligand (TRAIL) is described.
That molecule is referred to
as DR5 (it has also been alternatively referred to as Apo-2). Like DR4, DR5 is
reported to contain a
cytoplasmic death domain and be capable of signaling apoptosis.
In Sheridan et al., sera, a receptor called DcR1 (or alternatively, Apo-2DcR)
is disclosed as being
a potential decoy receptor for Apo-2 ligand (TRAIL). Sheridan et al. report
that DcRI can inhibit Apo-2
ligand function in vitro. See also, Pan et al., supr, for disclosure on the
decoy receptor referred to as TRID.
As presently understood, the cell death program contains at least three
important elements - activators,
inhibitors, and effectors; in C. elegans, these elements are encoded
respectively by three genes, Ced-4, Ced-9
and Ced-3 [Steller, Science, 267:1445 (1995); Chinnaiyan et al., Science,
275:1122-1126 (1997); Wang et al.,
Cell, 90:1-20 (1997)]. Two of the TNFR family members, TNFR1 and Fas/Apol
(CD95), can activate
apoptotic cell death [Chinnaiyan and Dixit, Current Biology, 6:555-562 (1996);
Fraser and Evan, Cell; 85:781-
784 (1996)]. TNFR1 is also known to mediate activation of the transcription
factor, NF-icB [Tartaglia et al.,
Cell, 74:845-853 (1993); Hsu et al., Cell, 84:299-308 (1996)]. In addition to
some ECD homology, these two
receptors share homology in their intracellular domain (ICD) in an
oligomerization interface known as the
death domain [Tartaglia et al., supr; Nagata, Cell, 88:355 (1997)]. Death
domains are also found in several
metazoan proteins that regulate apoptosis, namely, the Drosophila protein,
Reaper, and the mammalian proteins
referred to as FADD/MORT1, TRADD, and RIP [Cleaveland and Ihle, Cell, 81:479-
482 (1995)]. Using the
yeast-two hybrid system, Raven et al. report the identification of protein,
wsl-1, which binds to the TNFR1
death domain [Raven et al., Programmed Cell Death Meeting, September 20-24,
1995, Abstract at page 127;
Raven et al., European Cytokine Network, 7:Abstr. 82 at page 210 (April-June
1996); see also, Kitson et al.,
Nature, 384:372-375 (1996)]. The wsl-1 protein is described as being
homologous to TNFR1 (48% identity)
CA 02372511 2001-11-23
WO 00/77037 PCT/US00/14042
and having a restricted tissue distribution. According to Raven et al., the
tissue distribution of wsl-1 is
significantly different from the TNFR1 binding protein, TRADD.
Upon ligand binding and receptor clustering, TNFR1 and CD95 are believed to
recruit FADD into
a death-inducing signalling complex. CD95 purportedly binds FADD directly,
while TNFR1 binds FADD
indirectly via TRADD [Chinnaiyan et al., Cell, 81:505-512 (1995); Boldin et
al., J. Biol. Chem., 270:387-391
(1995); Hsu et al., supra; Chinnaiyan et al., J. Biol. Chem., 271:4961-4965
(1996)]. It has been reported that
FADD serves as an adaptor protein which recruits the Ced-3-related protease,
MACHa/FLICE (caspase 8),
into the death signalling complex [Boldin et al., Cell, 85:803-815 (1996);
Muzio et al., Cell, 85:817-827
(1996)]. MACHa/FLICE appears to be the trigger that sets off a cascade of
apoptotic proteases, including
the interleukin-1 R converting enzyme (ICE) and CPP32/Yama, which may execute
some critical aspects of the
cell death programme [Fraser and Evan, su ra .
It was recently disclosed that programmed cell death involves the activity of
members of a family of
cysteine proteases related to the C. elegans cell death gene, ced-3, and to
the mammalian IL-1-converting
enzyme, ICE. The activity of the ICE and CPP32/Yama proteases can be inhibited
by the product of the
cowpox virus gene, crmA [Ray et al., Cell, 69:597-604 (1992); Tewari et al.,
Cell, 81:801-809 (1995)].
Recent studies show that CrmA can inhibit TNFR1- and CD95-induced cell death
[Enari et al., Nature, 375:78-
81 (1995); Tewari et al., J. Biol. Chem., 270:3255-3260 (1995)].
As reviewed recently by Tewari et al., TNFR1, TNFR2 and CD40 modulate the
expression of
proinflammatory and costimulatory cytokines, cytokine receptors, and cell
adhesion molecules through
activation of the transcription factor, NF-KB [Tewari et al., Curr. Op. Genet.
Develop., 6:39-44 (1996)]. NF-
xB is the prototype of a family of dimeric transcription factors whose
subunits contain conserved Rel regions
[Verma et al., Genes Develop., 9:2723-2735 (1996); Baldwin, Ann. Rev.
Immunol., 14:649-681 (1996)].
In its latent form, NF-KB is complexed with members of the IxB inhibitor
family; upon inactivation of the IiB
in response to certain stimuli, released NF-icB translocates to the nucleus
where it binds to specific DNA
sequences and activates gene transcription.
For a review of the TNF family of cytokines and their receptors, see Gruss and
Dower, supra.
9. PR0365
Polypeptides such as human 2-19 protein may function as cytokines. Cytokines
are low molecular
weight proteins which function to stimulate or inhibit the differentiation,
proliferation or function of immune
cells. Cytokines often act as intercellular messengers and have multiple
physiological effects. Given the
physiological importance of immune mechanisms in vivo, efforts are currently
being under taken to identify
new, native proteins which are involved in effecting the immune system. We
describe herein the identification
of a novel polypeptide which has homology to the human 2-19 protein.
10. PRO1361
Efforts are being undertaken by both industry and academia to identify new,
native transmembrane
receptor proteins. Many efforts are focused on the screening of mammalian
recombinant DNA libraries to
11
CA 02372511 2001-11-23
WO 00/77037 PCT/US00/14042
identify the coding sequences for novel receptor proteins. We herein describe
the identification and
characterization of novel transmembrane polypeptides, designated herein as
PRO1361 polypeptides.
11. PRO1308
Follistatin is a secreted protein that regulates secretion of pituitary
follicle-stimulating hormone (FSH).
It functions by binding to, and thereby inhibiting, proteins such as activin
and other members of the
transforming growth factor beta (TGFR) family, that stimulate the production
and secretion of FSH from the
anterior pituitary. Follistatin is also involved in mechanisms that control
basic development, including the
induction of neural development. Follistatin also exhibits angiogenic
properties, particularly in combination
with basic fibroblast growth factor (bFGF). As such, there is strong interest
in identifying new members of
the follistatin family of proteins. The identification and characterization of
follistatins is the topic of the
following references which are incorporated herein by reference: Sugino et al.
J. Med Invest (1997) 44:0-2): 1-
14; Mather et al., Proc. Soc. Exp. biol. Med. (1997) 215(3):209-222; Thomsen,
G.H., Trends Genet (1997)
13(6): 209-211; DePaolo, L.V., Proc. Soc. Exp. Biol. Med. (1997) 214(4):328-
339; Peng etal., Biol. Signals
(1996) 5:81-89, and Halvorson et al. Fertil Steril (1996) 65(3):459-469.
12. PRO1183
Protoporphyrinogen oxidase catalyzes the penultimate step in the heme
biosynthetic pathway.
Deficiency in activity of this enzyme results in the human genetic disease
variegate porphyria. Thus,
protoporphyrinogen oxidases and molecules which either modulate or are related
to these oxidases are of
interest. Moreover, oxidases, and related molecules in general are also of
interest. Oxidases are further
described in at least Birchfield, et al., Biochemistry, 37(19):6905-6910
(1998); Fingar, et al., Cancer Res.,
57(20):4551-4556 (1997); Arnould, et al., Biochemistry, 36(33):10178-10184
(1997);.and Dailey and Dailey,
Cell Mol. Biol., 43(1):67-73 (1997).
13. PRO1272
The cement gland is an ectodermal organ in the head of frog embryos, lying
anterior to any neural
tissue. The cement gland, like neural tissue, has been shown to be induced by
the dorsal mesoderm. XAG-1
is a cement gland specific protein that is useful as a marker of cement gland
induction during development.
See, Sive, et al., Cell, 58(l):171-180 (1989); Itoh, et al., Development,
121(12):3979-3988 (1995). XAG-2
and other proteins related to the XAG family are further described in Aberger,
et al., Mech. Dev., 72(1-
2):115-130 (1998) and Gammill and Sive, Development, 124(2):471-481 (1997).
Thus, novel polypeptides
having sequence identity with XAG proteins are of interest.
14. PRO1419
Efforts are being undertaken by both industry and academia to identify new,
native secreted proteins.
Many efforts are focused on the screening of mammalian recombinant DNA
libraries to identify the coding
sequences for novel secreted proteins. We herein describe the identification
and characterization of novel
12
CA 02372511 2001-11-23
WO 00/77037 PCT/US00/14042
secreted polypeptides, designated herein as PRO1419 polypeptides.
15. PRO4999
Uromodulin is synthesized in the kidney and is the most abundant protein in
normal human urine.
The amino acid sequence encoded by one of the exons of the uromodulin gene has
homology to the low-
density-lipoprotein receptor and the epidermal growth factor precursor.
Pennica et al., Science 236:83-88
(1987). The function of uromodulin is not known; however, it may function as a
unique renal regulatory
glycoprotein that specifically binds to and regulates the circulating activity
of a number of potent cytokines,
as it binds to IL-1, IL-2 and TNF with high affinity. See Hession et al.,
Science 237:1479-1484 (1987). Su
et al. suggest that uromodulin plays a significant role in the innate immunity
of the urinary system and that the
immunostimulatory activity of uromodulin is potentially useful for
immunotherapy. Suet al., J. Immunology,
158:3449-3456 (1997).
We herein describe the identification and characterization of novel
polypeptides having sequence
similarity to uromodulin, designated herein as PR04999 polypeptides.
16. PR07170
Efforts are being undertaken by both industry and academia to identify new,
native secreted proteins.
Many efforts are focused on the screening of mammalian recombinant DNA
libraries to identify the coding
sequences for novel secreted proteins. We herein describe the identification
and characterization of novel
secreted polypeptides, designated herein as PR07170 polypeptides.
17. PRO248
Cytokines have been implicated in the pathogenesis of a number of brain
diseases in which
neurological dysfuntion has been attributed to a change in amino acid
neurotransmitter metabolism. In
particular, members of the transforming growth factors (TGFB) have been
implicated. Transforming growth
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, TGFB 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.
The TGFB family includes basic myelin proteins (BMP-2, BMP-4, BMP-5, BMP-6,
BMP-7), activins
A & B, decapentaplegic (dpp), 60A, OP-2, dorsalin, growth differentiation
factors (GDFs) 1, 3, and 9, nodal,
MIS, Inhibin a, transforming growth factors betas (TGF-B1, TGF-62, TGF-83, TGF-
B5), and glial-derived
neurotrophic factor (GDNF), Atrisano, et al., J. Biochemica et Biophvsica
Acta. 1222:71-80 (1994). Of
particuar interest are the growth differentiation factors, for as their name
implies, these factors are implicated
in the differentiation of cells.
13
CA 02372511 2001-11-23
WO 00/77037 PCT/US00/14042
Thus, identifying proteins having homology to the TGFB family members,
particularly growth
differentiation factor (GDF) 3, is of importance to the medical and industrial
community. Generally, proteins
having homology to each other have similar function. It is also of interest
when proteins having homology do
not have similar functions, indicating that certain structural motifs identify
information other than function,
such as locality of function.
18. PRO353
The complement proteins comprise a large group of serum proteins some of which
act in an enzymatic
cascade, producing effector molecules involved in inflammation. The complement
proteins are of particular
importance in regulating movement and function of cells involved in
inflammation. Given the physiological
importance of inflammation and related mechanisms in vivo, efforts are
currently being under taken to identify
new, native proteins which are involved in inflamation. We describe herein the
identification and
characterization of novel polypeptides which have homology to complement
proteins, designated herein as
PR0353 polypeptides.
19. PR0533
Growth factors are molecular signals or mediators that enhance cell growth or
proliferation, alone or
in concert, by binding to specific cell surface receptors. however, there are
other cellular reactions than only
growth upon expression to growth factors. As a result, growth factors are
better characterized as
multifunctional and potent cellular regulators. Their biological
effects*include proliferation, chemotaxis and
stimulation of extracellular matrix production. Growth factors can have both
stimulatory and inhibitory effects.
For example, transforming growth factors (TGF-R) is highly pleiotropic and can
stimulate proliferation in some
cells, especially connective tissues, while being a potent inhibitor of
proliferation in others, such as
lymphocytes and epithelial cells.
The physiological effect of growth stimulation or inhibition by growth factors
depends upon the state
of development and differentiation of the target tissue. The mechanism of
local cellular regulation by classical
endocrine molecules comprehends autocrine (same cell), juxtacrine (neighbor
cell), and paracrine (adjacent
cell) pathways. Peptide growth factors are elements of a complex biological
language, providing the basis for
intercellular communication. They permit cells to convey information between
each other, mediate interaction
between cells and change gene expression. the effect of these multifunctional
and pluripotent factors is
dependent on the presence or absence of other peptides.
Fibroblast growth factors (FGFs) are a family of heparin-binding, potent
mitogens for both normal
diploid fibroblasts and established cell lines, Godpodarowicz, D. et at.
(1984), Proc. Natl. Acad. Sci. USA
81: 6983. the FGF family comprises acidic FGF (FGF-1), basic FGF (FGF-2), INT-
2 (FGF-3), K-FGF/HST
(FGF-4), FGF-5, FGF-6, KGF (FGF-7), AIGF (FGF-8) among others. All FGFs have
two conserved
cysteine residues and share 30-50% sequence homology at the amino acid level.
These factors are mitogenic
for a wide variety of normal diploid mesoderm-derived and neural crest-derived
cells, inducing granulosa cells,
adrenal cortical cells, chrondocytes, myoblasts, corneal and vascular
endothelial cells (bovine or human),
14
CA 02372511 2004-05-18
WO 00/77037 PCT/US00/14042
vascular smooth muscle cells, lens, retina and prostatic epithelial cells,
oligodendrocytes, astrocytes,
chrondocytes, myoblasts and osteoblasts.
Fibroblast growth factors can also stimulate a large number of cell types in a
non-mitogenic manner.
These activities include promotion of cell migration into a wound area
(chemotaxis), initiation of new blood
vessel formulation (angiogenesis), modulation of nerve regeneration and
survival (neurotrophism), modulation
of endocrine functions, and stimulation or suppression of specific cellular
protein expression, extracellular
matrix production and cell survival. Baird, A. & Bohlen, P., Handbook of Exp.
Phrrnacol. 25(l): 369-418
(1990). These properties provide a basis for using fibroblast growth factors
in therapeutic approaches to
accelerate wound healing, nerve repair, collateral blood vessel formation, and
the like. For example, fibroblast
growth factors, have been suggested to minimize myocardium damage in heart
disease and surgery (U.S.P.
4,378,437).
We herein describe the identification and characterization of novel
polypeptides having homology to
FGF, herein designated PRO533 polypeptides.
20. PR0301
The widespread occurrence of cancer has prompted the devotion of considerable
resources and
discovering new treatments of treatment. One particular method involves the
creation of tumor or cancer
specific monoclonal antibodies (mAbs) which are specific to tumor antigens.
Such mAbs, which can
distinguish between normal and cancerous cells are useful in the diagnosis,
prognosis and treatment of the
disease. Particular antigens are known to be associated with neoplastic
diseases, such as colorectal cancer.
One particular antigen, the A33 antigen is expressed in more than 90% of
primary or metastatic colon
cancers as well as normal colon epithelium. Since colon cancer is a widespread
disease, early diagnosis and
treatment is an important medical goal. Diagnosis and treatment of colon
cancer can be implemented using
monoclonal antibodies (mAbs) specific therefore having fluorescent, nuclear
magnetic or radioactive tags.
Radioactive gene, toxins and/or drug tagged mAbs can be used for treatment in
situ with minimal patient
description. mAbs can also be used to diagnose during the diagnosis and
treatment of colon cancers. For
example, when the serum levels of the A33 antigen are elevated in a patient, a
drop of the levels after surgery
would indicate the tumor resection was successful. On the other hand, a
subsequent rise in serum A33 antigen
levels after surgery would indicate that metastases of the original tumor may
have formed or that new primary
tumors may have appeared. Such monoclonal antibodies can be used in lieu of,
or in conjunction
with surgery and/or other chemotherapies. For example, U.S.P. 4,579,827 and
(E.P. 199,141) are
directed to therapeutic administration of monoclonal antibodies, the latter of
which relates to the
application of anti-A33 mAb.
Many cancers of epithelial origin have adenovirus receptors. In fact,
adenovirus-derived vectors have
been proposed as a means of inserting antisense nucleic acids into tumors
(U.S.P. 5,518,885). Thus, the
association of viral receptors with neoplastic tumors is not unexpected.
We herein describe the identification and characterization of novel
polypeptides having homology to
certain cancer-associated antigens, designated herein as PRO301 polypeptides.
CA 02372511 2001-11-23
WO 00/77037 PCT/US00/14042
21. PRO187
Growth factors are molecular signals or mediators that enhance cell growth or
proliferation, alone or
in concert, by binding to specific cell surface receptors. However, there are
other cellular reactions than only
growth upon expression to growth factors. As a result, growth factors are
better characterized as
multifunctional and potent cellular regulators. Their biological effects
include proliferation, chemotaxis and
stimulation of extracellular matrix production. Growth factors can have both
stimulatory and inhibitory effects.
For example, transforming growth factor (TGF-(3) is highly pleiotropic and can
stimulate proliferation in some
cells, especially connective tissue, while being a potent inhibitor of
proliferation in others, such as lymphocytes
and epithelial cells.
The physiological effect of growth stimulation or inhibition by growth factors
depends upon the state
of development and differentiation of the target tissue. The mechanism of
local cellular regulation by classical
endocrine molecules involves comprehends autocrine (same cell), juxtacrine
(neighbor cell), and paracrine
(adjacent cells) pathways. Peptide growth factors are elements of a complex
biological language, providing
the basis for intercellular communication. They permit cells to convey
information between each other,
mediate interaction between cells and change gene expression. The effect of
these multifunctional and
pluripotent factors is dependent on the presence or absence of other peptides.
FGF-8 is a member of the fibroblast growth factors (FGFs) which are a family
of heparin-binding,
potent mitogens for both normal diploid fibroblasts and established cell
lines, Gospodarowicz et al. (1984),
Proc. Natl. Acad. Sci. USA 81:6963. The FGF family comprises acidic FGF (FGF-
1), basic FGF (FGF-2),
INT-2 (FGF-3), K-FGF/HST (FGF-4), FGF-5, FGF-6, KGF (FGF-7), AIGF (FGF-8)
among others. All
FGFs have two conserved cysteine residues and share 30-50% sequence homology
at the amino acid level.
These factors are mitogenic for a wide variety of normal diploid mesoderm-
derived and neural crest-derived
cells, including granulosa cells, adrenal cortical cells, chondrocytes,
myoblasts, corneal and vascular
endothelial cells (bovine or human), vascular smooth muscle cells, lens,
retina and prostatic epithelial cells,
oligodendrocytes, astrocytes, chrondocytes, myoblasts and osteoblasts.
Fibroblast growth factors can also stimulate a large number of cell types in a
non-mitogenic manner.
These activities include promotion of cell migration into wound area
(chemotaxis), initiation of new blood
vessel formulation (angiogenesis), modulation of nerve regeneration and
survival (neurotrophism), modulation
of endocrine functions, and stimulation or suppression of specific cellular
protein expression, extracellular
matrix production and cell survival. Baird & Bohlen, Handbook of Exp.
Pharmacol. 95(1): 369-418, Springer,
(1990). These properties provide a basis for using fibroblast growth factors
in therapeutic approaches to
accelerate wound healing, nerve repair, collateral blood vessel formation, and
the like. For example, fibroblast
growth factors have been suggested to minimize myocardium damage in heart
disease and surgery (U.S.P.
4,378,347).
FGF-8, also known as androgen-induced growth factor (AIGF), is a 215 amino
acid protein which
shares 30-40 % sequence homology with the other members of the FGF family. FGF-
8 has been proposed to
be under androgenic regulation and induction in the mouse mammary carcinoma
cell line SC3. Tanaka et al.,
Proc. Natl. Acad. Sci. USA 89: 8928-8932 (1992); Sato et al., J. Steroid
Biochem. Molec. Biol. 47: 91-98
16
CA 02372511 2001-11-23
WO 00/77037 PCT/US00/14042
(1993). As a result, FGF-8 may have a local role in the prostate, which is
known to be an androgen-
responsive organ. FGF-8 can also be oncogenic, as it displays transforming
activity when transfected into NIH-
3T3 fibroblasts. Kouhara et al., Oncogene 9 455-462 (1994). While FGF-8 has
been detected in heart, brain,
lung, kidney, testis, prostate and ovary, expression was also detected in the
absence of exogenous androgens.
Schmitt et at., J. Steroid Biochem. Mol. Biol. 57 (3-4): 173-78 (1996).
FGF-8 shares the property with several other FGFs of being expressed at a
variety of stages of murine
embryogenesis, which supports the theory that the various FGFs have multiple
and perhaps coordinated roles
in differentiation and embryogenesis. Moreover, FGF-8 has also been identified
as a protooncogene that
cooperates with Wnt-1 in the process of mammary tumorigenesis (Shackleford et
al., Proc. Natl. Acad. Sci.
USA 90, 740-744 (1993); Heikinheimo et al., Mech. Dev. 48: 129-138 (1994)).
In contrast to the other FGFs, FGF-8 exists as three protein isoforms, as a
result of alternative splicing
of the primary transcript. Tanaka et al., supra. Normal adult expression of
FGF-8 is weak and confined to
gonadal tissue, however northern blot analysis has indicated that FGF-8 mRNA
is present from day 10 through
day 12 or murine gestation, which suggests that FGF-8 is important to normal
development. Heikinheimo et
al., Mech Dev. 48(2): 129-38 (1994). Further in situ hybridization assays
between day 8 and 16 of gestation
indicated initial expression in the surface ectoderm of the first bronchial
arches, the frontonasal process, the
forebrain and the midbrain-hindbrain junction. At days 10-12, FGF-8 was
expressed in the surface ectoderm
of the forelimb and hindlimb buds, the nasal its and nasopharynx, the
infundibulum and in the telencephalon,
diencephalon and metencephalon. Expression continues in the developing
hindlimbs through day 13 of
gestation, but is undetectable thereafter. The results suggest that FGF-8 has
a unique temporal and spatial
pattern in embryogenesis and suggests a role for this growth factor in
multiple regions of ectodermal
differentiation in the post-gastrulation embryo.
We herein describe the identification of novel poypeptides having homology to
FGF-8, wherein those
polypeptides are heein designated PRO 187 polypeptides.
22. PRO337
Neuronal development in higher vertebrates is characterized by processes that
must successfully
navigate distinct cellular environment en route to their synaptic targets. The
result is a functionally precise
formation of neural circuits. The precision is believed to result form
mechanisms that regulate growth cone
pathfinding and target recognition, followed by latter refinement and
remodeling of such projections by events
that require neuronal activity, Goodman and Shatz, Cell/Neuron [Suppl.]
72(10): 77-98 (1993). It is further
evident that different neurons extend nerve fibers that are biochemically
distinct and rely on specific guidance
cues provided by cell-cell, cell-matrix, and chemotrophic interactions to
reach their appropriate synaptic
targets, Goodman et al., supra.
One particular means by which diversity of the neuronal cell surface may be
generated is through
differential expression of cell surface proteins referred to as cell adhesion
molecules (CAMs). Neuronally
expressed CAMs have been implicated in diverse developmental processes,
including migration of neurons
along radial glial cells, providing permissive or repulsive substrates for
neurite extension, and in promoting
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the selective fasciculation of axons in projectional pathways. Jessel, Neuron
1: 3-13 (1988); Edelman and
Crossin, Annu. Rev. Biochem. 60: 155-190 (1991). Interactions between CAMs
present on the growth cone
membrane and molecules on opposing cell membranes or in the extracellular
matrix are thought to provide the
specific guidance cues that direct nerve fiber outgrowth along appropriate
projectional pathways. Such
interactions are likely to result in the activation of various second
messenger systems within the growth cone
that regulate neurite outgrowth. Doherty and Walsh, Curr. Opin Neurobiol. 2:
595-601 (1992).
In higher vertebrates, most neural CAMs have been found to be members of three
major structural
families of proteins: the integrins, the cadherins, and the immunoglobulin
gene superfamily (IgSF). Jessel,
supra.; Takeichi, Annu. Rev. Biochem. 59: 237-252 (1990); Reichardt and
Tomaselli, Annu. Rev. Neurosci.
14: 531-570 (1991). Cell adhesion molecules of the IgSF (or Ig-CAMs), in
particular, constitute a large family
of proteins frequently implicated in neural cell interactions and nerve fiber
outgrowth during development,
Salzer and Colman, Dev. Neurosci. 11: 377-390 (1989); Brummendorf and Rathjen,
J. Neurochem. 61: 1207-
1219 (1993). However, the majority of mammalian Ig-CAMs appear to be too
widely expressed to specify
navigational pathways or synaptic targets suggesting that other CAMs, yet to
be identified, have role in these
more selective interactions of neurons.
Many of the known neural Ig-CAMs have been found to be attached to the plasma
membrane via a
glycosylphosphatidylinositol (GPI) anchor. Additionally, many studies have
implicated GPI-anchored proteins
in providing specific guidance cues during the outgrowth on neurons in
specific pathways. In studies of the
grasshopper nervous system, treatment of embryos with phosphatidylinositol-
specific phopholipase C (PIPLC),
which selectively removes GPI-anchored proteins from the surfaces of cells,
resulted in misdirection and faulty
navigation among subsets of pioneering growth cones, as well as inhibited
migratory patterns of a subset of
early neurons, Chang et al., Devel. 114: 507-519 (1992). The projection of
retinal fibers to the optic tectum
appears to depend, in part, on a 33 kDa GPI-anchored protein, however, the
precise nature of this protein is
unknown. Stahl et al., Neuron 5: 735-743 (1990).
The expression of various GPI-anchored proteins has been characterized amongst
the different
populations of primary rat neurons amongst dorsal root ganglion, sympathetic
neurons of the cervical ganglion,
sympathetic neurons of the superior cervical ganglion, and cerebellar granule
neurons. Rosen et al., J. Cell
Biol. 117: 617-627 (1992). In contrast to the similar pattern of total
membrane protein expression by these
different types of neurons, striking differences were observed in the
expression of GPI-anchored proteins
between these neurons. Recently, a 65 kDa protein band known as neurotrimin
was discovered and found to
be differentially expressed by primary neurons (Rosen et al., supra), and
restricted to the nervous system and
found to be the most abundant and earliest expressed of the GPI-anchored
species in the CNS. Struyk et al.,
J. Neuroscience 15(3): 2141-2156 (1995). The discovery of neurotrimin has
further lead to the identification
of a family of IgSF members, each containing three Ig-like domains that share
significant amino acid identity,
now termed IgLON. Struyk et al., supra; Pimenta et al., Gene 170(2): 189-95
(1996).
Additional members of the IgLON subfamily include opiate binding cell adhesion
molecule (OBCAM),
Schofield et al., EMBO J. 8: 489-495 (1989); limbic associated membrane
protein (LAMP), Pimenta et al.,
supra; CEPU-1; GP55, Wilson et al., J. Cell Sci. 109: 3129-3138 (1996); Eur.
J. Neurosci. 9(2): 334-41
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(1997); and AvGp50, Hancox et al., Brain Res. Mol. Brain Res. 44(2): 273-85
(1997).
While the expression of neurotrimin appears to be widespread, it does appear
to correlated with the
development of several neural circuits. For example, between E18 and P10,
neurotimin mRNA expression
within the forebrain is maintained at high levels in neurons of the developing
thalamus, cortical subplate, and
cortex, particularly laminae V and VI (with less intense expression in II, 11,
and IV, and minimal expression
in lamina 1). Cortical subplate neurons may provide an early, temporary
scaffold for the ingrowing thalamic
afferents en route to their final synaptic targets in the cortex. Allendoerfer
and Shatz, Annu. Rev. Neurosci.
17: 185-218 (1994). Conversely, subplate neurons have been suggested to be
required for cortical neurons
from layer V to select VI to grow into the thalamus, and neurons from layer V
to select their targets in the
colliculus, pons, and spinal cord (McConnell et al., J. Neurosci. 14: 1892-
1907 (1994). The high level
expression of neurotrimin in many of these projections suggests that it could
be involved in their development.
In the hindbrain, high levels of neurotrimin message expression were observed
within the pontine
nucleus and by the internal granule cells and Purkinje cells of the
cerebellum. The pontine nucleus received
afferent input from a variety of sources including corticopontine fibers of
layer V, and is a major source of
afferent input, via mossy fibers, to the granule cells which, in turn, are a
major source of afferent input via
parallel fibers to Purkinje cells. [Palay and Chan-Palay, The cerebellar
cortex: cytology and organization.
New York: Springer (1974]. High level expression of neurotrimin these neurons
again suggests potential
involvement in the establishment of these circuits.
Neurotrimin also exhibits a graded expression pattern in the early postnatal
striatum. Increased
neurotrimin expression is found overlying the dorsolateral striatum of the
rat, while lesser hybridization
intensity is seen overlying the ventromedial striatum. Struyk et al., supra.
This region of higher neurotrimin
hybridization intensity does not correspond to a cytoarchitecturally
differentiable region, rather it corresponds
to the primary area of afferent input from layer VI of the contralateral
sensorimotor cortex (Gerfen, Nature
311: 461-464 (1984); Donoghue and Herkenham, Brain Res. 365: 397-403 (1986)).
The ventromedial
striatum, by contrast, receives the majority of its afferent input from the
perirhinal and association cortex. It
is noteworthy that a complementary graded pattern of LAMP expression, has been
observed within the
striatium, with highest expression in ventromedial regions, and lowest
expression dorsolaterally. Levitt,
Science 223: 299-301 (1985); Chesselet et al., Neuroscience 40: 725-733
(1991).
23. PRO1411
Efforts are being undertaken by both industry and academia to identify new,
native secreted proteins.
Many efforts are focused on the screening of mammalian recombinant DNA
libraries to identify the coding
sequences for novel secreted proteins. We herein describe the identification
and characterization of a novel
secreted protein designated herein as PRO 1411.
24. PRO4356
Glycosylphosphatidylinositol (GPI) anchored proteoglycans are generally
localized to the cell surface
and are thus known to be involved in the regulation of responses of cells to
numerous growth factors, cell
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adhesion molecules and extracellular matrix components. The metastasis-
associated GPI-anchored protein
(MAGPIAP) is one of these cell surface proteins which appears to be involved
in metastasis. Metastasis is the
form of cancer wherein the transformed or malignant cells are traveling and
spreading the cancer from one site
to another. Therefore, identifying the polypeptides related to metastasis and
MAGPIAP is of interest.
25. PRO246
The cell surface protein HCAR is a membrane-bound protein that acts as a
receptor for subgroup C
of the adenoviruses and subgroup B of the coxsackieviruses. Thus, HCAR may
provide a means for mediating
viral infection of cells in that the presence of the HCAR receptor on the
cellular surface provides a binding site
for viral particles, thereby facilitating viral infection.
In light of the physiological importance of membrane-bound proteins and
specficially those which
serve a cell surface receptor for viruses, efforts are currently being
undertaken by both industry and academia
to identify new, native membrane-bound receptor proteins. Many of these
efforts are focused on the screening
of mammalian recombinant DNA libraries to identify the coding sequences for
novel receptor proteins. We
herein describe a novel membrane-bound polypeptide (designated herein as
PR0246) having homology to the
cell surface protein HCAR and to various tumor antigens including A33 and
carcinoembryonic antigen,
wherein this polypeptide may be a novel cell surface virus receptor or tumor
antigen.
26. PRO265
Protein-protein interactions include receptor and antigen complexes and
signaling mechanisms. As
more is known about the structural and functional mechanisms underlying
protein-protein interactions, protein-
protein interactions can be more easily manipulated to regulate the particular
result of the protein-protein
interaction. Thus, the underlying mechanisms of protein-protein interactions
are of interest to the scientific
and medical community.
All proteins containing leucine-rich repeats are thought to be involved in
protein-protein interactions.
Leucine-rich repeats are short sequence motifs present in a number of proteins
with diverse functions and
cellular locations. The crystal structure of ribonuclease inhibitor protein
has revealed that leucine-rich repeats
correspond to beta-alpha structural units. These units are arranged so that
they form a parallel beta-sheet with
one surface exposed to solvent, so that the protein acquires an unusual,
nonglubular shape. These two features
have been indicated as responsible for the protein-binding functions of
proteins containing leucine-rich repeats.
See, Kobe and Deisenhofer, Trends Biochem. Sci., 19(10):415-421 (Oct. 1994).
A study has been reported on leucine-rich proteoglycans which serve as tissue
organizers, orienting
and ordering collagen fibrils during ontogeny and are involved in pathological
processes such as wound
healing, tissue repair, and tumor stroma formation. Iozzo, R. V., Crit. Rev.
Biochem. Mol. Biol., 32(2):141-
174 (1997). Others studies implicating leucine rich proteins in wound healing
and tissue repair are De La
Salle, C., et al., Vouv. Rev. Fr. Hematol. (Germany), 37(4):215-222 (1995),
reporting mutations in the
leucine rich motif in a complex associated with the bleeding disorder Bernard-
Soulier syndrome and
Chlemetson, K. J., Thromb. Haemost. (Germany), 74(1):111-116 (July 1995),
reporting that platelets have
CA 02372511 2001-11-23
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leucine rich repeats. Another protein of particular interest which has been
reported to have leucine-rich repeats
is the SLIT protein which has been reported to be useful in treating neuro-
degenerative diseases such as
Alzheimer's disease, nerve damage such as in Parkinson's disease, and for
diagnosis of cancer, see,
Artavanistsakonas, S. and Rothberg, J. M., W09210518-A1 by Yale University.
Other studies reporting on
the biological functions of proteins having leucine-rich repeats include:
Tayar, N., et al., Mol. Cell
Endocrinol., (Ireland), 125(1-2):65-70 (Dec. 1996) (gonadotropin receptor
involvement); Miura, Y., et al.,
Nippon Rinsho (Japan), 54(7):1784-1789 (July 1996) (apoptosis involvement);
Harris, P. C., et al., J. Am.
Soc. Nephrol., 6(4):1125-1133 (Oct. 1995) (kidney disease involvement); and
Ruoslahti, E. I., et al.,
W09110727-A by La Jolla Cancer Research Foundation (decorin binding to
transforming growth factor-R
involvement for treatment for cancer, wound healing and scarring). Also of
particular interest is fibromodulin
and its use to prevent or reduce dermal scarring. A study of fibromodulin is
found in U.S. Patent No.
5,654,270 to Ruoslahti, et al.
Efforts are therefore being undertaken by both industry and academia to
identify new proteins having
leucine rich repeats to better understand protein-protein interactions. Of
particular interest are those proteins
having leucine rich repeats and homology to known proteins having leucine rich
repeats such as fibromodulin,
the SLIT protein and platelet glycoprotein V. Many efforts are focused on the
screening of mammalian
recombinant DNA libraries to identify the coding sequences for novel secreted
and membrane-bound proteins
having leucine rich repeats. We herein describe the identification and
characterization of novel polypeptides
having homology to fibromodulin, herein designated as PRO265 polypeptides.
27. PRO941
Cadherins are a large family of transmembrane proteins. Cadherins comprise a
family of calcium-
dependent glycoproteins that function in mediating cell-cell adhesion in
virtually all solid tissues of multicellular
organisms. At least cadherins 1-13 as well as types B, E, EP, M, N, P and R
have been identified and
characterized. Among the functions cadherins are known for, with some
exceptions, are that cadherins
participate in cell aggregation and are associated with cell-cell adhesion
sites. Recently, it has been reported
that while all cadherins share multiple repeats of a cadherin specific motif
believed to correspond to folding
of extracellular domains, members of the cadherin superfamily have divergent
structures and, possibly,
functions. In particular it has been reported that members of the cadherin
superfamily are involved in signal
transduction. See, Suzuki, J. Cell Biochem., 61(4):531-542 (1996). Cadherins
are further described in
Tanihara et al., J. Cell Sci., 107(6):1697-1704 (1994), Aberle et al., J. Cell
Biochem., 61(4):514-523 (1996)
and Tanihara et al., Cell Adhes. Commun., 2(1):15-26 (1994). We herein
describe the identification and
characterization of a novel polypeptide having homology to a cadherin protein,
designated herein as PRO941.
28. PRO10096
Interleukin-10 (IL-10) is a pleiotropic immunosuppressive cytokine that has
been implicated as an
important regulator of the functions of myeloid and lymphoid cells. It has
been demonstrated that IL-10
functions as a potent inhibitor of the activation of the synthesis of various
inflammatory cytokines including,
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for example, IL-1, IL-6, IFN-y and TNF-a (Gesser et al., Proc. Natl. Acad.
Sci. USA 94:14620-14625
(1997)). Moreover, IL-10 has been demonstrated to strongly inhibit several of
the accessory activities of
macrophages, thereby functioning as a potent suppressor of the effector
functions of macrophages, T-cells and
NK cells (Kuhn et al., Cell 75:263-274 (1993)). Furthermore, IL-10 has been
strongly implicated in the
regulation of B-cell, mast cell and thymocyte differentiation.
IL-10 was independently identified in two separate lines of experiments.
First, cDNA clones encoding
murine IL-10 were identified based upon the expression of cytokine synthesis
inhibitory factor (Moore et al.,
Science 248:1230-1234 (1990)), wherein the human IL-10 counterpart cDNAs were
subsequently identified
by cross-hybridization with the murine IL-10 cDNA (Viera et al., Proc. Natl.
Acad. Sci. USA 88:1172-1176
(1991)). Additionally, IL-10 was independently identified as a B-cell-derived
mediator which functioned to
co-stimulate active thymocytes (Suda et al., Cell Immunol. 129:228 (1990)).
We herein describe the identification and characterization of novel
polypeptides having sequence
similarity to IL-10, designated herein as PRO10096 polypeptides.
29. PR06003
Efforts are being undertaken by both industry and academia to identify new,
native receptor or
membrane-bound proteins. Many efforts are focused on the screening of
mammalian recombinant DNA
libraries to identify the coding sequences for novel receptor or membrane-
bound proteins. We herein describe
the identification and characterization of novel polypeptides designated
herein as PR06003 polypeptides.
SUMMARY OF THE INVENTION
In one embodiments of the present invention, the invention provides vectors
comprising DNA
encoding any of the herein described polypeptides. Host cell comprising any
such vector are also provided.
By way of example, the host cells may be CHO cells, E. coli, or yeast. A
process for producing any of the
herein described polypeptides is further provided and comprises culturing host
cells under conditions suitable
for expression of the desired polypeptide and recovering the desired
polypeptide from the cell culture.
In other embodiments, the invention provides chimeric molecules comprising any
of the herein
described polypeptides fused to a heterologous polypeptide or amino acid
sequence. Example of such chimeric
molecules comprise any of the herein described polypeptides fused to an
epitope tag sequence or a Fc region
of an immunoglobulin.
In another embodiment, the invention provides an antibody which specifically
binds to any of the
above or below described polypeptides. Optionally, the antibody is a
monoclonal antibody, humanized
antibody, antibody fragment or single-chain antibody.
In yet other embodiments, the invention provides oligonucleotide probes useful
for isolating genomic
and cDNA nucleotide sequences or as antisense probes, wherein those probes may
be derived from any of the
above or below described nucleotide sequences.
In other embodiments, the invention provides an isolated nucleic acid molecule
comprising a
nucleotide sequence that encodes a PRO polypeptide.
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In one aspect, the isolated nucleic acid molecule comprises a nucleotide
sequence having at least about
80% nucleic acid sequence identity, alternatively at least about 81% nucleic
acid sequence identity,
alternatively at least about 82 % nucleic acid sequence identity,
alternatively at least about 83 % nucleic acid
sequence identity, alternatively at least about 84% nucleic acid sequence
identity, alternatively at least about
85% nucleic acid sequence identity, alternatively at least about 86% nucleic
acid sequence identity,
alternatively at least about 87% nucleic acid sequence identity, alternatively
at least about 88% nucleic acid
sequence identity, alternatively at least about 89% nucleic acid sequence
identity, alternatively at least about
90% nucleic acid sequence identity, alternatively at least about 91% nucleic
acid sequence identity,
alternatively at least about 92% nucleic acid sequence identity, alternatively
at least about 93% nucleic acid
sequence identity, alternatively at least about 94% nucleic acid sequence
identity, alternatively at least about
95% nucleic acid sequence identity, alternatively at least about 96% nucleic
acid sequence identity,
alternatively at least about 97% nucleic acid sequence identity, alternatively-
at least about 98% nucleic acid
sequence identity and alternatively at least about 99% nucleic acid sequence
identity to (a) a DNA molecule
encoding a PRO polypeptide having a full-length amino acid sequence as
disclosed herein, an amino acid
sequence lacking the signal peptide as disclosed herein, an extracellular
domain of a transmembrane protein,
with or without the signal peptide, as disclosed herein or any other
specifically defined fragment of the full-
length amino acid sequence as disclosed herein, or (b) the complement of the
DNA molecule of (a).
In other aspects, the isolated nucleic acid molecule comprises a nucleotide
sequence having at least
about 80 % nucleic acid sequence identity, alternatively at least about 81 %
nucleic acid sequence identity,
alternatively at least about 82% nucleic acid sequence identity, alternatively
at least about 83% nucleic acid
sequence identity, alternatively at least about 84% nucleic acid sequence
identity, alternatively at least about
85% nucleic acid sequence identity, alternatively at least about 86% nucleic
acid sequence identity,
alternatively at least about 87% nucleic acid sequence identity, alternatively
at least about 88% nucleic acid
sequence identity, alternatively at least about 89% nucleic acid sequence
identity, alternatively at least about
90 % nucleic acid sequence identity, alternatively at least about 91 % nucleic
acid sequence identity,
alternatively at least about 92% nucleic acid sequence identity, alternatively
at least about 93% nucleic acid
sequence identity, alternatively at least about 94 % nucleic acid sequence
identity, alternatively at least about
95% nucleic acid sequence identity, alternatively at least about 96% nucleic
acid sequence identity,
alternatively at least about 97 % nucleic acid sequence identity,
alternatively at least about 98 % nucleic acid
sequence identity and alternatively at least about 99% nucleic acid sequence
identity to (a) a DNA molecule
comprising the coding sequence of a full-length PRO polypeptide cDNA as
disclosed herein, the coding
sequence of a PRO polypeptide lacking the signal peptide as disclosed herein,
the coding sequence of an
extracellular domain of a transmembrane PRO polypeptide, with or without the
signal peptide, as disclosed
herein or the coding sequence of any other specifically defined fragment of
the full-length amino acid sequence
as disclosed herein, or (b) the complement of the DNA molecule of (a).
In a further aspect, the invention concerns an isolated nucleic acid molecule
comprising a nucleotide
sequence having at least about 80 % nucleic acid sequence identity,
alternatively at least about 81 % nucleic acid
sequence identity, alternatively at least about 82% nucleic acid sequence
identity, alternatively at least about
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83 % nucleic acid sequence identity, alternatively at least about 84 % nucleic
acid sequence identity,
alternatively at least about 85% nucleic acid sequence identity, alternatively
at least about 86% nucleic acid
sequence identity, alternatively at least about 87% nucleic acid sequence
identity, alternatively at least about
88% nucleic acid sequence identity, alternatively at least about 89% nucleic
acid sequence identity,
alternatively at least about 90% nucleic acid sequence identity, alternatively
at least about 91 % nucleic acid
sequence identity, alternatively at least about 92% nucleic acid sequence
identity, alternatively at least about
93% nucleic acid sequence identity, alternatively at least about 94% nucleic
acid sequence identity,
alternatively at least about 95 % nucleic acid sequence identity,
alternatively at least about 96 % nucleic acid
sequence identity, alternatively at least about 97 % nucleic acid sequence
identity, alternatively at least about
98 % nucleic acid sequence identity and alternatively at least about 99 %
nucleic acid sequence identity to (a)
a DNA molecule that encodes the same mature polypeptide encoded by any of the
human protein cDNAs
deposited with the ATCC as disclosed herein, or (b) the complement of the DNA
molecule of (a).
Another aspect the invention provides an isolated nucleic acid molecule
comprising a nucleotide
sequence encoding a PRO polypeptide which is either transmembrane domain-
deleted or transmembrane
domain-inactivated, or is complementary to such encoding nucleotide sequence,
wherein the transmembrane
domain(s) of such polypeptide are disclosed herein. Therefore, soluble
extracellular domains of the herein
described PRO polypeptides are contemplated.
Another embodiment is directed to fragments of a PRO polypeptide coding
sequence, or the
complement thereof, that may find use as, for example, hybridization probes,
for encoding fragments of a PRO
polypeptide that may optionally encode a polypeptide comprising a binding site
for an anti-PRO antibody or
as antisense oligonucleotide probes. Such nucleic acid fragments are usually
at least about 20 nucleotides in
length, alternatively at least about 30 nucleotides in length, alternatively
at least about 40 nucleotides in length,
alternatively at least about 50 nucleotides in length, alternatively at least
about 60 nucleotides in length,
alternatively at least about 70 nucleotides in length, alternatively at least
about 80 nucleotides in length,
alternatively at least about 90 nucleotides in length, alternatively at least
about 100 nucleotides in length,
alternatively at least about 110 nucleotides in length, alternatively at least
about 120 nucleotides in length,
alternatively at least about 130 nucleotides in length, alternatively at least
about 140 nucleotides in length,
alternatively at least about 150 nucleotides in length, alternatively at least
about 160 nucleotides in length,
alternatively at least about 170 nucleotides in length, alternatively at least
about 180 nucleotides in length,
alternatively at least about 190 nucleotides in length, alternatively at least
about 200 nucleotides in length,
alternatively at least about 250 nucleotides in length, alternatively at least
about 300 nucleotides in length,
alternatively at least about 350 nucleotides in length, alternatively at least
about 400 nucleotides in length,
alternatively at least about 450 nucleotides in length, alternatively at least
about 500 nucleotides in length,
alternatively at least about 600 nucleotides in length, alternatively at least
about 700 nucleotides in length,
alternatively at least about 800 nucleotides in length, alternatively at least
about 900 nucleotides in length and
alternatively at least about 1000 nucleotides in length, wherein in this
context the term "about" means the
referenced nucleotide sequence length plus or minus 10% of that referenced
length. It is noted that novel
fragments of a PRO polypeptide-encoding nucleotide sequence may be determined
in a routine manner by
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aligning the PRO polypeptide-encoding nucleotide sequence with other known
nucleotide sequences using any
of a number of well known sequence alignment programs and determining which
PRO polypeptide-encoding
nucleotide sequence fragment(s) are novel. All of such PRO polypeptide-
encoding nucleotide sequences are
contemplated herein. Also contemplated are the PRO polypeptide fragments
encoded by these nucleotide
molecule fragments, preferably those PRO polypeptide fragments that comprise a
binding site for an anti-PRO
antibody.
In another embodiment, the invention provides isolated PRO polypeptide encoded
by any of the
isolated nucleic acid sequences hereinabove identified.
In a certain aspect, the invention concerns an isolated PRO polypeptide,
comprising an amino acid
sequence having at least about 80 % amino acid sequence identity,
alternatively at least about 81 % amino acid
sequence identity, alternatively at least about 82% amino acid sequence
identity, alternatively at least about
83 % amino acid sequence identity, alternatively at least about 84 % amino
acid sequence identity, alternatively
at least about 85 % amino acid sequence identity, alternatively at least about
86 % amino acid sequence identity,
alternatively at least about 87 % amino acid sequence identity, alternatively
at least about 88 % amino acid
sequence identity, alternatively at least about 89% amino acid sequence
identity, alternatively at least about
90 % amino acid sequence identity, alternatively at least about 91 % amino
acid sequence identity, alternatively
at least about 92 % amino acid sequence identity, alternatively at least about
93 % amino acid sequence identity,
alternatively at least about 94% amino acid sequence identity, alternatively
at least about 95% amino acid
sequence identity, alternatively at least about 96% amino acid sequence
identity, alternatively at least about
97% amino acid sequence identity, alternatively at least about 98% amino acid
sequence identity and
alternatively at least about 99 % amino acid sequence identity to a PRO
polypeptide having a full-length amino
acid sequence as disclosed herein, an amino acid sequence lacking the signal
peptide as disclosed herein, an
extracellular domain of a transmembrane protein, with or without the signal
peptide, as disclosed herein or any
other specifically defined fragment of the full-length amino acid sequence as
disclosed herein.
In a further aspect, the invention concerns an isolated PRO polypeptide
comprising an amino acid
sequence having at least about 80% amino acid sequence identity, alternatively
at least about 81 % amino acid
sequence identity, alternatively at least about 82% amino acid sequence
identity, alternatively at least about
83 % amino acid sequence identity, alternatively at least about 84 % amino
acid sequence identity, alternatively
at least about 85 % amino acid sequence identity, alternatively at least about
86 % amino acid sequence identity,
alternatively at least about 87% amino acid sequence identity, alternatively
at least about 88% amino acid
sequence identity, alternatively at least about 89% amino acid sequence
identity, alternatively at least about
90 % amino acid sequence identity, alternatively at least about 91 % amino
acid sequence identity, alternatively
at least about 92 % amino acid sequence identity, alternatively at least about
93 % amino acid sequence identity,
alternatively at least about 94 % amino acid sequence identity, alternatively
at least about 95 % amino acid
sequence identity, alternatively at least about 96% amino acid sequence
identity, alternatively at least about
97 % amino acid sequence identity, alternatively at least about 98 % amino
acid sequence identity and
alternatively at least about 99% amino acid sequence identity to an amino acid
sequence encoded by any of the
human protein cDNAs deposited with the ATCC as disclosed herein.
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In a further aspect, the invention concerns an isolated PRO polypeptide
comprising an amino acid
sequence scoring at least about 80% positives, alternatively at least about 81
% positives, alternatively at least
about 82% positives, alternatively at least about 83% positives, alternatively
at least about 84% positives,
alternatively at least about 85 % positives, alternatively at least about 86 %
positives, alternatively at least about
87% positives, alternatively at least about 88% positives, alternatively at
least about 89% positives,
alternatively at least about 90 % positives, alternatively at least about 91 %
positives, alternatively at least about
92% positives, alternatively at least about 93% positives, alternatively at
least about 94% positives,
alternatively at least about 95 % positives, alternatively at least about 96 %
positives, alternatively at least about
97 % positives, alternatively at least about 98 % positives and alternatively
at least about 99 % positives when
compared with the amino acid sequence of a PRO polypeptide having a full-
length amino acid sequence as
disclosed herein, an amino acid sequence lacking the signal peptide as
disclosed herein, an extracellular domain
of a transmembrane protein, with or without the signal peptide, as disclosed
herein or any other specifically
defined fragment of the full-length amino acid sequence as disclosed herein.
In a specific aspect, the invention provides an isolated PRO polypeptide
without the N-terminal signal
sequence and/or the initiating methionine and is encoded by a nucleotide
sequence that encodes such an amino
acid sequence as hereinbefore described. Processes for producing the same are
also herein described, wherein
those processes comprise culturing a host cell comprising a vector which
comprises the appropriate encoding
nucleic acid molecule under conditions suitable for expression of the PRO
polypeptide and recovering the PRO
polypeptide from the cell culture.
Another aspect the invention provides an isolated PRO polypeptide which is
either transmembrane
domain-deleted or transmembrane domain-inactivated. Processes for producing
the same are also herein
described, wherein those processes comprise culturing a host cell comprising a
vector which comprises the
appropriate encoding nucleic acid molecule under conditions suitable for
expression of the PRO polypeptide
and recovering the PRO polypeptide from the cell culture.
In yet another embodiment, the invention concerns agonists and antagonists of
a native PRO
polypeptide as defined herein. In a particular embodiment, the agonist or
antagonist is an anti-PRO antibody
or a small molecule.
In a further embodiment, the invention concerns a method of identifying
agonists or antagonists to a
PRO polypeptide which comprise contacting the PRO polypeptide with a candidate
molecule and monitoring
a biological activity mediated by said PRO polypeptide. Preferably, the PRO
polypeptide is a native PRO
polypeptide.
In a still further embodiment, the invention concerns a composition of matter
comprising a PRO
polypeptide, or an agonist or antagonist of a PRO polypeptide as herein
described, or an anti-PRO antibody,
in combination with a carrier. Optionally, the carrier is a pharmaceutically
acceptable carrier.
Another embodiment of the present invention is directed to the use of a PRO
polypeptide, or an
agonist or antagonist thereof as hereinbefore described, or an anti-PRO
antibody, for the preparation of a
medicament useful in the treatment of a condition which is responsive to the
PRO polypeptide, an agonist or
antagonist thereof or an anti-PRO antibody.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a nucleotide sequence (SEQ ID NO:3) of a native sequence PRO
196 cDNA, wherein
SEQ ID NO:3 is a clone designated herein as "DNA22779-1130".
Figure 2 shows the amino acid sequence (SEQ ID NO:4) derived from the coding
sequence of SEQ
ID NO:3 shown in Figure 1.
Figure 3 shows a nucleotide sequence (SEQ ID NO:8) of a native sequence PRO444
cDNA, wherein
SEQ ID NO:8 is a clone designated herein as "DNA26846-1397".
Figure 4 shows the amino acid sequence (SEQ ID NO:9) derived from the coding
sequence of SEQ
ID NO:8 shown in Figure 3.
Figure 5 shows a nucleotide sequence (SEQ ID NO: 10) of a native sequence
PRO183 cDNA, wherein
SEQ ID NO: 10 is a clone designated herein as "DNA28498".
Figure 6 shows the amino acid sequence (SEQ ID NO: 11) derived from the coding
sequence of SEQ
ID NO:10 shown in Figure 5.
Figure 7 shows a nucleotide sequence (SEQ ID NO: 12) of a native sequence PRO
185 cDNA, wherein
SEQ ID NO: 12 is a clone designated herein as "DNA28503".
Figure 8 shows the amino acid sequence (SEQ ID NO: 13) derived from the coding
sequence of SEQ
ID NO:12 shown in Figure 7.
Figure 9 shows a nucleotide sequence (SEQ ID NO: 14) of a native sequence
PRO210 cDNA, wherein
SEQ ID NO: 14 is a clone designated herein as "DNA32279-1131".
Figure 10 shows the amino acid sequence (SEQ ID NO: 15) derived from the
coding sequence of SEQ
ID NO:14 shown in Figure 9.
Figure 11 shows a nucleotide sequence (SEQ ID NO: 16) of a native sequence
PRO215 cDNA,
wherein SEQ ID NO: 16 is a clone designated herein as "DNA32288-1132".
Figure 12 shows the amino acid sequence (SEQ ID NO: 17) derived from the
coding sequence of SEQ
ID NO: 16 shown in Figure 11.
Figure 13 shows a nucleotide sequence (SEQ ID NO:21) of a native sequence
PRO217 cDNA,
wherein SEQ ID NO:21 is a clone designated herein as "DNA33094-1131".
Figure 14 shows the amino acid sequence (SEQ ID NO:22) derived from the coding
sequence of SEQ
ID NO:21 shown in Figure 13.
Figure 15 shows a nucleotide sequence (SEQ ID NO:23) of a native sequence
PRO242 cDNA,
wherein SEQ ID NO:23 is a clone designated herein as "DNA33785-1143".
Figure 16 shows the amino acid sequence (SEQ ID NO:24) derived from the coding
sequence of SEQ
ID NO:23 shown in Figure 15.
Figure 17 shows a nucleotide sequence (SEQ ID NO:28) of a native sequence
PRO288 cDNA,
wherein SEQ ID NO:28 is a clone designated herein as "DNA35663-1129".
Figure 18 shows the amino acid sequence (SEQ ID NO:29) derived from the coding
sequence of SEQ
ID NO:28 shown in Figure 17.
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Figure 19 shows a nucleotide sequence (SEQ ID NO:31) of a native sequence
PR0365 cDNA,
wherein SEQ ID NO:31 is a clone designated herein as "DNA46777-1253".
Figure 20 shows the amino acid sequence (SEQ ID NO:32) derived from the coding
sequence of SEQ
ID NO:31 shown in Figure 19.
Figure 21 shows a nucleotide sequence (SEQ ID NO:38) of a native sequence
PRO1361 cDNA,
wherein SEQ ID NO:38 is a clone designated herein as "DNA60783-161 1".
Figure 22 shows the amino acid sequence (SEQ ID NO:39) derived from the coding
sequence of SEQ
ID NO:38 shown in Figure 21.
Figure 23 shows a nucleotide sequence (SEQ ID NO:40) of a native sequence
PRO1308 cDNA,
wherein SEQ ID NO:40 is a clone designated herein as "DNA62306-1570".
Figure 24 shows the amino acid sequence (SEQ ID NO:41) derived from the coding
sequence of SEQ
ID NO:40 shown in Figure 23.
Figure 25 shows a nucleotide sequence (SEQ ID NO:51) of a native sequence PRO
1183 cDNA,
wherein SEQ ID NO:51 is a clone designated herein as "DNA62880-1513".
Figure 26 shows the amino acid sequence (SEQ ID NO:52) derived from the coding
sequence of SEQ
ID NO:51 shown in Figure 25.
Figure 27 shows a nucleotide sequence (SEQ ID NO:53) of a native sequence
PRO1272 cDNA,
wherein SEQ ID NO:53 is a clone designated herein as "DNA64896-1539".
Figure 28 shows the amino acid sequence (SEQ ID NO:54) derived from the coding
sequence of SEQ
ID NO:53 shown in Figure 27.
Figure 29 shows a nucleotide sequence (SEQ ID NO:55) of a native sequence
PRO1419 cDNA,
wherein SEQ ID NO:55 is a clone designated herein as "DNA71290-1630".
Figure 30 shows the amino acid sequence (SEQ ID NO:56) derived from the coding
sequence of SEQ
ID NO:55 shown in Figure 29.
Figure 31 shows a nucleotide sequence (SEQ ID NO:57) of a native sequence
PR04999 cDNA,
wherein SEQ ID NO:57 is a clone designated herein as "DNA96031-2664".
Figure 32 shows the amino acid sequence (SEQ ID NO:58) derived from the coding
sequence of SEQ
ID NO:57 shown in Figure 31.
Figure 33 shows a nucleotide sequence (SEQ ID NO:62) of a native sequence
PR07170 cDNA,
wherein SEQ ID NO:62 is a clone designated herein as "DNA108722-2743".
Figure 34 shows the amino acid sequence (SEQ ID NO:63) derived from the coding
sequence of SEQ
ID NO:62 shown in Figure 33.
Figure 35 shows a nucleotide sequence (SEQ ID NO:64) of a native sequence
PR0248 cDNA,
wherein SEQ ID NO:64 is a clone designated herein as "DNA35674-1142".
Figure 36 shows the amino acid sequence (SEQ ID NO:65) derived from the coding
sequence of SEQ
ID NO:64 shown in Figure 35.
Figure 37 shows a nucleotide sequence (SEQ ID NO:72) of a native sequence
PR0353 cDNA,
wherein SEQ ID NO:72 is a clone designated herein as "DNA41234".
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Figure 38 shows the amino acid sequence (SEQ ID NO:73) derived from the coding
sequence of SEQ
ID NO:72 shown in Figure 37.
Figure 39 shows a nucleotide sequence (SEQ ID NO:77) of a native sequence
PRO1318 cDNA,
wherein SEQ ID NO:77 is a clone designated herein as "DNA73838-1674".
Figure 40 shows the amino acid sequence (SEQ ID NO:78) derived from the coding
sequence of SEQ
ID NO:77 shown in Figure 39.
Figure 41 shows a nucleotide sequence (SEQ ID NO:79) of a native sequence
PRO1600 cDNA,
wherein SEQ ID NO:79 is a clone designated herein as "DNA77503-1686".
Figure 42 shows the amino acid sequence (SEQ ID NO:80) derived from the coding
sequence of SEQ
ID NO:79 shown in Figure 41.
Figure 43 shows a nucleotide sequence (SEQ ID NO:83) of a native sequence
PR09940 cDNA,
wherein SEQ ID NO:83 is a clone designated herein as "DNA92282".
Figure 44 shows the amino acid sequence (SEQ ID NO:84) derived from the coding
sequence of SEQ
ID NO: 83 shown in Figure 43.
Figure 45 shows a nucleotide sequence (SEQ ID NO:85) of a native sequence
PR0533 cDNA,
wherein SEQ ID NO:85 is a clone designated herein as "DNA49435-1219".
Figure 46 shows the amino acid sequence (SEQ ID NO:86) derived from the coding
sequence of SEQ
ID NO:85 shown in Figure 45.
Figure 47 shows a nucleotide sequence (SEQ ID NO:90) of a native sequence
PR0301 cDNA,
wherein SEQ ID NO:90 is a clone designated herein as "DNA40628-1216".
Figure 48 shows the amino acid sequence (SEQ ID NO:91) derived from the coding
sequence of SEQ
ID NO:90 shown in Figure 47.
Figure 49 shows a nucleotide sequence (SEQ ID NO:98) of a native sequence
PRO187 cDNA,
wherein SEQ ID NO:98 is a clone designated herein as "DNA27864-1155".
Figure 50 shows the amino acid sequence (SEQ ID NO:99) derived from the coding
sequence of SEQ
ID NO:98 shown in Figure 49.
Figure 51 shows a nucleotide sequence (SEQ ID NO: 103) of a native sequence
PR0337 cDNA,
wherein SEQ ID NO: 103 is a clone designated herein as "DNA43316-1237".
Figure 52 shows the amino acid sequence (SEQ ID NO: 104) derived from the
coding sequence of SEQ
ID NO: 103 shown in Figure 51.
Figure 53 shows a nucleotide sequence (SEQ ID NO: 105) of a native sequence
PRO 1411 cDNA,
wherein SEQ ID NO: 105 is a clone designated herein as "DNA59212-1627".
Figure 54 shows the amino acid sequence (SEQ ID NO: 106) derived from the
coding sequence of SEQ
ID NO: 105 shown in Figure 53.
Figure 55 shows a nucleotide sequence (SEQ ID NO: 107) of a native sequence
PR04356 cDNA,
wherein SEQ ID NO: 107 is a clone designated herein as "DNA86576-2595".
Figure 56 shows the amino acid sequence (SEQ ID NO: 108) derived from the
coding sequence of SEQ
ID NO:107 shown in Figure 55.
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Figure 57 shows a nucleotide sequence (SEQ ID NO: 109) of a native sequence
PR0246 cDNA,
wherein SEQ ID NO: 109 is a clone designated herein as "DNA35639-1172".
Figure 58 shows the amino acid sequence (SEQ ID NO: 110) derived from the
coding sequence of SEQ
ID NO:109 shown in Figure 57.
Figure 59 shows a nucleotide sequence (SEQ ID NO: 114) of a native sequence
PR0265 cDNA,
wherein SEQ ID NO:114 is a clone designated herein as "DNA36350-1158".
Figure 60 shows the amino acid sequence (SEQ ID NO: 115) derived from the
coding sequence of SEQ
ID NO: 114 shown in Figure 59.
Figure 61 shows a nucleotide sequence (SEQ ID NO: 120) of a native sequence
PR0941 cDNA,
wherein SEQ ID NO: 120 is a clone designated herein as "DNA53906-1368".
Figure 62 shows the amino acid sequence (SEQ ID NO: 121) derived from the
coding sequence of SEQ
ID NO:120 shown in Figure 61.
Figure 63 shows a nucleotide sequence (SEQ ID NO: 125) of a native sequence
PRO10096 cDNA,
wherein SEQ ID NO: 125 is a clone designated herein as "DNA125185-2806".
Figure 64 shows the amino acid sequence (SEQ ID NO: 126) derived from the
coding sequence of SEQ
ID NO: 125 shown in Figure 63.
Figure 65 shows a nucleotide sequence (SEQ ID NO: 127) of a native sequence
PRO6003 cDNA,
wherein SEQ ID NO: 127 is a clone designated herein as "DNA83568-2692".
Figure 66 shows the amino acid sequence (SEQ ID NO: 128) derived from the
coding sequence of SEQ
ID NO: 127 shown in Figure 65.
Figures 67A-B show a nucleotide sequence (SEQ ID NO: 129) of a native sequence
PRO6004 cDNA,
wherein SEQ ID NO: 129 is a clone designated herein as "DNA92259".
Figure 68 shows the amino acid sequence (SEQ ID NO: 130) derived from the
coding sequence of SEQ
ID NO:129 shown in Figures 67A-B.
Figure 69 shows a nucleotide sequence (SEQ ID NO: 131) of a native sequence
PR0350 cDNA,
wherein SEQ ID NO: 131 is a clone designated herein as "DNA44175-1314".
Figure 70 shows the amino acid sequence (SEQ ID NO: 132) derived from the
coding sequence of SEQ
ID NO: 131 shown in Figure 69.
Figure 71 shows a nucleotide sequence (SEQ ID NO: 136) of a native sequence
PR02630 cDNA,
wherein SEQ ID NO: 136 is a clone designated herein as "DNA8355 1".
Figure 72 shows the amino acid sequence (SEQ ID NO: 137) derived from the
coding sequence of SEQ
ID NO:136 shown in Figure 71.
Figure 73 shows a nucleotide sequence (SEQ ID NO: 138) of a native sequence
PR06309 cDNA,
wherein SEQ ID NO:138 is a clone designated herein as "DNA 116510".
Figure 74 shows the amino acid sequence (SEQ ID NO: 139) derived from the
coding sequence of SEQ
ID NO:138 shown in Figure 73.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
1. Definitions
The terms "PRO polypeptide" and "PRO" as used herein and when immediately
followed by a
numerical designation refer to various polypeptides, wherein the complete
designation (i.e., PRO/number)
refers to specific polypeptide sequences as described herein. The terms
"PRO/number polypeptide" and
"PRO/number" wherein the term "number" is provided as an actual numerical
designation as used herein
encompass native sequence polypeptides and polypeptide variants (which are
further defined herein). The PRO
polypeptides described herein 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. The term
"PRO polypeptide" refers
to each individual PRO/number polypeptide disclosed herein. All disclosures in
this specification which refer
to the "PRO polypeptide" refer to each of the polypeptides individually as
well as jointly. For example,
descriptions of the preparation of, purification of, derivation of, formation
of antibodies to or against,
administration of, compositions containing, treatment of a disease with, etc.,
pertain to each polypeptide of
the invention individually. The term "PRO polypeptide" also includes variants
of the PRO/number
polypeptides disclosed herein.
A "native sequence PRO polypeptide" comprises a polypeptide having the same
amino acid sequence
as the corresponding PRO polypeptide derived from nature. Such native sequence
PRO polypeptides can be
isolated from nature or can be produced by recombinant or synthetic means. The
term "native sequence PRO
polypeptide" specifically encompasses naturally-occurring truncated or
secreted forms of the specific PRO
polypeptide (e.g., an extracellular domain sequence), naturally-occurring
variant forms (e.g., alternatively
spliced forms) and naturally-occurring allelic variants of the polypeptide. In
various embodiments of the
invention, the native sequence PRO polypeptides disclosed herein are mature or
full-length native sequence
polypeptides comprising the full-length amino acids sequences shown in the
accompanying figures. Start and
stop codons are shown in bold font and underlined in the figures. However,
while the PRO polypeptide
disclosed in the accompanying figures are shown to begin with methionine
residues designated herein as amino
acid position 1 in the figures, it is conceivable and possible that = other
methionine residues located either
upstream or downstream from the amino acid position 1 in the figures may be
employed as the starting amino
acid residue for the PRO polypeptides.
The PRO polypeptide "extracellular domain" or "ECD" refers to a form of the
PRO polypeptide
which is essentially free of the transmembrane and cytoplasmic domains.
Ordinarily, a PRO polypeptide ECD
will have less than 1 % of such transmembrane and/or cytoplasmic domains and
preferably, will have less than
0.5% of such domains. It will be understood that any transmembrane domains
identified for the PRO
polypeptides of the present invention are identified pursuant to criteria
routinely employed in the art for
identifying that type of hydrophobic domain. The exact boundaries of a
transmembrane domain may vary but
most likely by no more than about 5 amino acids at either end of the domain as
initially identified herein.
Optionally, therefore, an extracellular domain of a PRO polypeptide may
contain from about 5 or fewer amino
acids on either side of the transmembrane domain/extracellular domain boundary
as identified in the Examples
or specification and such polypeptides, with or without the associated signal
peptide, and nucleic acid encoding
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them, are contemplated by the present invention.
The approximate location of the "signal peptides" of the various PRO
polypeptides disclosed herein
are shown in the present specification and/or the accompanying figures. It is
noted, however, that the C-
terminal boundary of a signal peptide may vary, but most likely by no more
than about 5 amino acids on either
side of the signal peptide C-terminal boundary as initially identified herein,
wherein the C-terminal boundary
of the signal peptide may be identified pursuant to criteria routinely
employed in the art for identifying that type
of amino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1-6 (1997)
and von Heinje et al., Nucl.
Acids. Res. 14:4683-4690 (1986)). Moreover, it is also recognized that, in
some cases, cleavage of a signal
sequence from a secreted polypeptide is not entirely uniform, resulting in
more than one secreted species.
These mature polypeptides, where the signal peptide is cleaved within no more
than about 5 amino acids on
either side of the C-terminal boundary of the signal peptide as identified
herein, and the polynucleotides
encoding them, are contemplated by the present invention.
"PRO polypeptide variant" means an active PRO polypeptide as defined above or
below having at least
about 80% amino acid sequence identity with a full-length native sequence PRO
polypeptide sequence as
disclosed herein, a PRO polypeptide sequence lacking the signal peptide as
disclosed herein, an extracellular
domain of a PRO polypeptide, with or without the signal peptide, as disclosed
herein or any other fragment
of a full-length PRO polypeptide sequence as disclosed herein. Such PRO
polypeptide variants include, for
instance, PRO polypeptides wherein one or more amino acid residues are added,
or deleted, at the N- or C-
terminus of the full-length native amino acid sequence. Ordinarily, a PRO
polypeptide variant will have at
least about 80% amino acid sequence identity, alternatively at least about 81
% amino acid sequence identity,
alternatively at least about 82 % amino acid sequence identity, alternatively
at least about 83 % amino acid
sequence identity, alternatively at least about 84% amino acid sequence
identity, alternatively at least about
85 % amino acid sequence identity, alternatively at least about 86 % amino
acid sequence identity, alternatively
at least about 87 % amino acid sequence identity, alternatively at least about
88 % amino acid sequence identity,
alternatively at least about 89% amino acid sequence identity, alternatively
at least about 90% amino acid
sequence identity, alternatively at least about 91 % amino acid sequence
identity, alternatively at least about
92 % amino acid sequence identity, alternatively at least about 93 % amino
acid sequence identity, alternatively
at least about 94 % amino acid sequence identity, alternatively at least about
95 % amino acid sequence identity,
alternatively at least about 96% amino acid sequence identity, alternatively
at least about 97% amino acid
sequence identity, alternatively at least about 98 % amino acid sequence
identity and alternatively at least about
99% amino acid sequence identity to a full-length native sequence PRO
polypeptide sequence as disclosed
herein, a PRO polypeptide sequence lacking the signal peptide as disclosed
herein, an extracellular domain of
a PRO polypeptide, with or without the signal peptide, as disclosed herein or
any other specifically defined
fragment of a full-length PRO polypeptide sequence as disclosed herein.
Ordinarily, PRO variant polypeptides
are at least about 10 amino acids in length, alternatively at least about 20
amino acids in length, alternatively
at least about 30 amino acids in length, alternatively at least about 40 amino
acids in length, alternatively at
least about 50 amino acids in length, alternatively at least about 60 amino
acids in length, alternatively at least
about 70 amino acids in length, alternatively at least about 80 amino acids in
length, alternatively at least about
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90 amino acids in length, alternatively at least about 100 amino acids in
length, alternatively at least about 150
amino acids in length, alternatively at least about 200 amino acids in length,
alternatively at least about 300
amino acids in length, or more.
"Percent (%) amino acid sequence identity" with respect to the PRO polypeptide
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 the specific PRO 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, BLAST-2, 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. For
purposes herein, however, % amino acid sequence identity values are generated
using the sequence comparison
computer program ALIGN-2, wherein the complete source code for the ALIGN-2
program is provided in
Table 1 below. The ALIGN-2 sequence comparison computer program was authored
by Genentech, Inc. and
the source code shown in Table 1 below has been filed with user documentation
in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The
ALIGN-2 program is publicly available through Genentech, Inc., South San
Francisco, California or may be
compiled from the source code provided in Table 1 below. The ALIGN-2 program
should be compiled for
use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence
comparison parameters are
set by the ALIGN-2 program and do not vary.
In situations where ALIGN-2 is employed for amino acid sequence comparisons,
the % amino acid
sequence identity of a given amino acid sequence A to, with, or against a
given amino acid sequence B (which
can alternatively be phrased as a given amino acid sequence A that has or
comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence B) is
calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by
the sequence alignment program
ALIGN-2 in that program's alignment of A and B, and where Y is the total
number of amino acid residues in
B. It will be appreciated that where the length of amino acid sequence A is
not equal to the length of amino
acid sequence B, the % amino acid sequence identity of A to B will not equal
the % amino acid sequence
identity of B to A. As examples of % amino acid sequence identity calculations
using this method, Tables 2
and 3 demonstrate how to calculate the % amino acid sequence identity of the
amino acid sequence designated
"Comparison Protein" to the amino acid sequence designated "PRO", wherein
"PRO" represents the amino
acid sequence of a hypothetical PRO polypeptide of interest, "Comparison
Protein" represents the amino acid
sequence of a polypeptide against which the "PRO" polypeptide of interest is
being compared, and "X, "Y"
and "Z" each represent different hypothetical amino acid residues.
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Unless specifically stated otherwise, all % amino acid sequence identity
values used herein are
obtained as described in the immediately preceding paragraph using the ALIGN-2
computer program.
However, % amino acid sequence identity values may also be obtained as
described below by using the WU-
BLAST-2 computer program (Altschul et al., Methods in Enzymology 266:460-480
(1996)). Most of the WU-
BLAST-2 search parameters are set to the default values. Those not set to
default values, i.e., the adjustable
parameters, are set with the following values: overlap span = 1, overlap
fraction = 0.125, word threshold
(T) = 11, and scoring matrix = BLOSUM62. When WU-BLAST-2 is employed, a %
amino acid sequence
identity value is determined by dividing (a) the number of matching identical
amino acid residues between the
amino acid sequence of the PRO polypeptide of interest having a sequence
derived from the native PRO
polypeptide and the comparison amino acid sequence of interest (i.e., the
sequence against which the PRO
polypeptide of interest is being compared which may be a PRO variant
polypeptide) as determined by WU-
BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptide
of interest. For example,
in the statement "a polypeptide comprising an the amino acid sequence A which
has or having at least 80%
amino acid sequence identity to the amino acid sequence B", the amino acid
sequence A is the comparison
amino acid sequence of interest and the amino acid sequence B is the amino
acid sequence of the PRO
polypeptide of interest.
Percent amino acid sequence identity may also be determined using the sequence
comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-
3402 (1997)). The
NCBI-BLAST2 sequence comparison program may be obtained from the National
Institute of Health,
Bethesda, MD. NCBI-BLAST2 uses several search parameters, wherein all of those
search
parameters are set to default values including, for example, unmask = yes,
strand = all, expected
occurrences = 10; minimum low complexity length = 15/5, multi-pass e-value =
0.01, constant for
multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix =
BLOSUM62.
In situations where NCBI-BLAST2 is employed for amino acid sequence
comparisons, the % amino
acid sequence identity of a given amino acid sequence A to, with, or against a
given amino acid sequence B
(which can alternatively be phrased as a given amino acid sequence A that has
or comprises a certain % amino
acid sequence identity to, with, or against a given amino acid sequence B) is
calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by
the sequence alignment program
NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total
number of amino acid
residues in B. It will be appreciated that where the length of amino acid
sequence A is not equal to the length
of amino acid sequence B, the % amino acid sequence identity of A to B will
not equal the % amino acid
sequence identity of B to A.
"PRO variant polynucleotide" or "PRO variant nucleic acid sequence" means a
nucleic acid molecule
which encodes an active PRO polypeptide as defined below and which has at
least about 80% nucleic acid
sequence identity with a nucleotide acid sequence encoding a full-length
native sequence PRO polypeptide
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sequence as disclosed herein, a full-length native sequence PRO polypeptide
sequence lacking the signal peptide
as disclosed herein, an extracellular domain of a PRO polypeptide, with or
without the signal peptide, as
disclosed herein or any other fragment of a full-length PRO polypeptide
sequence as disclosed herein.
Ordinarily, a PRO variant polynucleotide will have at least about 80 % nucleic
acid sequence identity,
alternatively at least about 81% nucleic acid sequence identity, alternatively
at least about 82% nucleic acid
sequence identity, alternatively at least about 83 % nucleic acid sequence
identity, alternatively at least about
84% nucleic acid sequence identity, alternatively at least about 85% nucleic
acid sequence identity,
alternatively at least about 86% nucleic acid sequence identity, alternatively
at least about 87% nucleic acid
sequence identity, alternatively at least about 88% nucleic acid sequence
identity, alternatively at least about
89 % nucleic acid sequence identity, alternatively at least about 90 % nucleic
acid sequence identity,
alternatively at least about 91 % nucleic acid sequence identity,
alternatively at least about 92 % nucleic acid
sequence identity, alternatively at least about 93 % nucleic acid sequence
identity; alternatively at least about
94 % nucleic acid sequence identity, alternatively at least about 95 % nucleic
acid sequence identity,
alternatively at least about 96% nucleic acid sequence identity, alternatively
at least about 97% nucleic acid
sequence identity, alternatively at least about 98 % nucleic acid sequence
identity and alternatively at least about
99% nucleic acid sequence identity with a nucleic acid sequence encoding a
full-length native sequence PRO
polypeptide sequence as disclosed herein, a full-length native sequence PRO
polypeptide sequence lacking the
signal peptide as disclosed herein, an extracellular domain of a PRO
polypeptide, with or without the signal
sequence, as disclosed herein or any other fragment of a full-length PRO
polypeptide sequence as disclosed
herein. Variants do not encompass the native nucleotide sequence.
Ordinarily, PRO variant polynucleotides are at least about 30 nucleotides in
length, alternatively at
least about 60 nucleotides in length, alternatively at least about 90
nucleotides in length, alternatively at least
about 120 nucleotides in length, alternatively at least about 150 nucleotides
in length, alternatively at least about
180 nucleotides in length, alternatively at least about 210 nucleotides in
length, alternatively at least about 240
nucleotides in length, alternatively at least about 270 nucleotides in length,
alternatively at least about 300
nucleotides in length, alternatively at least about 450 nucleotides in length,
alternatively at least about 600
nucleotides in length, alternatively at least about 900 nucleotides in length,
or more.
"Percent (%) nucleic acid sequence identity" with respect to PRO-encoding
nucleic acid sequences
identified herein is defined as the percentage of nucleotides in a candidate
sequence that are identical with the
nucleotides in the PRO nucleic acid sequence of interest, 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, BLAST-2,
ALIGN or Megalign
(DNASTAR) software. For purposes herein, however, % nucleic acid sequence
identity values are generated
using the sequence comparison computer program ALIGN-2, wherein the complete
source code for the
ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison
computer program was
authored by Genentech, Inc. and the source code shown in Table 1 below has
been filed with user
documentation in the U.S. Copyright Office, Washington D.C., 20559, where it
is registered under U.S.
CA 02372511 2001-11-23
WO 00/77037 PCT/US00/14042
Copyright Registration No. TXU510087. The ALIGN-2 program is publicly
available through Genentech,
Inc., South San Francisco, California or may be compiled from the source code
provided in Table 1 below.
The ALIGN-2 program should be compiled for use on a UNIX operating system,
preferably digital UNIX
V4.OD. All sequence comparison parameters are set by the ALIGN-2 program and
do not vary.
In situations where ALIGN-2 is employed for nucleic acid sequence comparisons,
the % nucleic acid
sequence identity of a given nucleic acid sequence C to, with, or against a
given nucleic acid sequence D
(which can alternatively be phrased as a given nucleic acid sequence C that
has or comprises a certain %
nucleic acid sequence identity to, with, or against a given nucleic acid
sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the
sequence alignment program ALIGN-
2 in that program's alignment of C and D, and where Z is the total number of
nucleotides in D. It will be
appreciated that where the length of nucleic acid sequence C is not equal to
the length of nucleic acid sequence
D, the % nucleic acid sequence identity of C to D will not equal the % nucleic
acid sequence identity of D to
C. As examples of % nucleic acid sequence identity calculations, Tables 4 and
5, demonstrate how to calculate
the % nucleic acid sequence identity of the nucleic acid sequence designated
"Comparison DNA" to the nucleic
acid sequence designated "PRO-DNA", wherein "PRO-DNA" represents a
hypothetical PRO-encoding nucleic
acid sequence of interest, "Comparison DNA" represents the nucleotide sequence
of a nucleic acid molecule
against which the "PRO-DNA" nucleic acid molecule of interest is being
compared, and "N", "L" and "V"
each represent different hypothetical nucleotides.
Unless specifically stated otherwise, all % nucleic acid sequence identity
values used herein are
obtained as described in the immediately preceding paragraph using the ALIGN-2
computer program.
However, % nucleic acid sequence identity values may also be obtained as
described below by using the WU-
BLAST-2 computer program (Altschul et al., Methods in Enzymology 266:460-480
(1996)). Most of the WU-
BLAST-2 search parameters are set to the default values. Those not set to
default values, i.e., the adjustable
parameters, are set with the following values: overlap span = 1, overlap
fraction = 0.125, word threshold
(T) = 11, and scoring matrix = BLOSUM62. When WU-BLAST-2 is employed, a %
nucleic acid sequence
identity value is determined by dividing (a) the number of matching identical
nucleotides between the nucleic
acid sequence of the PRO polypeptide-encoding nucleic acid molecule of
interest having a sequence derived
from the native sequence PRO polypeptide-encoding nucleic acid and the
comparison nucleic acid molecule
of interest (i.e., the sequence against which the PRO polypeptide-encoding
nucleic acid molecule of interest
is being compared which may be a variant PRO polynucleotide) as determined by
WU-BLAST-2 by (b) the
total number of nucleotides of the PRO polypeptide-encoding nucleic acid
molecule of interest. For example,
in the statement "an isolated nucleic acid molecule comprising a nucleic acid
sequence A which has or having
at least 80% nucleic acid sequence identity to the nucleic acid sequence B",
the nucleic acid sequence A is the
comparison nucleic acid molecule of interest and the nucleic acid sequence B
is the nucleic acid sequence of
the PRO polypeptide-encoding nucleic acid molecule of interest.
36
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Percent nucleic acid sequence identity may also be determined using the
sequence
comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-
3402 (1997)). The
NCBI-BLAST2 sequence comparison program may be obtained from the National
Institute of Health,
Bethesda, MD. NCBI-BLAST2 uses several search parameters, wherein all of those
search
parameters are set to default values including, for example, unmask = yes,
strand = all, expected
occurrences = 10, minimum low complexity length = 1515, multi-pass e-value =
0.01, constant for
multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix =
BLOSUM62.
In situations where NCBI-BLAST2 is employed for sequence comparisons, the %
nucleic acid
sequence identity of a given nucleic acid sequence C to, with, or against a
given nucleic acid sequence D
(which can alternatively be phrased as a given nucleic acid sequence C that
has or comprises a certain %
nucleic acid sequence identity to, with, or against a given nucleic acid
sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the
sequence alignment program NCBI-
BLAST2 in that program's alignment of C and D, and where Z is the total number
of nucleotides in D. It will
be appreciated that where the length of nucleic acid sequence C is not equal
to the length of nucleic acid
sequence D, the % nucleic acid sequence identity of C to D will not equal the
% nucleic acid sequence identity
of D to C.
In other embodiments, PRO variant polynucleotides are nucleic acid molecules
that encode an active
PRO polypeptide and which are capable of hybridizing, preferably under
stringent hybridization and wash
conditions, to nucleotide sequences encoding a full-length PRO polypeptide as
disclosed herein. PRO variant
polypeptides may be those that are encoded by a PRO variant polyaucleotide.
The term "positives", in the context of sequence comparison performed as
described above, includes
residues in the sequences compared that are not identical but have similar
properties (e.g. as a result of
conservative substitutions, see Table 6 below). For purposes herein, the %
value of positives is determined
by dividing (a) the number of amino acid residues scoring a positive value
between the PRO polypeptide amino
acid sequence of interest having a sequence derived from the native PRO
polypeptide sequence and the
comparison amino acid sequence of interest (i.e., the amino acid sequence
against which the PRO polypeptide
sequence is being compared) as determined in the BLOSUM62 matrix of WU-BLAST-2
by (b) the total
number of amino acid residues of the PRO polypeptide of interest.
Unless specifically stated otherwise, the % value of positives is calculated
as described in the
immediately preceding paragraph. However, in the context of the amino acid
sequence identity comparisons
performed as described for ALIGN-2 and NCBI-BLAST-2 above, includes amino acid
residues in the
sequences compared that are not only identical, but also those that have
similar properties. Amino acid
residues that score a positive value to an amino acid residue of interest are
those that are either identical to the
amino acid residue of interest or are a preferred substitution (as defined in
Table 6 below) of the amino acid
residue of interest.
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For amino acid sequence comparisons using ALIGN-2 or NCBI-BLAST2, the % value
of positives
of a given amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively
be phrased as a given amino acid sequence A that has or comprises a certain %
positives to, with, or against
a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scoring a positive value as
defined above by the sequence
alignment program ALIGN-2 or NCBI-BLAST2 in that program's alignment of A and
B, and where Y is the
total number of amino acid residues in B. It will be appreciated that where
the length of amino acid sequence
A is not equal to the length of amino acid sequence B, the % positives of A to
B will not equal the % positives
of B to A.
"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 PRO polypeptide natural environment will not be present.
Ordinarily, however, isolated
polypeptide will be prepared by at least one purification step.
An "isolated" PRO polypeptide-encoding nucleic acid or other polypeptide-
encoding nucleic acid 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 polypeptide-
encoding nucleic acid. An isolated
polypeptide-encoding nucleic acid molecule is other than in the form or
setting in which it is found in nature.
Isolated polypeptide-encoding nucleic acid molecules therefore are
distinguished from the specific polypeptide-
encoding nucleic acid molecule as it exists in natural cells. However, an
isolated polypeptide-encoding nucleic
acid molecule includes polypeptide-encoding nucleic acid molecules contained
in cells that ordinarily express
the polypeptide where, for example, the nucleic acid molecule is in a
chromosomal location different from that
of natural cells.
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.
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 polypeptide; a promoter
38
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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.
The term "antibody" is used in the broadest sense and specifically covers, for
example, single anti-
PRO monoclonal antibodies (including agonist, antagonist, and neutralizing
antibodies), anti-PRO antibody
compositions with polyepitopic specificity, single chain anti-PRO antibodies,
and fragments of anti-PRO
antibodies (see below). 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.
"Stringency" of hybridization reactions is readily determinable by one of
ordinary skill in the art, and
generally is an empirical calculation dependent upon probe length, washing
temperature, and salt concentration.
In general, longer probes require higher temperatures for proper annealing,
while shorter probes need lower
temperatures. Hybridization generally depends on the ability of denatured DNA
to reanneal when
complementary strands are present in an environment below their melting
temperature. The higher the degree
of desired homology between the probe and hybridizable sequence, the higher
the relative temperature which
can be used. As a result, it follows that higher relative temperatures would
tend to make the reaction
conditions more stringent, while lower temperatures less so. For additional
details and explanation of
stringency of hybridization reactions, see Ausubel et al., Current Protocols
in Molecular Biology, Wiley
Interscience Publishers, (1995).
"Stringent conditions" or "high stringency conditions", as defined herein, may
be identified by 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 C; (2)
employ during hybridization a
denaturing agent, such as formamide, for example, 50% (v/v) formamide with
0.1% bovine serum
*
albumin/0.1 % Ficoll/0. 1 % polyvinylpyrrolidone/50mM sodium phosphate buffer
at pH 6.5 with 750 mM
sodium chloride, 75 mM sodium citrate at 42 C; or (3) employ 50% formamide, 5
x SSC (0.75 M NaCl,
0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium
pyrophosphate, 5 x Denhardt's
solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10%
dextransulfate at 42 C, with washes
at 42 C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55
C, followed by a high-
stringency wash consisting of 0.1 x SSC containing EDTA at 55 C.
"Moderately stringent conditions" may be identified as described by Sambrook
et al., Molecular
Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and
include the use of washing
solution and hybridization conditions (e.g., temperature, ionic strength and
%SDS) less stringent that those
described above. An example of moderately stringent conditions is overnight
incubation at 37 C in a solution
comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate
(pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured
sheared salmon sperm DNA,
39
*-trademark
CA 02372511 2001-11-23
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followed by washing the filters in 1 x SSC at about 37-50 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.
The term "epitope tagged" when used herein refers to a chimeric polypeptide
comprising a PRO
polypeptide fused to a "tag polypeptide". The tag polypeptide has enough
residues to provide an epitope
against which an antibody can be made, yet is short enough such that it does
not interfere with activity of the
polypeptide to which it is fused. The tag polypeptide preferably also is
fairly unique so that the antibody does
not substantially cross-react with other epitopes. Suitable tag polypeptides
generally have at least six amino
acid residues and usually between about 8 and 50 amino acid residues
(preferably, between about 10 and 20
amino acid residues).
As used herein, the term "immunoadhesin" designates antibody-like molecules
which combine the
binding specificity of a heterologous protein (an "adhesin") with the effector
functions of immunoglobulin
constant domains. Structurally, the immunoadhesins comprise a fusion of an
amino acid sequence with the
desired binding specificity which is other than the antigen recognition and
binding site of an antibody (i.e., is
"heterologous"), and an immunoglobulin constant domain sequence. The adhesin
part of an immunoadhesin
molecule typically is a contiguous amino acid sequence comprising at least the
binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the immunoadhesin may
be obtained from any
immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including
IgA-1 and IgA-2), IgE, IgD
or IgM.
"Active" or "activity" for the purposes herein refers to form(s) of a PRO
polypeptide which retain
a biological and/or an immunological activity of native or naturally-occurring
PRO, wherein "biological"
activity refers to a biological function (either inhibitory or stimulatory)
caused by a native or naturally-
occurring PRO other than the ability to induce the production of an antibody
against an antigenic epitope
possessed by a native or naturally-occurring PRO and an "immunological"
activity refers to the ability to
induce the production of an antibody against an antigenic epitope possessed by
a native or naturally-occurring
PRO.
The term "antagonist" is used in the broadest sense, and includes any molecule
that partially or fully
blocks, inhibits, or neutralizes a biological activity of a native PRO
polypeptide disclosed herein. In a similar
manner, the term "agonist" is used in the broadest sense and includes any
molecule that mimics a biological
activity of a native PRO polypeptide disclosed herein. Suitable agonist or
antagonist molecules specifically
include agonist or antagonist antibodies or antibody fragments, fragments or
amino acid sequence variants of
native PRO polypeptides, peptides, antisense oligonucleotides, small organic
molecules, etc. Methods for
identifying agonists or antagonists of a PRO polypeptide may comprise
contacting a PRO polypeptide with a
candidate agonist or antagonist molecule and measuring a detectable change in
one or more biological activities
normally associated with the PRO polypeptide.
"Treatment" refers to both therapeutic treatment and prophylactic or
preventative measures, wherein
the object is to prevent or slow down (lessen) the targeted pathologic
condition or disorder. Those in need of
treatment include those already with the disorder as well as those prone to
have the disorder or those in whom
the disorder is to be prevented.
CA 02372511 2001-11-23
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"Chronic" administration refers to administration of the agent(s) in a
continuous mode as opposed to
an acute mode, so as to maintain the initial therapeutic effect (activity) for
an extended period of time.
"Intermittent" administration is treatment that is not consecutively done
without interruption, but rather is
cyclic in nature.
"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,
cats, cattle, horses, sheep, pigs,
goats, rabbits, etc. Preferably, the mammal is human.
Administration "in combination with" one or more further therapeutic agents
includes simultaneous
(concurrent) and consecutive administration in any order.
"Carriers" as used herein include pharmaceutically acceptable carriers,
excipients, or stabilizers which
are nontoxic to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often
the physiologically acceptable carrier is an aqueous pH buffered solution.
Examples of physiologically
acceptable carriers include buffers such as phosphate, citrate, and other
organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues) polypeptide;
proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, arginine or lysine; monosaccharides,
disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar
alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants
such as TWEEN', polyethylene
glycol (PEG), and PLURONICS".
"Antibody fragments" comprise a portion of an intact antibody, preferably the
antigen binding or
variable region of the intact antibody. Examples of antibody fragments include
Fab, Fab', F(ab')2, and Fv
fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):
1057-1062 [1995]); single-chain
antibody molecules; and multispecific antibodies formed from antibody
fragments.
Papain digestion of antibodies produces two identical antigen-binding
fragments, called "Fab"
fragments, each with a single antigen-binding site, and a residual "Fc"
fragment, a designation reflecting the
ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment
that has two antigen-combining sites
and is still capable of cross-linking antigen.
"Fv" is the minimum antibody fragment which contains a complete antigen-
recognition and -binding
site. This region consists of a dimer of one heavy- and one light-chain
variable domain in tight, non-covalent
association. It is in this configuration that the three CDRs of each variable
domain interact to define an
antigen-binding site on the surface of the VH-V,, dimer. Collectively, the six
CDRs confer antigen-binding
specificity to the antibody. However, even a single variable domain (or half
of an Fv comprising only three
CDRs specific for an antigen) has the ability to recognize and bind antigen,
although at a lower affinity than
the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the
first constant domain
(CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the
addition of a few residues at the
carboxy terminus of the heavy chain CH1 domain including one or more cysteines
from the antibody hinge
region. Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains
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bear a free thiol group. F(ab')2 antibody fragments originally were produced
as pairs of Fab' fragments which
have hinge cysteines between them. Other chemical couplings of antibody
fragments are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species
can be assigned to
one of two clearly distinct types, called kappa and lambda, based on the amino
acid sequences of their constant
domains.
Depending on the amino acid sequence of the constant domain of their heavy
chains, immunoglobulins
can be assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG,
and IgM, and several of these may be further divided into subclasses
(isotypes), e.g., IgGI, IgG2, IgG3, IgG4,
IgA, and IgA2.
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains
of antibody, wherein
these domains are present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a
polypeptide linker between the VH and VL domains which enables the sFv to form
the desired structure for
antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of
Monoclonal Antibodies, vol.
113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term "diabodies" refers to small antibody fragments with two antigen-
binding sites, which
fragments comprise a heavy-chain variable domain (VH) connected to a light-
chain variable domain (V,) in the
same polypeptide chain (VH-VL). By using a linker that is too short to allow
pairing between the two domains
on the same chain, the domains are forced to pair with the complementary
domains of another chain and create
two antigen-binding sites. Diabodies are described more fully in, for example,
EP 404,097; WO 93/11161;
and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
An "isolated" antibody is one which has been identified and separated and/or
recovered from a
component of its natural environment. Contaminant components of its natural
environment are materials which
would interfere with diagnostic or therapeutic uses for the antibody, and may
include enzymes, hormones, and
other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the
antibody will be purified (1)
to greater than 95 % by weight of antibody as determined by the Lowry method,
and most preferably more than
99% by weight, (2) 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 (3) to homogeneity by SDS-
PAGE under reducing or
nonreducing conditions using Coomassie blue or, preferably, silver stain.
Isolated antibody includes the
antibody in situ within recombinant cells since at least one component of the
antibody's natural environment
will not be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
The word "label" when used herein refers to a detectable compound or
composition which is
conjugated directly or indirectly to the antibody so as to generate a
"labeled" antibody. The label may be
detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in
the case of an enzymatic label, may
catalyze chemical alteration of a substrate compound or composition which is
detectable.
By "solid phase" is meant a non-aqueous matrix to which the antibody of the
present invention can
adhere. Examples of solid phases encompassed herein include those formed
partially or entirely of glass (e.g.,
controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides,
polystyrene, polyvinyl alcohol and
silicones. In certain embodiments, depending on the context, the solid phase
can comprise the well of an assay
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plate; in others it is a purification column (e.g., an affinity chromatography
column). This term also includes
a discontinuous solid phase of discrete particles, such as those described in
U.S. Patent No. 4,275,149.
A "liposome" is a small vesicle composed of various types of lipids,
phospholipids and/or surfactant
which is useful for delivery of a drug (such as a PRO polypeptide or antibody
thereto) to a mammal. The
components of the liposome are commonly arranged in a bilayer formation,
similar to the lipid arrangement
of biological membranes.
A "small molecule" is defined herein to have a molecular weight below about
500 Daltons.
"FGFR-1", "FGFR-2", "FGFR-3" and FGFR-4" refer to the fibroblast growth factor
receptors 1,
2, 3 and 4, respectively, as disclosed by Isacchi et al., Nuc. Acids Res.
18(7):1906 (1990), Dionne et al.,
EMBO J. 9(9):2685-2692 (1990), Keegan et al., Proc. Natl. Acad. Sci. USA
88:1095-1099 (1991) and
Partanen et al., EMBO J. 10(6):1347-1354 (1991), respectively.
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Table 1
1*
*
* C-C increased from 12 to 15
* Z is average of EQ
* B is average of ND
* match with stop is _M; stop-stop = 0; J (joker) match = 0
*1
#define _M -8 /* value of a match with a stop
int _day[26][26] = {
ABCDEFGHIJKLMNOPQRSTUV WXYZ*/
1* A */ { 2, 0,-2, 0, 0,-4, 0,-1,-2,-1, 0,_M, 1, 0,-2, 1, 1, 0, 0,-6, 0,-3,
0),
/* B */ { 0, 3,-4, 3, 2,-5,0,1,-2,0,0,-3,-2,2,-M,-I, 1, 0, 0, 0, 0,-2,-5, 0,-
3, 1),
/* C {-2,-4,15,-5,-5,-4,-3,-3,-2, 0,-5,-6,-5,-4,_M,-3,-5,-4, 0,-2, 0,-2,-8, 0,
0,-5),
/* D */ { 0, 3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2,_M,-1, 2,-1, 0, 0, 0,-2,-7,
0,-4, 2},
/* E *1 { 0, 2,-5, 3, 4,-5, 0, 1,-2, 0, 0,-3,-2, 1,_M,-1, 2,-1, 0, 0, 0,-2,-7,
0,-4, 31,
/* F */ {-4,-5,-4,-6,-5, 9,-5,-2, 1, 0,-5, 2, 0,-4,_M,-5,-5,-4,-3,-3, 0,-1, 0,
0, 7,-5},
/* G */ { 1, 0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, 0,_M,-1,-1,-3, 1, 0, 0,-1,-7,
0,-5, 01,
/* H {-1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2,_M, 0, 3, 2,-1,-1, 0,-2,-3, 0,
0, 2),
/* I *1 {-1,-2,-2,-2,-2, 1,-3,-2, 5, 0,-2, 2, 2,-2, M,-2,-2,-2,-1, 0, 0, 4,-5,
0,-1,-2},
/*J*/ {0,0,0,0,0,0,0,0,0,0,0,0, 0,0,_M,0,0,0,0,0,0,0,0,0,0,0},
/* K */ {-1, 0,-5, 0, 0,-5,-2, 0,-2, 0, 5,-3, 0, 1,_M,-1, 1, 3, 0, 0, 0,-2,-3,
0,-4, 01,
/* L {-2,-3,-6,-4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3,_M,-3,-2,-3,-3,-1, 0, 2,-2, 0,-
1,-2},
/* M {-1,-2,-5,-3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2, M,-2,-1, 0,-2,-1, 0, 2,-4, 0,-
2,-1),
/* N */ { 0, 2,-4, 2, 1,-4, 0, 2,-2, 0, 1,-3,-2, 2,_M,-1, 1, 0, 1, 0, 0,-2,-4,
0,-2, 1),
, M,- M,- M,- M,- M,- M},
/* O */ M, M,- M,- M,- M, 0,- M,- M,- M, - M-
, M-
- - - - - - - - -
/* P 0,-2, 0,-1,-3,-2,-1,_M, 6, 0, 0, 1, 0, 0,-1,-6, 0,-5, 0},
/* Q */ { 0, 1,-5, 2, 2,-5,-1, 3,-2, 0, 1,-2,-1, 1,_M, 0, 4, 0,-2,-5, 0,4, 3),
/* R {-2, 0,-4,-1,-1,-4,-3, 2,-2, 0, 3,-3, 0, 0, M, 0, 1, 6, 0,-1, 0,-2, 2,
0,4, 0),
1* S */ { 1, 0, 0, 0, 0,-3, 0, 0,-3,-2, 1,_M, 1,-1, 0, 2, 1, 0,-1,-2, 0,-3,
0),
/* T */ { 1, 0,-2, 0, 0,-3, 0,-1, 0, 0, 0,-1,-1, 0,_M, 0,-1,-1, 1, 3, 0, 0,-5,
0,-3, 0),
/*U*/ {0,0,0,0,0,0,0,0,0,0,0,0, 0,0,M,0,0,0,0,0,0,0,0,0,0,0},
/* V */ { 0,-2,-2,-2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2_,_M,-1,-2,-2,-1, 0, 0, 4,-
6, 0,-2,-2},
/* W *1 {-6,-5,-8,-7,-7, 0,-7,-3,-5, 0,-3,-2,-4,-4, M,-6,-5, 2,-2,-5, 0,-6,17,
0, 0,-6),
/*x{ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,0,M,0,0,0,0,0,0,0,0,0,0,0},
/* Y {-3,-3, 0,4,-4, 7,-5, 0,-1, 0,4,-1,-2,-2__M,-5,-4,-4,-3,-3, 0,-2, 0,
0,10,4},
/* Z */ { 0, 1,-5, 2, 3,-5, 0, 2,-2, 0, 0,-2,-1, 1,_M, 0, 3, 0, 0, 0, 0,-2,-6,
0,-4, 4)
50
44
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Table 1 (cont')
#include < stdio.h >
#include <ctype.h>
#define MAXJMP 16 /* max jumps in a diag
#define MAXGAP 24 /* don't continue to penalize gaps larger than this
#define JMPS 1024 /* max jmps in an path */
#define MX 4 /* save if there's at least MX-1 bases since last jmp
#define DMAT 3 /* value of matching bases */
#define DMIS 0 /* penalty for mismatched bases */
#define DINSO 8 /* penalty for a gap
#define DINS1 1 /* penalty per base
#define PINSO 8 /* penalty for a gap
#define PINS1 4 /* penalty per residue */
struct jmp {
short n[MAXJMP]; /* size of jmp (neg for dely)
unsigned short x[MAXJMP]; /* base no. of jmp in seq x
/* limits seq to 2^16 -1 *1
struct diag {
int score; /* score at last jmp
long offset; /* offset of prey block
short ijmp; /* current jmp index
struct jmp jp; /* list of jmps
struct path {
int spc; /* number of leading spaces
short n[JMPS]; /* size of jmp (gap) */
int x[JMPS]; /* loc of jmp (last elem before gap) */
char *ofile; /* output file name
char *namex[2]; /* seq names: getseqsO char *prog; /* prog name for err msgs
char *segx[2]; /* seqs: getsegs(
int dmax; /* best diag: nwQ
int dmax0; /* final diag */
int dna; /* set if dna: main()
int endgaps; /* set if penalizing end gaps
int gapx, gapy; /* total gaps in seqs
int lenO, lenl; /* seq lens */
int ngapx, ngapy; /* total size of gaps
int smax; /* max score: nwQ
int *xbm; /* bitmap for matching
long offset; /* current offset in jmp file */
struct diag *dx; /* holds diagonals */
struct path pp[2]; /* holds path for seqs
char *callocO, *mallocO, *indexO, *strcpyO;
char *getsegQ, *g_calloco;
CA 02372511 2001-11-23
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Table 1 (cont')
/* Needleman-Wunsch alignment program
*
* usage: progs file! file2
* where file! and filet are two dna or two protein sequences.
* The sequences can be in upper- or lower-case an may contain ambiguity
* Any lines beginning with ';', '>' or '<' are ignored
* Max file length is 65535 (limited by unsigned short x in the jmp struct)
* A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
* Output is in the file "align.out"
* The program may create a tmp file in /tmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650
/include "nw.h"
!include "day.h"
static _dbval[26]
1, 14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0, 10,0
static _pbval[26]
1, 210 < <('D'-'A')) I (l < <('N'-'A')), 4, 8, 16, 32, 64,
128, 256, OxFFFFFFF, 1 < < 10, 1 < < 11, 1 < < 12, 1 < < 13, 1 < < 14,
1< < 15, 1< < 16, 1< < 17, 1< < 18, 1< < 19, 1< <20, 1< <21, 1 22,
1 < <23, 1 < <24, 1 < <251(1 < <('E'-'A'))J(l < <('Q'-'A'))
main(ac, av) main
int ac;
char *av[];
{
prog = av[0];
if (ac != 3) {
fprintf(stderr, "usage: %s file l fle2\n", prog);
fprintf(stderr,"where file! and file2 are two dna or two protein
sequences.\n");
fprintf(stderr, "The sequences can be in upper- or lower-case\n");
fprintf(stderr,"Any lines beginning with ';' or ' <' are ignored\n");
fprintf(stderr,"Output is in the file \"align.out\"\n");
exit(1);
}
namex[0] = av[1];
namex[1] = av[2];
segx[0] = getseq(namex[0], &len0);
segx[l] = getseq(namex[l], &lenl);
xbm = (dna)? dbval :.pbval;
endgaps = 0; /* 1 to penalize endgaps
ofile = "align.out"; /* output file */
nwO; /* fill in the matrix, get the possible jmps
readjmpsO; /* get the actual jmps */
print(); /* print stats, alignment */
cleanup(0); /* unlink any tmp files */
}
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Table 1 (cont')
/* do the alignment, return best score: main()
* dna: values in Fitch and Smith, PNAS, 80, 1382-1386, 1983
* pro: PAM 250 values
* When scores are equal, we prefer mismatches to any gap, prefer
* a new gap to extending an ongoing gap, and prefer a gap in seqx
* to a gap in seq y.
nw() nW
{
char *px, *py; /* seqs and ptrs
int *ndely, *dely; /* keep track of dely
int ndelx, delx; /* keep track of delx
int *tmp; /* for swapping rowO, rowl
int mis; /* score for each type
int insO, insl; /* insertion penalties */
register id; /* diagonal index */
register ij; /* jmp index */
register *col0, *coll; /* score for curr, last row
register xx, yy; /* index into seqs
dx = (struct diag *)g_calloc("to get diags", len0+lenl+1, sizeof(struct
diag));
ndely = (int *)g_calloc("to get ndely", lenl+1, sizeof(int));
dely = (int *)g calloc("to get dely", lent + 1, sizeof(int));
col0 = (int *)g calloc("to get col0", lent + 1, sizeof(int));
col l = (int *)g calloc("to get col l ", lenl + 1, sizeof(int));
insO = (dna)? DINSO : PINSO;
insl = (dna)? DINS1 : PINS!;
smax = -10000;
if (endgaps) {
for (colO[0] = dely[0] = -insO, yy = 1; yy < = lenl; yy++) {
colO[yy] = dely[yy] = col0[yy-1] - insl;
ndely[yy] = yy;
1
colO[0] = 0; /* Waterman Bull Math Biol 84 */
1
else
for (yy = 1; yy <= lenl; yy++)
dely[yy] = -insO;
/* fill in match matrix
for (px = segx[0], xx = 1; xx <= lenO; px+ +, xx+ +)
{
1* initialize first entry in col
if (endgaps) {
if (xx = = 1)
coll[0] = delx = -(ins0+insl);
else
coll[0] = delx = colO[0] - insl;
ndelx = xx;
1
else {
col l [0] = 0;
delx = -ins0;
ndelx = 0;
1
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Table 1 (cont')
...nw
for (py = segx[1], yy = 1; yy < = lenl; py+ +, yy++) {
mis = co10[yy-l];
if (dna)
mis +_ (xbm[*px-'A']&xbm[*py-'A'])? DMAT : DMIS;
else
mis +_ _day[*px-'A'][*py-'A'];
/* update penalty for del in x seq;
* favor new del over ongong del
* ignore MAXGAP if weighting endgaps
if (endgaps I I ndely[yy] < MAXGAP) {
if (colO[yy] - insO > = dely[yy]) {
dely[yy] = colO[yy] - (insO+insl);
ndely[yy] = 1;
}else{
dely[yy] -= insl;
ndely[yy] + +;
}
} else {
if (colO[yy] - (insO+insl) > = dely[yy]) {
dely[yy] = colO[yy] - (ins0+insl);
ndely[yy] = 1;
} else
ndely[yy] + +;
}
/* update penalty for del in y seq;
* favor new del over ongong del
if (endgaps ndelx < MAXGAP) {
if (coll[yy-1] - insO > = delx) {
delx = coll[yy-1] - (insO+insl);
ndelx = 1;
} else {
delx -= ins1;
ndelx+ +;
} else { }
if (coll[yy-1] - (insO+insl) > = delx) {
delx = coll[yy-1] - (insO+insl);
ndelx = 1;
} else
ndelx+ +;
}
/* pick the maximum score; we're favoring
* mis over any del and delx over dely
55
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Table 1 (cont')
...nw
id =xx-yy+lenl-1;
if (mis > = delx && mis > = dely[yy])
coll[yy] = mis;
else if (delx > = dely[yy]) {
coll[yy] = delx;
ij = dx[id].ijmp;
if (dx[id].jp.n[O] && (!dna I I (ndelx > = MAXJMP
&& xx > dx[id].jp.x[ij]+MX) mis > dx[id].score+DINSO)) {
dx[id]. ijmp+ +;
if (++ij > = MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset + = sizeof(struct jmp) + sizeof(offset);
}
}
dx[id].jp.n[ij] = ndelx;
dx[id].jp.x[ij] = xx;
dx[id].score = delx;
}
else {
coll[yy] = dely[yy];
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (!dna I I (ndely[yy] > = MAXJMP
{
&& xx > dx[id].jp.x[ij]+MX) I I mis > dx[id].score + DINSO))
dx[id]. ijmp+ +;
if (++ij > = MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset + = sizeof(struct jmp) + sizeof(offset);
}
}
dx[id].jp.n[ij] = -ndely[yy];
dx[id].jp.x[ij] = xx;
dx[id].score = dely[yy];
}
if (xx = = len0 && yy < len I) {
/* last col
if (endgaps)
coll[yy] -= ins0+ins1*(lenl-yy);
if (col 1 [yy] > smax) {
smax = coll[yy];
dmax = id;
}
}
if (endgaps && xx < len0)
coll[yy-1] -= ins0+insl*(len0-xx);
if (coI l [yy-1 ] > smax) {
smax = coll[yy-1];
dmax = id;
}
Imp = cotO; colO = coil; coil = tmp;
}
(void) free((char *)ndely);
(void) free((char *)dely);
(void) free((char *)co10);
(void) free((char *)coll); }
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Table 1 (cont')
*
* print() -- only routine visible outside this module
*
* static:
* getmat() -- trace back best path, count matches: print()
* pr_alignO -- print alignment of described in array pQ: print()
* dumpblockp -- dump a block of lines with numbers, stars: pr_alignO
* nums() -- put out a number line: dumpblock()
* putlinep -- put out a line (name, [num], seq, [num]): dumpblock()
* stars() - -put a line of stars: dumpblockO
* stripname() -- strip any path and prefix from a seqname
#include "nw.h"
#define SPC 3
#define P_LINE 256 /* maximum output line
#define P_SPC 3 /* space between name or num and seq
extern _day[26][26];
int olen; /* set output line length */
FILE *fx; /* output file */
print() print
{
int lx, ly, firstgap, lastgap; /* overlap */
if ((fx = fopen(ofile, "w")) = = 0) {
fprintf(stderr,"%s: can't write %s\n", prog, ofile);
cleanup(!);
I
fprintf(fx, " < first sequence: %s (length = %d)\n", namex[0], lenO);
fprintf(fx, "<second sequence: %s (length = %d)\n", namex[1], lent);
olen = 60;
lx = lenO;
ly = lent;
firstgap = lastgap = 0;
if (dmax < lent - 1) { /* leading gap in x
pp[0].spc = firstgap = lent - dmax - 1;
ly -= PP[0].spc;
I
else if (dmax > lent - 1) { /* leading gap in y
pp[l].spc = firstgap = dmax - (lent - 1);
lx -= pp[l].spc;
}
if (dmax0 < lenO - 1) { /* trailing gap in x
lastgap = lenO - dmax0 -1;
lx -= lastgap;
1
else if (dmax0 > lenO - 1) { /* trailing gap in y
lastgap = dmax0 - (lenO - 1);
ly - = lastgap;
}
getmat(lx, ly, firstgap, lastgap);
pr_align0;
}
CA 02372511 2001-11-23
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Table 1 (cont')
* trace back the best path, count matches
static
getmat(lx, ly, firstgap, lastgap) getmat
int lx, ly; /* "core" (minus endgaps) *1
int firstgap, lastgap; /* leading trailing overlap
{
int nm, i0, il, sizO, sizl;
char outx[32];
double pct;
register n0, n l;
register char *pO, *pl;
/* get total matches, score
i0=il=siz0=sizl=0;
p0 = segx[O] + pp[1].spc;
pl = segx[1] + pp[O].spc;
nO = pp[l].spc + 1;
nl = pp[0].spc + 1;
nn=0;
while (*po && *p l) {
if (sizo) {
pl++;
nl++;
sizo--;
}
else if (sizi) {
p0++;
nO+ +;
sizl--;
}
else {
if (xbm[*p0-'A']&xbm[*pl-'A'])
nm++;
if (n0++ _= pp[0].x[io])
sizO = pp[0].n[iO++];
if (nl++ _= pp[l].x[il])
sizi = pp[l].n[il++];
po++;
pl++;
}
}
/* pct homology:
* if penalizing endgaps, base is the shorter seq
* else, knock off overhangs and take shorter core
*/
if (endgaps)
lx = (lenO < lenl)? lenO : lenl;
else
lx = (lx < ly)? Ix : ly;
pct = 100.*(double)nm/(double)lx;
fprintf(fx, "\n");
fprintf(fx, "< %d match%s in an overlap of %d: %.2f percent similarity\n",
nm, (nm == 1)? "" : "es", lx, pct);
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Table 1 (cont')
fprintf(fx, " <gaps in first sequence: %d", gapx); ...getmat
if (gapx) {
(void) sprintf(outx, " (%d %s%s)",
ngapx, (dna)? "base":"residue", (ngapx = = 1)? -:"S");
fprintf(fx," %s", outx);
fprintf(fx, ", gaps in second sequence: %d", gapy);
if (gapy) {
(void) sprintf(outx, " (%d %s%s)",
ngapy, (dna)? "base":"residue", (ngapy = = 1)? "":"s");
fprintf(fx,"%s", outx);
}
if (dna)
fprintf(fx,
"\n<score: %d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n",
smax, DMAT, DMIS, DINSO, DINS1);
else
fprintf(fx,
"\n < score: %d (Dayhoff PAM 250 matrix, gap penalty = %d + %d per
residue)\n",
smax, PINSO, PINS1);
if (endgaps)
fprintf(fx,
" <endgaps penalized. left endgap: %d %s%s, right endgap: %d %s%s\n",
firstgap, (dna)? "base" : "residue", (firstgap = = 1)? : "s",
lastgap, (dna)? "base" : "residue", (lastgap == 1)? "" : "s");
else
fprintf(fx, " <endgaps not penalized\n");
}
static nm; /* matches in core -- for checking */
static Imax; /* lengths of stripped file names
static ij[2]; /* jmp index for a path */
static nc[2]; /* number at start of current line */
static ni[2]; /* current elem number -- for gapping
static siz[2];
static char *ps[2]; /* ptr to current element */
static char *po[2]; /* ptr to next output char slot */
static char out[2][P_LINE]; /* output line */
static char star[P_LINE]; /* set by stars()
* print alignment of described in struct path pp[]
static
pr_align() pr align
{
int nn; /* char count
int more;
register i;
for (i = O, lmax = 0; i < 2; i++) {
nn = stripname(namex[i]);
if (nn > lmax)
Imax = nn;
nc[i] = 1;
ni[i] = 1;
siz[i] = ij[i] = 0;
ps[i] = seqx[i];
po[i] = out[i]; }
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Table 1 (cont')
for (nn = nm = 0, more = 1; more; ) { ...pr_align
for(i=more =0;i <2;i++){
* do we have more of this sequence?
if (!*ps[i])
continue;
more++;
if (pp[i].spc) { /* leading space
*po[i]++
PP[il. spc
}
else if (siz[i]) { /* in a gap
*po[i]++ _
siz[i]--;
}
else { /* we're putting a seq element
*Po[i] = *PS[i];
if (islower(*ps[i]))
*ps[i] = toupper(*ps[i]);
po[i]++;
ps[i] + +;
* are we at next gap for this seq?
if (ni[i] == PP[i].x[ij[ill) {
* we need to merge all gaps
* at this location
*/
siz[i] = pp[i].n[ij[i] + +];
while (ni[i] _= pp[i].x[ij[i]])
siz[i] += pp[i].n[ij[i] + +];
}
ni[i]++;
}
}
if (++nn == olen I !more && nn) {
dumpblockO;
for (i = 0; i < 2; i++)
Po[i] = out[i];
nn=0;
}
}
}
* dump a block of lines, including numbers, stars: pr_align()
static
dumpblock() dumpblock
{
register i;
for (i = 0; i < 2; i++)
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Table 1 (cont')
...dumpblock
(void) putc('\n', fx);
for(i=0;i<2;i++){
if (*out[i] && (*out[i] *(po[i]) ! _ ' ')) {
if (i = = 0)
nums(i);
if (i == 0 && *out[1])
starsO;
putline(i);
if (i == 0 && *out[1])
fprintf(fx, star);
if (i = = 1)
nums(i);
}
}
}
* put out a number line: dumpblock()
static
nums(ix) nums
int ix; /* index in out[] holding seq line */
{
char nline[P_LINE];
register i, j;
register char *pn, *px, *py;
for (pn = mine, i = 0; i < lmax+P SPC; i++, pn++)
*pn =
for (i = nc[ix], py = out[ix]; *py; py++, pn++) {
if(*py ' ' I I *py =_ -)
*pn =
else {
if (i% 10 == 0 I (i == 1 && nc[ix] != 1)) {
j=(i<0)?-i:i;
for (px = pn; j; j /= 10, px--)
*px=j%10+'V';
if (i < 0)
*px =
}
else
*pn =
i++;
}
}
*pn = '\0';
nc[ix] = i;
for (pn = nline; *pn; pn+ +)
(void) putc(*pn, fx);
(void) putc('\n', fx);
}
/*
* put out a line (name, [num], seq, [num]): dumpblock()
static
putline(ix) putline
int ix; {
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Table 1 (cont')
...putline
int i;
register char *px;
for (px = namex[ix], i = 0; *px && *px px++, i++)
(void) putc(*px,fx);
for (; i < lmax+P_SPC; i++)
(void) putc(' ', fx);
/* these count from 1:
* nip is current element (from 1)
* nc[] is number at start of current line
for (px = out[ix]; *px; px+ +)
(void) putc(*px&Ox7F, fx);
(void) putc('\n', fx);
}
* put a line of stars (seqs always in out[0], out[1]): dumpblock()
static
starsO stars
{
int i;
register char *p0, *pl, cx, *px;
if (!*out[O] I I (*out[O] __ && *(po[OD =_ ' ') I I
!*out[1] I I (*out[1] _ _ ' ' && *(po[1]) _ _ ' '))
return;
px = star;
for (i = lmax+P SPC; i; i--)
*px++ _ ,
for (p0 = out[0], pl. = out[1]; *p0 && *pl; p0++, pl++) {
if (isalpha(*p0) && isalpha(*pl)) {
if (xbm[*p0-'A']&xbm[*p1-'A']) {
cx
nm+ +;
}
else if (!dna && _day[*p0-'A'][*pl-'A'] > 0)
cx =
else
cx
}
else
cx
*px++ = cx;
}
*px++ = '\n';
*px = \0';
}
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Table 1 (cont')
* strip path or prefix from pn, return len: pr_align()
static
stripname(pn) stripname
char *pn; /* file name (may be path) */
{
register char *px, *py;
py = 0;
for (px = pn; *px; px++)
if (*px = _ '/')
py=px+1;
if (pY)
(void) strcpy(pn, py);
return(strlen(pn));
}
25
35
45
55
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Table 1 (cont')
* cleanup() -- cleanup any tmp file
* getse90 -- read in seq, set dna, len, maxlen
* g_callocO -- calloc() with error checkin
* readjmps() -- get the good jmps, from tmp file if necessary
* writejmpsO -- write a filled array of jmps to a tmp file: nw()
#include "nw.h"
#include <sys/file.h>
char *jname = "/tmp/homgXXXXXX"; /* tmp file for jmps
FILE *fj;
int cleanupO; /* cleanup tmp file */
long Iseeko;
* remove any tmp file if we blow
cleanup(i) cleanup
int i;
{
if (fj)
(void) unlink(jname);
exit(i);
}
* read, return ptr to seq, set dna, len, maxlen
* skip lines starting with';', '<', or ' > '
* seq in upper or lower case
char
getseq(file, len) getseq
char *file; /* file name
int *len; /* seq len */
{
char line[1024], *pseq;
register char *px, *py;
int natgc, tlen;
FILE *fp;
if ((fp = fopen(file, "r")) 0) {
fprintf(stderr,"%s: can't read %s\n", prog, file);
exit(1);
}
den = natgc = 0;
while (fgets(line, 1024, fp)) {
if(*line== ';' II *line Nine
continue;
for (px = line; *px '\n'; px++)
if (isupper(*px) I I islower(*px))
tlen+ +;
}
if ((pseq = malloc((unsigned)(tlen+6))) 0) {
fprintf(stderr,"%s: malloc() failed to get %d bytes for %s\n", prog, tlen+6,
file);
exit(l);
}
pseq[O] = pSeq[11 = pseq[2] = pseq[3] = '\O';
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Table 1 (cont')
...getseq
py = pseq + 4;
*len = tlen;
rewind(fp);
while (fgets(line, 1024, fp)) {
if (*line = = ';' I I *line *line
continue;
for (px = line; *px '\n'; px++) {
if (isupper(*px))
*py++ = *px;
else if (islower(*px))
*py + + = toupper(*px);
if (index("ATGCU",*(py-1)))
natgc+ +;
}
}
*py++ _ '\0';
*py='\0';
(void) fclose(fp);
dna = natgc > (tlen/3);
return(pseq+4);
}
char *
g_calloc(msg, nx, sz) g_calloc
char *msg; /* program, calling routine
int nx, sz; /* number and size of elements
{
char *px, *callocO;
if ((px = calloc((unsigned)nx, (unsigned)sz)) 0) {
if (*msg) {
fprintf(stderr, "%s: g_calloco failed %s (n=%d, sz=%d)\n", prog, msg, nx, sz);
exit(l);
}
}
return(px);
}
* get final jmps from dx[] or tmp file, set pp[], reset dmax: main()
readjmps() readjmps
{
int fd = -1;
int siz, i0, il;
register i, j, xx;
if(fj){
(void) fclose(fj);
if ((fd = open(jname, O_RDONLY, 0)) < 0) {
fprintf(stderr, "%s: can't open() %s\n", prog, jname);
cleanup(l);
}
}
for (i = iO = it = 0, dmaxO = dmax, xx = lenO; ; i++) {
while (1) {
for (j = dx[dmax].ijmp; j > = 0 && dx[dmax].jp.x[j] > = xx; j--)
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Table 1 (cont')
...readjmps
{
if (j < 0 && dx[dmax].offset && fj)
(void) lseek(fd, dx[dmax].offset, 0);
(void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp));
(void) read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));
dx[dmaxl.ijmp = MAXJMP-1;
}
else
break;
}
if (i > = JMPS) {
fprintf(stderr, "%s: too many gaps in alignment\n", prog);
cleanup(1);
}
if(j >=0){
siz = dx[dmax].jp=nU];
xx = dx[dmax].jp.xU];
dmax + = siz;
if (siz < 0) { /* gap in second seq
pp[1].n[il] = -siz;
xx + = siz;
/*id=xx-yy+lenl-1
pp[1].x[il] = xx - dmax + lenl - 1;
gapy+ +;
ngapy -= siz;
/* ignore MAXGAP when doing endgaps */
siz = (-siz < MAXGAP endgaps)? -siz : MAXGAP;
il++;
}
else if (siz > 0) { /* gap in first seq
pp[0].n[i0] = siz;
pp[0].x[i0] = xx;
gapx+ +;
ngapx + = siz;
/* ignore MAXGAP when doing endgaps */
siz = (siz < MAXGAP I I endgaps)? siz : MAXGAP;
i0++;
}
}
else
break;
}
/* reverse the order of jmps
for (j = 0, i0--; j < i0; j + +, i0--) {
i = pp[0l=n[]; pp[0].nU] = pp[0].n[i0]; pp[0].n[i0] = i;
i = pp[0].x[]; pp[0].x[] = pp[0].x[i0]; pp[0].x[i0] = i;
}
for (j = 0, it--; j < il; j++, it--) {
i = pp[1].nU]; pp[1].nU] = pp[1].n[il]; pp[1].n[il] = i;
i = pp[1].x[j]; pp[1].x[j] = pp[l].x[il]; pp[l].x[il] = i;
}
if (fd > = 0)
(void) close(fd);
if (fj) {
(void) unlink(jname);
fj = 0;
offset = 0;
} }
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Table 1 (cont')
1*
* write a filled jmp struct offset of the prey one (if any): nw()
*1
writejmps(ix) writejmps
int ix;
{
char *mktempo ;
if(!fj){
if (mktemp(jname) < 0) {
fprintf(stderr, "%s: can't mktemp() %s\n", prog, jname);
cleanup(1);
}
if ((fj = fopen(jname, "w")) 0) {
fprintf(stderr, "%s: can't write %s\n", prog, jname);
exit(1);
}
(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj);
(void) fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj);
}
30
40
50
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Table 2
PRO XXXXXXXXXXXXXXX (Length = 15 amino acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two
polypeptide sequences as determined
by ALIGN-2) divided by (the total number of amino acid residues of the PRO
polypeptide) _
5 divided by 15 = 33.3 %
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Table 3
PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two
polypeptide sequences as determined
by ALIGN-2) divided by (the total number of amino acid residues of the PRO
polypeptide) _
5 divided by 10 = 50%
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Table 4
PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)
Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid
sequences as determined by
ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic
acid sequence) _
6 divided by 14 = 42.9%
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Table 5
PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides)
Comparison DNA NNNNLLLVV (Length = 9 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid
sequences as determined by
ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic
acid sequence) _
4 divided by 12 = 33.3 %
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if. Compositions and Methods of the Invention
A. Full-Length PRO Polypeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding
polypeptides referred to in the present application as PRO polypeptides. In
particular, cDNAs encoding
various PRO polypeptides have been identified and isolated, as disclosed in
further detail in the Examples
below. It is noted that proteins produced in separate expression rounds may be
given different PRO numbers
but the UNQ number is unique for any given DNA and the encoded protein, and
will not be changed.
However, for sake of simplicity, in the present specification the protein
encoded by the full length native
nucleic acid molecules disclosed herein as well as all further native
homologues and variants included in the
foregoing definition of PRO, will be referred to as "PRO/number", regardless
of their origin or mode of
preparation.
As disclosed in the Examples below, various cDNA clones have been deposited
with the ATCC. The
actual nucleotide sequences of those clones can readily be determined by the
skilled artisan by sequencing of
the deposited clone using routine methods in the art. The predicted amino acid
sequence can be determined
from the nucleotide sequence using routine skill. For the PRO polypeptides and
encoding nucleic acids
described herein, Applicants have identified what is believed to be the
reading frame best identifiable with the
sequence information available at the time.
1. Full-length PRO196 Polvpentides
The present invention provides newly identified and isolated nucleotide
sequences encoding
polypeptides referred to in the present application as PRO 196. In particular,
Applicants have identified and
isolated cDNAs encoding a PRO196 polypeptide, as disclosed in further detail
in the Examples below. Using
BLAST and FastA sequence alignment computer programs, Applicants found that a
cDNA sequence encoding
full-length native sequence PRO196 encodes for a polypeptide having an amino
acid sequence which has
identity with the amino acid sequence of various TIE ligand polypeptides.
2. Full-length PRO444 Poly tides
The DNA26846-1397 clone was isolated from a human fetal lung library using a
trapping technique
which selects for nucleotide sequences encoding secreted proteins. Thus, the
DNA26846-1397 clone encodes
a secreted factor. As far as is known, the DNA26846-1397 sequence encodes a
novel factor designated herein
as PRO444. Although, using WU-BLAST2 sequence alignment computer programs,
some sequence identities
with known proteins were revealed.
3. Full-length PRO183 Polypeptides
The DNA28498 clone was isolated from a human tissue library. As far as is
known, the DNA28498
sequence encodes a novel factor designated herein as PRO183. Although, using
WU-BLAST2 sequence
alignment computer programs, some sequence identities with known proteins were
revealed.
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4. Full-length PRO185 Polypeptides
The DNA28503 clone was isolated from a human tissue library. As far as is
known, the DNA28503
sequence encodes a novel factor designated herein as PRO 185. Although, using
WU-BLAST2 sequence
alignment computer programs, some sequence identities with known proteins were
revealed.
5. Full-length PRO210 and PRO217 Polypeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding
polypeptides referred to in the present application as PR0210 and PR0217. In
particular, Applicants have
identified and isolated cDNAs encoding a PR0210 and PR0217 polypeptide, as
disclosed in further detail in
the Examples below. Using BLAST (FastA format) sequence alignment computer
programs, Applicants found
that cDNAs sequence encoding full-length native sequence PR0210 and PR0217
have homologies to known
proteins having EGF-like domains. Accordingly, it is presently believed that
the PR0210 and PR0217
polypeptides disclosed in the present application is a newly identified member
of the EGF-like family and
possesses properties typical of the EGF-like protein family.
6. Full-length PRO215 Polypeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding
polypeptides referred to in the present application as PR0215. In particular,
Applicants have identified and
isolated cDNAs encoding a PR0215 polypeptide, as disclosed in further detail
in the Examples below. Using
BLAST and FastA sequence alignment computer programs, Applicants found that a
cDNA sequence encoding
full-length native sequence PR0215 (shown in Figure 11 and SEQ ID NO: 16)
encodes for a polypeptide having
an amino acid sequence which has identity with the amino acid sequence of the
SLIT protein precursor.
PR0215 also has identity with a leucine rich repeat protein.
7. Full-length PRO242 Polypeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding
polypeptides referred to in the present application as PR0242. In particular,
Applicants have identified and
isolated cDNA encoding a PRO242 polypeptide, as disclosed in further detail in
the Examples below. Using
BLAST and FastA sequence alignment computer programs, Applicants found that a
cDNA sequence encoding
full-length native sequence PRO242 (shown in Figure 15 and SEQ ID NO:23) has
amino acid sequence identity
with human macrophage inflammatory protein 1-alpha, rabbit macrophage
inflammatory protein 1-beta, human
LD78 and rabbit immune activation gene 2. Accordingly, it is presently
believed that PR0242 polypeptide
disclosed in the present application is a newly identified member of the
chemokine family and possesses activity
typical of the chemokine family.
8. Full-length PRO288 Polypeptides
The present invention provides newly identified and isolated PR0288
polypeptides. In particular,
Applicants have identified and isolated various human PR0288 polypeptides. The
properties and
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characteristics of some of these PRO288 polypeptides are described in further
detail in the Examples below.
Based upon the properties and characteristics of the PR0288 polypeptides
disclosed herein, it is Applicants'
present belief that PR0288 is a member of the TNFR family, and particularly,
is a receptor for Apo-2 ligand.
9. Full-length PRO365 Polyggp iides
The present invention provides newly identified and isolated nucleotide
sequences encoding
polypeptides referred to in the present application as PR0365. In particular,
Applicants have identified and
isolated cDNA encoding a PRO365 polypeptide, as disclosed in further detail in
the Examples below. Using
BLAST and FastA sequence alignment computer programs, Applicants found that
various portions of the
PR0365 polypeptide have significant homology with the human 2-19 protein.
Accordingly, it is presently
believed that PR0365 polypeptide disclosed in the present application is a
newly identified member of the
human 2-19 protein family.
10. Full-length PRO1361 Polypentides
The DNA60783-1611 clone was isolated from a human B cell library. As far as is
known, the
DNA60783-1611 sequence encodes a novel factor designated herein as PRO1361;
using the WU-BLAST2
sequence alignment computer program, no sequence identities to any known
proteins were revealed.
11. Full-length PRO1308 Polypetides
Using WU-BLAST2 sequence alignment computer programs, it has been found that
PRO1308 shares
certain amino acid sequence identity with the amino acid sequence of the
follistatin protein designated
"S55369" in the Dayhoff database. Accordingly, it is presently believed that
PRO1308 disclosed in the present
application is a newly identified member of the follistatin protein family and
may possess activity or properties
typical of that family of proteins.
12. Full-length PRO1183 Polypentides
Using WU-BLAST2 sequence alignment computer programs, it has been found that a
full-length native
sequence PRO 1183 (shown in Figure 26 and SEQ ID NO:52) has certain amino acid
sequence identity with
protoporphyrinogen oxidase. Accordingly, it is presently believed that PRO
1183 disclosed in the present
application is a newly identified member of the oxidase family and may possess
enzymatic activity typical of
oxidases.
13. Full-length PRO1272 Polypeptides
Using WU-BLAST2 sequence alignment computer programs, it has been found that a
full-length native
sequence PRO 1272 (shown in Figure 28 and SEQ ID NO:54) has certain amino acid
sequence identity with
cement gland-specific protein from Xenopus laevis. Accordingly, it is
presently believed that PRO 1272
disclosed in the present application is a newly identified member of the XAG
family and may share at least one
mechanism with the XAG proteins.
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14. Full-length PRO1419 Polvoeptides
As far as is known, the DNA71290-1630 sequence encodes a novel factor
designated herein as
PRO1419. Using WU-BLAST2 sequence alignment computer programs, minimal
sequence identities to known
proteins were revealed.
15. Full-length PRO4999 Polypeptides
Using the ALIGN-2 sequence alignment computer program referenced above, it has
been found that
the full-length native sequence PR04999 (shown in Figure 32 and SEQ ID NO:58)
has certain amino acid
sequence identity with UROM_HUMAN. Accordingly, it is presently believed that
the PR04999 polypeptide
disclosed in the present application is a newly identified member of the
uromodulin protein family and may
possess one or more biological and/or immunological activities or properties
typical of that protein family.
16. Full-length PR07170 Polypeptides
The DNA108722-2743 clone was isolated from a human library as described in the
Examples below.
As far as is known, the DNA 108722-2743 nucleotide sequence encodes a novel
factor designated herein as
PR07170; using the ALIGN-2 sequence alignment computer program, no significant
sequence identities to any
known proteins were revealed.
17. Full-length PRO248 Polypeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding
polypeptides referred to in the present application as PR0248. In particular,
Applicants have identified and
isolated cDNA encoding a PR0248 polypeptide, as disclosed in further detail in
the Examples below. Using
known programs such as BLAST and FastA sequence alignment computer programs,
Applicants found that
a cDNA sequence encoding full-length native sequence PR0248 (amino acid
sequence shown in Figure 36 and
SEQ ID NO:65) has certain amino acid sequence identity with growth
differentiation factor 3, from mouse and
from homo sapiens. Accordingly, it is presently believed that PRO248
polypeptide disclosed in the present
application is a newly identified member of the transforming growth factor (3
family and possesses growth and
differentiation capabilities typical of the this family.
18. Full-length PR0353 Polvpeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding
polypeptides referred to in the present application as PR0353. In particular,
Applicants have identified and
isolated cDNA encoding PR0353 polypeptides, as disclosed in further detail in
the Examples below. Using
BLAST and, FastA sequence alignment computer programs, Applicants found that
various portions of the
PR0353 polypeptides have certain homology with the human and mouse complement
proteins. Accordingly,
it is presently believed that the PR0353 polypeptides disclosed in the present
application are newly identified
members of the complement protein family and possesses the ability to effect
the inflammation process as is
typical of the complement family of proteins.
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19. Full-length PRO1318 and PRO1600 Polypeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding
polypeptides referred to in the present application as PRO 1318 and PRO 1600.
In particular, Applicants have
identified and isolated cDNAs encoding PRO1318 and PRO1600 polypeptides, as
disclosed in further detail
in the Examples below. Using BLAST and FastA sequence alignment computer
programs, Applicants found
that cDNA sequence encoding full-length native sequence PRO1318 and PRO1600
(shown in Figure 40 and
SEQ ID NO:78 and Figure 42 and SEQ ID NO:80, respectively) have amino acid
sequence identity with one
or more chemokines. Accordingly, it is presently believed that the PRO1318 and
PRO1600 polypeptides
disclosed in the present application are newly identified members of the
chemokine family and possesses
activity typical of the chemokine family.
20. Full-length PRO533 Polypeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding
polypeptides referred to in the present application as PR0533. In particular,
Applicants have identified and
isolated cDNA encoding a PR0533 polypeptide, as disclosed in further detail in
the Examples below. Using
BLAST-2 and FastA sequence alignment computer programs, Applicants found that
a full-length native
sequence PRO533 (shown in Figure 46 and SEQ ID NO:86) has a Blast score of 509
and 53 % amino acid
sequence identity with fibroblast growth factor (FGF). Accordingly, it is
presently believed that PR0533
disclosed in the present application is a newly identified member of the
fibroblast growth factor family and may
possess activity typical of such polypeptides.
21. Full-length PRO301 Polypeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding
polypeptides referred to in the present application as PRO301. In particular,
Applicants have identified and
isolated cDNA encoding a PRO301 polypeptide, as disclosed in further detail in
the Examples below. Using
BLAST and FastA sequence alignment computer programs, Applicants found that a
full-length native sequence
PR0301 (shown in Figure 48 and SEQ ID NO:91) has a Blast score of 246
corresponding to 30% amino acid
sequence identity with human A33 antigen precursor. Accordingly, it is
presently believed that PRO301
disclosed in the present application is a newly identified member of the A33
antigen protein family and may
be expressed in human neoplastic diseases such as colorectal cancer.
22. Full-length PRO187 Polypeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding
polypeptides referred to in the present application as PRO187. In particular,
Applicants have identified and
isolated cDNA encoding a PRO 187 polypeptide, as disclosed in further detail
in the Examples below. Using
BLAST and FastA sequence alignment computer programs, Applicants found that a
full-length native sequence
PRO 187 (shown in Figure 50) has 74 % amino acid sequence identity and BLAST
score of 310 with various
androgen-induced growth factors and FGF-8. Accordingly, it is presently
believed that PRO 187 polypeptide
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disclosed in the present application is a newly identified member of the FGF-8
protein family and may possess
identify activity or property typical of the FGF-8-like protein family.
23. Full-length PR0337 Polypeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding
polypeptides referred to in the present application as PR0337. In particular,
Applicants have identified and
isolated cDNA encoding a PR0337 polypeptide, as disclosed in further detail in
the Examples below. Using
BLAST, BLAST-2 and FastA sequence alignment computer programs, Applicants
found that a full-length
native sequence PR0337 has 97 % amino acid sequence identity with rat
neurotrimin, 85 % sequence identity
with chicken CEPU, 73 % sequence identity with chicken G55, 59 % homology with
human LAMP and 84 %
homology with human OPCAM. Accordingly, it is presently believed that PR0337
disclosed in the present
application is a newly identified member of the IgLON sub family of the
immunoglobulin superfamily and may
possess neurite growth and differentiation potentiating properties.
24. Full-length PRO1411 Polypeptides
As far as is known, the DNA59212-1627 sequence encodes a novel factor
designated herein as
PRO 1411. However, using WU-BLAST2 sequence alignment computer programs, some
sequence identities
to known proteins were revealed.
25. Full-length PRO4356 Polypeptides
Using WU-BLAST2 sequence alignment computer programs, it has been found that a
full-length native
sequence PRO4356 (shown in Figure 56 and SEQ ID NO: 108) has certain amino
acid sequence identity with
metastasis associated GPI-anchored protein. Accordingly, it is presently
believed that PR04356 disclosed in
the present application is a newly identified member of this family and shares
similar mechanisms.
26. Full-length PRO246 Polypeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding
polypeptides referred to in the present application as PR0246. In particular,
Applicants have identified and
isolated cDNA encoding a PR0246 polypeptide, as disclosed in further detail in
the Examples below. Using
BLAST and FastA sequence alignment computer programs, Applicants found that a
portion of the PR0246
polypeptide has significant homology with the human cell surface protein HCAR.
Accordingly, it is presently
believed that PRO246 polypeptide disclosed in the present application may be a
newly identified membrane-
bound virus receptor or tumor cell-specific antigen.
27. Full-length PRO265 Polypentides
The present invention provides newly identified and isolated nucleotide
sequences encoding
polypeptides referred to in the present application as PR0265. In particular,
Applicants have identified and
isolated cDNA encoding a PR0265 polypeptide, as disclosed in further detail in
the Examples below. Using
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programs such as BLAST and FastA sequence alignment computer programs,
Applicants found that various
portions of the PR0265 polypeptide have significant homology with the
fibromodulin protein and fibromodulin
precursor protein. Applicants have also found that the DNA encoding the PR0265
polypeptide has significant
homology with platelet glycoprotein V, a member of the leucine rich related
protein family involved in skin
and wound repair. Accordingly, it is presently believed that PR0265
polypeptide disclosed in the present
application is a newly identified member of the leucine rich repeat family and
possesses protein protein binding
capabilities, as well as be involved in skin and wound repair as typical of
this family.
28. Full-length PRO941 Polypeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding
polypeptides referred to in the present application as PRO941. In particular,
Applicants have identified and
isolated cDNA encoding a PRO941 polypeptide, as disclosed in further detail in
the Examples below. Using
BLAST and FastA sequence alignment computer programs, Applicants found that
the PRO941 polypeptide has
significant similarity to one or more cadherin proteins. Accordingly, it is
presently believed that PR0941
polypeptide disclosed in the present application is a newly identified
cadherin homolog.
29. Full-length PRO10096 Polvoeptides
Using the ALIGN-2 sequence alignment computer program referenced above, it has
been found that
the full-length native sequence PRO 10096 (shown in Figure 64 and SEQ ID NO:
126) has certain amino acid
sequence identity with various interleukin-l0-related molecules. Accordingly,
it is presently believed that the
PRO10096 polypeptide disclosed in the present application is a newly
identified IL-10 homolog and may
possess one or more biological and/or immunological activities or properties
typical of that protein.
30. Full-length PR06003 Polypeptides
The DNA83568-2692 clone was isolated from a human fetal kidney library as
described in the
Examples below. As far as is known, the DNA83568-2692 nucleotide sequence
encodes a novel factor
designated herein as PRO6003; using the ALIGN-2 sequence alignment computer
program, no significant
sequence identities to any known proteins were revealed.
B. PRO Polypeptide Variants
In addition to the full-length native sequence PRO polypeptides described
herein, it is contemplated
that PRO variants can be prepared. PRO variants can be prepared by introducing
appropriate nucleotide
changes into the PRO DNA, and/or by synthesis of the desired PRO polypeptide.
Those skilled in the art will
appreciate that amino acid changes may alter post-translational processes of
the PRO, such as changing the
number or position of glycosylation sites or altering the membrane anchoring
characteristics.
Variations in the native full-length sequence PRO or in various domains of the
PRO 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. Patent No. 5,364,934. Variations
may be a substitution, deletion or
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insertion of one or more codons encoding the PRO that results in a change in
the amino acid sequence of the
PRO as compared with the native sequence PRO. Optionally the variation is by
substitution of at least one
amino acid with any other amino acid in one or more of the domains of the PRO.
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 PRO with that of homologous
known protein molecules 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 about
1 to 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 exhibited by the
full-length or mature native
sequence.
PRO polypeptide fragments are provided herein. Such fragments may be truncated
at the N-terminus
or C-terminus, or may lack internal residues, for example, when compared with
a full length native protein.
Certain fragments lack amino acid residues that are not essential for a
desired biological activity of the PRO
polypeptide.
PRO fragments may be prepared by any of a number of conventional techniques.
Desired peptide
fragments may be chemically synthesized. An alternative approach involves
generating PRO fragments by
enzymatic digestion, e.g., by treating the protein with an enzyme known to
cleave proteins at sites defined by
particular amino acid residues, or by digesting the DNA with suitable
restriction enzymes and isolating the
desired fragment. Yet another suitable technique involves isolating and
amplifying a DNA fragment encoding
a desired polypeptide fragment, by polymerase chain reaction (PCR).
Oligonucleotides that define the desired
termini of the DNA fragment are employed at the 5' and 3' primers in the PCR.
Preferably, PRO polypeptide
fragments share at least one biological and/or immunological activity with the
native PRO polypeptide disclosed
herein.
In particular embodiments, conservative substitutions of interest are shown in
Table 6 under the
heading of preferred substitutions. If such substitutions result in a change
in biological activity, then more
substantial changes, denominated exemplary substitutions in Table 6, or as
further described below in reference
to amino acid classes, are introduced and the products screened.
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Table 6
Original Exemplary Preferred
Residue Substitutions Substitutions
Ala (A) val; leu; ile val
Arg (R) lys; gin; asn lys
Asn (N) gin; his; lys; arg gin
Asp (D) glu glu
Cys (C) ser ser
Gln (Q) asn asn
Glu (E) asp asp
Gly (G) pro; ala ala
His (H) asn; gin; lys; arg arg
Ile (I) leu; val; met; ala; phe;
norleucine leu
Leu (L) norleucine; ile; val;
met; ala; phe ile
Lys (K) arg; gin; asn arg
Met (M) leu; phe; ile leu
Phe (F) leu; val; ile; ala; tyr leu
Pro (P) ala ala
Ser (S) thr thr
Thr (T) ser ser
Trp (W) tyr; phe tyr
Tyr (Y) trp; phe; thr; ser phe
Val (V) ile; leu; met; phe;
ala; norleucine leu
Substantial modifications in function or immunological identity of the PRO
polypeptide are
accomplished by selecting substitutions that differ significantly in their
effect on maintaining (a) the structure
of the polypeptide backbone in the area of the substitution, for example, as a
sheet or helical conformation,
(b) the charge or hydrophobicity of the molecule at the target site, or (c)
the bulk of the side chain. Naturally
occurring residues are divided into groups based on common side-chain
properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gin, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these
classes for another
class. Such substituted residues also may be introduced into the conservative
substitution sites or, more
preferably, into the remaining (non-conserved) sites.
The variations can be made using methods known in the art such as
oligonucleotide-mediated (site-
directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed
mutagenesis [Carteret 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)], restriction selection mutagenesis [Wells et al.,
Philos. Trans. R. Soc. London SerA,
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317:415 (1986)] or other known techniques can be performed on the cloned DNA
to produce the PRO variant
DNA.
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 [Cunningham and Wells,
Science, 244: 1081-1085 (1989)].
Alanine is also typically preferred because it is the most common amino acid.
Further, it is frequently found
in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman &
Co., N.Y.); Chothia, J.
Mol. Biol., 150:1(1976)]. If alanine substitution does not yield adequate
amounts of variant, an isoteric amino
acid can be used.
C. Modifications of PRO
Covalent modifications of PRO are included within the scope of this invention.
One type of covalent
modification includes reacting targeted amino acid residues of a PRO
polypeptide with an organic derivatizing
agent that is capable of reacting with selected side chains or the N- or C-
terminal residues of the PRO.
Derivatization with bifunctional agents is useful, for instance, for
crosslinking PRO to a water-insoluble
support matrix or surface for use in the method for purifying anti-PRO
antibodies, and vice-versa. 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.
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-amino groups of
lysine, arginine, and histidine side
chains [T. E. Creighton, Proteins: Structure and Molecular Properties, W.H.
Freeman & Co., San Francisco,
pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-
terminal carboxyl group.
Another type of covalent modification of the PRO 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 native sequence PRO (either by removing the underlying glycosylation
site or by deleting the
glycosylation by chemical and/or enzymatic means), and/or adding one or more
glycosylation sites that are not
present in the native sequence PRO. In addition, the phrase includes
qualitative changes in the glycosylation
of the native proteins, involving a change in the nature and proportions of
the various carbohydrate moieties
present.
Addition of glycosylation sites to the PRO polypeptide 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 PRO (for O-linked
glycosylation sites). The PRO amino
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acid sequence may optionally be altered through changes at the DNA level,
particularly by mutating the DNA
encoding the PRO polypeptide at preselected bases such that codons are
generated that will translate into the
desired amino acids.
Another means of increasing the number of carbohydrate moieties on the PRO
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 September 1987, and in Aplin and Wriston, CRC
Crit. Rev. Biochem., pp. 259-
306 (1981).
Removal of carbohydrate moieties present on the PRO polypeptide may be
accomplished chemically
or enzymatically or by mutational substitution of codons encoding for 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 exo-glycosidases as described by Thotakura et al., Meth.
Enzymol., 138:350 (1987).
Another type of covalent modification of PRO comprises linking the PRO
polypeptide to one of a
variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG),
polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835;
4,496,689; 4,301,144; 4,670,417;
4,791,192 or 4,179,337.
The PRO of the present invention may also be modified in a way to form a
chimeric molecule
comprising PRO fused to another, heterologous polypeptide or amino acid
sequence.
In one embodiment, such a chimeric molecule comprises a fusion of the PRO 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 the PRO. The presence of such
epitope-tagged forms of the PRO
can be detected using an antibody against the tag polypeptide. Also, provision
of the epitope tag enables the
PRO to be readily purified by affinity purification using an anti-tag antibody
or another type of affinity matrix
that binds to the epitope tag. 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, 6E10, 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, 3(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)].
In an alternative embodiment, the chimeric molecule may comprise a fusion of
the PRO with an
immunoglobulin or a particular region of an immunoglobulin. For a bivalent
form of the chimeric molecule
(also referred to as an "immunoadhesin"), such a fusion could be to the Fc
region of an IgG molecule. The
Ig fusions preferably include the substitution of a soluble (transmembrane
domain deleted or inactivated) form
of a PRO polypeptide in place of at least one variable region within an Ig
molecule. In a particularly preferred
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embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the
hinge, CHI, CH2 and CH3
regions of an IgG1 molecule. For the production of immunoglobulin fusions see
also US Patent No. 5,428,130
issued June 27, 1995.
D. Preparation of PRO
The description below relates primarily to production of PRO by culturing
cells transformed or
transfected with a vector containing PRO nucleic acid. It is, of course,
contemplated that alternative methods,
which are well known in the art, may be employed to prepare PRO. For instance,
the PRO 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, CA
(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, CA) using manufacturer's instructions. Various
portions of the PRO may be
chemically synthesized separately and combined using chemical or enzymatic
methods to produce the full-
length PRO.
1. Isolation of DNA Encoding PRO
DNA encoding PRO may be obtained from a cDNA library prepared from tissue
believed to possess
the PRO mRNA and to express it at a detectable level. Accordingly, human PRO
DNA can be conveniently
obtained from a cDNA library prepared from human tissue, such as described in
the Examples. The PRO-
encoding gene may also be obtained from a genomic library or by known
synthetic procedures (e.g., automated
nucleic acid synthesis).
Libraries can be screened with probes (such as antibodies to the PRO 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., Molecular Cloning: A Laboratory Manual (New York: Cold Spring
Harbor Laboratory Press,
1989). An alternative means to isolate the gene encoding PRO is to use PCR
methodology [Sambrook et al.,
supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor
Laboratory Press, 1995)].
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 '2P-labeled ATP, biotinylation or enzyme labeling.
Hybridization conditions, including
moderate stringency and high stringency, are provided in Sambrook et al.,
supra.
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 using methods known in the art and
as described herein.
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Nucleic acid having protein coding sequence may be obtained by screening
selected cDNA or genomic
libraries using the deduced amino acid sequence disclosed herein for the first
time, and, if necessary, using
conventional primer extension procedures as described in Sambrook et al., sue,
to detect precursors and
processing intermediates of mRNA that may not have been reverse-transcribed
into cDNA.
2. Selection and Transformation of Host Cells
Host cells are transfected or transformed with expression or cloning vectors
described herein for PRO
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.
Methods of eukaryotic cell transfection and prokaryotic cell transformation
are known to the ordinarily
skilled artisan, for example, CaCl2, CaPO4, liposome-mediated 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., sW, or
electroporation is generally
used for prokaryotes. 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 June 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
transfections have been described in U.S. Patent No. 4,399,216.
Transformations into yeast are typically
carried out according to the method of Van Solingen et al., J. Bact., 130:946
(1977) 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, 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).
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
E. coll. 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 W31 10 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable
prokaryotic host cells
include 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 41 P
disclosed in DD 266,710 published 12 April
1989), Pseudomonas such as P. aeruginosa, and Streptomyces. 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
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enzymes. For example, strain W3110 may be modified to effect a genetic
mutation in the genes encoding
proteins endogenous 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
W31 10 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3
phoA E15 (argF-lac)169
degP ompT kan`; E. coli W3110 strain 37D6, which has the complete genotype
tonA ptr3 phoA E15 (argF-
lac)169 degP ompT rbs7 ilvG kan ; 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.
Patent No. 4,946,783 issued 7 August 1990. Alternatively, in vitro methods of
cloning, e.g., PCR or other
nucleic acid polymerase reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or
yeast are suitable cloning
or expression hosts for PRO-encoding vectors. Saccharomyces cerevisiae is a
commonly used lower
eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach
and Nurse, Nature, 290:
140 [1981]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent
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., 154(2):737-742 [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
October 1990); and
filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO
91/00357 published 10 January
1991), and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem.
Bionhvs. Res. Commun., 112:284-
289 [1983]; Tilbum 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).
Suitable host cells for the expression of glycosylated PRO 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 CV 1 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 CCL5 1). The selection of the appropriate host cell is
deemed to be within the skill in
the art.
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3. Selection and Use of a Replicable Vector
The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO 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.
The PRO may be produced recombinantly not only directly, but also as a fusion
polypeptide with a
heterologous polypeptide, which may be a signal sequence or other polypeptide
having a specific cleavage site
at the N-terminus of the mature protein or polypeptide. In general, the signal
sequence may be a component
of the vector, or it may be a part of the PRO-encoding DNA that is inserted
into the vector. The signal
sequence may be a prokaryotic signal sequence selected, for example, from the
group of 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. Patent No. 5,010,182), or acid
phosphatase leader, the C. albicans
glucoamylase leader (EP 362,179 published 4 April 1990), or the signal
described in WO 90/13646 published
15 November 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.
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 21A plasmid
origin is suitable for yeast, and various viral origins (SV40, polyoma,
adenovirus, VSV or BPV) are useful
for cloning vectors in mammalian cells.
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.
An example of suitable selectable markers for mammalian cells are those that
enable the identification
of cells competent to take up the PRO-encoding nucleic acid, 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 trpl 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
trpl gene provides a selection marker for a mutant strain of yeast lacking the
ability to grow in tryptophan,
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for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
Expression and cloning vectors usually contain a promoter operably linked to
the PRO-encoding
nucleic acid sequence 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 (3-
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. Nat]. Acad. Sci. USA,
80:21-25 (1983)]. Promoters
for use in bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably linked to the DNA
encoding PRO.
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 Reg., 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.
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.
PRO 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 July
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.
Transcription of a DNA encoding the PRO 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, a-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 PRO coding sequence, but is preferably located at a
site 5' from the promoter.
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
CA 02372511 2004-05-18
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segments transcribed as polyadenylated fragments in the untranslated portion
of the mRNA encoding PRO.
Still other. methods, vectors, and host cells suitable for adaptation to the
synthesis of PRO 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.
4. Detecting Gene Amplification/Expression
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 maybe labeled
and the assay maybe 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.
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 PRO polypeptide or
against a synthetic peptide based
on the DNA sequences provided herein or against exogenous sequence fused to
PRO DNA and encoding a
specific antibody epitope.
5. Purification of Polypeptide
Forms of PRO 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 PRO can be disrupted by various
physical or chemical means, such
as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing
agents.
It may be desired to purify PRO 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 G-75; protein A Sepharose columns to remove contaminants such as IgG;
and metal chelating
columns to bind epitope-tagged forms of the PRO. 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); Scopes, Protein Purification: Principles and Practice, Springer-
Verlag, New York (1982). The
purification step(s) selected will depend, for example, on the nature of the
production process used and the
particular PRO produced.
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E. Uses for PRO
Nucleotide sequences (or their complement) encoding PRO 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. PRO nucleic acid will also be useful for
the preparation of PRO
polypeptides by the recombinant techniques described herein.
The full-length native sequence PRO gene, or portions thereof, may be used as
hybridization probes
for a cDNA library to isolate the full-length PRO cDNA or to isolate still
other cDNAs (for instance, those
encoding naturally-occurring variants of PRO or PRO from other species) which
have a desired sequence
identity to the native PRO sequence disclosed herein. Optionally, the length
of the probes will be about 20 to
about 50 bases. The hybridization probes may be derived from at least
partially novel regions of the full length
native nucleotide sequence wherein those regions may be determined without
undue experimentation or from
genomic sequences including promoters, enhancer elements and introns of native
sequence PRO. By way of
example, a screening method will comprise isolating the coding region of the
PRO 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 32P or 355, 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 the PRO gene of the present invention can be used to screen
libraries of human cDNA, genomic
DNA or mRNA to determine which members of such libraries the probe hybridizes
to. Hybridization
techniques are described in further detail in the Examples below.
Any EST sequences disclosed in the present application may similarly be
employed as probes, using
the methods disclosed herein.
Other useful fragments of the PRO nucleic acids include antisense or sense
oligonucleotides
comprising a singe-stranded nucleic acid sequence (either RNA or DNA) capable
of binding to target PRO
mRNA (sense) or PRO DNA (antisense) sequences. Antisense or sense
oligonucleotides, according to the
present invention, comprise a fragment of the coding region of PRO DNA. Such a
fragment generally
comprises at least about 14 nucleotides, preferably from about 14 to 30
nucleotides. The ability to derive an
antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a
given protein is described in,
for example, Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et
al. (BioTechniques 6:958,
1988).
Binding of antisense or sense oligonucleotides to target nucleic acid
sequences results in the formation
of duplexes that block transcription or translation of the target sequence by
one of several means, including
enhanced degradation of the duplexes, premature termination of transcription
or translation, or by other means.
The antisense oligonucleotides thus may be used to block expression of PRO
proteins. Antisense or sense
oligonucleotides further comprise oligonucleotides having modified sugar-
phosphodiester backbones (or other
sugar linkages, such as those described in WO 91/06629) and wherein such sugar
linkages are resistant to
endogenous nucleases. Such oligonucleotides with resistant sugar linkages are
stable in vivo (i.e., capable of
resisting enzymatic degradation) but retain sequence specificity to be able to
bind to target nucleotide
sequences.
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Other examples of sense or antisense oligonucleotides include those
oligonucleotides which are
covalently linked to organic moieties, such as those described in WO 90/10048,
and other moieties that
increases affinity of the oligonucleotide for a target nucleic acid sequence,
such as poly-(L-lysine). Further still,
intercalating agents, such as ellipticine, and alkylating agents or metal
complexes may be attached to sense or
antisense oligonucleotides to modify binding specificities of the antisense or
sense oligonucleotide for the target
nucleotide sequence.
Antisense or sense oligonucleotides may be introduced into a cell containing
the target nucleic acid
sequence by any gene transfer method, including, for example, CaPO4-mediated
DNA transfection,
electroporation, or by using gene transfer vectors such as Epstein-Barr virus.
In a preferred procedure, an
antisense or sense oligonucleotide is inserted into a suitable retroviral
vector. A cell containing the target
10. nucleic acid sequence is contacted with the recombinant retroviral vector,
either in vivo or ex vivo. Suitable
retroviral vectors include, but are not limited to, those derived from the
murine retrovirus M-MuLV, N2 (a
retrovirus derived from M-MuLV), or the double copy vectors designated DCTSA,
DCT5B and DCT5C (see
WO 90/13641).
Sense or antisense oligonucleotides also may be introduced into a cell
containing the target nucleotide
sequence by formation of a conjugate with a ligand binding molecule, as
described in WO 91/04753. Suitable
ligand binding molecules include, but are not limited to, cell surface
receptors, growth factors, other cytokines,
or other ligands that bind to cell surface receptors. Preferably, conjugation
of the ligand binding molecule does
not substantially interfere with the ability of the ligand binding molecule to
bind to its corresponding molecule
or receptor, or block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell.
Alternatively, a sense or an antisense oligonucleotide may be introduced into
a cell containing the
target nucleic acid sequence by formation of an oligonucleotide-lipid complex,
as described in WO 90/10448.
The sense or antisense oligonucleotide-lipid complex is preferably dissociated
within the cell by an endogenous
lipase.
Antisense or sense RNA or DNA molecules are generally at least about 5 bases
in length, about 10
bases in length, about 15 bases in length, about 20 bases in length, about 25
bases in length, about 30 bases
in length, about 35 bases in length, about 40 bases in length, about 45 bases
in length, about 50 bases in length,
about 55 bases in length, about 60 bases in length, about 65 bases in length,
about 70 bases in length, about
75 bases in length, about 80 bases in length, about 85 bases in length, about
90 bases in length, about 95 bases
in length, about 100 bases in length, or more.
The probes may also be employed in PCR techniques to generate a pool of
sequences for identification
of closely related PRO coding sequences.
Nucleotide sequences encoding a PRO can also be used to construct
hybridization probes for mapping
the gene which encodes that PRO 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.
When the coding sequences for PRO encode a protein which binds to another
protein (example, where
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the PRO is a receptor), the PRO can be used in assays to identify the other
proteins or molecules involved in
the binding interaction. By such methods, inhibitors of the receptor/ligand
binding interaction can be
identified. Proteins involved in such binding interactions can also be used to
screen for peptide or small
molecule inhibitors or agonists of the binding interaction. Also, the receptor
PRO can be used to isolate
correlative ligand(s). Screening assays can be designed to find lead compounds
that mimic the biological
activity of a native PRO or a receptor for PRO. Such screening assays will
include assays amenable to high-
throughput screening of chemical libraries, making them particularly suitable
for identifying small molecule
drug candidates. Small molecules contemplated include synthetic organic or
inorganic compounds. 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.
Nucleic acids which encode PRO or its 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 PRO can be used to clone genomic
DNA encoding PRO in
accordance with established techniques and the genomic sequences used to
generate transgenic animals that
contain cells which express DNA encoding PRO. 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.
Patent Nos. 4,736,866 and 4,870,009. Typically, particular cells would be
targeted for PRO transgene
incorporation with tissue-specific enhancers. Transgenic animals that include
a copy of a transgene encoding
PRO 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 PRO. 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
reagent 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.
Alternatively, non-human homologues of PRO can be used to construct a PRO
"knock out" animal
which has a defective or altered gene encoding PRO as a result of homologous
recombination between the
endogenous gene encoding PRO and altered genomic DNA encoding PRO introduced
into an embryonic stem
cell of the animal. For example, cDNA encoding PRO can be used to clone
genomic DNA encoding PRO in
accordance with established techniques. A portion of the genomic DNA encoding
PRO 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
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(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, for their ability to
defend against certain pathological conditions and for their development of
pathological conditions due to
absence of the PRO polypeptide.
Nucleic acid encoding the PRO polypeptides may also be used in gene therapy.
In gene therapy
applications, genes are introduced into cells in order to achieve in vivo
synthesis of a therapeutically effective
genetic product, for example for replacement of a defective gene. "Gene
therapy" includes both conventional
gene therapy where a lasting effect is achieved by a single treatment, and the
administration of gene therapeutic
agents, which involves the one time or repeated administration of a
therapeutically effective DNA or mRNA.
Antisense RNAs and DNAs can be used as therapeutic agents for blocking the
expression of certain genes in
vivo. It has already been shown that short antisense oligonucleotides can be
imported into cells where they act
as inhibitors, despite their low intracellular concentrations caused by their
restricted uptake by the cell
membrane. (Zamecnik et al., Prop. Natl. Acad. Sci. USA 83:4143-4146 [1986]).
The oligonucleotides can
be modified to enhance their uptake, e.g. by substituting their negatively
charged phosphodiester groups by
uncharged groups.
There are a variety of techniques available for introducing nucleic acids into
viable cells. The
techniques vary depending upon whether the nucleic acid is transferred into
cultured cells in vitro, or in vivo
in the cells of the intended host. Techniques suitable for the transfer of
nucleic acid into mammalian cells in
vitro include the use of liposomes, electroporation, microinjection, cell
fusion, DEAE-dextran, the calcium
phosphate precipitation method, etc. The currently preferred in vivo gene
transfer techniques include
transfection with viral (typically retroviral) vectors and viral coat protein-
liposome mediated transfection (Dzau
et al., Trends in Biotechnology 11, 205-210 [1993]). In some situations it is
desirable to provide the nucleic
acid source with an agent that targets the target cells, such as an antibody
specific for a cell surface membrane
protein or the target cell, a ligand for a receptor on the target cell, etc.
Where liposomes are employed,
proteins which bind to a cell surface membrane protein associated with
endocytosis may be used for targeting
and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic
for a particular cell type, antibodies
for proteins which undergo internalization in cycling, proteins that target
intracellular localization and enhance
intracellular half-life. The technique of receptor-mediated endocytosis is
described, for example, by Wu et
al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.
Acad. Sci. USA 87, 3410-3414
(1990). For review of gene marking and gene therapy protocols see Anderson et
al., Science 256, 808-813
(1992).
The PRO polypeptides described herein may also be employed as molecular weight
markers for
protein electrophoresis purposes and the isolated nucleic acid sequences may
be used for recombinantly
expressing those markers.
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The nucleic acid molecules encoding the PRO polypeptides or fragments thereof
described herein are
useful for chromosome identification. In this regard, there exists an ongoing
need to identify new chromosome
markers, since relatively few chromosome marking reagents, based upon actual
sequence data are presently
available. Each PRO nucleic acid molecule of the present invention can be used
as a chromosome marker.
The PRO polypeptides and nucleic acid molecules of the present invention may
also be used for tissue
typing, wherein the PRO polypeptides of the present invention may be
differentially expressed in one tissue
as compared to another. PRO nucleic acid molecules will find use for
generating probes for PCR, Northern
analysis, Southern analysis and Western analysis.
The PRO polypeptides described herein may also be employed as therapeutic
agents. The PRO
polypeptides of the present invention can be formulated according to known
methods to prepare
pharmaceutically useful compositions, whereby the PRO product hereof is
combined in admixture with a
pharmaceutically acceptable carrier vehicle. Therapeutic formulations are
prepared for storage by mixing the
active ingredient having the desired degree of purity with optional
physiologically 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; 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,
arginine or lysine; monosaccharides,
disaccharides and other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions
such as sodium; and/or nonionic
surfactants such as TWEENTM, PLURONICSTM or PEG.
The formulations to be used for in vivo administration must be sterile. This
is readily accomplished
by filtration through sterile filtration membranes, prior to or following
lyophilization and reconstitution.
Therapeutic compositions herein generally are placed into a container having a
sterile access port, for
example, an intravenous solution bag or vial having a stopper pierceable by a
hypodermic injection needle.
The route of administration is in accord with known methods, e.g. injection or
infusion by
intravenous, intraperitoneal, intracerebral, intramuscular, intraocular,
intraarterial or intralesional routes,
topical administration, or by sustained release systems.
Dosages and desired drug concentrations of pharmaceutical compositions of the
present invention may
vary depending on the particular use envisioned. The determination of the
appropriate dosage or route of
administration is well within the skill of an ordinary physician. Animal
experiments provide reliable guidance
for the determination of effective doses for human therapy. Interspecies
scaling of effective doses can be
performed following the principles laid down by Mordenti, J. and Chappell, W.
"The use of interspecies
scaling in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi
et al., Eds., Pergamon Press,
New York 1989, pp. 42-96.
When in vivo administration of a PRO polypeptide or agonist or antagonist
thereof is employed,
normal dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of
mammal body weight or more
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per day, preferably about 1 g/kg/day to 10 mg/kg/day, depending upon the
route of administration. Guidance
as to particular dosages and methods of delivery is provided in the
literature; see, for example, U.S. Pat. Nos.
4,657,760; 5,206,344; or 5,225,212. It is anticipated that different
formulations will be effective for different
treatment compounds and different disorders, that administration targeting one
organ or tissue, for example,
may necessitate delivery in a manner different from that to another organ or
tissue.
Where sustained-release administration of a PRO polypeptide is desired in a
formulation with release
characteristics suitable for the treatment of any disease or disorder
requiring administration of the PRO
polypeptide, microencapsulation of the PRO polypeptide is contemplated.
Microencapsulation of recombinant
proteins for sustained release has been successfully performed with human
growth hormone (rhGH), interferon-
(rhIFN- ), interleukin-2, and MN rgp120. Johnson et al., Nat. Med., 2:795-799
(1996); Yasuda, Biomed.
Ther., 27:1221-1223 (1993); Horaetal., Bio/Technology. 8:755-758(1990);
Cleland, "Design and Production
of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere
Systems," in Vaccine Design:
The Subunit and Adjuvant Approach, Powell and Newman, eds, (Plenum Press: New
York, 1995), pp. 439-
462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat. No. 5,654,010.
The sustained-release formulations of these proteins were developed using poly-
lactic-coglycolic acid
(PLGA) polymer due to its biocompatibility and wide range of biodegradable
properties. The degradation
products of PLGA, lactic and glycolic acids, can be cleared quickly within the
human body. Moreover, the
degradability of this polymer can be adjusted from months to years depending
on its molecular weight and
composition. Lewis, "Controlled release of bioactive agents from
lactide/glycolide polymer," in: M. Chasin
and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel
Dekker: New York, 1990),
pp. 1-41.
This invention encompasses methods of screening compounds to identify those
that mimic the PRO
polypeptide (agonists) or prevent the effect of the PRO polypeptide
(antagonists). Screening assays for
antagonist drug candidates are designed to identify compounds that bind or
complex with the PRO 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.
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.
All assays for antagonists are common in that they call for contacting the
drug candidate with a PRO
polypeptide encoded by a nucleic acid identified herein under conditions and
for a time sufficient to allow these
two components to interact.
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 PRO 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 PRO polypeptide and drying. Alternatively, an immobilized antibody,
e.g., a monoclonal antibody,
specific for the PRO polypeptide to be immobilized can be used to anchor it to
a solid surface. The assay is
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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.
If the candidate compound interacts with but does not bind to a particular PRO
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 GALL-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 (3-galactosidase.
A complete kit (MATCHMAKER") 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.
Compounds that interfere with the interaction of a gene encoding a PRO
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.
To assay for antagonists, the PRO polypeptide may be added to a cell along
with the compound to be
screened for a particular activity and the ability of the compound to inhibit
the activity of interest in the
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presence of the PRO polypeptide indicates that the compound is an antagonist
to the PRO polypeptide.
Alternatively, antagonists may be detected by combining the PRO polypeptide
and a potential antagonist with
membrane-bound PRO polypeptide receptors or recombinant receptors under
appropriate conditions for a
competitive inhibition assay. The PRO polypeptide can be labeled, such as by
radioactivity, such that the
number of PRO 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 PRO 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 PRO polypeptide.
Transfected cells that are grown on glass slides are exposed to labeled PRO
polypeptide. The PRO 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.
As an alternative approach for receptor identification, labeled PRO
polypeptide can be photoaffinity-
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.
In another assay for antagonists, mammalian cells or a membrane preparation
expressing the receptor
would be incubated with labeled PRO polypeptide in the presence of the
candidate compound. The ability of
the compound to enhance or block this interaction could then be measured.
More specific examples of potential antagonists include an oligonucleotide
that binds to the fusions
of immunoglobulin with PRO polypeptide, 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 PRO polypeptide that recognizes the receptor but imparts no effect,
thereby competitively inhibiting the
action of the PRO polypeptide.
Another potential PRO 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 DNA or RNA. For example,
the 5' coding portion of
the polynucleotide sequence, which encodes the mature PRO 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
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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 PRO polypeptide. The
antisense RNA oligonucleotide
hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule
into the PRO polypeptide
(antisense - Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as
Antisense Inhibitors of Gene
Expression (CRC Press: Boca Raton, FL, 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 PRO
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.
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 PRO polypeptide, thereby
blocking the normal biological
activity of the PRO 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.
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 September 18, 1997).
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.
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.
Uses of the herein disclosed molecules may also be based upon the positive
functional assay hits
disclosed and described below. Methods based upon those assay hits are also
encompassed by the present
invention.
F. Anti-PRO Antibodies
The present invention further provides anti-PRO antibodies. Exemplary
antibodies include polyclonal,
monoclonal, humanized, bispecific, and heteroconjugate antibodies.
1. Polyclonal Antibodies
The anti-PRO antibodies 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
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agent and/or adjuvant will be injected in the mammal by multiple subcutaneous
or intraperitoneal injections.
The immunizing agent may include the PRO 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.
2. Monoclonal Antibodies
The anti-PRO antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may
be prepared using hybridoma methods, such as those described by Kohler and
Milstein, Nature, 256:495
(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.
The immunizing agent will typically include the PRO 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 polyethylene glycol, to form
a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice,
Academic Press, (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.
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, California and the
American Type Culture Collection,
Manassas, Virginia. 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].
The culture medium in which the hybridoma cells are cultured can then be
assayed for the presence
of monoclonal antibodies directed against PRO. Preferably, the binding
specificity of monoclonal antibodies
produced by the hybridoma cells is determined by immunoprecipitation or by an
in vitro binding assay, such
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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).
After the desired hybridoma cells are identified, the clones may be subcloned
by limiting dilution
procedures and grown by standard methods [Goding, su ra . 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.
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.
The monoclonal antibodies may also be made by recombinant DNA methods, such as
those described
in U.S. Patent 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, Chinese hamster ovary
(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. Patent No. 4,816,567; Morrison et al., su ra 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.
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.
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.
3. Human and Humanized Antibodies
The anti-PRO 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')2 or
other antigen-binding
subsequences of antibodies) which contain minimal sequence derived from non-
human immunoglobulin.
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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 optimally also will comprise at least a portion of an immunoglobulin
constant region (Fc), typically
that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986);
Riechmann et al., Nature,
332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
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. Patent 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.
Human antibodies can also be produced 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 Boerner et al. are also
available for the preparation of
human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, p. 77
(1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)]. Similarly, human
antibodies can be made by
introducing of human immunoglobulin loci into transgenic animals, e.g., mice
in which the endogenous
immunoglobulin genes have been partially or completely inactivated. Upon
challenge, human antibody
production is observed, which closely resembles that seen in humans in all
respects, including gene
rearrangement, assembly, and antibody repertoire. This approach is described,
for example, in U.S. Patent
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in
the following scientific
publications: Marks et al., Bio/Technology ] 0, 779-783 (1992); Lonberg et
al., Nature 368 856-859 (1994);
Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14,
845-51 (1996); Neuberger,
Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol.
13 65-93 (1995).
The antibodies may also be affinity matured using known selection and/or
mutagenesis methods as
described above. Preferred affinity matured antibodies have an affinity which
is five times, more preferably
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times, even more preferably 20 or 30 times greater than the starting antibody
(generally murine, humanized
or human) from which the matured antibody is prepared.
4. Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies that have binding
5 specificities for at least two different antigens. In the present case, one
of the binding specificities is for the
PRO, the other one is for any other antigen, and preferably for a cell-surface
protein or receptor or receptor
subunit.
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-
10 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).
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 (CH 1) 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).
According to another approach described in WO 96/27011, the interface between
a pair of antibody
molecules can be engineered to maximize the percentage of heterodimers which
are recovered from
recombinant cell culture. The preferred interface comprises at least a part of
the CH3 region of an antibody
constant domain. In this method, one or more small amino acid side chains from
the interface of the first
antibody molecule are replaced with larger side chains (e.g. tyrosine or
tryptophan). Compensatory "cavities"
of identical or similar size to the large side chain(s) are created on the
interface of the second antibody
molecule by replacing large amino acid side chains with smaller ones (e. g.
alanine or threonine). This provides
a mechanism for increasing the yield of the heterodimer over other unwanted
end-products such as
homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody
fragments (e.g. F(ab')2
bispecific antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been
described in the literature. For example, bispecific antibodies can be
prepared can be prepared using chemical
linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein
intact antibodies are
proteolytically cleaved to generate F(ab')2 fragments. These fragments are
reduced in the presence of the
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dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and
prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of
the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction
with mercaptoethylamine and is
mixed with an equimolar amount of the other Fab'-TNB derivative to form the
bispecific antibody. The
bispecific antibodies produced can be used as agents for the selective
immobilization of enzymes.
Fab' fragments may be directly recovered from E. coli and chemically coupled
to form bispecific
antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the
production of a fully humanized
bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately
secreted from E. coli and subjected
to directed chemical coupling in vitro to form the bispecific antibody. The
bispecific antibody thus formed was
able to bind to cells overexpressing the ErbB2 receptor and normal human T
cells, as well as trigger the lytic
activity of human cytotoxic lymphocytes against human breast tumor targets.
Various technique for making and isolating bispecific antibody fragments
directly from recombinant
cell culture have also been described. For example, bispecific antibodies have
been produced using leucine
zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine
zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different antibodies
by gene fusion. The antibody
homodimers were reduced at the hinge region to form monomers and then re-
oxidized to form the antibody
heterodimers. This method can also be utilized for the production of antibody
homodimers. The "diabody"
technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-
6448 (1993) has provided an
alternative mechanism for making bispecific antibody fragments. The fragments
comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain (VL) by a
linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the VH and VL
domains of one fragment
are forced to pair with the complementary VL and V. domains of another
fragment, thereby forming two
antigen-binding sites. Another strategy for making bispecific antibody
fragments by the use of single-chain
Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol.
152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example,
trispecific antibodies can be
prepared. Tutt et al., J. Immunol. 147:60 (1991).
Exemplary bispecific antibodies may bind to two different epitopes on a given
PRO polypeptide
herein. Alternatively, an anti-PRO polypeptide arm may be combined with an arm
which binds to a triggering
molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3,
CD28, or B7), or Fc receptors
for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) so as
to focus cellular defense
mechanisms to the cell expressing the particular PRO polypeptide. Bispecific
antibodies may also be used to
localize cytotoxic agents to cells which express a particular PRO polypeptide.
These antibodies possess a
PRO-binding arm and an arm which binds a cytotoxic agent or a radionuclide
chelator, such as EOTUBE,
DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the PRO
polypeptide and further binds
tissue factor (TF).
5. Heteroconjuaaate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate
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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. Patent 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 crosslinking
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. Patent No.
4,676,980.
6. Effector Function Engineering
It may be desirable to modify the antibody of the invention with respect to
effector function, so as to
enhance, e.g., the effectiveness of the antibody in treating cancer. For
example, cysteine residue(s) may be
introduced into the Fc region, thereby allowing interchain disulfide bond
formation in this region. The
homodimeric antibody thus generated may have improved internalization
capability and/or increased
complement-mediated cell killing and antibody-dependent cellular cytotoxicity
(ADCC). See Caron et al., J.
Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922
(1992). Homodimeric antibodies
with enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in
Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody
can be engineered that has
dual Fc regions and may thereby have enhanced complement lysis and ADCC
capabilities. See Stevenson et
al., Anti-Cancer Drug Design. 3: 219-230 (1989).
7. Immunoconju ates
The invention also pertains to immunoconjugates comprising an antibody
conjugated to a cytotoxic
agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active
toxin of bacterial, fungal, plant,
or animal origin, or fragments thereof), or a radioactive isotope (i.e., a
radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
been described
above. Enzymatically active toxins and fragments thereof that can be used
include diphtheria A chain,
nonbinding active fragments of diphtheria toxin, exotoxin A chain (from
Pseudomonas aeruginosa), ricin A
chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii
proteins, dianthin proteins, Phytolaca
americans proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,
enomycin, and the tricothecenes. A variety
of radionuclides are available for the production of radioconjugated
antibodies. Examples include 212Bi, 1311,
131In, 90Y, and "Re. Conjugates of the antibody and cytotoxic agent are made
using a variety of
bifunctional protein-coupling agents such as N-succinimidyl-3-(2-
pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl
adipimidate HCL), active esters
(such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-
azido compounds (such as bis (p-
azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-
diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine
compounds (such as 1,5-difluoro-2,4-
dinitrobenzene). For example, a ricin immunotoxin can be prepared as described
in Vitetta et al., Science,
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238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene
triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide
to the antibody. See
WO94/11026.
In another embodiment, the antibody may be conjugated to a "receptor" (such
streptavidin) for
utilization in tumor pretargeting wherein the antibody-receptor conjugate is
administered to the patient,
followed by removal of unbound conjugate from the circulation using a clearing
agent and then administration
of a "ligand" (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., a
radionucleotide).
8. Immunoliposomes
The antibodies disclosed herein may also be formulated as immunoliposomes.
Liposomes containing
the antibody are prepared by methods known in the art, such as described in
Epstein et al., Proc. Natl. Acad.
Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030
(1980); and U.S. Pat. Nos.
4,485,045 and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Patent No.
5,013,556.
Particularly useful liposomes can be generated by the reverse-phase
evaporation method with a lipid
composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine
(PEG-PE). Liposomes are extruded through filters of defined-pore size to yield
liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention can be
conjugated to the liposomes as
described in Martin et al ., J. Biol. Chem., 257: 286-288 (1982) via a
disulfide-interchange reaction. A
chemotherapeutic agent (such as Doxorubicin) is optionally contained within
the liposome. See Gabizon et al. ,
J. National Cancer Inst., 81(19): 1484 (1989).
9. Pharmaceutical Compositions of Antibodies
Antibodies specifically binding a PRO polypeptide identified herein, as well
as other molecules
identified by the screening assays disclosed hereinbefore, can be administered
for the treatment of various
disorders in the form of pharmaceutical compositions.
If the PRO polypeptide is intracellular and whole antibodies are used as
inhibitors, internalizing
antibodies are preferred. However, lipofections or liposomes can also be used
to deliver the antibody, or an
antibody fragment, into cells. Where antibody fragments are used, the smallest
inhibitory fragment that
specifically binds to the binding domain of the target protein is preferred.
For example, based upon the
variable-region sequences of an antibody, peptide molecules can be designed
that retain the ability to bind the
target protein sequence. Such peptides can be synthesized chemically and/or
produced by recombinant DNA
technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-
7893 (1993). T h e
formulation herein may also contain more than one active compound as necessary
for the particular indication
being treated, preferably those with complementary activities that do not
adversely affect each other.
Alternatively, or in addition, the composition may comprise an agent that
enhances its function, such as, for
example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-
inhibitory agent. Such molecules
are suitably present in combination in amounts that are effective for the
purpose intended.
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The active ingredients may also be entrapped in microcapsules prepared, for
example, by coacervation
techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsules and
poly-(methylmethacylate) microcapsules, respectively, in colloidal drug
delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles, and
nanocapsules) or in macroemulsions.
Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.
The formulations to be used for in vivo administration must be sterile. This
is readily accomplished
by filtration through sterile filtration membranes.
Sustained-release preparations may be prepared. Suitable examples of sustained-
release preparations
include semipermeable matrices of solid hydrophobic polymers containing the
antibody, 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 T" (injectable
microspheres composed of lactic acid-glycolic acid copolymer and leuprolide
acetate), and poly-D-(-)-3-
hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins for shorter
time periods. When encapsulated
antibodies remain in the body for a long time, they may denature or aggregate
as a result of exposure to
moisture at 37 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 sulthydryl residues,
lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and developing
specific polymer matrix compositions.
G. Uses for anti-PRO Antibodies
The anti-PRO antibodies of the invention have various utilities. For example,
anti-PRO antibodies
may be used in diagnostic assays for PRO, e.g., detecting its expression in
specific cells, tissues, or serum.
Various diagnostic assay techniques known in the art may be used, such as
competitive binding assays, direct
or indirect sandwich assays and immunoprecipitation assays conducted in either
heterogeneous or homogeneous
phases [Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc.
(1987) pp. 147-158]. The
antibodies used in the diagnostic assays can be labeled with a detectable
moiety. The detectable moiety should
be capable of producing, either directly or indirectly, a detectable signal.
For example, the detectable moiety
may be a radioisotope, such as 3H, '4C, 32P, 35S, or 125 1, a fluorescent or
chemiluminescent compound, such
as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as
alkaline phosphatase, beta-
galactosidase or horseradish peroxidase. Any method known in the art for
conjugating the antibody to the
detectable moiety may be employed, including those methods described by Hunter
et al., Nature, 144:945
(1962); David et al., Biochemistry, 13:1014 (1974); Pain et al., J. Immunol.
Meth., 40:219 (1981); and
Nygren, J. Histochem. and Cytochem., 30:407 (1982).
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Anti-PRO antibodies also are useful for the affinity purification of PRO from
recombinant cell culture
or natural sources. In this process, the antibodies against PRO are
immobilized on a suitable support, such
a Sephadex resin or filter paper, using methods well known in the art. The
immobilized antibody then is
contacted with a sample containing the PRO 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 PRO, which is bound to the
immobilized antibody. Finally, the support is washed with another suitable
solvent that will release the PRO
from the antibody.
The following examples are offered for illustrative purposes only, and are not
intended to limit the
scope of the present invention in any way.
All patent and literature references cited in the present specification are
hereby incorporated by
reference in their entirety.
EXAMPLES
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, Manassas,
VA.
EXAMPLE 1: Extracellular Domain Homology Screening to Identify Novel
Polvoentides and cDNA Encoding
Therefor
The extracellular domain (ECD) sequences (including the secretion signal
sequence, if any) from about
950 known secreted proteins from the Swiss-Prot public database were used to
search EST databases. The
EST databases included public databases (e.g., . Dayhoff, GenBank), and
proprietary databases (e.g.
LIFESEQI, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed
using the computer program
BLAST or BLAST-2 (Altschul 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 sequences. Those
comparisons with 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,
WA).
Using this extracellular domain homology screen, consensus DNA sequences were
assembled relative
to the other identified EST sequences using phrap. In addition, the consensus
DNA sequences obtained were
often (but not always) extended using repeated cycles of BLAST or BLAST-2 and
phrap to extend the
consensus sequence as far as possible using the sources of EST sequences
discussed above.
Based upon the consensus sequences obtained as described above,
oligonucleotides were then
synthesized and used to identify by PCR a cDNA library that contained the
sequence of interest and for use
as probes to isolate a clone of the full-length coding sequence for a PRO
polypeptide. Forward and reverse
PCR primers generally range from 20 to 30 nucleotides and are often designed
to give a PCR product of about
100-1000 bp in length. The probe sequences are typically 40-55 bp in length.
In some cases, additional
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oligonucleotides are synthesized when the consensus sequence is greater than
about 1-1.5kbp. In order to
screen several libraries for a full-length clone, DNA from the libraries was
screened by PCR amplification,
as per Ausubel et al., Current Protocols in Molecular Biology, with the PCR
primer pair. A positive library
was then used to isolate clones encoding the gene of interest using the probe
oligonucleotide and one of the
primer pairs.
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, CA.
The cDNA was primed with
oligo dT containing a Not! site, linked with blunt to Sail hemikinased
adaptors, cleaved with NotI, sized
appropriately by gel electrophoresis, and cloned in a defined orientation into
a suitable cloning, vector (such
as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfit
site; see, Holmes et al.,
Science, 253:1278-1280 (1991)) in the unique Xhol and Not! sites.
EXAMPLE 2: Isolation of cDNA clones by Amylase Screening
1. Preparation of oligo dT mimed cDNA library
mRNA was isolated from a human tissue of interest using reagents and protocols
from Invitrogen, San
Diego, CA (Fast Track 2). This RNA was used to generate an oligo dT primed
cDNA library in the vector
pRK5D using reagents and protocols from Life Technologies, Gaithersburg, MD
(Super Script Plasmid
System). In this procedure, the double stranded cDNA was sized to greater than
1000 bp and the SalUNotl
tinkered cDNA was cloned into Xhol/Notl cleaved vector. pRK5D is a cloning
vector that has an sp6
transcription initiation site followed by an Sfil restriction enzyme site
preceding the Xhol/Notl cDNA cloning
sites.
2. Preparation of random primed cDNA library
A secondary cDNA library was generated in order to preferentially represent
the 5' ends of the
primary cDNA clones. Sp6 RNA was generated from the primary library (described
above), and this RNA
was used to generate a random primed cDNA library in the vector pSST-AMY.0
using reagents and protocols
from Life Technologies (Super Script Plasmid System, referenced above). In
this procedure the double
stranded cDNA was sized to 500-1000 bp, tinkered with blunt to Not! adaptors,
cleaved with Sf!, and cloned
into Sfi!/Notl cleaved vector. pSST-AMY.0 is a cloning vector that has a yeast
alcohol dehydrogenase
promoter preceding the cDNA cloning sites and the mouse amylase sequence (the
mature sequence without the
secretion signal) followed by the yeast alcohol dehydrogenase terminator,
after the cloning sites. Thus, cDNAs
cloned into this vector that are fused in frame with amylase sequence will
lead to the secretion of amylase from
appropriately transfected yeast colonies.
3. Transformation and Detection
DNA from the library described in paragraph 2 above was chilled on ice to
which was added
electrocompetent DHIOB bacteria (Life Technologies, 20 ml). The bacteria and
vector mixture was then
electroporated as recommended by the manufacturer. Subsequently, SOC media
(Life Technologies, 1 ml)
was added and the mixture was incubated at 37 C for 30 minutes. The
transformants were then plated onto
20 standard 150 mm LB plates containing ampicillin and incubated for 16 hours
(37 C). Positive colonies
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were scraped off the plates and the DNA was isolated from the bacterial pellet
using standard protocols, e.g.
CsCI-gradient. The purified DNA was then carried on to the yeast protocols
below.
The yeast methods were divided into three categories: (1) Transformation of
yeast with the
plasmid/cDNA combined vector; (2) Detection and isolation of yeast clones
secreting amylase; and (3) PCR
amplification of the insert directly from the yeast colony and purification of
the DNA for sequencing and
further analysis.
The yeast strain used was HD56-5A (ATCC-90785). This strain has the following
genotype: MAT
alpha, ura3-52, leu2-3, leu2-112, his3-11, his3-15, MALI, SUC+, GAL'.
Preferably, yeast mutants can be
employed that have deficient post-translational pathways. Such mutants may
have translocation deficient alleles
in sec7l, sec72, sec62, with truncated sec7l being most preferred.
Alternatively, antagonists (including
antisense nucleotides and/or ligands) which interfere with the normal
operation of these genes, other proteins
implicated in this post translation pathway (e.g., SEC61p, SEC72p, SEC62p,
SEC63p, TDJlp or SSAlp-4p)
or the complex formation of these proteins may also be preferably employed in
combination with the amylase-
expressing yeast.
Transformation was performed based on the protocol outlined by Gietz et al.,
Nucl. Acid. Res.,
20:1425 (1992). Transformed cells were then inoculated from agar into YEPD
complex media broth (100 ml)
and grown overnight at 30 C. The YEPD broth was prepared as described in
Kaiser et al., Methods in Yeast
Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 207 (1994). The
overnight culture was then
diluted to about 2 x 106 cells/ml (approx. ODwO =0.1) into fresh YEPD broth
(500 ml) and regrown to 1 x 10'
cells/ml (approx. OD6,=0.4-0.5).
The cells were then harvested and prepared for transformation by transfer into
GS3 rotor bottles in
a Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and
then resuspended into sterile
water, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman
GS-6KR centrifuge. The
supernatant was discarded and the cells were subsequently washed with LiAc/TE
(10 ml, 10 mM Tris-HCI,
1 mM EDTA pH 7.5, 100 mM Li2OOCCH3), and resuspended into LiAc/TE (2.5 ml).
Transformation took place by mixing the prepared cells (100 l) with freshly
denatured single stranded
salmon testes DNA (Lofstrand Labs, Gaithersburg, MD) and transforming DNA (1
g, vol. < 10 l) in
microfuge tubes. The mixture was mixed briefly by vortexing, then 40 % PEG/TE
(600 l, 40 % polyethylene
glycol-4000, 10 mM Tris-HCI, 1 mM EDTA, 100 mM Li2OOCCH3, pH 7.5) was added.
This mixture was
gently mixed and incubated at 30 C while agitating for 30 minutes. The cells
were then heat shocked at 42 C
for 15 minutes, and the reaction vessel centrifuged in a microfuge at 12,000
rpm for 5-10 seconds, decanted
and resuspended into TE (500 Id, 10 mM Tris-HCI, 1 mM EDTA pH 7.5) followed by
recentrifugation. The
cells were then diluted into TE (1 ml) and aliquots (200 l) were spread onto
the selective media previously
prepared in 150 mm growth plates (VWR).
Alternatively, instead of multiple small reactions, the transformation was
performed using a single,
large scale reaction, wherein reagent amounts were scaled up accordingly.
The selective media used was a synthetic complete dextrose agar lacking uracil
(SCD-Ura) prepared
as described in Kaiser et al., Methods in Yeast Genetics, Cold Spring Harbor
Press, Cold Spring Harbor, NY,
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p. 208-210 (1994). Transformants were grown at 30 C for 2-3 days.
The detection of colonies secreting amylase was performed by including red
starch in the selective
growth media. Starch was coupled to the red dye (Reactive Red-120, Sigma) as
per the procedure described
by Biely et al., Anal. Biochem., 172:176-179 (1988). The coupled starch was
incorporated into the SCD-Ura
agar plates at a final concentration of 0.15 % (w/v), and was buffered with
potassium phosphate to a pH of 7.0
(50-100 mM final concentration).
The positive colonies were picked and streaked across fresh selective media
(onto 150 nun plates) in
order to obtain well isolated and identifiable single colonies. Well isolated
single colonies positive for amylase
secretion were detected by direct incorporation of red starch into buffered
SCD-Ura agar. Positive colonies
were determined by their ability to break down starch resulting in a clear
halo around the positive colony
visualized directly.
4. Isolation of DNA by PCR Amplification
When a positive colony was isolated, a portion of it was picked by a toothpick
and diluted into sterile
water (30 pl) in a 96 well plate. At this tilne, the positive colonies were
either frozen and stored for
subsequent analysis or immediately amplified. An aliquot of cells (5 pl) was
used as a template for the PCR
reaction in a 25 al volume containing: 0.5 pl Klentaq (Clontech, Palo Alto,
CA); 4.0 pl 10 mM dNTP's
(Perkin Elmer-Cetus); 2.5 Eel Kentaq buffer (Clontech); 0.25 pl forward oligo
1; 0.25 pl reverse oligo 2; 12.5
pl distilled water. The sequence of the forward oligonucleotide 1 was,
5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3' (SEQ ID NO: 1)
The sequence of reverse oligonucleotide 2 was:
5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3' (SEQ ID NO:2)
PCR was then performed as follows:
a. Denature 92 C, 5 minutes
b. 3 cycles of: Denature 92 C, 30 seconds
Anneal 59 C, 30 seconds
Extend 72 C, 60 seconds
c. 3 cycles of. Denature 92 C, 30 seconds
Anneal 57 C, 30 seconds
Extend 72 C, 60 seconds
d. 25 cycles of. Denature 92 C, 30 seconds
Anneal 55 C, 30 seconds
Extend 72 C, 60 seconds
e. Hold 4 C
The underlined regions of the oligonucleotides annealed to the ADH promoter
region and the amylase
region, respectively, and amplified a 307 bp region from vector pSST-AMY.0
when no insert was present.
Typically, the first 18 nucleotides of the 5' end of these oligonucleotides
contained annealing sites for the
sequencing primers. Thus, the total product of the PCR reaction from an empty
vector was 343 bp. However,
signal sequence-fused cDNA resulted in considerably longer nucleotide
sequences.
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Following the PCR, an aliquot of the reaction (5 !d) was examined by agarose
gel electrophoresis in
a 1 % agarose gel using a Tris-Borate-EDTA (TBE) buffering system as described
by Sambrook et al., sLpra.
Clones resulting in a single strong PCR product larger than 400 bp were
further analyzed by DNA sequencing
*
after purification with a 96 Qiaquick PCR clean-up column (Qiagen Inc.,
Chatsworth, CA).
EXAMPLE 3: Isolation of cDNA Clones Using Signal Algorithm Analysis
Various polypeptide-encoding nucleic acid sequences were identified by
applying a proprietary signal
sequence finding algorithm developed by Genentech, Inc. (South San Francisco,
CA) upon ESTs as well as
clustered and assembled EST fragments from public (e.g., GenBank) and/or
private (LIFESEQ , Incyte
Pharmaceuticals, Inc., Palo Alto, CA) databases. The signal sequence algorithm
computes a secretion signal
score based on the character of the DNA nucleotides surrounding the first and
optionally the second methionine
codon(s) (ATG) at the 5'-end of the sequence or sequence fragment under
consideration. The nucleotides
following the first ATG must code for at least 35 unambiguous amino acids
without any stop codons. If the
first ATG has the required amino acids, the second is not examined. If neither
meets the requirement, the
candidate sequence is not scored. In order to determine whether the EST
sequence contains an authentic signal
sequence, the DNA and corresponding amino acid sequences surrounding the ATG
codon are scored using a
set of seven sensors (evaluation parameters) known to be associated with
secretion signals. Use of this
algorithm resulted in the identification of numerous polypeptide-encoding
nucleic acid sequences.
EXAMPLE 4: Isolation of cDNA clones Encoding Human PRO196
PRO196 was identified by screening the GenBank database using the computer
program BLAST
(Altshul et al., Methods in Enzymology 266:460-480 (1996). The PRO196 sequence
shows homology with
known expressed sequence tag (EST) sequences T35448, T11442, and W77823. None
of the known EST
sequences have been identified as full length sequences, or described as
ligands associated with the TIE
receptors.
Following its identification, NLI was cloned from a human fetal lung library
prepared from mRNA
purchased from Clontech, Inc. (Palo Alto, CA, USA), catalog # 6528-1,
following the manufacturer's
instructions. The library was screened by hybridization with synthetic
oligonucleotide probes:
(a) 5'-GCTGACGAACCAAGGCAACTACAAACTCCTGGT-3' (SEQ ID NO:5);
(b) 5' TGCGGCCGGACCAGTCCTCCATGGTCACCAGGAGTTTGTAG-3' (SEQ ID NO:6);
(c) 5'-GGTGGTGAACTGCTTGCCGTTGTGCCATGTAAA-3' ' (SEQ ID NO:7).
based on the ESTs found in the GenBank database. cDNA sequences were sequenced
in their entireties.
The nucleotide and amino acid sequences of PRO196 are shown in Figure 1 (SEQ
ID NO:3) and
Figure 2 (SEQ ID NO:4), respectively. PRO196 shows significant sequence
identity with both the TIE1 and
the TIE2 ligand.
A clone of PRO196 was deposited with the American Type Culture Collection,
10801 University
Blvd., Manassas, VA 20110-2209, USA (ATCC) on September 18, 1997 under the
terms of the Budapest
Treaty, and has been assigned the deposit number 209280.
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EXAMPLE 5: Isolation of cDNA clones Encoding Human PR0444
A cDNA sequence isolated in the amylase screen described in Example 2 above
was designated
DNA13121. Oligonucleotide probes were generated to this sequence and used to
screen a human fetal lung
library (LIB25) prepared as described in paragraph 1 of Example 2 above. The
cloning vector was pRK5B
(pRK5B is a precursor of pRK5D that does not contain the Sfil site; see,
Holmes et al., Science, 253:1278-
1280 (1991)), and the cDNA size cut was less than 2800 bp.
A full length clone was identified that contained a single open reading frame
with an apparent
translational initiation site at nucleotide positions 608-610 and ending at
the stop codon found at nucleotide
positions 959-961 (Figure 3, SEQ ID NO:8). The predicted polypeptide precursor
is 117 amino acids long,
has a calculated molecular weight of approximately 12,692 daltons and an
estimated pI of approximately 7.50.
Analysis of the full-length PRO444 sequence shown in Figure 4 (SEQ ID NO:9)
evidences the presence of a
signal peptide at amino acid 1 to about amino acid 16. An analysis of the
Dayhoff database (version 35.45
SwissProt 35) evidenced homology between the PR0444 amino acid sequence and
the following Dayhoff
sequences: CEF44D12 8, P -R88452, YNE1_CAEEL, A47312, AF009957_1, and
A06133_1.
Clone DNA26846-1397 was deposited with the ATCC on October 27, 1998 and is
assigned ATCC
deposit no. 203406.
EXAMPLE 6: Isolation of cDNA clones Encoding Human PR0183. PR0185. PR09940.
PRO2630 and
PR06309
DNA molecules encoding the PRO 183, PRO185, PR09940, PR02630 and PR06309
polypeptides
shown in the accompanying figures were obtained through GenBank.
EXAMPLE 7: Isolation of cDNA clones Encoding Human PR0210 and PRO217
A consensus DNA sequence was assembled using phrap as described in Example 1
above. In some
cases, the consensus DNA sequence as extended using repeated cycles of blast
and phrap to extend the
consensus sequence as far as possible using the sources of EST sequences
listed above. Based on this
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. The
library used to isolate DNA32279-1131 was fetal kidney.
cDNA clones were sequenced in their entirety. The entire nucleotide sequence
of DNA32279-1131
is shown in Figure 9 (SEQ ID NO: 14) and amino acid sequence of PRO210 is
shown in Figure 10 (SEQ ID
NO: 15). The entire nucleotide sequence of DNA33094-1131 is shown in Figure 13
(SEQ ID NO:21) and
amino acid sequence of PR0217 is shown in Figure 14 (SEQ ID NO:22).
EXAMPLE 8: Isolation of cDNA clones Encoding Human PR0215
A consensus DNA sequence was assembled relative to the other identified EST
sequences as described
in Example 1 above, wherein the consensus sequence was designated herein as
DNA28748. Based on the
DNA28748 consensus sequence, oligonucleotides were synthesized to identify by
PCR a cDNA library that
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contained the sequence of interest and for use as probes to isolate a clone of
the full-length coding sequence
for PR0215.
A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer 5'-GTGGCTGGCACACAATGAGATC-3' (SEQ ID NO:18)
reverse PCR primer 5'-CCAATGTGTGCAAGCGGTTGTG-3' (SEQ ID NO: 19)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA28748
sequence which had the following nucleotide sequence:
hybridization probe
5'-TCAAGAGCCTGGACCTCAGCCACAATCTCATCTCTGACTTTGCCTGGAGC-3' (SEQ ID NO:20).
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was
screened by PCR amplification with the PCR primer pair identified above. A
positive library was then used
to isolate clones encoding the PR0215 gene using the probe oligonucleotide and
one of the PCR primers.
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, CA. The cDNA was primed
with oligo dT containing a
Notl site, linked with blunt to Sall hemikinased adaptors, cleaved with Notl,
sized appropriately by gel
electrophoresis, and cloned in a defined orientation into a suitable cloning
vector (such as pRKB or pRKD;
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 Notl sites.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for
PRO215 [herein designated as DNA32288-1132 and the derived protein sequence
for PRO215.
The entire nucleotide sequence of DNA32288-1132 is shown in Figure 11 (SEQ ID
NO: 16). Clone
DNA32288-1132 contains a single open reading frame with an apparent
translational initiation site at
nucleotide positions 308-310 and ending at the stop codon at nucleotide
positions 1591-1593 (Figure 11, the
initiation and stop codons are circled). The predicted polypeptide precursor
is 428 amino acids long (Figure
12). Clone DNA32288-1132 has been deposited with ATCC and is assigned ATCC
deposit no. 209261.
Analysis of the amino acid sequence of the full-length PRO215 shows it has
homology to member of
the leucine rich repeat protein superfamily, including the leucine rich repeat
protein and the SLIT protein.
EXAMPLE 9: Isolation of cDNA clones Encoding Human PRO242
An expressed sequence tag (EST) DNA database (LIFESEQTM, Incyte
Pharmaceuticals, Palo Alto,
CA) was searched and an EST was identified which showed homology to a
chemokine. Based on this sequence,
oligonucleotides were synthesized to identify by PCR a cDNA library that
contained the sequence of interest
and for use as probes to isolate a clone of the full-length coding sequence
for PRO242.
A pair of PCR primer (forward and reverse) were synthesized:
forward PCR primer 5'-GGATCAGGCAGGAGGAGTTTGGG-3' (SEQ ID NO:25)
reverse PCR primer 5'-GGATGGGTACAGACTTTCTTGCC-3' (SEQ ID NO:26)
Additionaly, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA28709
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sequence which had the following nucleotide sequence:
hybridization probe
5'-ATGATGGGCCTCTCCTTGGCCTCTGCTGTGCTCCTGGCCTCCCTCCTGAG-3" (SEQ ID NO:27)
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was
screened by PCR amplification with the PCR primer pair identified above. A
positive library was then used
to isolate clones encoding the PR0242 gene using the probe oligonucleotide and
one of the PCR primers.
RNA for construction of the cDNA libraries was isolated from human fetal lung
tissue. A cDNA
clone was sequenced in entirety. The entire nucleotide sequence of DNA33785-
1143 is shown in Figure 15
(SEQ ID NO:23). Clone DNA33785-1143 contains a single open reading frame with
an apparent translational
initiation site at nucleotide positions 333-335 and ending at the stop codon
at nucleotide positions 615-617
(Figure 16; SEQ ID NO:24). The predicted polypeptide precursor is 94 amino
acids long (Figure 16).
Based on a BLAST and FastA sequence alignment analysis (using the ALIGN
computer program) of
the full-length sequence, PR0242 shows amino acid sequence identity to human
macrophage inflammatory
protein 1-alpha, rabbitt macrophage inflammatory protein 1-beta, human LD78
and rabbit immune activation
gene 2.
EXAMPLE 10: Isolation of cDNA clones Encoding Human PR0288
d having
A synthetic probe based on the sequence encoding the DcR1 ECD [Sheridan et
al., supra an
the following sequence:
5'-CATAAAAGTTCCTGCACCATGACCAGAGACACAGTGTGTCAGTGTAAAGA-3' (SEQ ID NO:30)
was used to screen a human fetal lung cDNA library. To prepare the cDNA
library, mRNA was isolated from
human fetal lung tissue using reagents and protocols from Invitrogen, San
Diego, CA (Fast Track 2). This
RNA was used to generate an oligo dT primed cDNA library in the vector pRK5D
using reagents and protocols
from Life Technologies, Gaithersburg, MD (Super Script Plasmid System). In
this procedure, the double
stranded cDNA was sized to greater than 1000 bp and the Sall/NotI linkered
cDNA was cloned into XhoI/Notl
cleaved vector. pRK5D is a cloning vector that has an sp6 transcription
initiation site followed by an Sfil
restriction enzyme site preceding the XhoI/NotI cDNA cloning sites.
A full length clone was identified (DNA35663-1129) that contained a single
open reading frame with
an apparent translational initiation site at nucleotide positions 157-159 and
ending at the stop codon found at
nucleotide positions 1315-1317 (Figure 17; SEQ ID NO:28). The clone is
referred to as pRK5-35663 and is
deposited as ATCC No. 209201.
The predicted polypeptide precursor is 386 amino acids long and has a
calculated molecular weight
of approximately 41.8 kDa. Sequence analysis indicated a N-terminal signal
peptide (amino acids 1-55),
followed by an ECD (amino acids 56-212), transmembrane domain (amino acids 213-
232) and.intracellular
region (amino acids 233-386). (Figure 18). The signal peptide cleavage site
was confirmed by N-terminal
protein sequencing of a PR0288 ECD immunoadhesin (not shown). This structure
suggests that PRO288 is
a type I transmembrane protein. PR0288 contains 3 potential N-linked
glycosylation sites, at amino acid
positions 127, 171 and 182. (Figure 18)
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TNF receptor family proteins are typically characterized by the presence of
multiple (usually four)
cysteine-rich domains in their extracellular regions -- each cysteine-rich
domain being approximately 45 amino
acids long and containing approximately 6, regularly spaced, cysteine
residues. Based on the crystal structure
of the type 1 TNF receptor, the cysteines in each domain typically form three
disulfide bonds in which usually
cysteines 1 and 2, 3 and 5, and 4 and 6 are paired together. Like DR4, DRS,
and DcRI, PR0288 contains
two extracellular cysteine-rich pseudorepeats, whereas other identified
mammalian TNFR family members
contain three or more such domains [Smith et al., Cell, 76:959 (1994)].
Based on an alignment analysis of the PR0288 sequence shown in Figure 18 (SEQ
ID NO:29),
PR0288 shows more sequence identity to the ECD of DR4, DR5, or DcR1 than to
other apoptosis-linked
receptors, such as TNFR1, Fas/Apo-1 or DR3. The predicted intracellular
sequence of PR0288 also shows
more homology to the corresponding region of DR4 or DR5 as compared to TNFR1,
Fas or DR3. The
intracellular region of PR0288 is about 50 residues shorter than the
intracellular regions identified for DR4
or DR5. It is presently believed that PR0288 may contain an truncated death
domain (amino acids 340-364),
which corresponds to the carboxy-terminal portion of the death domain
sequences of DR4 and DR5. Five out
of six amino acids that are essential for signaling by TNFR1 [Tartaglia et
al., supraa and that are conserved
or semi-conserved in DR4 and DR5, are absent in PR0288.
EXAMPLE 11: Isolation of cDNA clones Encoding Human PR0365
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described
in Example 1 above. This consensus sequence is herein designated DNA35613.
Based on the DNA35613
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
PRO365.
Forward and reverse PCR primers were synthesized:
forward PCR primer 5'-GGCTGGCCTGCAGAGATC-3' (SEQ ID NO:33)
forward PCR primer 5'-AATGTGACCACTGGACTCCC-3' (SEQ ID NO:34)
forward PCR primer 5'-AGGCTTGGAACTCCCTTC-3' (SEQ ID NO:35)
reverse PCR primer 5'-AAGATTCTTGAGCGATTCCAGCTG-3' (SEQ ID NO:36)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA35613
sequence which had the following nucleotide sequence
hybridization probe
5'-AATCCCTGCTCTTCATGGTGACCTATGACGACGGAAGCACAAGACTG-3' (SEQ ID NO:37)
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was
screened by PCR amplification with one of the PCR primer pairs identified
above. A positive library was then
used to isolate clones encoding the PR0365 gene using the probe
oligonucleotide and one of the PCR
primers.RNA for construction of the cDNA libraries was isolated from human
fetal kidney tissue.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for
PR0365 [herein designated as DNA46777-1253] (SEQ ID NO:31) and the derived
protein sequence for
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PR0365.
The entire nucleotide sequence of DNA46777-1253 is shown in Figure 19 (SEQ ID
NO:3 1). Clone
DNA46777-1253 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 15-17 and ending at the stop codon at nucleotide positions 720-722
(Figure 19). The predicted
polypeptide precursor is 235 amino acids long (Figure 20). Important regions
of the polypeptide sequence
encoded by clone DNA46777-1253 have been identified and include the following:
a signal peptide
corresponding to amino acids 1-20 and multiple potential N-glycosylation
sites. Clone DNA46777-1253 has
been deposited with ATCC and is assigned ATCC deposit no. 209619.
Analysis of the amino acid sequence of the full-length PR0365 polypeptide
suggests that portions of
it possess significant homology to the human 2-19 protein, thereby indicating
that PR0365 may be a novel
human 2-19 protein homolog.
EXAMPLE 12: Isolation of cDNA clones Encoding Human PRO1361
Use of the signal sequence algorithm described in Example 3 above allowed
identification of an EST
cluster sequence from the Incyte database, designated Incyte cluster sequence
10685. This EST cluster
sequence was then compared to a variety of expressed sequence tag (EST)
databases which included public
EST databases (e.g., GenBank) and a proprietary EST DNA database (Lifeseq ,
Incyte Pharmaceuticals, Palo
Alto, CA) to identify existing homologies. The homology search was performed
using the computer program
BLAST or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480 (1996)).
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 a consensus DNA sequence with the program "phrap"
(Phil Green, University
of Washington, Seattle, Washington). The consensus sequence obtained therefrom
is herein designated
DNA58839.
In light of an observed sequence homology between the DNA58839 sequence and an
EST sequence
contained within the Incyte EST clone no. 2967927, the Incyte EST clone no.
2967927 was purchased and the
cDNA insert was obtained and sequenced. The sequence of this cDNA insert is
shown in Figure 21 and is
herein designated as DNA60783-161 1.
Clone DNA60783-1611 contains a single open reading frame with an apparent
translational initiation
site at nucleotide positions 142-144 and ending at the stop codon at
nucleotide positions 1132-1134 (Figure 21).
The predicted polypeptide precursor is 330 amino acids long (Figure 22). The
full-length PRO 1361 protein
shown in Figure 22 has an estimated molecular weight of about 36,840 daltons
and a pl of about 4.84.
Analysis of the full-length PRO1361 sequence shown in Figure 22 (SEQ ID NO:39)
evidences the presence
of the following: a signal peptide from about amino acid 1 to about amino acid
23, a transmembrane domain
from about amino acid 266 to about amino acid 284, a leucine zipper pattern
sequence from about amino acid
155 to about amino acid 176 and potential N-glycosylation sites from about
amino acid 46 to about amino acid
.35 49, from about amino acid 64 to about amino acid 67, from about amino acid
166 to about amino acid 169 and
from about amino acid 191 to about amino acid 194. Clone DNA60783-1611 has
been deposited with ATCC
on August 18, 1998 and is assigned ATCC deposit no. 203130.
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An analysis of the Dayhoff database (version 35.45 SwissProt 35), using a WU-
BLAST2 sequence
alignment analysis of the full-length sequence shown in Figure 22 (SEQ ID
NO:39), evidenced significant
homology between the PRO 1361 amino acid sequence and the following Dayhoff
sequences: 150620, G64876,
PMCMSG102B 2MSGI04, HUMIGLVXY 1 and PH1370.
EXAMPLE 13: Isolation of cDNA clones Encoding Human PRO 1308
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described
in Example 1 above. The consensus sequence was extended then using repeated
cycles of BLAST and phrap
to extend the consensus sequence as far as possible using the sources of EST
sequences discussed above. The
extended consensus sequence is designated herein as "DNA35726". Based on the
DNA35726 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 PRO 1308.
The following PCR primers (forward and reverse) were synthesized:
forward PCR primers 5'-TCCTGTGAGCACGTGGTGTG-3' (SEQ ID NO:42);
5'-GGGTGGGATAGACCTGCG-3' (SEQ ID NO:43);
5'-AAGGCCAAGAAGGCTGCC-3' (SEQ ID NO:44); and
5'-CCAGGCCTGCAGACCCAG-3' (SEQ ID NO:45).
reverse PCR primers 5'-CTTCCTCAGTCCTTCCAGGATATC-3' (SEQ ID NO:46);
5'-AAGCTGGATATCCTCCGTGTTGTC-3' (SEQ ID NO:47);
5'-CCTGAAGAGGCATGACTGCTTTTCTCA-3' (SEQ ID NO:48); and
5'-GGGGATAAACCTATTAATTATTGCTAC-3' (SEQ ID NO:49).
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA35726
sequence which had the following nucleotide sequence:
hybridization probe: 5' -AACGTCACCTACATCTCCTCGTGCCACATGCGCCAGGCCACCTG-3' (SEQ
ID NO:50).
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was
screened by PCR amplification with the PCR primer pair identified above. A
positive library was then used
to isolate clones encoding the PRO1308 gene using the probe oligonucleotide
and one of the PCR primers.
RNA for construction of the cDNA libraries was isolated from a human SK-Lu-1
adenocarcinoma cell line.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for
PRO1308 (designated herein as DNA62306-1570 [Figure 23, SEQ ID NO:40]; and the
derived protein
sequence for PRO1308.
The entire coding sequence of PRO1308 is shown in Figure 23 (SEQ ID NO:40).
Clone DNA62306-
1570 contains a single open reading frame with an apparent translational
initiation site at nucleotide positions
17-19 and an apparent stop codon at nucleotide positions 806-808. The
predicted polypeptide precursor is 263
amino acids long. The full-length PRO1308 protein shown in Figure 24 has an
estimated molecular weight
of about 27,663 daltons and a pI of about 6.77. Additional features include a
signal peptide at about amino
acids 1-20, potential N-glycosylation sites at about amino acids 73-76 and 215-
218, and regions of homology
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with osteonectin domains at about amino acids 97-129 and 169-201.
An analysis of the Dayhoff database (version 35.45 SwissProt 35), using a WU-
BLAST2 sequence
alignment analysis of the full-length sequence shown in Figure 24 (SEQ ID
NO:41), revealed significant
homology between the PRO1308 amino acid sequence and Dayhoff sequence S55369.
Homology was also
revealed between the PRO 1308 amino acid sequence and the following Dayhoff
sequences: FSA_HUMAN,
P1120063, CELT13C2_1, AGRI RAT, p_W09406, 601639, SCI-RAT, S60062, S51362, and
IOV7 CHICK.
Clone DNA62306-1570 has been deposited with ATCC and is assigned ATCC deposit
no. 203254.
EXAMPLE 14: Isolation of cDNA clones Encoding Human PRO1183
Use of the signal sequence algorithm described in Example 3 above allowed
identification of an EST
cluster sequence from the Incyte database. This EST cluster sequence was then
compared to a variety of
expressed sequence tag (EST) databases which included public EST databases
(e.g., GenBank) and a
proprietary EST DNA database (LIFESEQ , Incyte Pharmaceuticals, Palo Alto, CA)
to identify existing
homologies. The homology search was performed using the computer program BLAST
or BLAST2 (Altshul
et al., Methods in Enzymology 266:460-480 (1996)). 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 a
consensus DNA sequence with the program "phrap" (Phil Green, University of
Washington, Seattle,
Washington). The consensus sequence obtained therefrom is herein designated
DNA56037.
In light of an observed sequence homology between the DNA56037 sequence and an
EST sequence
*
contained within the Incyte EST 1645856 (from a library constructed from
prostate tumor tissue), the clone
which includes EST 1645856 was purchased and the cDNA insert was obtained and
sequenced. The sequence
of this cDNA insert is shown in Figure 25 and is herein designated as DNA62880-
1513.
The full length clone shown in Figure 25 contained a single open reading frame
with an apparent
translational initiation site at nucleotide positions 20-22 and ending at the
stop codon found at nucleotide
positions 1535-1537 (Figure 25; SEQ ID NO:5 1). The predicted polypeptide
precursor (Figure 26, SEQ ID
NO:52) is 505 amino acids long. The signal peptide is approximately at amino
acids 1-23 of SEQ ID NO:52.
PRO1183 has a calculated molecular weight of approximately 56,640 daltons and
an estimated pl of
approximately 6.1. Clone DNA62880-1513 was deposited with the ATCC on August
4, 1998 and is assigned
ATCC deposit no. 203097.
An analysis of the Dayhoff database (version 35.45 SwissProt 35), using a WU-
BLAST2 sequence
alignment analysis of the full-length sequence shown in Figure 26 (SEQ ID
NO:52), revealed sequence identity
between the PROI 183 amino acid sequence and the following Dayhoff sequences:
MTVO10 1, P -W41604,
S54021, AOFB_HUMAN, NPAJ4683_1, S74689, GEN13608, ACHC_ACHFU, AB011173_1 and
PUO_MICRU. It is believed that administration of PRO1183 or regulators thereof
may treat certain oxidase
disorders such as variegate porphyria.
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EXAMPLE 15: Isolation of cDNA clones Encoding Human PRO 1272
Use of the signal sequence algorithm described in Example 3 above allowed
identification of an EST
cluster sequence from the Incyte database. This EST cluster sequence was then
compared to a variety of
expressed sequence tag (EST) databases which included public EST databases
(e.g., GenBank) and a
proprietary EST DNA database (LIFESEQ , Incyte Pharmaceuticals, Palo Alto, CA)
to identify existing
homologies. The homology search was performed using the computer program BLAST
or BLAST2 (Altshul
et al., Methods in Enzymology 266:460-480 (1996)). 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 a
consensus DNA sequence with the program "phrap" (Phil Green, University of
Washington, Seattle,
Washington). The consensus sequence obtained therefrom is herein designated
DNA58753.
In light of an observed sequence homology between the DNA58753 sequence and an
EST sequence
contained witin the EST clone 3049165, the Incyte clone (from a lung library)
including EST 3049165 was
purchased and the cDNA insert was obtained and sequenced. The sequence of this
cDNA insert is shown in
Figure 27 and is herein designated as DNA64896-1539.
The full length clone shown in Figure 27 contained a single open reading frame
with an apparent
translational initiation site at nucleotide positions 58-60 and ending at the
stop codon found at nucleotide
positions 556-558 (Figure 27; SEQ ID NO:53). The predicted polypeptide
precursor (Figure 28, SEQ ID
NO:54) is 166 amino acids long. The signal peptide is at about amino acids 1-
23 of SEQ ID NO:54.
PRO1272 has a calculated molecular weight of approximately 19,171 daltons and
an estimated pI of
approximately 8.26. Clone DNA64896-1539 was deposited with the ATCC on
September 9, 1998 and is
assigned ATCC deposit no. 203238.
An analysis of the Dayhoff database (version 35.45 SwissProt 35), using a WU-
BLAST2 sequence
alignment analysis of the full-length sequence shown in Figure 28 (SEQ ID
NO:54), revealed sequence identity
between the PRO 1272 amino acid sequence and the following Dayhoff sequences
(information from database
incorporated herein): AF025474_1, D69100, AE000757_10, H69466, CELC50E3_12,
XLRANBPI_1,
YD67 SCHPO, B69459, H36856, and FRU40755 1.
EXAMPLE 16: Isolation of cDNA clones Encoding Human PRO1419
Use of the signal sequence algorithm described in Example 3 above allowed
identification of an EST
cluster sequence from the Incyte database. This EST cluster sequence was then
compared to a variety of
expressed sequence tag (EST) databases which included public EST databases
(e.g., GenBank) and a
proprietary EST DNA database (LIFESEQ , Incyte Pharmaceuticals, Palo Alto, CA)
to identify existing
homologies. One or more of the ESTs was derived from a diseased tonsil tissue
library. The homology search
was performed using the computer program BLAST or BLAST2 (Altshul et al.,
Methods in Enzymology
266:460-480 (1996)). 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 a
consensus DNA sequence with the
program "phrap" (Phil Green, University of Washington, Seattle, Washington).
The consensus sequence
obtained therefrom is herein designated DNA59761.
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In light of an observed sequence homology between the DNA59761 sequence and an
EST sequence
contained within the Incyte EST 3815008, the clone including this EST was
purchased and the cDNA insert
was obtained and sequenced. The sequence of this cDNA insert is shown in
Figure 29 and is herein designated
as DNA71290-1630.
The full length clone shown in Figure 29 contained a single open reading frame
with an apparent
translational initiation site at nucleotide positions 86-88 and ending at the
stop codon found at nucleotide
positions 341-343 (Figure 29; SEQ ID NO:55). The predicted polypeptide
precursor (Figure 30, SEQ ID
NO:56) is 85 amino acids long with the signal peptide at about amino acids 1-
17 of SEQ ID N0:56. PRO 1419
has a calculated molecular weight of approximately 9,700 daltons and an
estimated pl of approximately 9.55.
Clone DNA71290-1630 was deposited with the ATCC on September 22, 1998 and is
assigned ATCC deposit
no. 203275.
An analysis of the Dayhoff database (version 35.45 SwissProt 35), using a WU-
BLAST2 sequence
alignment analysis of the full-length sequence shown in Figure 30 (SEQ ID
NO:56), revealed sequence identity
between the PR01419 amino acid sequence and the following Dayhoff sequences
(data incorporated herein):
S07975 (B3-hordein), C48232, HOR7_HORVU, GEN11764, S14970, AF020312_i,
STAJ3220 1,
CER07E3 1, CEY37A1B 4, and ATAC00423810.
EXAMPLE 17: Isolation of cDNA clones Encoding Human PR04999
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described
in Example 1 above. This consensus sequence is herein designated DNA86634.
Based on the DNA86634
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
PR04999.
PCR primers (forward and reverse) were synthesized:
forward PCR primer 5'-CCACTTGCCATGAACATGCCAC-3' (SEQ ID NO:59)
reverse PCR primer 5'-CCTCTTGACAGACATAGCGAGCCAC-3' (SEQ ID NO:60)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA86634
sequence which had the following nucleotide sequence
hybridization probe
5'-CACTCTTGTCTGTGGGAACCACACATCTTGCCACAACTGTGGC-3' (SEQ ID NO:61)
RNA for construction of the cDNA libraries was isolated from human testis
tissue. DNA sequencing
of the clones isolated as described above gave the full-length DNA sequence
for a full-length PRO4999
polypeptide (designated herein as DNA96031-2664 [Figure 31, SEQ ID NO:57]) and
the derived protein
sequence for that PRO4999 polypeptide.
The full length clone identified above contained a single open reading frame
with an apparent
translational initiation site at nucleotide positions 42-44 and a stop signal
at nucleotide positions 2283-2285
(Figure 31, SEQ ID NO:57). The predicted polypeptide precursor is 747 amino
acids long, has a calculated
molecular weight of approximately 82,710 daltons and an estimated pI of
approximately 6.36. Analysis of the
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full-length PR04999 sequence shown in Figure 32 (SEQ ID NO:58) evidences the
presence of a variety of
important polypeptide domains as shown in Figure 32, wherein the locations
given for those important
polypeptide domains are approximate as described above. Clone DNA96031-2664
has been deposited with
ATCC on June 15, 1999 and is assigned ATCC deposit no. 237-PTA.
An analysis of the Dayhoff database (version 35.45 SwissProt 35), using the
ALIGN-2 sequence
alignment analysis of the full-length sequence shown in Figure 32 (SEQ ID
NO:58), evidenced sequence
identity between the PR04999 amino acid sequence and the following Dayhoff
sequences: UROM_HUMAN;
FBN1 HUMAN; GGU88872 1; S52111; GEN12408; P R79478; P _W48756; P _R53087; P
R14584; and
S78549.
EXAMPLE 18: Isolation of cDNA clones Encoding Human PRO7170
Use of the signal sequence algorithm described in Example 3 above allowed
identification of an EST
cluster sequence from the LIFESEQ database, Incyte Pharmaceuticals, Palo
Alto, designated herein as
CLU57836. This EST cluster sequence was then compared to a variety of
expressed sequence tag (EST)
databases which included public EST databases (e.g., Genbank) and a
proprietary EST DNA database
(LIFESEQ , Incyte Pharmaceuticals, Palo Alto, CA) to identify existing
homologies. The homology search
was performed using the computer program BLAST or BLAST2 (Altshul et al.,
Methods in Enzymology
266:460-480 (1996)). 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 a
consensus DNA sequence with the
program "phrap" (Phil Green, University of Washington, Seattle, Washington).
The consensus sequence
obtained therefrom is herein designated DNA58756.
In light of an observed sequence homology between the DNA58756 sequence and an
EST sequence
encompassed within clone no. 2251462 from the LIFESEQ database, Incyte
Pharmaceuticals, Palo Alto, CA,
clone no. 2251462 was purchased and the cDNA insert was obtained and
sequenced. It was found herein that
that cDNA insert encoded a full-length protein. The sequence of this cDNA
insert is shown in Figure 33 and
is herein designated as DNA108722-2743.
Clone DNA 108722-2743 contains a single open reading frame with an apparent
translational initiation
site at nucleotide positions 60-62 and ending at the stop codon at nucleotide
positions 1506-1508 (Figure 33).
The predicted polypeptide precursor is 482 amino acids long (Figure 34). The
full-length PRO7170 protein
shown in Figure 34 has an estimated molecular weight of about 49,060 daltons
and a pI of about 4.74.
Analysis of the full-length PRO7170 sequence shown in Figure 34 (SEQ ID NO:63)
evidences the presence
of a variety of important polypeptide domains as shown in Figure 34, wherein
the locations given for those
important polypeptide domains are approximate as described above. Clone DNA
108722-2743 has been
deposited with ATCC on August 17, 1999 and is assigned ATCC Deposit No. 552-
PTA.
An analysis of the Dayhoff database (version 35.45 SwissProt 35), using the
ALIGN-2 sequence
alignment analysis of the full-length sequence shown in Figure 34 (SEQ ID
NO:63), evidenced sequence
identity between the PRO7170 amino acid sequence and the following Dayhoff
sequences: P_Y 12291, 14714 1,
D88733_1, DMC56G7_1, P Y11606, HWP1_CANAL, HSMUC5BEX_1, HSU78550_1, HSU70136
1, and
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SGS3_DROME.
EXAMPLE 19: Isolation of cDNA clones Encoding Human PR0248
A consensus DNA sequence was assembled relative to the other identified EST
sequences as described
in Example 1 above, wherein the consensus sequence is designated herein as
DNA33481. Based on the
DNA33481 consensus sequence, oligonucleotides were synthesized to identify by
PCR a cDNA library that
contained the sequence of interest and for use as probes to isolate a clone of
the full-length coding sequence
for PR0248. Specifically, the following primers were used:
Forward primer 1 (SEO ID NO:66): 5'-GTCTGACAGCCACTCCAGAG-3'
Hybridization probe (SEO ID NO:67):
5'-TCTCCAATTTCTGGGCTTAGATAAGGCGCCTTCACCCCAGAAGTTCC-3'
Reverse primer 1 (SEO ID NO:68): 5'-GTCCCAGGTTATAGTAAGAATTGG-3'
Forward primer 2 (SEO ID NO:69): 5'-GTGTTGCGGTCAGTCCCATG-3'
Forward primer 3 (SEO ID NO:70): 5'-GCTGTCTCCCATTTCCATGC-3'
Reverse primer 2 (SEQ ID NO:71): 5'-CGACTACCATGTCTTCATAATGTC-3'
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was
screened by PCR amplification with the PCR primer pair identified above. A
positive library was then used
to isolate clones encoding the PR0248 gene using the probe oligonucleotide and
one of the PCR primers.
RNA for construction of the cDNA libraries was isolated from human fetal
kidney tissue.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for
PR0248 [herein designated as DNA35674-1142] and the derived protein sequence
for PR0248.
The entire nucleotide sequence of DNA35674-1142 is shown in Figure 35 (SEQ ID
NO:64). Clone
DNA35674-1142 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 66-68 and ending at the stop codon at nucleotide positions 1217-1219
(Figure 35; SEQ ID NO:64).
The predicted polypeptide precursor is 364 amino acids long (Figure 36). Clone
DNA35674-1142 has been
deposited on October 28, 1997 with ATCC and is assigned ATCC deposit no.
209416.
Analysis of the amino acid sequence of the full-length PR0248 suggests that it
has certain amino acid
sequence identity with growth differentiation factor 3 from human and mouse.
EXAMPLE 20: Isolation of cDNA clones Encoding Human PR0353
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described
in Example 1 above. This consensus sequences is herein designated DNA36363.
The consensus DNA
sequence was extended using repeated cycles of BLAST and phrap to extend the
consensus sequence as far as
possible using the sources of EST sequences discussed above. Based on the
DNA36363 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 PR0353.
Based on the DNA36363 consensus sequence, forward and reverse PCR primers were
synthesized
as follows:
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forward PCR primer 5'-TACAGGCCCAGTCAGGACCAGGGG-3' (SEQ ID NO:74)
reverse PCR primer 5'-CTGAAGAAGTAGAGGCCGGGCACG-3' (SEQ ID NO:75).
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the DNA36363 consensus
sequence which had the following nucleotide sequence:
hybridization probe
5'-CCCGGTGCTTGCGCTGCTGTGACCCCGGTACCTCCATGTACCCGG-3' (SEQ ID NO:76)
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was
screened by PCR amplification with one of the PCR primer pairs identified
above. A positive library was then
used to isolate clones encoding the PR0353 gene using the probe
oligonucleotide and one of the PCR primers.
RNA for construction of the cDNA libraries was isolated from human fetal
kidney tissue.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for
PR0353 [herein designated as DNA41234-1242] (SEQ ID NO:72) and the derived
protein sequence for
PR0353.
The entire nucleotide sequence of DNA41234-1242 is shown in Figure 37 (SEQ ID
NO:72). Clone
DNA41234-1242 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 305-307 and ending at the stop codon at nucleotide positions 1148-
1150 (Figure 37). The predicted
polypeptide precursor is 281 amino acids long (Figure 38). Important regions
of the amino acid sequence
encoded by PR0353 include the signal peptide, corresponding to amino acids 1-
26, the start of the mature
protein at amino acid position 27, a potential N-glycosylation site,
corresponding to amino acids 93-98 and a
region which has homology to a 30 kd adipocyte complement-related protein
precursor, corresponding to amino
acids 99-281. Clone DNA41234-1242 has been deposited with the ATCC and is
assigned ATCC deposit no.
209618.
Analysis of the amino acid sequence of the full-length PR0353 polypeptides
suggests that portions
of them possess significant homology to portions of human and murine
complement proteins, thereby indicating
that PR0353 may be a novel complement protein.
EXAMPLE 21: Isolation of cDNA clones Encoding Human PRO1318
The cDNA molecule corresponding to DNA73838-1674 as shown in Figure 39 (SEQ ID
NO:77) was
obtained from Curagen, Inc.
EXAMPLE 22: Isolation of cDNA clones Encoding Human PRO1600
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described
in Example 1 above. This consensus sequences is herein designated DNA75516.
The consensus DNA
sequence was extended using repeated cycles of BLAST and phrap to extend the
consensus sequence as far as
possible using the sources of EST sequences discussed above. Based on the
DNA75516 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 PRO1600.
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Based on the DNA75516 consensus sequence, oligonucleotide probes were
synthesized as follows:
5'-AGACATGGCTCAGTCACTGG-3' (SEQ ID NO:81)
5'-GACCCCTAAAGGGCCATAG-3' (SEQ ID NO:82).
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was
screened with the probes identified above. RNA for construction of the cDNA
libraries was isolated from
human fetal heart tissue.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for
PRO1600 [herein designated as DNA77503-1686] (SEQ ID NO:79) and the derived
protein sequence for
PRO1600.
The entire nucleotide sequence of DNA77503-1686 is shown in Figure 41 (SEQ ID
NO:79). Clone
DNA77503-1686 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 6-8 and ending at the stop codon at nucleotide positions 408-410
(Figure 41). The predicted
polypeptide precursor is 134 amino acids long (Figure 42). Important regions
of the amino acid sequence of
PRO1600 are shown in Figure 42. Clone DNA77503-1686 has been deposited with
the ATCC and is assigned
ATCC deposit no. 203362.
EXAMPLE 23: Isolation of cDNA clones Encoding Human PR0533
The EST sequence accession number AF007268, a murine fibroblast growth factor
(FGF- 15) was used
to search various public EST databases (e.g., GenBank, Dayhoff, etc.). The
search was performed using the
computer program BLAST or BLAST2 [Altschul 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
sequences. The search resulted
in a hit with GenBank EST AA220994, which has been identified as stratagene
NT2 neuronal precursor
937230.
Based on the Genbank EST AA220994 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. Forward and reverse PCR primers may range
from 20 to 30 nucleotides
(typically about 24), and are designed to give a PCR product of 100-1000 bp in
length. The probe sequences
are typically 40-55 bp (typically about 50) in length. In order to screen
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, with the PCR primer pair. A positive library
was then used to isolate clones
encoding the gene of interest using the probe oligonucleotide and one of the
PCR primers.
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was
screened by PCR amplification with the PCR primer pair identified below. A
positive library was then used
to isolate clones encoding the PR0533 gene using the probe oligonucleotide and
one of the PCR primers.
RNA for construction of the cDNA libraries was isolated from human fetal
retina. The cDNA
libraries used to isolated the cDNA clones were constructed by standard
methods using commercially available
reagents (e.g., Invitrogen, San Diego, CA; Clontech, etc.) The cDNA was primed
with oligo dT containing
a Notl site, linked with blunt to SaII hemikinased adaptors, cleaved with
Not!, sized appropriately by gel
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electrophoresis, and cloned in a defined orientation into a suitable cloning
vector (such as pRKB or pRKD;
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 Not! sites.
A cDNA clone was sequenced in its entirety. The full length nucleotide
sequence of PR0533 is
shown in Figure 45 (SEQ ID NO:85). Clone DNA49435-1219 contains a single open
reading frame with an
apparent translational initiation site at nucleotide positions 459-461 (Figure
45; SEQ ID NO:85). The predicted
polypeptide precursor is 216 amino acids long. Clone DNA47412-1219 has been
deposited with ATCC and
is assigned ATCC deposit no. ATCC 209480.
Based on a BLAST-2 and FastA sequence alignment analysis of the full-length
sequence, PR0533
shows amino acid sequence identity to fibroblast growth factor (53%).
The oligonucleotide sequences used in the above procedure were the following:
FGF15.forward: 5'-ATCCGCCCAGATGGCTACAATGTGTA-3' (SEQ ID NO:87);
FGF15.probe: 5'-GCCTCCCGGTCTCCCTGAGCAGTGCCAAACAGCGGCAGTGTA-3' (SEQ ID NO:88);
FGF15.reverse: 5'-CCAGTCCGGTGACAAGCCCAAA-3' (SEQ ID NO:89).
EXAMPLE 24: Isolation of cDNA clones Encoding Human PRO301
A consensus DNA sequence designated herein as DNA35936 was assembled using
phrap as described
in Example 1 above. Based on this 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.
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was
screened by PCR amplification with the PCR primer pair identified below. A
positive library was then used
to isolate clones encoding the PRO301 gene using the probe oligonucleotide and
one of the PCR primers.
RNA for construction of the cDNA libraries was isolated from human fetal
kidney.
A cDNA clone was sequenced in its entirety. The full length nucleotide
sequence of native sequence
PRO301 is shown in Figure 47 (SEQ ID NO:90). Clone DNA40628-1216 contains a
single open reading
frame with an apparent translational initiation site at nucleotide positions
52-54 (Figure 47; SEQ ID NO:90).
The predicted polypeptide precursor is 299 amino acids long with a predicted
molecular weight of 32,583
daltons and pI of 8.29. Clone DNA40628-1216 has been deposited with ATCC and
is assigned ATCC deposit
No. ATCC 209432.
Based on a BLAST and FastA sequence alignment analysis of the full-length
sequence, PRO301 shows
amino acid sequence identity to A33 antigen precursor (30%) and coxsackie and
adenovirus receptor protein
(29%).
The oligonucleotide sequences used in the above procedure were the following:
OLI2162 (35936.fl) 5'-TCGCGGAGCTGTGTTCTGTTTCCC-3' (SEQ ID NO:92)
OL12163 (35936.pl)
5'-TGATCGCGATGGGGACAAAGGCGCAAGCTCGAGAGGAAACTGTTGTGCCT-3' (SEQ ID NO:93)
OLI2164 (35936.12)
5'-ACACCTGGTTCAAAGATGGG-3' (SEQ ID NO:94)
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OLI2165 (35936.rl)
5'-TAGGAAGAGTTGCTGAAGGCACGG-3' (SEQ ID NO:95)
OLI2166 (35936.f3)
5'-TTGCCTTACTCAGGTGCTAC-3' (SEQ ID NO:96)
OLI2167 (35936.r2)
5'-ACTCAGCAGTGGTAGGAAAG-3' (SEQ ID NO:97)
EXAMPLE 25: Isolation of cDNA clones Encoding Human PRO187
A proprietary expressed sequence tag (EST) DNA database (LIFESEQTM, Incyte
Pharmaceuticals,
Palo Alto, CA) was searched and an EST (#843193) was identified which showed
homology to fibroblast
growth factor (FGF-8) also known as androgen-induced growth factor. mRNA was
isolated from human fetal
lung tissue using reagents and protocols from Invitrogen, San Diego, CA (Fast
Track 2). The cDNA libraries
used to isolate the cDNA clones were constructed by standard methods using
commercially available reagents
(e.g., Invitrogen, San Diego, CA, Life Technologies, Gaithersburg, MD). The
cDNA was primed with oligo
dT containing a Not! site, linked with blunt to Sall hemikinased adaptors,
cleaved with Notl, sized
appropriately by gel electrophoresis, and cloned in a defined orientation into
the cloning vector pRK5D using
reagents and protocols from Life Technologies, Gaithersburg, MD (Super Script
Plasmid System). The double-
stranded cDNA was sized to greater than 1000 bp and the SaII/NotI linkered
cDNA was cloned into Xhol/Notl
cleaved vector. pRK5D is a cloning vector that has an sp6 transcription
initiation site followed by an SfiI
restriction enzyme site preceding the Xhol/Notl cDNA cloning sites.
Several libraries from various tissue sources were screened by PCR
amplification with the following
oligonucleotide probes:
IN843193.f (0LI315) (SEO ID NO: 100)
5'-CAGTACGTGAGGGACCAGGGCGCCATGA-3'
IN843193.r (OLI 317) (SEO ID NO: 101)
5'-CCGGTGACCTGCACGTGCTTGCCA-3'
A positive library was then used to isolate clones encoding the PRO187 gene
using one of the above
oligonucleotides and the following oligonucleotide probe:
IN843193.p (OLI 316) (SEO ID NO:102)
5'-GCGGATCTGCCGCCTGCTCANCTGGTCGGTCATGGCGCCCT-3'
A cDNA clone was sequenced in entirety. The entire nucleotide sequence of
PRO187 (DNA27864-
1155) is shown in Figure 49 (SEQ ID NO:98). Clone DNA27864-1155 contains a
single open reading frame
with an apparent translational initiation site at nucleotide position 1
(Figure 49; SEQ ID NO:98). The
predicted polypeptide precursor is 205 amino acids long. Clone DNA27864-1155
has been deposited with the
ATCC (designation: DNA27864-1155) and is assigned ATCC deposit no. ATCC
209375.
Based on a BLAST and FastA sequence alignment analysis (using the ALIGN
computer program) of
the full-length sequence, the PRO 187 polypeptide shows 74 % amino acid
sequence identity (Blast score 310)
to human fibroblast growth factor-8 (androgen-induced growth factor).
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EXAMPLE 26: Isolation of cDNA clones Encoding Human PR0337
A cDNA sequence identified in the amylase screen described in Example 2 above
is herein designated
DNA42301. The DNA42301 sequence was then compared to other EST sequences using
phrap as described
in Example 1 above and a consensus sequence designated herein as DNA28761 was
identified. Based on this
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. In order
to screen several libraries for a source of a full-length clone, DNA from the
libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive library
was then used to isolate clones
encoding the PR0337 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction
of the cDNA libraries was isolated from human fetal brain.
A cDNA clone was sequenced in its entirety. The full length nucleotide
sequence of DNA43316-1237
is shown in Figure 51 (SEQ ID NO: 103). Clone DNA43316-1237 contains a single
open reading frame with
an apparent translational initiation site at nucleotide positions 134-136
(Figure 51; SEQ ID NO: 103). The
predicted polypeptide precursor is 344 amino acids long. Clone DNA43316-1237
has been deposited with
ATCC and is assigned ATCC deposit no. 209487
Based on a BLAST-2 and FastA sequence alignment analysis of the full-length
sequence, PR0337
shows amino acid sequence identity to rat neurotrimin (97%).
EXAMPLE 27: Isolation of cDNA clones Encoding Human PRO1411
Use of the signal sequence algorithm described in Example 3 above allowed
identification of an EST
cluster sequence from an Incyte database. This EST cluster sequence was then
compared to a variety of
expressed sequence tag (EST) databases which included public EST databases
(e.g., GenBank) and a
proprietary EST DNA database (LIFESEQ , Incyte Pharmaceuticals, Palo Alto, CA)
to identify existing
homologies. One or more of the ESTs were derived from a thryroid tissue
library. The homology search was
performed using the computer program BLAST or BLAST2 (Altshul et al., Methods
in Enzymology 266:460-
480 (1996)). 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 a consensus DNA
sequence with the program
"phrap" (Phil Green, University of Washington, Seattle, Washington). The
consensus sequence obtained
therefrom is herein designated DNA56013.
In light of the sequence homology between the DNA56013 sequence and an EST
sequence contained
within the Incyte EST 1444225, the clone including this EST was purchased and
the cDNA insert was obtained
and sequenced. The sequence of this cDNA insert is shown in Figure 53 and is
herein designated as
DNA59212-1627.
The full length clone shown in Figure 53 contained a single open reading frame
with an apparent
translational initiation site at nucleotide positions 184-186 and ending at
the stop codon found at nucleotide
positions 1504-1506 (Figure 53; SEQ ID NO: 105). The predicted polypeptide
precursor (Figure 54, SEQ ID
NO: 106) is 440 amino acids long. The signal peptide is at about amino acids 1-
21, and the cell attachment site
is at about amino acids 301-303 of SEQ ID NO:106. PRO1411 has a calculated
molecular weight of
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approximately 42,208 daltons and an estimated pI of approximately 6.36. Clone
DNA59212-1627 was
deposited with the ATCC on September 9, 1998 and is assigned ATCC deposit no.
203245.
An analysis of the Dayhoff database (version 35.45 SwissProt 35), using a WU-
BLAST2 sequence
alignment analysis of the full-length sequence shown in Figure 54 (SEQ ID NO:
106), revealed sequence
identity between the PRO 1411 amino acid sequence and the following Dayhoff
sequences (data from database
incorporated herein): MTV023_19, P _R05307, P W26348, P P82962, AF000949_1,
EBNI_EBV, P _R95107,
GRP2 PHAVU, P R81318, and S744391.
EXAMPLE 28: Isolation of cDNA clones Encoding Human PRO4356
A consensus DNA sequence was assembled relative to other EST sequences using
phrap asdescribed
in Example 1 above. This consensus sequence is designated herein "DNA80200".
Based upon an observed
homology between the DNA80200 consensus sequence and an EST sequence contained
within Merck EST
clone 248287, Merck EST clone 248287 was purchased and its insert obtained and
sequenced, thereby
providing DNA86576-2595.
The entire coding sequence of PR04356 is shown in Figure 55 (SEQ ID NO: 107).
Clone
DNA86576-2595 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 55-57, and an apparent stop codon at nucleotide positions 808-810.
The predicted polypeptide
precursor is 251 amino acids long. Clone DNA86576-2595 has been deposited with
ATCC and is assigned
ATCC deposit no. 203868. The full-length PRO4356 protein shown in Figure 56
has an estimated molecular
weight of about 26,935 daltons and a pI of about 7.42.
An analysis of the Dayhoff database (version 35.45 SwissProt 35), using a WU-
BLAST2 sequence
alignment analysis of the full-length sequence shown in Figure 56 (SEQ ID NO:
108), revealed homology
between the PRO4356 amino acid sequence and the following Dayhoff sequences
incorporated herein:
RNMAGPIAN 1, UPAR BOVIN, 542152, AF007789 1, UPAR RAT, UPAR MOUSE, P W31165,
P W31168, P _R44423 and P W26359.
EXAMPLE 29: Isolation of cDNA clones Encoding Human PRO246
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described
in Example 1 above. This consensus sequence is herein designated DNA30955.
Based on the DNA30955
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
PRO246.
A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer 5'-AGGGTCTCCAGGAGAAAGACTC-3' (SEQ ID NO:111)
reverse PCR primer 5'-ATTGTGGGCCTTGCAGACATAGAC-3' (SEQ ID NO: 112)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA30955
sequence which had the following nucleotide sequence
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hybridization probe
5'-GGCCACAGCATCAAAACCTTAGAACTCAATGTACTGGTTCCTCCAGCTCC-3' (SEQ ID NO: 113)
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was
screened by PCR amplification with the PCR primer pair identified above. A
positive library was then used
to isolate clones encoding the PR0246 gene using the probe oligonucleotide and
one of the PCR primers.
RNA for construction of the cDNA libraries was isolated from human fetal liver
tissue. DNA
sequencing of the clones isolated as described above gave the full-length DNA
sequence for PR0246 [herein
designated as DNA35639-1172] (SEQ ID NO: 109) and the derived protein sequence
for PR0246.
The entire nucleotide sequence of DNA35639-1172 is shown in Figure 57 (SEQ ID
NO: 109). Clone
DNA35639-1172 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 126-128 and ending at the stop codon at nucleotide positions 1296-
1298 (Figure 57). The predicted
polypeptide precursor is 390 amino acids long (Figure 58). Clone DNA35639-1172
has been deposited with
ATCC and is assigned ATCC deposit no. ATCC 209396.
Analysis of the amino acid sequence of the full-length PRO246 polypeptide
suggests that it possess
significant homology to the human cell surface protein HCAR, thereby
indicating that PR0246 may be a novel
cell surface virus receptor.
EXAMPLE 30: Isolation of cDNA clones Encoding Human PRO265
A consensus DNA sequence was assembled relative to other EST sequences as
described in Example
1 above using phrap. This consensus sequence is herein designated DNA33679.
Based on the DNA33679
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
PR0265.
PCR primers (two forward and one reverse) were synthesized:
forward PCR primer A: 5'-CGGTCTACCTGTATGGCAACC-3' (SEQ ID NO:116);
forward PCR primer B: 5'-GCAGGACAACCAGATAAACCAC-3' (SEQ ID NO:117);
reverse PCR primer 5'-ACGCAGATTTGAGAAGGCTGTC-3' (SEQ ID NO: 118)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA33679
sequence which had the following nucleotide sequence
hybridization probe
5'-TTCACGGGCTGCTCTTGCCCAGCTCTTGAAGCTTGAAGAGCTGCAC-3' (SEQ ID NO:119)
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was
screened by PCR amplification with PCR primer pairs identified above. A
positive library was then used to
isolate clones encoding the PRO265 gene using the probe oligonucleotide and
one of the PCR primers.
RNA for construction of the cDNA libraries was isolated from human a fetal
brain library.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for
PR0265 [herein designated as DNA36350-1158] (SEQ ID NO: 114) and the derived
protein sequence for
PR0265.
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The entire nucleotide sequence of DNA36350-1158 is shown in Figure 59 (SEQ ID
NO: 114). Clone
DNA36350-1158 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 352-354 and ending at the stop codon at positions 2332-2334 (Figure
59). The predicted polypeptide
precursor is 660 amino acids long (Figure 60). Clone DNA36350-1158 has been
deposited with ATCC and
is assigned ATCC deposit no. ATCC 209378.
Analysis of the amino acid sequence of the full-length PR0265 polypeptide
suggests that portions of
it possess significant homology to the fibromodulin and the fibromodulin
precursor, thereby indicating that
PR0265 may be a novel member of the leucine rich repeat family, particularly
related to fibromodulin.
EXAMPLE 31: Isolation of cDNA clones Encoding Human PRO941
A consensus sequence was obtained relative to a variety of EST sequences as
described in Example
1 above, wherein the consensus sequence obtained is herein designated
DNA35941. Based on the DNA35941
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
PR0941.
A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer 5'-CTTGACTGTCTCTGAATCTGCACCC-3' (SEQ ID NO:122)
reverse PCR primer 5'-AAGTGGTGGAAGCCTCCAGTGTGG-3' (SEQ ID NO: 123)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA35941
sequence which had the following nucleotide sequence
hybridization probe
5'-CCACTACGGTATTAGAGCAAAAGTTAAAAACCATCATGGTTCCTGGAGCAGC-3' (SEQ ID
NO: 124)
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was
screened by PCR amplification with the PCR primer pair identified above. A
positive library was then used
to isolate clones encoding the PRO941 gene using the probe oligonucleotide and
one of the PCR primers.
RNA for construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB227).
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for
PRO941 [herein designated as DNA53906-1368] (SEQ ID NO: 120) and the derived
protein sequence for
PR0941.
The entire nucleotide sequence of DNA53906-1368 is shown in Figure 61 (SEQ ID
NO: 120). Clone
DNA53906-1368 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 37-39 and ending at the stop codon at nucleotide positions 2353-2355
(Figure 61). The predicted
polypeptide precursor is 772 amino acids long (Figure 62). The full-length
PRO941 protein shown in Figure
62 has an estimated molecular weight of about 87,002 daltons and a pI of about
4.64. Analysis of the full-
length PRO941 sequence shown in Figure 62 (SEQ ID NO: 121) evidences the
presence of the following: a
signal peptide from about amino acid 1 to about amino acid 21, potential N-
glycosylation sites from about
amino acid 57 to about amino acid 60, from about amino acid 74 to about amino
acid 77, from about amino
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acid 419 to about amino acid 422, from about amino acid 437 to about amino
acid 440, from about amino acid
508 to about amino acid 511, from about amino acid 515 to about amino acid
518, from about amino acid 516
to about amino acid 519 and from about amino acid 534 to about amino acid 537,
and cadherin extracellular
repeated domain signature sequences from about amino acid 136 to about amino
acid 146 and from about amino
acid 244 to about amino acid 254. Clone DNA53906-1368 has been deposited with
ATCC on April 7, 1998
and is assigned ATCC deposit no. 209747.
Analysis of the amino acid sequence of the full-length PR0941 polypeptide
suggests that it possesses
significant sequence similarity to a cadherin protein, thereby indicating that
PR0941 may be a novel cadherin
protein family member. More specifically, an analysis of the Dayhoff database
(version 35.45 SwissProt 35)
evidenced significant homology between the PR0941 amino acid sequence and the
following Dayhoff
sequences, 150180, CADA_CHICK, 150178, GEN12782, CADC_HUMAN, P -W25637,
A38992, P R49731,
D38992 and G02678.
EXAMPLE 32: Isolation of cDNA clones Encoding Human PRO10096
Use of the signal sequence algorithm described in Example 3 above allowed
identification of an EST
cluster sequence from the Incyte database, designated herein as 5086173H1.
This EST cluster sequence was
then compared to a variety of expressed sequence tag (EST) databases which
included public EST databases
(e.g., GenBank) and a proprietary EST DNA database (LIFESEQ , Incyte
Pharmaceuticals, Palo Alto, CA)
to identify existing homologies. The homology search was performed using the
computer program BLAST
or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480 (1996)). 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 a consensus DNA sequence with the program "phrap" (Phil Green,
University of Washington,
Seattle, Washington). The consensus sequence obtained therefrom is herein
designated DNA! 10880.
In light of an observed sequence homology between the DNA 110880 sequence and
an EST sequence
encompassed within clone no. 5088384 from the Incyte database, clone no.
5088384 was purchased and the
cDNA insert was obtained and sequenced. It was found herein that that cDNA
insert encoded a full-length
protein. The sequence of this cDNA insert is shown in Figure 63 and is herein
designated as DNA125185-
2506.
Clone DNA 125185-2506 contains a single open reading frame withan apparent
translational initiation
site at nucleotide positions 58-60 and ending at the stop codon at nucleotide
positions 595-597 (Figure 63).
The predicted polypeptide precursor is 179 amino acids long (Figure 64). The
full-length PRO10096 protein
shown in Figure 64 has an estimated molecular weight of about 20,011 daltons
and a pl of about 8.10.
Analysis of the full-length PRO10096 sequence shown in Figure 64 (SEQ ID NO:
126) evidences the presence
of a variety of important polypeptide domains as shown in Figure 64, wherein
the locations given for those
important polypeptide domains are approximate as described above. Clone
DNA125185-2506 has been
deposited with ATCC on December 7, 1999 and is assigned ATCC deposit no. 1031-
PTA.
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EXAMPLE 33: Isolation of cDNA clones Encoding Human PR06003
A cDNA clone (DNA83568-2692) encoding a native human PR06003 polypeptide was
identified
using a yeast screen, in a human fetal kidney cDNA library that preferentially
represents the 5' ends of the
primary cDNA clones.
Clone DNA83568-2692 contains a single open reading frame with an apparent
translational initiation
site at nucleotide positions 638-640 and ending at the stop codon at
nucleotide positions 2225-2227 (Figure 65).
The predicted polypeptide precursor is 529 amino acids long (Figure 66). The
full-length PRO6003 protein
shown in Figure 66 has an estimated molecular weight of about 59,583 daltons
and a pI of about 6.36.
Analysis of the full-length PRO6003 sequence shown in Figure 66 (SEQ ID NO:
128) evidences the presence
of a variety of important polypeptide domains as shown in Figure 66, wherein
the locations given for those
important polypeptide domains are approximate as described above. Clone
DNA83568-2692 has been
deposited with ATCC on July 20, 1999 and is assigned ATCC Deposit No. 386-PTA.
An analysis of the Dayhoff database (version 35.45 SwissProt 35), using the
ALIGN-2 sequence
alignment analysis of the full-length sequence shown in Figure 66 (SEQ ID NO:
128), evidenced sequence
identity between the PRO6003 amino acid sequence and the following Dayhoff
sequences: P W58986,
PTND7_1, YKZ3_YEAST, CEK04B12_1, AB014464_1, PCU07059_1, S31213, CELF25E2_2,
AF036408 1, and AB007932 1.
EXAMPLE 34: Isolation of cDNA clones Encoding Human PRO6004
A consensus sequence was obtained relative to a variety of EST sequences as
described in Example
1 above, wherein the consensus sequence obtained is herein designated
DNA85042. Based upon an observed
homology between the DNA85402 consensus sequence and an EST sequence contained
within Incyte EST
clone no. 3078492, that clone was purchased and its insert obtained and
sequenced. The sequence of that insert
is herein designated as DNA92259 and is shown in Figures 67A-B (SEQ ID NO:
129).
Clone DNA92259 contains a single open reading frame with an apparent
translational initiation site
at nucleotide positions 16-18 and ending at the stop codon at nucleotide
positions 1078-1080 (Figures 67A-B).
The predicted polypeptide precursor is 354 amino acids long (Figure 68). The
full-length PRO6004 protein
shown in Figure 68 has an estimated molecular weight of about 38,719 daltons
and a pI of about 6.12.
Analysis of the full-length PR06004 sequence shown in Figure 68 (SEQ ID NO:
130) evidences the presence
of a variety of important polypeptide domains as shown in Figure 68, wherein
the locations given for those
important polypeptide domains are approximate as described above.
An analysis of the Dayhoff database (version 35.45 SwissProt 35), using the
ALIGN-2 sequence
alignment analysis of the full-length sequence shown in Figure 68 (SEQ ID NO:
130), evidenced sequence
identity between the PRO6004 amino acid sequence and the following Dayhoff
sequences: P _W05152,
LAMP HUMAN, P W05157, P W05155, I56551, OPCM_RAT, AMAL DROME,
DMU78177_1,137246
and NCA1_HUMAN.
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EXAMPLE 35: Isolation of cDNA clones Encoding Human PR0350
A consensus sequence was obtained relative to a variety of EST sequences as
described in Example
1 above, wherein the consensus sequence obtained is herein designated
DNA39493. Based on the DNA39493
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
PRO350.
A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer 5'-TCAGGGCTGCCAGGAAGGAAGAGC-3' (SEQ ID NO:133)
reverse PCR primer 5'-GCAGGAGGAGAAGGTCTTCCAGAAGAAG-3' (SEQ ID NO: 134)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA39493
sequence which had the following nucleotide sequence
hybridization probe
5'-AGAAGTTCCAGTCAGCCCACAAGATGCCATTGTCCCCCGGCCTCC-3' (SEQ ID NO: 135)
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was
screened by PCR amplification with the PCR primer pair identified above. A
positive library was then used
to isolate clones encoding the PR0350 gene using the probe oligonucleotide and
one of the PCR primers.
RNA for construction of the cDNA libraries was isolated from human fetal
kidney tissue.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for
PR0350 [herein designated as DNA44175-1314] (SEQ ID NO: 131) and the derived
protein sequence for
PRO350.
The entire nucleotide sequence of DNA44175-1314 is shown in Figure 69 (SEQ ID
NO: 131). Clone
DNA44175-1314 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 356-358 and ending at the stop codon at nucleotide positions 821-823
(Figure 69). The predicted
polypeptide precursor is 155 amino acids long (Figure 70). The full-length
PR0350 protein shown in Figure
70 has an estimated molecular weight of about 17,194 daltons and a pl of about
10.44. Analysis of the full-
length PRO350 sequence shown in Figure 70 (SEQ ID NO: 132) evidences the
presence of a variety of
important polypeptide domains as shown in Figure 70.
EXAMPLE 36: Use of PRO as a hybridization probe
The following method describes use of a nucleotide sequence encoding PRO as a
hybridization probe.
DNA comprising the coding sequence of full-length or mature PRO as disclosed
herein is employed
as a probe to screen for homologous DNAs (such as those encoding naturally-
occurring variants of PRO) in
human tissue cDNA libraries or human tissue genomic libraries.
Hybridization and washing of filters containing either library DNAs is
performed under the following
high stringency conditions. Hybridization of radiolabeled PRO-derived probe to
the filters is performed in a
solution of 50 % formamide, 5x SSC, 0.1 % SDS, 0.1 % sodium pyrophosphate, 50
mM sodium phosphate, pH
6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42 C for 20 hours.
Washing of the filters is performed
in an aqueous solution of 0. lx SSC and 0.1 % SDS at 42 C.
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DNAs having a desired sequence identity with the DNA encoding full-length
native sequence PRO
can then be identified using standard techniques known in the art.
EXAMPLE 37: Expression of PRO in E. coli
This example illustrates preparation of an unglycosylated form of PRO by
recombinant expression in
E. coll.
The DNA sequence encoding PRO 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 for an antibiotic resistance gene, a trp promoter, a polyhis leader
(including the first six STII codons,
polyhis sequence, and enterokinase cleavage site), the PRO coding region,
lambda transcriptional terminator,
and an argU gene.
The ligation mixture is then used to transform a selected E. coli strain using
the methods described
in Sambrook et al., supr. 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.
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.
After culturing the cells 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
solubilized PRO protein can then be purified using a metal chelating column
under conditions that allow tight
binding of the protein.
PRO may be expressed in E. coli in a poly-His tagged form, using the following
procedure. The
DNA encoding PRO is initially amplified using selected PCR primers. The
primers will contain restriction
enzyme sites which correspond to the restriction enzyme sites on the selected
expression vector, and other
useful sequences providing for efficient and reliable translation initiation,
rapid purification on a metal chelation
column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His
tagged sequences are then
ligated into an expression vector, which is used to transform an E. coil host
based on strain 52 (W3110
fuhA(tonA) Ion galE rpoHts(htpRts) clpP(laclq). Transformants are first grown
in LB containing 50 mg/ml
carbenicillin at 30 C with shaking until an O.D.600 of 3-5 is reached.
Cultures are then diluted 50-100 fold
into CRAP media (prepared by mixing 3.57 g (NH4)2SO4, 0.71 g sodium
citrate=2H20, 1.07 g KCI, 5.36 g
Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as well as
110 mM MPOS, pH 7.3, 0.55 %
(w/v) glucose and 7 mM MgSO4) and grown for approximately 20-30 hours at 30 C
with shaking. Samples
are removed to verify expression by SDS-PAGE analysis, and the bulk culture is
centrifuged to pellet the cells.
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Cell pellets are frozen until purification and refolding.
E. coil paste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in
10 volumes (w/v) in 7
M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium
tetrathionate is added to make final
concentrations of 0.1 M and 0.02 M, respectively, and the solution is stirred
overnight at 4 C. This step results
in a denatured protein with all cysteine residues blocked by sulfitolization.
The solution is centrifuged at 40,000
rpm in a Beckman Ultracentifuge for 30 min. The supernatant is diluted with 3-
5 volumes of metal chelate
column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22
micron filters to clarify. The
clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal chelate column
equilibrated in the metal chelate
column buffer. The column is washed with additional buffer containing 50 mM
imidazole (Calbiochem, Utrol
grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole.
Fractions containing the
desired protein are pooled and stored at 4 C. Protein concentration is
estimated by its absorbance at 280 nm
using the calculated extinction coefficient based on its amino acid sequence.
The proteins are refolded by diluting the sample slowly into freshly prepared
refolding buffer
consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCI, 2.5 M urea, 5 mM cysteine, 20
mM glycine and 1 mM
EDTA. Refolding volumes are chosen so that the final protein concentration is
between 50 to 100
micrograms/ml. The refolding solution is stirred gently at 4 C for 12-36
hours. The refolding reaction is
quenched by the addition of TFA to a final concentration of 0.4% (pH of
approximately 3). Before further
purification of the protein, the solution is filtered through a 0.22 micron
filter and acetonitrile is added to
2-10% final concentration. The refolded protein is chromatographed on a Poros
R1/H reversed phase column
using a mobile buffer of 0.1% TFA with elution with a gradient of acetonitrile
from 10 to 80%. Aliquots of
fractions with A280 absorbance are analyzed on SDS polyacrylamide gels and
fractions containing
homogeneous refolded protein are pooled. Generally, the properly refolded
species of most proteins are eluted
at the lowest concentrations of acetonitrile since those species are the most
compact with their hydrophobic
interiors shielded from interaction with the reversed phase resin. Aggregated
species are usually eluted at
higher acetonitrile concentrations. In addition to resolving misfolded forms
of proteins from the desired form,
the reversed phase step also removes endotoxin from the samples.
Fractions containing the desired folded PRO polypeptide are pooled and the
acetonitrile removed using
a gentle stream of nitrogen directed at the solution. Proteins are formulated
into 20 mM Hepes, pH 6.8 with
0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using
G25 Superfine (Pharmacia)
resins equilibrated in the formulation buffer and sterile filtered.
Many of the PRO polypeptides disclosed herein were successfully expressed as
described above.
EXAMPLE 38: Expression of PRO in mammalian cells
This example illustrates preparation of a potentially glycosylated form of PRO
by recombinant
expression in mammalian cells.
The vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as
the expression vector.
Optionally, the PRO DNA is ligated into pRK5 with selected restriction enzymes
to allow insertion of the PRO
DNA using ligation methods such as described in Sambrook et al., The resulting
vector is called pRK5-
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PRO.
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,ug pRK5-PRO
DNA is mixed with about
1 g DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)]
and dissolved in 500 Al
of 1 mM Tris-HCI, 0.1 mM EDTA, 0.227 M CaCl2. To this mixture is added,
dropwise, 500 l of 50 mM
HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO4, and a precipitate is allowed to
form for 10 minutes at
25 C. The precipitate is suspended and added to the 293 cells and allowed to
settle for about four hours at
37 C. The culture medium is aspirated off and 2 ml of 20% glycerol in 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.
Approximately 24 hours after the transfections, the culture medium is removed
and replaced with
culture medium (alone) or culture medium containing 200 Ci/m135S-cysteine and
200 Ci/ml 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 PRO polypeptide. The cultures containing transfected cells may
undergo further incubation
(in serum free medium) and the medium is tested in selected bioassays.
In an alternative technique, PRO 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 g pRK5-PRO 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 g/ml bovine insulin and
0.1 g/ml bovine transferrin. After about four days, the conditioned media is
centrifuged and filtered to
remove cells and debris. The sample containing expressed PRO can then be
concentrated and purified by any
selected method, such as dialysis and/or column chromatography.
In another embodiment, PRO can be expressed in CHO cells. The pRK5-PRO can be
transfected into
CHO cells using known reagents such as CaPO4 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 35S-methionine. After determining the presence of PRO 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 PRO can then be
concentrated and purified by
any selected method.
Epitope-tagged PRO may also be expressed in host CHO cells. The PRO may be
subcloned out of
the pRK5 vector. 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 PRO
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
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performed, as described above, to verify expression. The culture medium
containing the expressed poly-His
tagged PRO can then be concentrated and purified by any selected method, such
as by Nit+-chelate affinity
chromatography.
PRO may also be expressed in CHO and/or COS cells by a transient expression
procedure or in CHO
cells by another stable expression procedure.
Stable expression in CHO cells is performed using the following procedure. The
proteins are
expressed as an IgG construct (immunoadhesin), in which the coding sequences
for the soluble forms (e.g.
extracellular domains) of the respective proteins are fused to an IgGI
constant region sequence containing the
hinge, CH2 and CH2 domains and/or is a poly-His tagged form.
Following PCR amplification, the respective DNAs are subcloned in a CHO
expression vector using
standard techniques as described in Ausubel et al., Current Protocols of
Molecular Biology, Unit 3.16, John
Wiley and Sons (1997). CHO expression vectors are constructed to have
compatible restriction sites 5' and
3' of the DNA of interest to allow the convenient shuttling of cDNA's. The
vector used expression in CHO
cells is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779
(1996), and uses the SV40 early
promoter/enhancer to drive expression of the cDNA of interest and
dihydrofolate reductase (DHFR). DHFR
expression permits selection for stable maintenance of the plasmid following
transfection.
Twelve micrograms of the desired plasmid DNA is introduced into approximately
10 million CHO
cells using commercially available transfection reagents Superfect (Quiagen),
Dosper or Fugene (Boehringer
Mannheim). The cells are grown as described in Lucas et al., supra.
Approximately 3 x 10' cells are frozen
in an ampule for further growth and production as described below.
The ampules containing the plasmid DNA are thawed by placement into water bath
and mixed by
vortexing. The contents are pipetted into a centrifuge tube containing 10 mLs
of media and centrifuged at 1000
rpm for 5 minutes. The supernatant is aspirated and the cells are resuspended
in 10 mL of selective media (0.2
m filtered PS20 with 5 % 0.2 pm diafiltered fetal bovine serum). The cells are
then aliquoted into a 100 mL
spinner containing 90 mL of selective media. After 1-2 days, the cells are
transferred into a 250 mL spinner
filled with 150 mL selective growth medium and incubated at 37 C. After
another 2-3 days, 250 mL, 500 mL
and 2000 mL spinners are seeded with 3 x 105 cells/mL. The cell media is
exchanged with fresh media by
centrifugation and resuspension in production medium. Although any suitable
CHO media may be employed,
a production medium described in U.S. Patent No. 5,122,469, issued June 16,
1992 may actually be used.
A 3L production spinner is seeded at 1.2 x 106 cells/mL. On day 0, the cell
number pH ie determined. On
day 1, the spinner is sampled and sparging with filtered air is commenced. On
day 2, the spinner is sampled,
the temperature shifted to 33 C, and 30 mL of 500 g/L glucose and 0.6 mL of
10% antifoam (e.g., 35%
polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken.
Throughout the
production, the pH is adjusted as necessary to keep it at around 7.2. After 10
days, or until the viability
dropped below 70%, the cell culture is harvested by centrifugation and
filtering through a 0.22,um filter. The
filtrate was either stored at 4 C or immediately loaded onto columns for
purification.
For the poly-His tagged constructs, the proteins are purified using a Ni-NTA
column (Qiagen).
Before purification, imidazole is added to the conditioned media to a
concentration of 5 mM. The conditioned
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media is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,
buffer containing 0.3
M NaCI and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4 C. After loading,
the column is washed with
additional equilibration buffer and the protein eluted with equilibration
buffer containing 0.25 M imidazole.
The highly purified protein is 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 (Pharmacia) column
and stored at -80 C.
Immunoadhesin (Fc-containing) constructs are purified from the conditioned
media as follows. The
conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which
had been equilibrated in 20
mM Na phosphate buffer, pH 6.8. After loading, the column is washed
extensively with equilibration buffer
before elution with 100 mM citric acid, pH 3.5. The eluted protein is
immediately neutralized by collecting
1 ml fractions into tubes containing 275 gL of 1 M Tris buffer, pH 9. The
highly purified protein is
subsequently desalted into storage buffer as described above for the poly-His
tagged proteins. The homogeneity
is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing
by Edman degradation.
Many of the PRO polypeptides disclosed herein were successfully expressed as
described above.
EXAMPLE 39: Expression of PRO in Yeast
The following method describes recombinant expression of PRO in yeast.
First, yeast expression vectors are constructed for intracellular production
or secretion of PRO from
the ADH2/GAPDH promoter. DNA encoding PRO and the promoter is inserted into
suitable restriction
enzyme sites in the selected plasmid to direct intracellular expression of
PRO. For secretion, DNA encoding
PRO can be cloned into the selected plasmid, together with DNA encoding the
ADH2/GAPDH promoter, a
native PRO signal peptide or other mammalian signal peptide, or, for example,
a yeast alpha-factor or invertase
secretory signal/leader sequence, and linker sequences (if needed) for
expression of PRO.
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.
Recombinant PRO 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 PRO may further be purified using selected column
chromatography resins.
Many of the PRO polypeptides disclosed herein were successfully expressed as
described above.
EXAMPLE 40: Expression of PRO in Baculovirus-Infected Insect Cells
The following method describes recombinant expression of PRO in Baculovirus-
infected insect cells.
The sequence coding for PRO 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 PRO or the desired
portion of the coding sequence
of PRO such as the sequence encoding the extracellular domain of a
transmembrane protein or the sequence
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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.
Recombinant baculovirus is generated by co-transfecting the above plasmid and
BaculoGoldTM 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 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).
Expressed poly-his tagged PRO can then be purified, for example, by Nil'-
chelate affinity
chromatography as follows. Extracts are prepared from recombinant virus-
infected Sf9 cells as described by
Rupert et al., Nature, M:175-179 (1993). Briefly, Sf9 cells are washed,
resuspended in sonication buffer
(25 mL Hepes, pH 7.9; 12.5 mM MgCl2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40;
0.4 M KCI), 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 NaCI, 10% glycerol,
pH 7.8) and filtered
through a 0.45 gin filter. A NiZ+-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 A290
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 NaCI, 10% glycerol, pH 6.0), which elutes
nonspecifically bound
protein. After reaching AZ30baseline 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 Nil'-NTA-conjugated to alkaline phosphatase (Qiagen).
Fractions containing the eluted
Hislo-tagged PRO are pooled and dialyzed against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) PRO can be
performed using known
chromatography techniques, including for instance, Protein A or protein G
column chromatography.
Many of the PRO polypeptides disclosed herein were successfully expressed as
described above.
EXAMPLE 41: Preparation of Antibodies that Bind PRO
This example illustrates preparation of monoclonal antibodies which can
specifically bind PRO.
Techniques for producing the monoclonal antibodies are known in the art and
are described, for
instance, in Coding, M. Immunogens that may be employed include purified PRO,
fusion proteins
containing PRO, and cells expressing recombinant PRO on the cell surface.
Selection of the immunogen can
be made by the skilled artisan without undue experimentation.
Mice, such as Balb/c, are immunized with the PRO immunogen emulsified in
complete Freund's
adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-
100 micrograms. Alternatively,
the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research,
Hamilton, MT) 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
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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 anti-PRO
antibodies.
After a suitable antibody titer has been detected, the animals "positive" for
antibodies can be injected
with a final intravenous injection of PRO. Three to four days later, the mice
are sacrificed and the spleen cells
are harvested. The spleen cells are then fused (using 35% polyethylene glycol)
to a selected murine myeloma
cell line such as P3X63AgU. 1, available from ATCC, No. CRL 1597. 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.
The hybridoma cells will be screened in an ELISA for reactivity against PRO.
Determination of
"positive" hybridoma cells secreting the desired monoclonal antibodies against
PRO is within the skill in the
art.
The positive hybridoma cells can be injected intraperitoneally into syngeneic
Balb/c mice to produce
ascites containing the anti-PRO 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.
EXAMPLE 43: Purification of PRO Polypeptides Using Specific Antibodies
Native or recombinant PRO polypeptides may be purified by a variety of
standard techniques in the
art of protein purification. For example, pro-PRO polypeptide, mature PRO
polypeptide, or pre-PRO
polypeptide is purified by immunoaffinity chromatography using antibodies
specific for the PRO polypeptide
of interest. In general, an immunoaffinity column is constructed by covalently
coupling the anti-PRO
polypeptide antibody to an activated chromatographic resin.
Polyclonal immunoglobulins are prepared from immune sera either by
precipitation with ammonium
sulfate or by purification on immobilized Protein A (Pharmacia LKB
Biotechnology, Piscataway, N.J.).
Likewise, monoclonal antibodies are prepared from mouse ascites fluid by
ammonium sulfate precipitation or
chromatography on immobilized Protein A. Partially purified immunoglobulin is
covalently attached to a
chromatographic resin such as CnBr-activated SEPHAROSETM (Pharmacia LKB
Biotechnology). The antibody
is coupled to the resin, the resin is blocked, and the derivative resin is
washed according to the manufacturer's
instructions.
Such an immunoaffinity column is utilized in the purification of PRO
polypeptide by preparing a
fraction from cells containing PRO polypeptide in a soluble form. This
preparation is derived by solubilization
of the whole cell or of a subcellular fraction obtained via differential
centrifugation by the addition of detergent
or by other methods well known in the art. Alternatively, soluble PRO
polypeptide containing a signal
sequence may be secreted in useful quantity into the medium in which the cells
are grown.
A soluble PRO polypeptide-containing preparation is passed over the
immunoaffinity column, and the
column is washed under conditions that allow the preferential absorbance of
PRO polypeptide (e.g., high ionic
strength buffers in the, presence of detergent). Then, the column is eluted
under conditions that disrupt
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antibody/PRO polypeptide binding (e.g., a low pH buffer such as approximately
pH 2-3, or a high
concentration of a chaotrope such as urea or thiocyanate ion), and PRO
polypeptide is collected.
EXAMPLE 44: Drug Screening
This invention is particularly useful for screening compounds by using PRO
polypeptides or binding
fragment thereof in any of a variety of drug screening techniques. The PRO
polypeptide or fragment employed
in such a test may either be free in solution, affixed to a solid support,
borne on a cell surface, or located
intracellularly. One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably
transformed with recombinant nucleic acids expressing the PRO polypeptide or
fragment. Drugs are screened
against such transformed cells in competitive binding assays. Such cells,
either in viable or fixed form, can
be used for standard binding assays. One may measure, for example, the
formation of complexes between PRO
polypeptide or a fragment and the agent being tested. Alternatively, one can
examine the diminution in complex
formation between the PRO polypeptide and its target cell or target receptors
caused by the agent being tested.
Thus, the present invention provides methods of screening for drugs or any
other agents which can
affect a PRO polypeptide-associated disease or disorder. These methods
comprise contacting such an agent
with an PRO polypeptide or fragment thereof and assaying (I) for the presence
of a complex between the agent
and the PRO polypeptide or fragment, or (ii) for the presence of a complex
between the PRO polypeptide or
fragment and the cell, by methods well known in the art. In such competitive
binding assays, the PRO
polypeptide or fragment is typically labeled. After suitable incubation, free
PRO polypeptide or fragment is
separated from that present in bound form, and the amount of free or
uncomplexed label is a measure of the
ability of the particular agent to bind to PRO polypeptide or to interfere
with the PRO polypeptide/cell
complex.
Another technique for drug screening provides high throughput screening for
compounds having
suitable binding affinity to a polypeptide and is described in detail in WO
84/03564, published on September
13, 1984. Briefly stated, large numbers of different small peptide test
compounds are synthesized on a solid
substrate, such as plastic pins or some other surface. As applied to a PRO
polypeptide, the peptide test
compounds are reacted with PRO polypeptide and washed. Bound PRO polypeptide
is detected by methods
well known in the art. Purified PRO polypeptide can also be coated directly
onto plates for use in the
aforementioned drug screening techniques. In addition, non-neutralizing
antibodies can be used to capture the
peptide and immobilize it on the solid support.
This invention also contemplates the use of competitive drug screening assays
in which neutralizing
antibodies capable of binding PRO polypeptide specifically compete with a test
compound for binding to PRO
polypeptide or fragments thereof. In this manner, the antibodies can be used
to detect the presence of any
peptide which shares one or more antigenic determinants with PRO polypeptide.
EXAMPLE 45: Rational Drug Design
The goal of rational drug design is to produce structural analogs of
biologically active polypeptide of
interest (i.e., a PRO polypeptide) or of small molecules with which they
interact, e.g., agonists, antagonists,
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or inhibitors. Any of these examples can be used to fashion drugs which are
more active or stable forms of
the PRO polypeptide or which enhance or interfere with the function of the PRO
polypeptide in vivo (c.f.,
Hodgson, Bio/Technoloey, 2: 19-21 (1991)).
In one approach, the three-dimensional structure of the PRO polypeptide, or of
an PRO
polypeptide-inhibitor complex, is determined by x-ray crystallography, by
computer modeling or, most
typically, by a combination of the two approaches. Both the shape and charges
of the PRO polypeptide must
be ascertained to elucidate the structure and to determine active site(s) of
the molecule. Less often, useful
information regarding the structure of the PRO polypeptide may be gained by
modeling based on the structure
of homologous proteins. In both cases, relevant structural information is used
to design analogous PRO
polypeptide-like molecules or to identify efficient inhibitors. Useful
examples of rational drug design may
include molecules which have improved activity or stability as shown by
Braxton and Wells, Biochemistry.
31:7796-7801 (1992) or which act as inhibitors, agonists, or antagonists of
native peptides as shown by
Athauda et a!., J. Biochem., 113,:742-746 (1993).
It is also possible to isolate a target-specific antibody, selected by
functional assay, as described above,
and then to solve its crystal structure. This approach, in principle, yields a
pharmacore upon which subsequent
drug design can be based. It is possible to bypass protein crystallography
altogether by generating
anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active
antibody. As a mirror image of a
mirror image, the binding site of the anti-ids would be expected to be an
analog of the original receptor. The
anti-id could then be used to identify and isolate peptides from banks of
chemically or biologically produced
peptides. The isolated peptides would then act as the pharmacore.
By virtue of the present invention, sufficient amounts of the PRO polypeptide
may be made available
to perform such analytical studies as X-ray crystallography. In addition,
knowledge of the PRO polypeptide
amino acid sequence provided herein will provide guidance to those employing
computer modeling techniques
in place of or in addition to x-ray crystallography.
EXAMPLE 46: Mouse KidneyMesangial Cell Proliferation Assay (Assay 92)
This assay shows that certain polypeptides of the invention act to induce
proliferation of mammalian
kidney mesangial cells and, therefore, are useful for treating kidney
disorders associated with decreased
mesangial cell function such as Berger disease or other nephropathies
associated with Schonlein-Henoch
purpura, celiac disease, dermatitis herpetiformis or Crohn disease. The assay
is performed as follows. On
day one, mouse kidney mesangial cells are plated on a 96 well plate in growth
media (3:1 mixture of
Dulbecco's modified Eagle's medium and Ham's F12 medium, 95% fetal bovine
serum, 5% supplemented
with 14 mM HEPES) and grown overnight. On day 2, PRO polypeptides are diluted
at 2 concentrations(1 %
and 0.1 %) in serum-free medium and added to the cells. Control samples are
serum-free medium alone. On
day 4, 2O0 of the Cell Titer 96 Aqueous one solution reagent (Progema) was
added to each well and the
colormetric reaction was allowed to proceed for 2 hours. The absorbance (OD)
is then measured at 490 nm.
A positive in the assay is anything that gives an absorbance reading which is
at least 15% above the control
reading.
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The following polypeptides tested positive in this assay: PRO1272.
EXAMPLE 47: Detection of PRO Polypeptides That Affect Glucose or FFA Uptake by
Primary Rat
Adipocytes (Assay 94)
This assay is designed to determine whether PRO polypeptides show the ability
to affect glucose or
FFA uptake by adipocyte cells. PRO polypeptides testing positive in this assay
would be expected to be useful
for the therapeutic treatment of disorders where either the stimulation or
inhibition of glucose uptake by
adipocytes would be beneficial including, for example, obesity, diabetes or
hyper- or hypo-insulinemia.
In a 96 well format, PRO polypeptides to be assayed are added to primary rat
adipocytes, and allowed
to incubate overnight. Samples are taken at 4 and 16 hours and assayed for
glycerol, glucose and FFA uptake.
After the 16 hour incubation, insulin is added to the media and allowed to
incubate for 4 hours. At this time,
a sample is taken and glycerol, glucose and FFA uptake is measured. Media
containing insulin without the
PRO polypeptide is used as a positive reference control. As the PRO
polypeptide being tested may either
stimulate or inhibit glucose and FFA uptake, results are scored as positive in
the assay if greater than 1.5 times
or less than 0.5 times the insulin control.
The following PRO polypeptides tested positive as either stimulators or
inhibitors of glucose and/or
FFA uptake in this assay: PR0196, PR0185, PR0210, PR0215, PR0242, PR0288,
PRO1183, PRO1419,
PR09940, PR0301, PR0337 and PR0265.
EXAMPLE 48: Stimulation of Adult Heart Hypertrophy (Assay 2)
This assay is designed to measure the ability of various PRO polypeptides to
stimulate hypertrophy
of adult heart. PRO polypeptides testing positive in this assay would be
expected to be useful for the
therapeutic treatment of various cardiac insufficiency disorders.
Ventricular myocytes freshly isolated from adult (250g) Sprague Dawley rats
are plated at 2000
cell/well in 180 l volume. Cells are isolated and plated on day 1, the PRO
polypeptide-containing test samples
or growth medium only (negative control) (20 l volume) is added on day 2 and
the cells are then fixed and
stained on day 5. After staining, cell size is visualized wherein cells
showing no growth enhancement as
compared to control cells are given a value of 0.0, cells showing small to
moderate growth enhancement as
compared to control cells are given a value of 1.0 and cells showing large
growth enhancement as compared
to control cells are given a value of 2Ø Any degree of growth enhancement as
compared to the negative
control cells is considered positive for the assay.
The following PRO polypeptides tested positive in this assay: PR0301.
EXAMPLE 49: Inhibition of Vascular Endothelial Growth Factor (VEGF) Stimulated
Proliferation of
Endothelial Cell Growth (Assay 9)
The ability of various PRO polypeptides to inhibit VEGF stimulated
proliferation of endothelial cells
was tested. Polypeptides testing positive in this assay are useful for
inhibiting endothelial cell growth in
mammals where such an effect would be beneficial, e.g., for inhibiting tumor
growth.
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Specifically, bovine adrenal cortical capillary endothelial cells (ACE) (from
primary culture, maximum
of 12-14 passages) were plated in 96-well plates at 500 cells/well per 100
microliter. Assay media included
low glucose DMEM, 10% calf serum, 2 mM glutamine, and 1X
penicillin/streptomycin/fungizone. Control
wells included the following: (1) no ACE cells added; (2) ACE cells alone; (3)
ACE cells plus 5 ng/ml FGF;
(4) ACE cells plus 3 ng/ml VEGF; (5) ACE cells plus 3 ng/ml VEGF plus 1 ng/ml
TGF-beta; and (6) ACE
cells plus 3 ng/ml VEGF plus 5 ng/ml LIF. The test samples, poly-his tagged
PRO polypeptides (in 100
microliter volumes), were then added to the wells (at dilutions of 1 %, 0.1 %
and 0.01 %, respectively). The
cell cultures were incubated for 6-7 days at 37 C/5 % CO2. After the
incubation, the media in the wells was
aspirated, and the cells were washed 1X with PBS. An acid phosphatase reaction
mixture (100 microliter;
0.1M sodium acetate, pH 5.5, 0.1 % Triton X-100, 10 mM p-nitrophenyl
phosphate) was then added to each
well. After a 2 hour incubation at 37 C, the reaction was stopped by addition
of 10 microliters 1N NaOH.
Optical density (OD) was measured on a microplate reader at 405 nm.
The activity of PRO polypeptides was calculated as the percent inhibition of
VEGF (3 ng/ml)
stimulated proliferation (as determined by measuring acid phosphatase activity
at OD 405 nm) relative to the
cells without stimulation. TGF-beta was employed as an activity reference at 1
ng/ml, since TGF-beta blocks
70-90% of VEGF-stimulated ACE cell proliferation. The results are indicative
of the utility of the PRO
polypeptides in cancer therapy and specifically in inhibiting tumor
angiogenesis. Numerical values (relative
inhibition) are determined by calculating the percent inhibition of VEGF
stimulated proliferation by the PRO
polypeptides relative to cells without stimulation and then dividing that
percentage into the percent inhibition
obtained by TGF-0 at 1 ng/ml which is known to block 70-90% of VEGF stimulated
cell proliferation. The
results are considered positive if the PRO polypeptide exhibits 30 % or
greater inhibition of VEGF stimulation
of endothelial cell growth (relative inhibition 30% or greater).
The following polypeptide tested positive in this assay: PRO301, PRO187 and
PR0246.
EXAMPLE 50: Stimulatory Activity in Mixed Lymphocyte Reaction (MLR) Assay
(Assay 24)
This example shows that certain polypeptides of the invention are active as a
stimulator of the
proliferation of stimulated T-lymphocytes. Compounds which stimulate
proliferation of lymphocytes are useful
therapeutically where enhancement of an immune response is beneficial. A
therapeutic agent may take the
form of antagonists of the polypeptide of the invention, for example, murine-
human chimeric, humanized or
human antibodies against the polypeptide.
The basic protocol for this assay is described in Current Protocols in
Immunology, unit 3.12; edited
by J E Coligan, A M Kruisbeek, D H Marglies, E M Shevach, W Strober, National
Insitutes of Health,
Published by John Wiley & Sons, Inc.
More specifically, in one assay variant, peripheral blood mononuclear cells
(PBMC) are isolated from
mammalian individuals, for example a human volunteer, by leukopheresis (one
donor will supply stimulator
PBMCs, the other donor will supply responder PBMCs). If desired, the cells are
frozen in fetal bovine serum
and DMSO after isolation. Frozen cells may be thawed overnight in assay media
(37 C, 5 % CO2) and then
washed and resuspended to 3x106 cells/ml of assay media (RPMI; 10% fetal
bovine serum, 1%
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penicillin/streptomycin, 1 % glutamine, 1% HEPES, 1 % non-essential amino
acids, 1% pyruvate). The
stimulator PBMCs are prepared by irradiating the cells (about 3000 Rads).
The assay is prepared by plating in triplicate wells a mixture of:
100:1 of test sample diluted to 1 % or to 0.1 %,
50 :1 of irradiated stimulator cells, and
50 :1 of responder PBMC cells.
100 microliters of cell culture media or 100 microliter of CD4-[gG is used as
the control. The wells are then
incubated at 37 C, 5% CO2 for 4 days. On day 5, each well is pulsed with
tritiated thymidine (1.0 mC/well;
Amersham). After 6 hours the cells are washed 3 times and then the uptake of
the label is evaluated.
In another variant of this assay, PBMCs are isolated from the spleens of
Balb/c mice and C57B6 mice.
The cells are teased from freshly harvested spleens in assay media (RPMI; 10%
fetal bovine serum, I%
penicillin/streptomycin, 1 % glutamine, 1 % HEPES, 1 % non-essential amino
acids, 1 % pyruvate) and the
PBMCs are isolated by overlaying these cells over Lympholyte M (Organon
Teknika), centrifuging at 2000
rpm for 20 minutes, collecting and washing the mononuclear cell layer in assay
media and resuspending the
cells to 1x107 cells/ml of assay media. The assay is then conducted as
described above.
Positive increases over control are considered positive with increases of
greater than or equal to 180%
being preferred. However, any value greater than control indicates a
stimulatory effect for the test protein.
The following PRO polypeptides tested positive in this assay: PRO533 and
PRO301.
EXAMPLE 51: PDB 12 Cell Proliferation (Assay 29)
This example demonstrates that various PRO polypeptides have efficacy in
inducing proliferation of
PDB 12 pancreatic ductal cells and are, therefore, useful in the therapeutic
treatment of disorders which involve
protein secretion by the pancreas, including diabetes, and the like.
PDB12 pancreatic ductal cells are plated on fibronectin coated 96 well plates
at 1.5x 10' cells per well
in 100 L/180 L of growth media. 100 L of growth media with the PRO
polypeptide test sample or negative
control lacking the PRO polypeptide is then added to well, for a final volume
of 200 L. Controls contain
growth medium containing a protein shown to be inactive in this assay. Cells
are incubated for 4 days at 37 C.
20 L of Alamar Blue Dye (AB) is then added to each well and the flourescent
reading is measured at 4 hours
post addition of AB, on a microtiter plate reader at 530 nm excitation and 590
mn emission. The standard
employed is cells without Bovine Pituitary Extract (BPE) and with various
concentrations of BPE. Buffer or
growth medium only controls from unknowns are run 2 times on each 96 well
plate.
Percent increase in protein production is calculated by comparing the Alamar
Blue Dye calculated
protein concentration produced by the PRO polypeptide-treated cells with the
Alamar Blue Dye calculated
protein concentration produced by the negative control cells. A percent
increase in protein production of
greater than or equal to 25% as compared to the negative control cells is
considered positive.
The following PRO polypeptides tested positive in this assay: PR0301.
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EXAMPLE 52: Guinea Pig Vascular Leak (Assay 32)
This assay is designed to determine whether PRO polypeptides of the present
invention show the
ability to induce vascular permeability. Polypeptides testing positive in this
assay are expected to be useful
for the therapeutic treatment of conditions which would benefit from enhanced
vascular permeability including,
for example, conditions which may benefit from enhanced local immune system
cell infiltration.
Hairless guinea pigs weighing 350 grams or more were anesthetized with
Ketamine (75-80 mg/kg)
and 5 mg/kg Xylazine intramuscularly. Test samples containing the PRO
polypeptide or a physiological buffer
without the test polypeptide are injected into skin on the back of the test
animals with 100 dl per injection site
intradermally. There were approximately 16-24 injection sites per animal. One
ml of Evans blue dye (1 %
in PBS) is then injected intracardially. Skin vascular permeability responses
to the compounds (i. e., blemishes
at the injection sites of injection) are visually scored by measuring the
diameter (in mm) of blue-colored leaks
from the site of injection at 1, 6 and 24 hours post administration of the
test materials. The mm diameter of
blueness at the site of injection is observed and recorded as well as the
severity of the vascular leakage.
Blemishes of at least 5 mm in diameter are considered positive for the assay
when testing purified proteins,
being indicative of the ability to induce vascular leakage or permeability. A
response greater than 7 mm
diameter is considered positive for conditioned media samples. Human VEGF at
0.1 g/100 p1 is used as a
positive control, inducing a response of 4-8 mm diameter.
The following PRO polypeptides tested positive in this assay: PRO533.
EXAMPLE 53: Retinal Neuron Survival (Assay 52)
This example demonstrates that certain PRO polypeptides have efficacy in
enhancing the survival of
retinal neuron cells and, therefore, are useful for the therapeutic treatment
of retinal disorders or injuries
including, for example, treating sight loss in mammals due to retinitis
pigmentosum, AMD, etc.
Sprague Dawley rat pups at postnatal day 7 (mixed population: glia and retinal
neuronal types) are
killed by decapitation following CO2 anesthesia and the eyes are removed under
sterile conditions. The neural
retina is dissected away from the pigment epithelium and other ocular tissue
and then dissociated into a single
cell suspension using 0.25% trypsin in Cat+, Mg2+-free PBS. The retinas are
incubated at 37 C for 7-10
minutes after which the trypsin is inactivated by adding 1 ml soybean trypsin
inhibitor. The cells are plated
at 100,000 cells per well in 96 well plates in DMEM/F12 supplemented with N2
and with or without the
specific test PRO polypeptide. Cells for all experiments are grown at 37 C in
a water saturated atmosphere
of 5% CO2. After 2-3 days in culture, cells are stained with calcein AM then
fixed using 4%
paraformaldehyde and stained with DAPI for determination of total cell count.
The total cells (fluorescent)
are quantified at 20X objective magnification using CCD camera and NIH image
software for Maclntosh.
Fields in the well are chosen at random.
The effect of various concentration of PRO polypeptides are reported herein
where percent survival
is calculated by dividing the total number of calcein AM positive cells at 2-3
days in culture by the total
number of DAPI-labeled cells at 2-3 days in culture. Anything above 30%
survival is considered positive.
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The following PRO polypeptides tested positive in this assay using polypeptide
concentrations within
the range of 0.01% to 1.0% in the assay: PR0350.
EXAMPLE 54: Proliferation of Rat Utricular Supporting Cells (Assay 54)
This assay shows that certain polypeptides of the invention act as potent
mitogens for inner ear
supporting cells which are auditory hair cell progenitors and, therefore, are
useful for inducing the regeneration
of auditory hair cells and treating hearing loss in mammals. The assay is
performed as follows. Rat UEC-4
utricular epithelial cells are aliquoted into 96 well plates with a density of
3000 cells/well in 200 l of serum-
containing medium at 33 C. The cells are cultured overnight and are then
switched to serum-free medium at
37 C. Various dilutions of PRO polypeptides (or nothing for a control) are
then added to the cultures and the
cells are incubated for 24 hours. After the 24 hour incubation, 3H-thymidine
(1 izCi/well) is added and the
cells are then cultured for an additional 24 hours. The cultures are then
washed to remove unincorporated
radiolabel, the cells harvested and Cpm per well determined. Cpm of at least
30% or greater in the PRO
polypeptide treated cultures as compared to the control cultures is considered
a positive in the assay.
The following polypeptides tested positive in this assay: PRO337.
EXAMPLE 55: Rod Photoreceptor Cell Survival (Assay 56)
This assay shows that certain polypeptides of the invention act to enhance the
survival/proliferation
of rod photoreceptor cells and, therefore, are useful for the therapeutic
treatment of retinal disorders or injuries
including, for example, treating sight loss in mammals due to retinitis
pigmentosum, AMD, etc.
Sprague Dawley rat pups at 7 day postnatal (mixed population: glia and retinal
neuronal cell types)
are killed by decapitation following CO2 anesthesis and the eyes are removed
under sterile conditions. The
neural retina is dissected away form the pigment epithelium and other ocular
tissue and then dissociated into
a single cell suspension using 0.25% trypsin in Cat+, Mg2+-free PBS. The
retinas are incubated at 37 C for
7-10 minutes after which the trypsin is inactivated by adding 1 ml soybean
trypsin inhibitor. The cells are
plated at 100,000 cells per well in 96 well plates in DMEM/F12 supplemented
with N2. Cells for all
experiments are grown at 37 C in a water saturated atmosphere of 5 % CO2.
After 2-3 days in culture, cells
are fixed using 4% paraformaldehyde, and then stained using CellTracker Green
CMFDA. Rho 4D2 (ascites
or IgG 1:100), a monoclonal antibody directed towards the visual pigment
rhodopsin is used to detect rod
photoreceptor cells by indirect immunofluorescence. The results are calculated
as % survival: total number
of calcein - rhodopsin positive cells at 2-3 days in culture, divided by the
total number of rhodopsin positive
cells at time 2-3 days in culture. The total cells (fluorescent) are
quantified at 20x objective magnification
using a CCD camera and NIH image software for Macintosh. Fields in the well
are chosen at random.
The following polypeptides tested positive in this assay: PRO350.
EXAMPLE 56: Skin Vascular Permeability Assay (Assay 64)
This assay shows that certain polypeptides of the invention stimulate an
immune response and induce
inflammation by inducing mononuclear cell, eosinophil and PMN infiltration at
the site of injection of the
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animal. Compounds which stimulate an immune response are useful
therapeutically where stimulation of an
immune response is beneficial. This skin vascular permeability assay is
conducted as follows. Hairless guinea
pigs weighing 350 grams or more are anesthetized with ketamine (75-80 mg/Kg)
and 5 mg/Kg xylazine
intramuscularly (IM). A sample of purified polypeptide of the invention or a
conditioned media test sample
is injected intradermally onto the backs of the test animals with 100 l per
injection site. It is possible to have
about 10-30, preferably about 16-24, injection sites per animal. One l of
Evans blue dye (1% in physiologic
buffered saline) is injected intracardially. Blemishes at the injection sites
are then measured (mm diameter)
at 1 hr and 6 hr post injection. Animals were sacrificed at 6 hrs after
injection. Each skin injection site is
biopsied and fixed in formalin. The skins are then prepared for
histopathologic evaluation. Each site is
evaluated for inflammatory cell infiltration into the skin. Sites with visible
inflammatory cell inflammation are
scored as positive. Inflammatory cells may be neutrophilic, eosinophilic,
monocytic or lymphocytic. At least
a minimal perivascular infiltrate at the injection site is scored as positve,
no infiltrate at the site of injection is
scored as negative.
The following polypeptide tested positive in this assay: PRO301.
EXAMPLE 57: Induction of Endothelial Cell Apoptosis (Assay 73)
The ability of PRO polypeptides to induce apoptosis in endothelial cells was
tested in human venous
umbilical vein endothelial cells (HUVEC, Cell Systems). A positive test in the
assay is indicative of the
usefulness of the polypeptide in therapeutically treating tumors as well as
vascular disorders where inducing
apoptosis of endothelial cells would be beneficial.
*
The cells were plated on 96-well microtiter plates (Amersham Life Science,
cytostar-T scintillating
microplate, RPNQ160, sterile, tissue-culture treated, individually wrapped),
in 10 % serum (CSG-medium, Cell
Systems), at a density of 2 x 10' cells per well in a total volume of 100 l.
On day 2, test samples containing
the PRO polypeptide were added in triplicate at dilutions of 1%, 0.33% and
0.11 %. Wells without cells were
used as a blank and wells with cells only were used as a negative control. As
a positive control 1:3 serial
dilutions of 501AI of a 3x stock of staurosporine were used. The ability of
the PRO polypeptide to induce
apoptosis was determined by processing of the 96 well plates for detection of
Annexin V, a member of the
calcium and phospholipid binding proteins, to detect apoptosis.
0.2 ml Annexin V - Biotin stock solution (10014g/ml) was diluted in 4.6 ml 2 x
CaZ+ binding buffer
and 2.5 % BSA (1:25 dilution). 50 Fcl of the diluted Annexin V - Biotin
solution was added to each well (except
controls) to a final concentration of 1.0 g/ml. The samples were incubated
for 10-15 minutes with Annexin-
Biotin prior to direct addition of 35S-Streptavidin. IS-Streptavidin was
diluted in 2x CaZ+ Binding buffer, 2.5%
BSA and was added to all wells at a final concentration of 3 x 10' cpm/well.
The plates were then sealed,
centrifuged at 1000 rpm for 15 minutes and placed on orbital shaker for 2
hours. The analysis was performed
on a 1450 Microbeta Trilux (Wallac). Percent above background represents the
percentage amount of counts
per minute above the negative controls. Percents greater than or equal to 30%
above background are
considered positive.
The following PRO polypeptides tested positive in this assay: PR0301.
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EXAMPLE 58: Induction of c-fos in Cortical Neurons (Assay 83)
This assay is designed to determine whether PRO polypeptides show the ability
to induce c-fos in
cortical neurons. PRO polypeptides testing positive in this assay would be
expected to be useful for the
therapeutic treatment of nervous system disorders and injuries where neuronal
proliferation would be
beneficial.
Cortical neurons are dissociated and plated in growth medium at 10,000 cells
per well in 96 well
plates. After aproximately 2 cellular divisions, the cells are treated for 30
minutes with the PRO polypeptide
or nothing (negative control). The cells are then fixed for 5 minutes with
cold methanol and stained with an
antibody directed against phosphorylated CREB. mRNA levels are then calculated
using chemiluminescence.
A positive in the assay is any factor that results in at least a 2-fold
increase in c-fos message as compared to
the negative controls.
The following PRO polypeptides tested positive in this assay: PRO288.
EXAMPLE 59: Induction of Pancreatic 13-Cell Precursor Differentiation (Assay
89)
This assay shows that certain polypeptides of the invention act to induce
differentiation of pancreatic
a-cell precursor cells into mature pancreatic a-cells and, therefore, are
useful for treating various insulin
deficient states in mammals, including diabetes mellitus. The assay is
performed as follows. The assay uses
a primary culture of mouse fetal pancreatic cells and the primary readout is
an alteration in the expression of
markers that represent either (3-cell precursors or mature p-cells. Marker
expression is measured by real time
quantitative PCR (RTQ-PCR); wherein the marker being evaluated is insulin.
The pancreata are dissected from E14 embryos (CD1 mice). The pancreata are
then digested with
collagenase/dispase in F 12/DMEM at 37 C for 40 to 60 minutes
(collagenase/dispase, 1.37 mg/ml, Boehringer
Mannheim, #1097113). The digestion is then neutralized with an equal volume of
5% BSA and the cells are
washed once with RPMI1640. At day 1, the cells are seeded into 12-well tissue
culture plates (pre-coated with
laminin, 20 g/ml in PBS, Boehringer Mannheim, #124317). Cells from pancreata
from 1-2 embryos are
distributed per well. The culture medium for this primary cuture is 14F/1640.
At day 2, the media is removed
and the attached cells washed with RPMI/1640. Two mis of minimal media are
added in addition to the protein
to be tested. At day 4, the media is removed and RNA prepared from the cells
and marker expression analyzed
by real time quantitative RT-PCR. A protein is considered to be active in the
assay if it increases the expression
of the relevant (3-cell marker as compared to untreated controls.
14F/1640 is RPMI1640 (Gibco) plus the following:
group A 1:1000
group B 1:1000
recombinant human insulin 10 g/ml
Aprotinin (50 g/ml) 1:2000 (Boehringer manheim #981532)
Bovine pituitary extract (BPE) 6014g/ml
Gentamycin 100 ng/ml
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Group A : (in 10ni1 PBS)
Transferrin, 100mg (Sigma T2252)
Epidermal Growth Factor, 1001ig (BRL 100004)
Triiodothyronine,10141 of 5x10 M (Sigma T5516)
Ethanolamine, l001A1 of 10' M (Sigma E0135)
Phosphoethalamine, l001A1 of 10-' M (Sigma P0503)
Selenium, 41LI of 10' M (Aesar #12574)
Group C : (in lOml 100% ethanol)
Hydrocortisone, 21AI of 5X10-3 M (Sigma #H0135)
Progesterone, 100 l of 1X10'3 M (Sigma #P6149)
Forskolin, 500 1 of 20mM (Calbiochem #344270)
Minimal media:
RPMI 1640 plus transferrin (10 g/ml), insulin (1 g/ml), gentamycin (100
ng/ml), aprotinin (50
g/ml) and BPE (15 g/ml).
Defined media:
RPMI 1640 plus transferrin (10 g/ml), insulin (1 g/ml), gentamycin (100
ng/ml) and aprotinin (50
g/ml).
The following polypeptides were positive in this assay: PRO1361, PRO1308,
PRO1600 and
PR04356.
EXAMPLE 60: Pericyte c-Fos Induction (Assay 93)
This assay shows that certain polypeptides of the invention act to induce the
expression of c-fos in
pericyte cells and, therefore, are useful not only as diagnostic markers for
particular types of pericyte-
associated tumors but also for giving rise to antagonists which would be
expected to be useful for the
therapeutic treatment of pericyte-associated tumors. Induction of c-fos
expression in pericytes is also indicative
of the induction of angiogenesis and, as such, PRO polypeptides capable of
inducing the expression of c-fos
would be expected to be useful for the treatment of conditions where induced
angiogenesis would be beneficial
including, for example, wound healing, and the like. Specifically, on day 1,
pericytes are received from VEC
Technologies and all but 5 ml of media is removed from flask. On day 2, the
pericytes are trypsinized,
washed, spun and then plated onto 96 well plates. On day 7, the media is
removed and the pericytes are
treated with 100 l of PRO polypeptide test samples and controls (positive
control = DME+5% serum +/-
PDGF at 500 ng/ml; negative control = protein 32). Replicates are averaged and
SD/CV are determined.
Fold increase over Protein 32 (buffer control) value indicated by
chemiluminescence units (RLU) luminometer
reading verses frequency is plotted on a histogram. Two-fold above Protein 32
value is considered positive for
the assay. ASY Matrix: Growth media = low glucose DMEM = 20% FBS + 1X pen
strep + 1X fungizone.
Assay Media = low glucose DMEM +5% FBS.
The following polypeptides tested positive in this assay: PRO444 and PRO217.
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EXAMPLE 61: Detection of Polypeptides That Affect Glucose or FFA Uptake in
Skeletal Muscle (Assay 106)
This assay is designed to determine whether PRO polypeptides show the ability
to affect glucose or
FFA uptake by skeletal muscle cells. PRO polypeptides testing positive in this
assay would be expected to be
useful for the therapeutic treatment of disorders where either the stimulation
or inhibition of glucose uptake
by skeletal muscle would be beneficial including, for example, diabetes or
hyper- or hypo-insulinemia.
In a 96 well format, PRO polypeptides to be assayed are added to primary rat
differentiated skeletal
muscle, and allowed to incubate overnight. Then fresh media with the PRO
polypeptide and +/- insulin are
added to the wells. The sample media is then monitored to determine glucose
and FFA uptake by the skeletal
muscle cells. The insulin will stimulate glucose and FFA uptake by the
skeletal muscle, and insulin in media
without the PRO polypeptide is used as a positive control, and a limit for
scoring. As the PRO polypeptide
being tested may either stimulate or inhibit glucose and FFA uptake, results
are scored as positive in the assay
if greater than 1.5 times or less than 0.5 times the insulin control.
The following PRO polypeptides tested positive as either stimulators or
inhibitors of glucose and/or
FFA uptake in this assay: PRO196, PRO183, PRO185, PR0215, PRO288, PRO1361,
PRO1600, PRO4999,
PR07170, PR0533 and PRO187.
EXAMPLE 62: Fetal Hemoglobin Induction in an Erythroblastic Cell Line (Assay
107)
This assay is useful for screening PRO polypeptides for the ability to induce
the switch from adult
hemoglobin to fetal hemoglobin in an erythroblastic cell line. Molecules
testing positive in this assay are
expected to be useful for therapeutically treating various mammalian
hemoglobin-associated disorders such as
the various thalassemias. The assay is performed as follows. Erythroblastic
cells are plated in standard growth
medium at 1000 cells/well in a 96 well format. PRO polypeptides are added to
the growth medium at a
concentration of 0.2% or 2% and the cells are incubated for 5 days at 37 C. As
a positive control, cells are
treated with 100 M heroin and as a negative control, the cells are untreated.
After 5 days, cell lysates are
prepared and analyzed for the expression of gamma globin (a fetal marker). A
positive in the assay is a gamma
globin level at least 2-fold above the negative control.
The following polypeptides tested positive in this assay: PRO1419.
EXAMPLE 63: Chondrocyte Re-differentiation Assay (Assay 110)
This assay shows that certain polypeptides of the invention act to induce
redifferentiation of
chondrocytes, therefore, are expected to be useful for the treatment of
various bone and/or cartilage disorders
such as, for example, sports injuries and arthritis. The assay is performed as
follows. Porcine chondrocytes
are isolated by overnight collagenase digestion of articulary cartilage of
metacarpophalangeal joints of 4-6
month old female pigs. The isolated cells are then seeded at 25,000 cells/cm2
in Ham F-12 containing 10%
FBS and 4 g/ml gentamycin. The culture media is changed every third day and
the cells are then seeded in
96 well plates at 5,000 cells/well in 100 1 of the same media without serum
and 100 l of the test PRO
polypeptide, 5 nM staurosporin (positive control) or medium alone (negative
control) is added to give a final
volume of 200 l/well. After 5 days of incubation at 37 C, a picture of each
well is taken and the
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differentiation state of the chondrocytes is determined. A positive result in
the assay occurs when the
redifferentiation of the chondrocytes is determined to be more similar to the
positive control than the negative
control.
The following polypeptide tested positive in this assay: PRO215, PR0353,
PRO365, PRO1272,
PRO301 and PR0337.
EXAMPLE 64: Chondrocyte Proliferation Assay (Assay 111)
This assay is designed to determine whether PRO polypeptides of the present
invention show the
ability to induce the proliferation and/or redifferentiation of chondrocytes
in culture. PRO polypeptides testing
positive in this assay would be expected to be useful for the therapeutic
treatment of various bone and/or
cartilage disorders such as, for example, sports injuries and arthritis.
Porcine chondrocytes are isolated by overnight collagenase digestion of
articular cartilage of the
metacarpophalangeal joint of 4-6 month old female pigs. The isolated cells are
then seeded at 25,000 cells/cm2
in Ham F-12 containing 10% FBS and 4 g/ml gentamycin. The culture media is
changed every third day and
the cells are reseeded to 25,000 cells/cm2 every five days. On day 12, the
cells are seeded in 96 well plates
at 5,000 cells/well in 10011 of the same media without serum and 100 l of
either serum-free medium (negative
control), staurosporin (final concentration of 5 nM; positive control) or the
test PRO polypeptide are added
to give a final volume of 200 l/well. After 5 days at 37 C, 20 l of Alamar
blue is added to each well and
the plates are incubated for an additional 3 hours at 37 C. The fluorescence
is then measured in each well
(Ex:530 nm; Em: 590 nm). The fluorescence of a plate containing 200 l of the
serum-free medium is
measured to obtain the background. A positive result in the assay is obtained
when the fluorescence of the
PRO polypeptide treated sample is more like that of the positive control than
the negative control.
The following PRO polypeptides tested positive in this assay: PRO215, PRO217,
PRO248, PRO1361,
PRO1419, PR0533 and PRO265.
EXAMPLE 65: Mouse Mesengial Cell Inhibition Assay (Assay 114)
This assay is designed to determine whether PRO polypeptides of the present
invention show the
ability to inhibit the proliferation of mouse mesengial cells in culture. PRO
polypeptides testing positive in
this assay would be expected to be useful for the therapeutic treatment of
such diseases or conditions where
inhibition of mesengial cell proliferation would be beneficial such as, for
example, cystic renal dysplasia,
polycystic kidney disease, or other kidney disease assoiciated with abnormal
mesengial cell proliferation, renal
tumors, and the like.
On day 1, mouse mesengial cells are plated on a 96 well plate in growth medium
(a 3:1 mixture of
Dulbecco's modified Eagle's medium and Ham's F12 medium, 95 %; fetal bovine
serum, 5 %; supplemented
with 14mM HEPES) and then are allowed to grow overnight. On day 2, the PRO
polypeptide is diluted at 2
different concentrations (1 %, 0.1 %) in serum-free medium and is added to the
cells. The negative control is
growth medium without added PRO polypeptide. After the cells are allowed to
incubate for 48 hours, 20 l
of the Cell Titer 96 Aqueous one solution reagent (Promega) is added to each
well and the colormetric reaction
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is allowed to proceed for 2 hours. The absorbance (OD) is then measured at 490
rim. A positive in the assay
is an absorbance reading which is at least 10% above the negative control.
The following PRO polypeptides tested positive in this assay: PRO 1318.
EXAMPLE 66: Induction of Pancreatic (3-Cell Precursor Proliferation (Assay
117)
This assay shows that certain polypeptides of the invention act to induce an
increase in the number
of pancreatic a-cell precursor cells and, therefore, are useful for treating
various insulin deficient states in
mammals, including diabetes mellitus. The assay is performed as follows. The
assay uses a primary culture
of mouse fetal pancreatic cells and the primary readout is an alteration in
the expression of markers that
represent either R-cell precursors or mature n-cells. Marker expression is
measured by real time quantitative
PCR (RTQ-PCR); wherein the marker being evaluated is a transcription factor
called Pdxl.
The pancreata are dissected from E14 embryos (CD1 mice). The pancreata are
then digested with
collagenase/dispase in F 12/DMEM at 37 C for 40 to 60 minutes
(collagenase/dispase, 1.37 mg/ml, Boehringer
Mannheim, #1097113). The digestion is then neutralized with an equal volume of
5% BSA and the cells are
washed once with RPMI1640. At day 1, the cells are seeded into 12-well tissue
culture plates (pre-coated with
laminin, 20 g/ml in PBS, Boehringer Mannheim, #124317). Cells from pancreata
from 1-2 embryos are
distributed per well. The culture medium for this primary cuture is 14F/1640.
At day 2, the media is removed
and the attached cells washed with RPMI/ 1640. Two mis of minimal media are
added in addition to the protein
to be tested. At day 4, the media is removed and RNA prepared from the cells
and marker expression analyzed
by real time quantitative RT-PCR. A protein is considered to be active in the
assay if it increases the expression
of the relevant P-cell marker as compared to untreated controls.
14F/1640 is RPMI1640 (Gibco) plus the following:
group A 1:1000
group B 1:1000
recombinant human insulin 10 g/ml
Aprotinin (50 g/ml) 1:2000 (Boehringer manheim #981532)
Bovine pituitary extract (BPE) 60 g/ml
Gentamycin 100 ng/ml
Group A : (in lOml PBS)
Transferrin, 100mg (Sigma T2252)
Epidermal Growth Factor, 1001Ag (BRL 100004)
Triiodothyronine,10 d of 5x101 M (Sigma T5516)
Ethanolamine, 1001A1 of 10'M (Sigma E0135)
Phosphoethalamine, 100141 of 10-' M (Sigma P0503)
Selenium, 41LI of 10-' M (Aesar #12574)
Group C : (in 10ml 100% ethanol)
Hydrocortisone, 21LI of 5X10-3 M (Sigma #H0135)
Progesterone, 100141 of 1X103 M (Sigma #P6149)
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Forskolin, 5O01A1 of 20mM (Calbiochem #344270)
Minimal media:
RPMI 1640 plus transferrin (10 g/ml), insulin (1 g/ml), gentamycin (100
ng/ml), aprotinin (50
g/ml) and BPE (15 g/ml).
Defined media:
RPMI 1640 plus transferrin (10 g/ml), insulin (1 g/ml), gentamycin (100
ng/ml) and aprotinin (50
g/ml).
The following polypeptides tested positive in this assay: PR0183, PR0185,
PR0288.
EXAMPLE 67: In Vitro Antitumor Assay (Assay 161)
The antiproliferative activity of various PRO polypeptides was determined in
the investigational,
disease-oriented in vitro anti-cancer drug discovery assay of the National
Cancer Institute (NCI), using a
sulforhodamine B (SRB) dye binding assay essentially as described by Skehan et
al., J. Natl. Cancer Inst.
82:1107-1112 (1990). The 60 tumor cell lines employed in this study ("the NCI
panel"), as well as conditions
for their maintenance and culture in vitro have been described by Monks et
al., J. Natl. Cancer Inst. 83:757-
766 (1991). The purpose of this screen is to initially evaluate the cytotoxic
and/or cytostatic activity of the test
compounds against different types of tumors (Monks et al., supra; Boyd,
Cancer: Princ. Pract. Oncol. Update
3(10):1-12 [1989]).
Cells from approximately 60 human tumor cell lines were harvested with
trypsin/EDTA (Gibco),
washed once, resuspended in IMEM and their viability was determined. The cell
suspensions were added by
pipet (100 L volume) into separate 96-well microtiter plates. The cell
density for the 6-day incubation was
less than for the 2-day incubation to prevent overgrowth. Inoculates were
allowed a preincubation period of
24 hours at 37 C for stabilization. Dilutions at twice the intended test
concentration were added at time zero
in 100 L aliquots to the microtiter plate wells (1:2 dilution). Test
compounds were evaluated at five half-log
dilutions (1000 to 100,000-fold). Incubations took place for two days and six
days in a 5% CO2 atmosphere
and 100% humidity.
After incubation, the medium was removed and the cells were fixed in 0.1 ml of
10 % trichloroacetic
acid at 40 C. The plates were rinsed five times with deionized water, dried,
stained for 30 minutes with 0.1
ml of 0.4% sulforhodamine B dye (Sigma) dissolved in 1 % acetic acid, rinsed
four times with 1 % acetic acid
to remove unbound dye, dried, and the stain was extracted for five minutes
with 0.1 ml of 10 mM Tris base
[tris(hydroxymethyl)aminomethane], pH 10.5. The absorbance (OD) of
sulforhodamine B at 492 nm was
measured using a computer-interfaced, 96-well microtiter plate reader.
A test sample is considered positive if it shows at least 50% growth
inhibitory effect at one or more
concentrations. The positive results are shown in the following Table 7.
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Table 7
Compound Tumor Type Designation
PRO301 NSCL NCI-H322M
PRO301 Leukemia MOLT-4; SR
PRO301 NSCL A549/ATCC; EKVX;
PRO301 NSCL NCI-H23; NCI-460; NCI-H226
PRO301 Colon COLO 205; HCC-2998;
PRO301 Colon HCT-15; KM12; HT29;
PRO301 Colon HCT-116
PRO301 CNS SF-268; SF-295; SNB-19
PRO301 Melanoma MALME-3M; SK-MEL-2;
PRO301 Melanoma SK-MEL-5;UACC-257
PRO301 Melanoma UACC-62
PRO301 Ovarian IGROVI; OVCAR-4
PRO301 Ovarian OVCAR-5
PRO301 Ovarian OVCAR-8; SKOOV-3
PRO301 Renal ACHN;CAKI-1; TK-10; UO-31
PRO301 Prostate PC-3; DU-145
PRO301 Breast NCI/ADR-RES; HS 578T
PRO301 Breast MDA-MB-435;MDA-N; T-47D
PRO301 Melanoma M14
PRO301 Leukemia CCRF-CEM;HL-60(TB); K-562
PRO301 Leukemia RPMI-8226
PRO301 Melanoma LOX IMVI
PRO301 Renal 786-0; SN12C
PRO301 Breast MCF7; MDA-MB-231/ATCC
PRO301 Breast BT-549
PRO301 NSCL HOP-62
PRO301 CNS SF-539
PRO301 Ovarian OVCAR-3
The results of these assays demonstrate that the positive testing PRO
polypeptides are useful for
inhibiting neoplastic growth in a number of different tumor cell types and may
be used therapeutically therefor.
Antibodies against these PRO polypeptides are useful for affinity purification
of these useful polypeptides.
Nucleic acids encoding these PRO polypeptides are useful for the recombinant
preparation of these
polypeptides.
EXAMPLE 68: Gene Amplification in Tumors
This example shows that certain PRO polypeptide-encoding genes are amplified
in the genome of
certain human lung, colon and/or breast cancers and/or cell lines.
Amplification is associated with
overexpression of the gene product, indicating that the polypeptides are
useful targets for therapeutic
intervention in certain cancers such as colon, lung, breast and other cancers
and diagnostic determination of
the presence of those cancers. Therapeutic agents may take the form of
antagonists of the PRO polypeptide,
for example, murine-human chimeric, humanized or human antibodies against a
PRO polypeptide.
The starting material for the screen was genomic DNA isolated from a variety
cancers. The DNA
is quantitated precisely, e.g., fluorometrically. As a negative control, DNA
was isolated from the cells of ten
normal healthy individuals which was pooled and used as assay controls for the
gene copy in healthy
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individuals (not shown). The 5' nuclease assay (for example, TagManTM) and
real-time quantitative PCR (for
example, ABI Prizm 7700 Sequence Detection SystemTM (Perkin Elmer, Applied
Biosystems Division, Foster
City, CA)), were used to find genes potentially amplified in certain cancers.
The results were used to
determine whether the DNA encoding the PRO polypeptide is 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 individuals with tumors of the type and stage as indicated
in Table 8. An explanation of
the abbreviations used for the designation of the primary tumors listed in
Table 8 and the primary tumors and
cell lines referred to throughout this example are given below.
The results of the TagManTM are reported in delta (A) Ct units. One unit
corresponds to I 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 and a
TagManTM fluorescent probe derived
from the PRO polypeptide-encoding gene. Regions of the PRO polypeptide-
encoding gene which are most
likely to contain unique nucleic acid sequences and which are least likely to
have spliced out introns are
preferred for the primer and probe derivation, e.g., 3'-untranslated regions.
The sequences for the primers
and probes (forward, reverse and probe) used for the PRO polypeptide gene
amplification analysis were as
follows:
PRO533 (DNA49435-1219)
forward: 5'-GGGACGTGCTTCTACAAGAACAG-3' (SEQ ID NO: 140)
reverse: 5'-CAGGCTTACAATGTTATGATCAGACA-3' (SEQ ID NO: 141)
probe: 5'-TATTCAGAGTTTTCCATTGGCAGTGCCAGTT-3' (SEQ ID NO: 142)
PRO187 (DNA27864-1155)
forward: 5'-GGCCTTGCAGACAACCGT-3' (SEQ ID NO: 143)
reverse: 5'-CAGACTGAGGGAGATCCGAGA-3' (SEQ ID NO: 144)
probe: 5'-GCAGATTTTGAGGACAGCCACCTCCA-3' (SEQ ID NO: 145)
forward2: 5'-CATCAAGCGCCTCTACCA-3' (SEQ ID NO: 146)
reverse2: 5'-CACAAACTCGAACTGCTTCTG-3' (SEQ ID NO: 147)
probe2: 5'-CAGCTGCCCTTCCCCAACCA-3' (SEQ ID NO: 148)
PRO246 (DNA35639-1172)
forward: 5'-GGCAGAGACTTCCAGTCACTGA-3' (SEQ ID NO: 149)
reverse: 5'-GCCAAGGGTGGTGTTAGATAGG-3' (SEQ ID NO: 150)
probe: 5'-CAGGCCCCCTTGATCTGTACCCCA-3' (SEQ ID NO:151)
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 (forward [.f] and reverse [.r]) are used to generate
an amplicon typical of a PCR
reaction. A third oligonucleotide, or probe (.p), 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.
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During the amplification reaction, the Taq DNA polymerise 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.
*
The 5' nuclease procedure is run on a real-time quantitative PCR device such
as the ABI Prism
7700TM Sequence Detection. The system consists of a thermocycler, 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.
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 ACt 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.
Table 8 describes the stage, T stage and N stage of various primary tumors
which were used to screen
the PRO polypeptide compounds of the invention.
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Table 8
Primary Lung and Colon Tumor Profiles
Primary Tumor Stage Stage Other Stage Dukes Stage T Stage N Stage
Human lung tumor AdenoCa (SRCC724) [LT I] IIA Ti Ni
Human lung tumor SqCCa (SRCC725) [LTIa] IIB T3 NO
Human lung tumor AdenoCa (SRCC726) [LT2] IB T2 NO
Human lung tumor AdenoCa (SRCC727) [LT3] IIIA Ti N2
Human lung tumor AdenoCa (SRCC728) [LT4] IB T2 NO
Human lung tumor SqCCa (SRCC729) [LT6] IB T2 NO
Human lung tumor Aden/SqCCa (SRCC730) [LT7] IA Ti NO
Human lung tumor AdenoCa (SRCC73 1) [LT9] IB T2 NO
Human lung tumor SqCCa (SRCC732) [LT10] IIB T2 N1
Human lung tumor SqCCa (SRCC733) [LT1 1] IIA Ti N1
Human lung tumor AdenoCa (SRCC734) [LT12] IV T2 NO
Human lung tumor AdenoSqCCa (SRCC735)[LT13]IB T2 NO
Human lung tumor SqCCa (SRCC736) [LT15] IB T2 NO
Human lung tumor SqCCa (SRCC737) [LT16] IB T2 NO
Human lung tumor SqCCa (SRCC738) [LT17] IIB T2 Ni
Human lung tumor SqCCa (SRCC739) [LT18] IB T2 NO
Human lung tumor SqCCa (SRCC740) [LT19] IB T2 NO
Human lung tumor LCCa (SRCC741) [LT21] IIB T3 Ni
Human lung AdenoCa (SRCC81 1) [LT22] 1A Ti NO
Human colon AdenoCa (SRCC742) [CT2] M1 D pT4 NO
Human colon AdenoCa (SRCC743) [CT3] B pT3 NO
Human colon AdenoCa (SRCC744) [CT8] B T3 NO
Human colon AdenoCa (SRCC745) [CT10] A pT2 NO
Human colon AdenoCa (SRCC746) [CT12] MO, R1 B T3 NO
Human colon AdenoCa (SRCC747) [CT14] pMO, RO B pT3 pNO
Human colon AdenoCa (SRCC748) [CT15] Ml, R2 D T4 N2
Human colon AdenoCa (SRCC749) [CT16] pMO B pT3 pNO
Human colon AdenoCa (SRCC750) [CT17] C1 pT3 pNl
Human colon AdenoCa (SRCC751) [CT I] MO, R1 B pT3 NO
Human colon AdenoCa (SRCC752) [CT4] B pT3 MO
Human colon AdenoCa (SRCC753) [CT5] G2 C 1 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) [CT 11] B T3 NO
Human colon AdenoCa (SRCC758) [CT18] MO, RO B pT3 pNO
DNA Preparation:
DNA was prepared from cultured cell lines, primary tumors, normal human blood.
The isolation was
performed using purification kit, buffer set and protease and all from
Quiagen, according to the manufacturer's
instructions and the description below.
Cell culture lysis:
Cells were washed and trypsinized at a concentration of 7.5 x 108 per tip and
pelleted by centrifuging
at 1000 rpm for 5 minutes at 4 C, followed by washing again with 1/2 volume of
PBS recentrifugation. The
pellets were washed a third time, the suspended cells collected and washed 2x
with PBS. The cells were then
suspended into 10 ml PBS. Buffer Cl was equilibrated at 4 C. Qiagen protease #
19155 was diluted into 6.25
ml cold ddH2O to a final concentration of 20 mg/ml and equilibrated at 4 C. 10
ml of G2 Buffer was prepared
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by diluting Qiagen RNAse A stock (100 mg/ml) to a final concentration of 200
gg/ml.
Buffer C1 (10 ml, 4 C) and ddH2O (40 ml, 4 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 swinging bucket rotor at 2500 rpm at 4 C for 15 minutes. The
supernatant was discarded and the
nuclei were suspended with a vortex into 2 ml Buffer C1 (at 4 C) and 6 ml
ddH2O, followed by a second 4 C
centrifugation at 2500 rpm for 15 minutes. The nuclei were then resuspended
into the residual buffer using
200 ml 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 C for 60
minutes. The incubation and
centrifugation was repeated until the lysates were clear (e.g., incubating
additional 30-60 minutes, pelleting
at 3000 x g for 10 min., 4 C).
Solid human tumor sample preparation and lysis:
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 ddH2O to a final concentration of 20
mg/ml and stored at 4 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 nil G2 buffer for 60 seconds
using the large tip of the
polytron in a laminar-flow TC hood in order to avoid inhalation of aerosols,
and held at room temperature.
Between samples, the polytron was cleaned by spinning at 2 x 30 seconds each
in 2L ddH2O, followed by G2
buffer (50 ml). If tissue was still present on the generator tip, the
apparatus was disassembled and cleaned.
Quiagen protease (prepared as indicated above, 1.0 ml) was added, followed by
vortexing and
incubation at 50 C for 3 hours. The incubation and centrifugation was repeated
until the lysates were clear
(e.g., incubating additional 30-60 minutes, pelleting at 3000 x g for 10 min.,
4 C).
Human blood preparation and lysis:
Blood was drawn from healthy volunteers using standard infectious agent
protocols and citrated into
10 ml samples per tip. Quiagen protease was freshly prepared by dilution into
6.25 ml cold ddH2O to a final
concentration of 20 mg/ml and stored at 4 C. G2 buffer was prepared by
diluting RNAse A to a final
concentration of 200 Mg/ml from 100 mg/ml stock. The blood (10 ml) was placed
into a 50 ml conical tube
and 10 nil Cl buffer and 30 ml ddH2O (both previously equilibrated to 4 C)
were added, and the components
mixed by inverting and held on ice for 10 minutes. The nuclei were pelleted
with a Beckman swinging bucket
rotor at 2500 rpm, 4 C for 15 minutes and the supernatant discarded. With a
vortex, the nuclei were
suspended into 2 ml Cl buffer (4 C) and 6 ml ddH2O (4 C). Vortexing was
repeated until the pellet was white.
The nuclei were then suspended into the residual buffer using a 200 jl tip. G2
buffer (10 ml) were added to
the suspended nuclei while gently vortexing, followed by vigorous vortexing
for 30 seconds. Quiagen protease
was added (200 Al) and incubated at 50 C for 60 minutes. The incubation and
centrifugation was repeated until
the lysates were clear (e.g., incubating additional 30-60 minutes, pelleting
at 3000 x g for 10 min., 4 C).
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Purification of cleared lysates:
(1) Isolation of genomic DNA:
Genomic DNA was equilibrated (1 sample per maxi tip preparation) with 10 nil
QBT buffer. QF
elution buffer was equilibrated at 50 C. The samples were vortexed for 30
seconds, then loaded onto
equilibrated tips and drained by gravity. The tips were washed with 2 x 15 ml
QC buffer. The DNA was
eluted into 30 ml silanized, autoclaved 30 ml Corey tubes with 15 ml QF buffer
(50 C). Isopropanol (10.5
nil) was added to each sample, the tubes covered with parafin and mixed by
repeated inversion until the DNA
precipitated. Samples were pelleted by centrifugation in the SS-34 rotor at
15,000 rpm for 10 minutes at 4 C.
The pellet location was marked, the supernatant discarded, and 10 nil 70%
ethanol (4 C) was added. Samples
were pelleted again by centrifugation on the SS-34 rotor at 10,000 rpm for 10
minutes at 4 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 C, taking care not to overdry the samples.
After drying, the pellets were dissolved into 1.0 nil TE (pH 8.5) and placed
at 50 C for 1-2 hours.
Samples were held overnight at 4 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 5x in order to shear the
DNA. Samples were then placed at 50 C for 1-2 hours.
(2) Ouantitation of genomic DNA and preparation for gene amplification assay:
The DNA levels in each tube were quantified by standard A2,O, A280
spectrophotometry on a 1:20
dilution (5 pl DNA + 95 ml ddH2O) using the 0.1 ml quartz cuvetts in the
Beckman DU640
spectrophotometer. A2,0/A2eo ratios were in the range of 1.8-1.9. Each DNA
samples was then diluted further
to approximately 200 ng/ml in TE (pH 8.5). If the original material was highly
concentrated (about 700
ng/ d), the material was placed at 50 C for several hours until resuspended.
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
fluorometer to warm-up for about 15 minutes. The Hoechst dye working solution
(#H33258, 10 ,u1, prepared
within 12 hours of use) was diluted into 100 ml 1 x THE buffer. A 2 ml cuvette
was filled with the
fluorometer solution, placed into the machine, and the machine was zeroed.
pGEM 3Zf(+) (2 ul, lot
#360851026) was added to 2 ml of fluorometer solution and calibrated at 200
units. An additional 2 k1 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.
The fluorometricly determined concentration was then used to dilute each
sample to 10 ng/ l in
ddH2O. This was done simultaneously on all template samples for a single
TaqMan plate assay, and with
enough material to run 500-1000 assays. The samples were tested in triplicate
with TagmanTM primers and
probe 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 C. Aliquots which
were subsequently to be used in the gene amplification assay were stored at 4
C. Each 1 ml aliquot is enough
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for 8-9 plates or 64 tests.
Gene amplification assay:
The PRO polypeptide compounds of the invention were screened in the following
primary tumors and
the resulting ACt values greater than or equal to 1.0 are reported in Table 9
below.
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Table 9
ACt values in lung and colon primary tumors and cell line models
Primary Tumors PRO187 PR0533 PRO246
or Cell Lines
LT7 1.04
LT13 2.74 1.63
2.98 1.68
2.44
LT3 1.06
LT12 2.70 2.47
2.90 1.74
2.27
LT30 1.67
LT21 1.50
LT-la 1.02
LT10 1.07
LT 11 1.09 3.43
1.41
LT15 3.75 2.11
3.92 1.56
3.49
LT16 2.10 1.66
LT17 1.32 2.68
1.69
LT19 4.05 1.67 1.91
3.99 1.68
1.16
CT2 3.56
CT8 1.01
CT10 1.81
CT14 1.82
CT1 1.24
1.34
CT5 2.96 1.33
2.99 2.39
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Table 9 (cont')
ACt values in lunz and colon primary tumors and cell line models
Primary Tumors PRO187 PR0533 PR0246
or Cell Lines
CT6 1.10
CT7 1.40
CT9 1.39 1.09
CT11 2.22 1.48
2.26 1.12
Because amplification of the various DNAs described above occurs in various
cancerous tumors and
tumor cell lines derived from various human tissues, these molecules likely
play a significant role in tumor
formation and/or growth. As a result, amplification and/or enhanced expression
of these molecules can serve
as a diagnostic for detecting the presence of tumor in an individual and
antagonists (e.g., antibodies) directed
against the proteins encoded by the above described DNA molecules would be
expected to have utility in cancer
therapy.
EXAMPLE 69: Gene Expression in Bovine Pericytes (Assay 105)
This assay is designed to identify gene expression patterns in pericytes
induced by the hits in assay
93 described above. Bovine pericytes are plated on 60mm culture dishes in
growth media for! week. On day
1, various PRO polypeptides are diluted (1 %) and incubated with the pericytes
for 1, 4 and 24 hr. timepoints.
The cells are harvested and the RNA isolated using TRI-Reagent following the
included instructions. The
RNA is then quantified by reading the 260/280 OD using a spectrophotometer.
The gene expression analysis
is done by TaqMan reactions using Perkin Elmer reagents and specially designed
bovine probes and primers.
Expression of the following genes is analyzed: GAPDH, beta-integrin,
connective tissue growth factor
(CTGF), ICAM-1, monocyte chemoattractant protein-1 (MCP-1), osteopontin,
transforming growth factor-beta
(TGF-beta), TGF-beta receptor, tissue inhibitor of metalloproteinase (TIMP),
tissue factor (TF), VEGF-a,
thrombospondin, VEGF-P, angiopoeitin-2, and collagenase. Replicates are then
averaged and the SD
determined. The gene expression levels are then normalized to GAPDH. These are
then normalized to the
expression levels obtained with a protein (PIN32) which does not significantly
induce gene expression in bovine
pericytes when compared to untreated controls. Any PRO polypeptide that gives
a gene expression level 2-fold
or higher over the PIN32 control is considered a positive hit.
The following PRO polypeptides tested positive in this assay: PR0217.
EXAMPLE 70: Cvtokine Release Assay (Assay 120)
This assay is designed to determine whether PRO polypeptides of the present
invention are capable
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of inducing the release of cytokines from peripheral blood mononuclear cells
(PBMCs). PRO polypeptides
capable of inducing the release of cytokines from PBMCs are useful from the
treatment of conditions which
would benefit from enhanced cytokine release and will be readily evident to
those of ordinary skill in the art.
Specifically, 1x106 cells/ml of peripheral blood mononuclear cells (PBMC) are
cultured with I% of a PRO
polypeptide for 3 days in complete RPMI media. The supernatant is then
harvested and tested for increased
concentrations of various cytokines by ELISA as compared to a human IgG
treated control. A positive in the
assay is a 10-fold or greater increase in cytokine concentration in the PRO
polypeptide treated sample as
compared to the human IgG treated control.
The following polypeptides tested positive in this assay: PRO9940.
EXAMPLE 71: Identification of PRO Polypeptides That Activate Pericytes (Assay
125)
This assay shows that certain polypeptides of the invention act to activate
proliferation of pericyte cells
and, therefore, are useful not only as diagnostic markers for particular types
of pericyte-associated tumors but
also for giving rise to antagonists which would be expected to be useful for
the therapeutic treatment of
pericyte-associated tumors. Activation of pericyte proliferation also
correlates with the induction of
angiogenesis and, as such, PRO polypeptides capable of inducing pericyte
proliferation would be expected to
be useful for the treatment of conditions where induced angiogenesis would be
beneficial including, for
example, wound healing, and the like. Specifically, on day 1, pericytes are
received from VEC Technologies,
and all but 5 ml media is removed from the flask. On day 2, the pericytes are
trypsinized, washed, spun and
plated on 96 well plates. On day 7, the media is removed and the pericytes are
treated with 1001d of either
the specific PRO polypeptide or control treatments (positive control =
DME+5%+/- PDGF @ 500ng/ l;
negative control=PIN32, a polypeptide determined to have no significant effect
on pericyte proliferation).
C-fos and GAPDH gene expression levels are then determined and the replicates
are averaged and the SD is
determined. The c-fos values are normalized to GAPDH and the results are
expressed as fold increase over
PIN2. Anything providing at least a 2-fold or higher response as compared to
the negative control is
considered positive for the assay.
The following polypeptides tested positive in this assay: PRO217.
EXAMPLE 72: Identification of Receptor/Ligand Interactions
In this assay, various PRO polypeptides are tested for ability to bind to a
panel of potential receptor
or ligand molecules for the purpose of identifying receptor/ligand
interactions. The identification of a ligand
for a known receptor, a receptor for a known ligand or a novel receptor/ligand
pair is useful for a variety of
indications including, for example, targeting bioactive molecules (linked to
the ligand or receptor) to a cell
known to express the receptor or ligand, use of the receptor or ligand as a
reagent to detect the presence of the
ligand or receptor in a composition suspected of containing the same, wherein
the composition may comprise
cells suspected of expressing the ligand or receptor, modulating the growth of
or another biological or
immunological activity of a cell known to express or respond to the receptor
or ligand, modulating the immune
response of cells or toward cells that express the receptor or ligand,
allowing the preparaion of agonists,
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WO 00/77037 PCT/US00/14042
antagonists and/or antibodies directed against the receptor or ligand which
will modulate the growth of or a
biological or immunological activity of a cell expressing the receptor or
ligand, and various other indications
which will be readily apparent to the ordinarily skilled artisan.
The assay is performed as follows. A PRO polypeptide of the present invention
suspected of being
a ligand for a receptor is expressed as a fusion protein containing the Fc
domain of human IgG (an
immunoadhesin). Receptor-ligand binding is detected by allowing interaction of
the immunoadhesin
polypeptide with cells (e.g. Cos cells) expressing candidate PRO polypeptide
receptors and visualization of
bound immunoadhesin with fluorescent reagents directed toward the Fc fusion
domain and examination by
microscope. Cells expressing candidate receptors are produced by transient
transfection, in parallel, of defined
subsets of a library of cDNA expression vectors encoding PRO polypeptides that
may function as receptor
molecules. Cells are then incubated for 1 hour in the presence of the PRO
polypeptide immunoadhesin being
tested for possible receptor binding. The cells are then washed and fixed with
paraformaldehyde. The cells
are then incubated with fluorescent conjugated antibody directed against the
Fc portion of the PRO polypeptide
immunoadhesin (e.g. FITC conjugated goat anti-human-Fc antibody). The cells
are then washed again and
examined by microscope. A positive interaction is judged by the presence of
fluorescent labeling of cells
transfected with cDNA encoding a particular PRO polypeptide receptor or pool
of receptors and an absence
of similar fluorescent labeling of similarly prepared cells that have been
transfected with other cDNA or pools
of cDNA. If a defined pool of cDNA expression vectors is judged to be positive
for interaction with a PRO
polypeptide immunoadhesin, the individual cDNA species that comprise the pool
are tested individually (the
pool is "broken down") to determine the specific cDNA that encodes a receptor
able to interact with the PRO
polypeptide immunoadhesin.
In another embodiment of this assay, an epitope-tagged potential ligand PRO
polypeptide (e.g. 8
histidine "His" tag) is allowed to interact with a panel of potential receptor
PRO polypeptide molecules that
have been expressed as fusions with the Fc domain of human IgG
(immunoadhesins). Following a 1 hour
co-incubation with the epitope tagged PRO polypeptide, the candidate receptors
are each immunoprecipitated
with protein A beads and the beads are washed. Potential ligand interaction is
determined by western blot
analysis of the immunoprecipitated complexes with antibody directed towards
the epitope tag. An interaction
is judged to occur if a band of the anticipated molecular weight of the
epitope tagged protein is observed in the
western blot analysis with a candidate receptor, but is not observed to occur
with the other members of the
panel of potential receptors.
Using these assays, the following receptor/ligand interactions have been
herein identified:
(1) PR0533 binds to the fibroblast growth factor receptor-4 (FGFR-4; see
Partanen et al., EMBO J.
10(6):1347-1354 (1991)).
(2) PRO301 binds to itself and, therefore, functions as an adhesion molecule.
(3) PRO187 binds to the fibroblast growth factor receptor-3 (FGFR-3; see
Keegan et al., Proc. Natl.
Acad. Sci. USA 88:1095-1099 (1991)) with high affinity and with lower affinity
to to FGFR-1, 2 and
4 (see Isacchi et al., Nuc. Acids Res. 18(7):1906 (1990), Dionne et al., EMBO
J. 9(9):2685-2692
(1990) and Partanen et al., EMBO J. 10(6):1347-1354 (1991), respectively).
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(4) PR0337 binds to PRO6004.
(5) PRO1411 binds to PR04356.
(6) PRO10096 binds to PR02630.
(7) PR0246 binds to itself and, therefore, functions as an adhesion molecule.
(8) PR06307 binds to PR0265.
(9) PR06003 binds to PRO941.
Deposit of Material
The following materials have been deposited with the American Type Culture
Collection, 10801
University Blvd., Manassas, VA 20110-2209, USA (ATCC):
Table 10
Material ATCC Dep. No. Deposit Date
DNA22779-1130 209280 September 18, 1997
DNA26846-1397 203406 October 27, 1998
DNA32279-1131 209259 September 16, 1997
DNA32288-1132 209261 September 16, 1997
DNA33094-1131 209256 September 16, 1997
DNA33785-1143 209417 October 28, 1997
DNA35663-1129 209201 June 18, 1997
DNA46777-1253 209619 February 5, 1998
DNA60783-1611 203130 August 18, 1998
DNA62306-1570 203254 September 9, 1998
DNA62880-1513 203097 August 4, 1998
DNA64896-1539 203238 September 9, 1998
DNA71290-1630 203275 September 22, 1998
DNA96031-2664 PTA-237 June 15, 1999
DNA108722-2743 PTA-552 August 17, 1999
DNA35674-1142 209416 October 28, 1997
DNA41234 209618 February 5, 1998
DNA77503-1686 203362 October 20, 1998
DNA49435-1219 209480 November 21, 1997
DNA40628-1216 209432 November 7, 1997
DNA27864-1155 209375 October 16, 1997
DNA43316-1237 209487 November 21, 1997
DNA59212-1627 203245 September 9, 1998
DNA86576-2595 203868 March 23, 1999
DNA35639-1172 209396 October 17, 1997
DNA36350-1158 209378 October 16, 1997
DNA53906-1368 209747 April 7, 1998
DNA125185-2806 PTA-1031 December 7, 1999
DNA83568-2692 PTA-386 July 20, 1999
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 a viable culture of
the deposit 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
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WO 00/77037 PCT/US00/14042
availability of the progeny of the culture of the deposit 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 122 and the Commissioner's rules
pursuant thereto (including 37 CFR
1.14 with particular reference to 886 OG 638).
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
material 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.
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 construct 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
deposit of material herein does 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.
159
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Sequence Listing
<110> Genentech, Inc.
Avi J. Ashkenazi
Kevin P. Baker
David A. Botstein
Luc Desnoyers
Dan L. Eaton
Napoleone Ferrara
Sherman Fong
Wei-Qiang Gao
Hanspeter Gerber
Mary E. Gerritsen
Audrey Goddard
Paul J. Godowski
Austin L. Gurney
Ivar J. Kljavin
Jennie P. Mather
Mary A. Napier
James Pan
Nicholas F. Paoni
Margaret Ann Roy
Timothy A. Stewart
Daniel Tumas
Colin K. Watanabe
P. Mickey Williams
William I. Wood
Zemin Zhang
<120> SECRETED AND TRANSMEMBRANE POLYPEPTIDES AND NUCLEIC
ACIDS ENCODING THE SAME
<130> P3130RlPCT
<140> PCT/US00/14042
<141> 2000-05-22
<150> US 60/139,695
<151> 1999-06-15
<150> US 60/145,070
<151> 1999-07-20
<150> US 60/145,698
<151> 1999-07-26
<150> US 60/149,396
<151> 1999-08-17
<150> PCT/US99/20111
<151> 1999-09-01
<150> PCT/US99/20594
<151> 1999-09-08
<150> PCT/US99/21090
<151> 1999-09-15
<150> PCT/US99/21547
<151> 1999-09-15
<150> PCT/US99/28313
<151> 1999-11-30
1
CA 02372511 2002-05-14
<150> PCT/US99/28301
<151> 1999-12-01
<150> PCT/US99/28565
<151> 1999-12-02
<150> US 60/169,495
<151> 1999-12-07
<150> PCT/USOO/00219
<151> 2000-01-05
<150> PCT/USOO/04341
<151> 2000-02-18
<150> PCT/USOO/04342
<151> 2000-02-18
<150> PCT/USOO/04414
<151> 2000-02-22
<150> PCT/USOO/05601
<151> 2000-03-01
<150> PCT/USOO/05841
<151> 2000-03-02
<150> PCT/USOO/07377
<151> 2000-03-20
<150> PCT/US00/08439
<151> 2000-03-30
<150> PCT/USOO/13358
<151> 2000-05-15
<150> PCT/USOO/13705
<151> 2000-05-17
<160> 151
<210> 1
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 1
tgtaaaacga cggccagtta aatagacctg caattattaa tct 43
<210> 2
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 2
caggaaacag ctatgaccac ctgcacacct gcaaatccat t 41
2
CA 02372511 2002-05-14
<210> 3
<211> 2290
<212> DNA
<213> Homo Sapien
<400> 3
ggctgagggg aggcccggag cctttctggg gcctggggga tcctcttgca 50
ctggtgggtg gagagaagcg cctgcagcca accagggtca ggctgtgctc 100
acagtttcct ctggcggcat gtaaaggctc cacaaaggag ttgggagttc 150
aaatgaggct gctgcggacg gcctgaggat ggaccccaag ccctggacct 200
gccgagcgtg gcactgaggc agcggctgac gctactgtga gggaaagaag 250
gttgtgagca gccccgcagg agccctggcc agccctggcc ccagcctctg 300
ccggagccct ctgtggaggc agagccagtg gagcccagtg aggcagggct 350
gcttggcagc caccggcctg caactcagga acccctccag aggccatgga 400
caggctgccc ctctgacggc cagggtgaag catgtgagga gccgccccgg 450
agccaagcag gagggaagag gctttcatag attctattca caaagaataa 500
ccaccatttt gcaaggacca tgaggccact gtgcgtgaca tgctggtggc 550
tcggactgct ggctgccatg ggagctgttg caggccagga ggacggtttt 600
gagggcactg aggagggctc gccaagagag ttcatttacc taaacaggta 650
caagcgggcg ggcgagtccc aggacaagtg cacctacacc ttcattgtgc 700
cccagcagcg ggtcacgggt gccatctgcg tcaactccaa ggagcctgag 750
gtgcttctgg agaaccgagt gcataagcag gagctagagc tgctcaacaa 800
tgagctgctc aagcagaagc ggcagatcga gacgctgcag cagctggtgg 850
aggtggacgg cggcattgtg agcgaggtga agctgctgcg caaggagagc 900
cgcaacatga actcgcgggt cacgcagctc tacatgcagc tcctgcacga 950
gatcatccgc aagcgggaca acgcgttgga gctctcccag ctggagaaca 1000
ggatcctgaa ccagacagcc gacatgctgc agctggccag caagtacaag 1050
gacctggagc acaagtacca gcacctggcc acactggccc acaaccaatc 1100
agagatcatc gcgcagcttg aggagcactg ccagagggtg ccctcggcca 1150
ggcccgtccc ccagccaccc cccgctgccc cgccccgggt ctaccaacca 1200
cccacctaca accgcatcat caaccagatc tctaccaacg agatccagag 1250
tgaccagaac ctgaaggtgc tgccaccccc tctgcccact atgcccactc 1300
tcaccagcct cccatcttcc accgacaagc cgtcgggccc atggagagac 1350
tgcctgcagg ccctggagga tggccacgac accagctcca tctacctggt 1400
gaagccggag aacaccaacc gcctcatgca ggtgtggtgc gaccagagac 1450
3
CA 02372511 2002-05-14
acgaccccgg gggctggacc gtcatccaga gacgcctgga tggctctgtt 1500
aacttcttca ggaactggga gacgtacaag caagggtttg ggaacattga 1550
cggcgaatac tggctgggcc tggagaacat ttactggctg acgaaccaag 1600
gcaactacaa actcctggtg accatggagg actggtccgg ccgcaaagtc 1650
tttgcagaat acgccagttt ccgcctggaa cctgagagcg agtattataa 1700
gctgcggctg gggcgctacc atggcaatgc gggtgactcc tttacatggc 1750
acaacggcaa gcagttcacc accctggaca gagatcatga tgtctacaca 1800
ggaaactgtg cccactacca gaagggaggc tggtgataaa acgcctgtgc 1850
ccactccaac ctcaacgggg tctggtaccg cgggggccat taccggagcc 1900
gctaccagga cggagtctac tgggctgagt tccgaggagg ctcttactca 1950
ctcaagaaag tggtgatgat gatccgaccg aaccccaaca ccttccacta 2000
agccagctcc ccctcctgac ctctcgtggc cattgccagg agcccaccct 2050
ggtcacgctg gccacagcac aaagaacaac tcctcaccag ttcatcctga 2100
ggctgggagg accgggatgc tggattctgt tttccgaagt cactgcagcg 2150
gatgatggaa ctgaatcgat acggtgtttt ctgtccctcc tactttcctt 2200
cacaccagac agcccatcat gtctccagga caggacagga ctccagacaa 2250
ctctttcttt aaataaatta agtctctaca ataaaaaaaa 2290
<210> 4
<211> 493
<212> PRT
<213> Homo Sapien
<400> 4
Met Arg Pro Leu Cys Val Thr Cys Trp Trp Leu Gly Leu Leu Ala
1 5 10 15
Ala Met Gly Ala Val Ala Gly Gln Glu Asp Gly Phe Glu Gly Thr
20 25 30
Glu Glu Gly Ser Pro Arg Glu Phe Ile Tyr Leu Asn Arg Tyr Lys
35 40 45
Arg Ala Gly Glu Ser Gln Asp Lys Cys Thr Tyr Thr Phe Ile Val
50 55 60
Pro Gin Gln Arg Val Thr Gly Ala Ile Cys Val Asn Ser Lys Glu
65 70 75
Pro Glu Val Leu Leu Glu Asn Arg Val His Lys Gln Glu Leu Glu
80 85 90
Leu Leu Asn Asn Glu Leu Leu Lys Gln Lys Arg Gln Ile Glu Thr
95 100 105
Leu Gln Gln Leu Val Glu Val Asp Gly Gly Ile Val Ser Glu Val
110 115 120
4
CA 02372511 2002-05-14
Lys Leu Leu Arg Lys Glu Ser Arg Asn Met Asn Ser Arg Val Thr
125 130 135
Gln Leu Tyr Met Gln Leu Leu His Glu Ile Ile Arg Lys Arg Asp
140 145 150
Asn Ala Leu Glu Leu Ser Gln Leu Glu Asn Arg Ile Leu Asn Gln
155 160 165
Thr Ala Asp Met Leu Gln Leu Ala Ser Lys Tyr Lys Asp Leu Glu
170 175 180
His Lys Tyr Gln His Leu Ala Thr Leu Ala His Asn Gln Ser Glu
185 190 195
Ile Ile Ala Gln Leu Glu Glu His Cys Gln Arg Val Pro Ser Ala
200 205 210
Arg Pro Val Pro Gln Pro Pro Pro Ala Ala Pro Pro Arg Val Tyr
215 220 225
Gln Pro Pro Thr Tyr Asn Arg Ile Ile Asn Gln Ile Ser Thr Asn
230 235 240
Glu Ile Gln Ser Asp Gln Asn Leu Lys Val Leu Pro Pro Pro Leu
245 250 255
Pro Thr Met Pro Thr Leu Thr Ser Leu Pro Ser Ser Thr Asp Lys
260 265 270
Pro Ser Gly Pro Trp Arg Asp Cys Leu Gln Ala Leu Glu Asp Gly
275 280 285
His Asp Thr Ser Ser Ile Tyr Leu Val Lys Pro Glu Asn Thr Asn
290 295 300
Arg Leu Met Gln Val Trp Cys Asp Gln Arg His Asp Pro Gly Gly
305 310 315
Trp Thr Val Ile Gln Arg Arg Leu Asp Gly Ser Val Asn Phe Phe
320 325 330
Arg Asn Trp Glu Thr Tyr Lys Gln Gly Phe Gly Asn Ile Asp Gly
335 340 345
Glu Tyr Trp Leu Gly Leu Glu Asn Ile Tyr Trp Leu Thr Asn Gln
350 355 360
Gly Asn Tyr Lys Leu Leu Val Thr Met Glu Asp Trp Ser Gly Arg
365 370 375
Lys Val Phe Ala Glu Tyr Ala Ser Phe Arg Leu Glu Pro Glu Ser
380 385 390
Glu Tyr Tyr Lys Leu Arg Leu Gly Arg Tyr His Gly Asn Ala Gly
395 400 405
Asp Ser Phe Thr Trp His Asn Gly Lys Gln Phe Thr Thr Leu Asp
410 415 420
Arg Asp His Asp Val Tyr Thr Gly Asn Cys Ala His Tyr Gln Lys
425 430 435
CA 02372511 2002-05-14
Gly Gly Trp Trp Tyr Asn Ala Cys Ala His Ser Asn Leu Asn Gly
440 445 450
Val Trp Tyr Arg Gly Gly His Tyr Arg Ser Arg Tyr Gln Asp Gly
455 460 465
Val Tyr Trp Ala Glu Phe Arg Gly Gly Ser Tyr Ser Leu Lys Lys
470 475 480
Val Val Met Met Ile Arg Pro Asn Pro Asn Thr Phe His
485 490
<210> 5
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 5
gctgacgaac caaggcaact acaaactcct ggt 33
<210> 6
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 6
tgcggccgga ccagtcctcc atggtcacca ggagtttgta g 41
<210> 7
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 7
ggtggtgaac tgcttgccgt tgtgccatgt aaa 33
<210> 8
<211> 1218
<212> DNA
<213> Homo Sapien
<400> 8
cccacgcgtc cggcgccgtg gcctcgcgtc catctttgcc gttctctcgg 50
acctgtcaca aaggagtcgc gccgccgccg ccgccccctc cctccggtgg 100
gcccgggagg tagagaaagt cagtgccaca gcccgaccgc gctgctctga 150
gccctgggca cgcggaacgg gagggagtct gagggttggg gacgtctgtg 200
agggagggga acagccgctc gagcctgggg cgggcggacc ggactggggc 250
cggggtaggc tctggaaagg gcccgggaga gaggtggcgt: tggtcagaac 300
6
CA 02372511 2002-05-14
ctgagaaaca gccgagaggt tttccaccga ggcccgcgct tgagggatct 350
gaagaggttc ctagaagagg gtgttccctc tttcgggggt cctcaccaga 400
agaggttctt gggggtcgcc cttctgagga ggctgcggct aacagggccc 450
agaactgcca ttggatgtcc agaatcccct gtagttgata atgttgggaa 500
taagctctgc aactttcttt ggcattcagt tgttaaaaac aaataggatg 550
caaattcctc aactccaggt tatgaaaaca gtacttggaa aactgaaaac 600
tacctaaatg atcgtctttg gttgggccgt gttcttagcg agcagaagcc 650
ttggccaggg tctgttgttg actctcgaag agcacatagc ccacttccta 700
gggactggag gtgccgctac taccatgggt aattcctgta tctgccgaga 750
tgacagtgga acagatgaca gtgttgacac ccaacagcaa caggccgaga 800
acagtgcagt acccactgct gacacaagga gccaaccacg ggaccctgtt 850
cggccaccaa ggaggggccg aggacctcat gagccaagga gaaagaaaca 900
aaatgtggat gggctagtgt tggacacact ggcagtaata cggactcttg 950
tagataagta agtatctgac tcacggtcac ctccagtgga atgaaaagtg 1000
ttctgcccgg aaccatgact ttaggactcc ttcagttcct ttaggacata 1050
ctcgccaagc cttgtgctca cagggcaaag gagaatattt taatgctccg 1100
ctgatggcag agtaaatgat aagatttgat gtttttgctt gctgtcatct 1150
actttgtctg gaaatgtcta aatgtttctg tagcagaaaa cacgataaag 1200
ctatgatctt tattagag 1218
<210> 9
<211> 117
<212> PRT
<213> Homo Sapien
<400> 9
Met Ile Val Phe Gly Trp Ala Val Phe Leu Ala Ser Arg Ser Leu
1 5 10 15
Gly Gln Gly Leu Leu Leu Thr Leu Glu Glu His Ile Ala His Phe
20 25 30
Leu Gly Thr Gly Gly Ala Ala Thr Thr Met Gly Asn Ser Cys Ile
35 40 45
Cys Arg Asp Asp Ser Gly Thr Asp Asp Ser Val Asp Thr Gln Gln
50 55 60
Gln Gln Ala Glu Asn Ser Ala Val Pro Thr Ala Asp Thr Arg Ser
65 70 75
Gln Pro Arg Asp Pro Val Arg Pro Pro Arg Arg Gly Arg Gly Pro
80 85 90
His Glu Pro Arg Arg Lys Lys Gln Asn Val Asp Gly Leu Val Leu
7
CA 02372511 2002-05-14
95 100 105
Asp Thr Leu Ala Val Ile Arg Thr Leu Val Asp Lys
110 115
<210> 10
<211> 1231
<212> DNA
<213> Homo Sapien
<400> 10
cccacgcgtc cgcgcagtcg cgcagttctg cctccgcctg ccagtctcgc 50
ccgcgatccc ggcccggggc tgtggcgtcg actccgaccc aggcagccag 100
cagcccgcgc gggagccgga ccgccgccgg aggagctcgg acggcatgct 150
gagccccctc ctttgctgaa gcccgagtgc ggagaagccc gggcaaacgc 200
aggctaagga gaccaaagcg gcgaagtcgc gagacagcgg acaagcagcg 250
gaggagaagg aggaggaggc gaacccagag aggggcagca aaagaagcgg 300
tggtggtggg cgtcgtggcc atggcggcgg ctatcgccag ctcgctcatc 350
cgtcagaaga ggcaagcccg cgagcgcgag aaatccaacg cctgcaagtg 400
tgtcagcagc cccagcaaag gcaagaccag ctgcgacaaa aacaagttaa 450
atgtcttttc ccgggtcaaa ctcttcggct ccaagaagag gcgcagaaga 500
agaccagagc ctcagcttaa gggtatagtt accaagctat acagccgaca 550
aggctaccac ttgcagctgc aggcggatgg aaccattgat ggcaccaaag 600
atgaggacag cacttacact ctgtttaacc tcatccctgt gggtctgcga 650
gtggtggcta tccaaggagt tcaaaccaag ctgtacttgg caatgaacag 700
tgagggatac ttgtacacct cggaactttt cacacctgag tgcaaattca 750
aagaatcagt gtttgaaaat tattatgtga catattcatc aatgatatac 800
cgtcagcagc agtcaggccg agggtggtat ctgggtctga acaaagaagg 850
agagatcatg aaaggcaacc atgtgaagaa gaacaagcct gcagctcatt 900
ttctgcctaa accactgaaa gtggccatgt acaaggagcc atcactgcac 950
gatctcacgg agttctcccg atctggaagc gggaccccaa ccaagagcag 1000
aagtgtctct ggcgtgctga acggaggcaa atccatgagc cacaatgaat 1050
caacgtagcc agtgagggca aaagaagggc tctgtaacag aaccttacct 1100
ccaggtgctg ttgaattctt ctagcagtcc ttcacccaaa agttcaaatt 1150
tgtcagtgac atttaccaaa caaacaggca gagttcacta ttctatctgc 1200
cattagacct tcttatcatc catactaaag c 1231
<210> 11
<211> 245
8
CA 02372511 2002-05-14
<212> PRT
<213> Homo Sapien
<400> 11
Met Ala Ala Ala Ile Ala Ser Ser Leu Ile Arg Gln Lys Arg Gln
1 5 10 15
Ala Arg Glu Arg Glu Lys Ser Asn Ala Cys Lys Cys Val Ser Ser
20 25 30
Pro Ser Lys Gly Lys Thr Ser Cys Asp Lys Asn Lys Leu Asn Val
35 40 45
Phe Ser Arg Val Lys Leu Phe Gly Ser Lys Lys Arg Arg Arg Arg
50 55 60
Arg Pro Glu Pro Gln Leu Lys Gly Ile Val Thr Lys Leu Tyr Ser
65 70 75
Arg Gln Gly Tyr His Leu Gln Leu Gln Ala Asp Gly Thr Ile Asp
80 85 90
Gly Thr Lys Asp Glu Asp Ser Thr Tyr Thr Leu Phe Asn Leu Ile
95 100 105
Pro Val Gly Leu Arg Val Val Ala Ile Gln Gly Val Gln Thr Lys
110 115 120
Leu Tyr Leu Ala Met Asn Ser Glu Gly Tyr Leu Tyr Thr Ser Glu
125 130 135
Leu Phe Thr Pro Glu Cys Lys Phe Lys Glu Ser Val Phe Glu Asn
140 145 150
Tyr Tyr Val Thr Tyr Ser Ser Met Ile Tyr Arg Gln Gln Gln Ser
155 160 165
Gly Arg Gly Trp Tyr Leu Gly Leu Asn Lys Glu Gly Glu Ile Met
170 175 180
Lys Gly Asn His Val Lys Lys Asn Lys Pro Ala Ala His Phe Leu
185 190 195
Pro Lys Pro Leu Lys Val Ala Met Tyr Lys Glu Pro Ser Leu His
200 205 210
Asp Leu Thr Glu Phe Ser Arg Ser Gly Ser Gly Thr Pro Thr Lys
215 220 225
Ser Arg Ser Val Ser Gly Val Leu Asn Gly Gly Lys Ser Met Ser
230 235 240
His Asn Glu Ser Thr
245
<210> 12
<211> 744
<212> DNA
<213> Homo Sapien
<400> 12
atggccgcgg ccatcgctag cggcttgatc cgccagaagc ggcaggcgcg 50
9
CA 02372511 2002-05-14
ggagcagcac tgggaccggc cgtctgccag caggaggcgg agcagcccca 100
gcaagaaccg cgggctctgc aacggcaacc tggtggatat cttctccaaa 150
gtgcgcatct tcggcctcaa gaagcgcagg ttgcggcgcc aagatcccca 200
gctcaagggt atagtgacca ggttatattg caggcaaggc tactacttgc 250
aaatgcaccc cgatggagct ctcgatggaa ccaaggatga cagcactaat 300
tctacactct tcaacctcat accagtggga ctacgtgttg ttgccatcca 350
gggagtgaaa acagggttgt atatagccat gaatggagaa ggttacctct 400
acccatcaga actttttacc cctgaatgca agtttaaaga atctgttttt 450
gaaaattatt atgtaatcta ctcatccatg ttgtacagac aacaggaatc 500
tggtagagcc tggtttttgg gattaaataa ggaagggcaa gctatgaaag 550
ggaacagagt aaagaaaacc aaaccagcag ctcattttct acccaagcca 600
ttggaagttg ccatgtaccg agaaccatct ttgcatgatg ttggggaaac 650
ggtcccgaag cctggggtga cgccaagtaa aagcacaagt gcgtctgcaa 700
taatgaatgg aggcaaacca gtcaacaaga gtaagacaac atag 744
<210> 13
<211> 247
<212> PRT
<213> Homo Sapien
<400> 13
Met Ala Ala Ala Ile Ala Ser Gly Leu Ile Arg Gln Lys Arg Gln
1 5 10 15
Ala Arg Glu Gln His Trp Asp Arg Pro Ser Ala Ser Arg Arg Arg
20 25 30
Ser Ser Pro Ser Lys Asn Arg Gly Leu Cys Asn Gly Asn Leu Val
35 40 45
Asp Ile Phe Ser Lys Val Arg Ile Phe Gly Leu Lys Lys Arg Arg
50 55 60
Leu Arg Arg Gln Asp Pro Gln Leu Lys Gly Ile Val Thr Arg Leu
65 70 75
Tyr Cys Arg Gln Gly Tyr Tyr Leu Gln Met His Pro Asp Gly Ala
80 85 90
Leu Asp Gly Thr Lys Asp Asp Ser Thr Asn Ser Thr Leu Phe Asn
95 100 105
Leu Ile Pro Val Gly Leu Arg Val Val Ala Ile Gln Gly Val Lys
110 115 120
Thr Gly Leu Tyr Ile Ala Met Asn Gly Glu Gly Tyr Leu Tyr Pro
125 130 135
Ser Glu Leu Phe Thr Pro Glu Cys Lys Phe Lys Glu Ser Val Phe
140 145 150
CA 02372511 2002-05-14
Glu Asn Tyr Tyr Val Ile Tyr Ser Ser Met Leu Tyr Arg Gln Gln
155 160 165
Glu Ser Gly Arg Ala Trp Phe Leu Gly Leu Asn Lys Glu Gly Gln
170 175 180
Ala Met Lys Gly Asn Arg Val Lys Lys Thr Lys Pro Ala Ala His
185 190 195
Phe Leu Pro Lys Pro Leu Glu Val Ala Met Tyr Arg Glu Pro Ser
200 205 210
Leu His Asp Val Gly Glu Thr Val Pro Lys Pro Gly Val Thr Pro
215 220 225
Ser Lys Ser Thr Ser Ala Ser Ala Ile Met Asn Gly Gly Lys Pro
230 235 240
Val Asn Lys Ser Lys Thr Thr
245
<210> 14
<211> 2609
<212> DNA
<213> Homo Sapien
<400> 14
ctcgcagccg agcgcggccg gggaagggct ctccttccag cgccgagcac 50
tgggccctgg cagacgcccc aagattgttg tgaggagtct agccagttgg 100
tgagcgctgt aatctgaacc agctgtgtcc agactgaggc cccatttgca 150
ttgtttaaca tacttagaaa atgaagtgtt catttttaac attcctcctc 200
caattggttt aatgctgaat tactgaagag ggctaagcaa aaccaggtgc 250
ttgcgctgag ggctctgcag tggctgggag gaccccggcg ctctccccgt 300
gtcctctcca cgactcgctc ggcccctctg gaataaaaca cccgcgagcc 350
ccgagggccc agaggaggcc gacgtgcccg agctcctccg ggggtcccgc 400
ccgcgagctt tcttctcgcc ttcgcatctc ctcctcgcgc gtcttggaca 450
tgccaggaat aaaaaggata ctcactgtta ccattctggc tctctgtctt 500
ccaagccctg ggaatgcaca ggcacagtgc acgaatggct ttgacctgga 550
tcgccagtca ggacagtgtt tagatattga tgaatgccca accatccccg 600
aggcctgccg aggagacatg atgtgtgtta accaaaatgg cgggtattta 650
tgcattcccc ggacaaaccc tgtgtatcga gggccctact cgaaccccta 700
ctcgaccccc tactcaggtc cgtacccagc agctgcccca ccactctcag 750
ctccaaacta tcccacgatc tccaggcctc ttatatgccg ctttggatac 800
cagatggatg aaagcaacca atgtgtggat gtggacgagt gtgcaacaga 850
ttcccaccag tgcaacccca cccagatctg catcaatact gaaggcgggt 900
11
CA 02372511 2002-05-14
acacctgctc ctgcaccgac ggatattggc ttctggaagg ccagtgctta 950
gacattgatg aatgtcgcta tggttactgc cagcagctct gtgcgaatgt 1000
tcctggatcc tattcttgta catgcaaccc tggttttacc ctcaatgagg 1050
atggaaggtc ttgccaagat gtgaacgagt gtgccaccga gaacccctgc 1100
gtgcaaacct gcgtcaacac ctacggctct ctcatctgcc gctgtgaccc 1150
aggatatgaa cttgaggaag atggcgttca ttgcagtgat atggacgagt 1200
gcagcttctc tgagttcctc tgccaacatg agtgtgtgaa ccagcccggc 1250
acatacttct gctcctgccc tccaggctac atcctgctgg atgacaaccg 1300
aagctgccaa gacatcaacg aatgtgagca caggaaccac acgtgcaacc 1350
tgcagcagac gtgctacaat ttacaagggg gcttcaaatg catcgacccc 1400
atccgctgtg aggagcctta tctgaggatc agtgataacc gctgtatgtg 1450
tcctgctgag aaccctggct gcagagacca gccctttacc atcttgtacc 1500
gggacatgga cgtggtgtca ggacgctccg ttcccgctga catcttccaa 1550
atgcaagcca cgacccgcta ccctggggcc tattacattt tccagatcaa 1600
atctgggaat gagggcagag aattttacat gcggcaaacg ggccccatca 1650
gtgccaccct ggtgatgaca cgccccatca aagggccccg ggaaatccag 1700
ctggacttgg aaatgatcac tgtcaacact gtcatcaact tcagaggcag 1750
ctccgtgatc cgactgcgga tatatgtgtc gcagtaccca ttctgagcct 1800
cgggctggag cctccgacgc tgcctctcat tggcaccaag ggacaggaga 1850
agagaggaaa taacagagag aatgagagcg acacagacgt taggcatttc 1900
ctgctgaacg tttccccgaa gagtcagccc cgacttcctg actctcacct 1950
gtactattgc agacctgtca ccctgcagga cttgcaaccc ccagttccta 2000
tgacacagtt atcaaaaagt attatcattg ctcccctgat agaagattgt 2050
tggtgaattt tcaaggcctt cagtttattt ccactatttt caaagaaaat 2100
agattaggtt tgcgggggtc tgagtctatg ttcaaagact gtgaacagct 2150
tgctgtcact tcttcacctc ttccactcct tctctcactg tgttactgct 2200
ttgcaaagac ctgggagctg gcggggaacc ctgggagtag ctagtttgct 2250
ttttgcgtac aaagagaacg ctatgtaaac aaaccacagc aggatcgaag 2300
ggtttttaga gaatgtgttt caaaaccatg cctggtattt tcaaccataa 2350
aagaagtttc agttgtcctt aaatttgtat aacggtttaa ttctgtcttg 2400
ttcattttga gtatttttaa aaaatatgtc gtagaattcc ttcgaaaggc 2450
cttcagacac atgctatgtt ctgtcttccc aaacccagtc tcctctccat 2500
12
CA 02372511 2002-05-14
tttagcccag tgttttcttt gaggacccct taatcttgct ttctttagaa 2550
tttttaccca attggattgg aatgcagagg tctccaaact gattaaatat 2600
ttgaagaga 2609
<210> 15
<211> 448
<212> PRT
<213> Homo Sapien
<400> 15
Met Pro Gly Ile Lys Arg Ile Leu Thr Val Thr Ile Leu Ala Leu
1 5 10 15
Cys Leu Pro Ser Pro Gly Asn Ala Gln Ala Gln Cys Thr Asn Gly
20 25 30
Phe Asp Leu Asp Arg Gln Ser Gly Gln Cys Leu Asp Ile Asp Glu
35 40 45
Cys Arg Thr Ile Pro Glu Ala Cys Arg Gly Asp Met Met Cys Val
50 55 60
Asn Gln Asn Gly Gly Tyr Leu Cys Ile Pro Arg Thr Asn Pro Val
65 70 75
Tyr Arg Gly Pro Tyr Ser Asn Pro Tyr Ser Thr Pro Tyr Ser Gly
80 85 90
Pro Tyr Pro Ala Ala Ala Pro Pro Leu Ser Ala Pro Asn Tyr Pro
95 100 105
Thr Ile Ser Arg Pro Leu Ile Cys Arg Phe Gly Tyr Gln Met Asp
110 115 120
Glu Ser Asn Gln Cys Val Asp Val Asp Glu Cys Ala Thr Asp Ser
125 130 135
His Gln Cys Asn Pro Thr Gln Ile Cys Ile Asn Thr Glu Gly Gly
140 145 150
Tyr Thr Cys Ser Cys Thr Asp Gly Tyr Trp Leu Leu Glu Gly Gln
155 160 165
Cys Leu Asp Ile Asp Glu Cys Arg Tyr Gly Tyr Cys Gln Gln Leu
170 175 180
Cys Ala Asn Val Pro Gly Ser Tyr Ser Cys Thr Cys Asn Pro Gly
185 190 195
Phe Thr Leu Asn Glu Asp Gly Arg Ser Cys Gln Asp Val Asn Glu
200 205 210
Cys Ala Thr Glu Asn Pro Cys Val Gln Thr Cys Val Asn Thr Tyr
215 220 225
Gly Ser Leu Ile Cys Arg Cys Asp Pro Gly Tyr Glu Leu Glu Glu
230 235 240
Asp Gly Val His Cys Ser Asp Met Asp Glu Cys Ser Phe Ser Glu
245 250 255
13
CA 02372511 2002-05-14
Phe Leu Cys Gln His Glu Cys Val Asn Gln Pro Gly Thr Tyr Phe
260 265 270
Cys Ser Cys Pro Pro Gly Tyr Ile Leu Leu Asp Asp Asn Arg Ser
275 280 285
Cys Gln Asp Ile Asn Glu Cys Glu His Arg Asn His Thr Cys Asn
290 295 300
Leu Gln Gln Thr Cys Tyr Asn Leu Gin Gly Gly Phe Lys Cys Ile
305 310 315
Asp Pro Ile Arg Cys Glu Glu Pro Tyr Leu Arg Ile Ser Asp Asn
320 325 330
Arg Cys Met Cys Pro Ala Glu Asn Pro Gly Cys Arg Asp Gln Pro
335 340 345
Phe Thr Ile Leu Tyr Arg Asp Met Asp Val Val Ser Gly Arg Ser
350 355 360
Val Pro Ala Asp Ile Phe Gln Met Gln Ala Thr Thr Arg Tyr Pro
365 370 375
Gly Ala Tyr Tyr Ile Phe Gln Ile Lys Ser Gly Asn Glu Gly Arg
380 385 390
Glu Phe Tyr Net Arg Gln Thr Gly Pro Ile Ser Ala Thr Leu Val
395 400 405
Met Thr Arg Pro Ile Lys Gly Pro Arg Glu Ile Gln Leu Asp Leu
410 415 420
Glu Met Ile Thr Val Asn Thr Val Ile Asn Phe Arg Gly Ser Ser
425 430 435
Val Ile Arg Leu Arg Ile Tyr Val Ser Gln Tyr Pro Phe
440 445
<210> 16
<211> 2447
<212> DNA
<213> Homo Sapien
<400> 16
caggtccaac tgcacctcgg ttctatcgat tgaattcccc ggggatcctc 50
tagagatccc tcgacctcga cccacgcgtc cgaacacagg tccttgttgc 100
tgcagagaag cagttgtttt gctggaagga gggagtgcgc gggctgcccc 150
gggctcctcc ctgccgcctc ctctcagtgg atggttccag gcaccctgtc 200
tggggcaggg agggcacagg cctgcacatc gaaggtgggg tgggaccagg 250
ctgcccctcg ccccagcatc caagtcctcc cttgggcgcc cgtggccctg 300
cagactctca gggctaaggt cctctgttgc tttttggttc caccttagaa 350
gaggctccgc ttgactaaga gtagcttgaa ggaggcacca tgcaggagct 400
gcatctgctc tggtgggcgc ttctcctggg cctggctcag gcctgccctg 450
14
CA 02372511 2002-05-14
agccctgcga ctgtggggaa aagtatggct tccagatcgc cgactgtgcc 500
taccgcgacc tagaatccgt gccgcctggc ttcccggcca atgtgactac 550
actgagcctg tcagccaacc ggctgccagg cttgccggag ggtgccttca 600
gggaggtgcc cctgctgcag tcgctgtggc tggcacacaa tgagatccgc 650
acggtggccg ccggagccct ggcctctctg agccatctca agagcctgga 700
cctcagccac aatctcatct ctgactttgc ctggagcgac ctgcacaacc 750
tcagtgccct ccaattgctc aagatggaca gcaacgagct gaccttcatc 800
ccccgcgacg ccttccgcag cctccgtgct ctgcgctcgc tgcaactcaa 850
ccacaaccgc ttgcacacat tggccgaggg caccttcacc ccgctcaccg 900
cgctgtccca cctgcagatc aacgagaacc ccttcgactg cacctgcggc 950
atcgtgtggc tcaagacatg ggccctgacc acggccgtgt ccatcccgga 1000
gcaggacaac atcgcctgca cctcacccaa tgtgctcaag ggtacaccgc 1050
tgagccgcct gccgccactg ccatgctcgg cgccctcagt gcagctcagc 1100
taccaaccca gccaggatgg tgccgagctg cggcctggtt ttgctctggc 1150
actgcactgt gatgtggacg ggcagccggc ccctcagctt cactggcaca 1200
tccagatacc cagtggcatt gtggagatca ccagccccaa cgtgggcact 1250
gatgggcgtg ccctgcctgg cacccctttg gccagctccc agccgcgctt 1300
ccaggccttt gccaatggca gcctgcttat ccccgacttt ggcaagctgg 1350
aggaaggcac ctacagctgc ctggccacca atgagctggg cagtgctgag 1400
agctcagtgg acgtggcact ggccacgccc ggtgagggtg gtgaggacac 1450
actgtggccc aggttccatg gcaaagcggt tgagggaaag ggctgctata 1500
cggttgacaa cgaggtgcag ccatcagggc cggaggacaa tgtggtcatc 1550
atctacctca gccgtgctgg gaaccctgag gctgcagtcg cagaaggggt 1600
ccctgggcag ctgcccccag gcctgctcct gctgggccaa agcctcctcc 1650
tcttcttctt cctcacctcc ttctagcccc acccagggct tccctaactc 1700
ctccccttgc ccctaccaat gcccctttaa gtgctgcagg ggtctggggt 1750
tggcaactcc tgaggcctgc atgggtgact tcacattttc ctacctctcc 1800
ttctaatctc ttctagagca cctgctatcc ccaacttcta gacctgctcc 1850
aaactagtga ctaggataga atttgatccc ctaactcact gtctgcggtg 1900
ctcattgctg ctaacagcat tgcctgtgct ctcctctcag gggcagcatg 1950
ctaacggggc gacgtcctaa tccaactggg agaagcctca gtggtggaat 2000
tccaggcact gtgactgtca agctggcaag ggccaggatt gggggaatgg 2050
CA 02372511 2002-05-14
agctggggct tagctgggag gtggtctgaa gcagacaggg aatgggagag 2100
gaggatggga agtagacagt ggctggtatg gctctgaggc tccctggggc 2150
ctgctcaagc tcctcctgct ccttgctgtt ttctgatgat ttgggggctt 2200
gggagtccct ttgtcctcat ctgagactga aatgtgggga tccaggatgg 2250
ccttccttcc tcttaccctt cctccctcag cctgcaacct ctatcctgga 2300
acctgtcctc cctttctccc caactatgca tctgttgtct gctcctctgc 2350
aaaggccagc cagcttggga gcagcagaga aataaacagc atttctgatg 2400
ccaaaaaaaa aaaaaaaaaa gggcggccgc gactctagag tcgacct 2447
<210> 17
<211> 428
<212> PRT
<213> Homo Sapien
<400> 17
Met Gln Glu Leu His Leu Leu Trp Trp Ala Leu Leu Leu Gly Leu
1 5 10 15
Ala Gln Ala Cys Pro Glu Pro Cys Asp Cys Gly Glu Lys Tyr Gly
20 25 30
Phe Gln Ile Ala Asp Cys Ala Tyr Arg Asp Leu Glu Ser Val Pro
35 40 45
Pro Gly Phe Pro Ala Asn Val Thr Thr Leu Ser Leu Ser Ala Asn
50 55 60
Arg Leu Pro Gly Leu Pro Glu Gly Ala Phe Arg Glu Val Pro Leu
65 70 75
Leu Gln Ser Leu Trp Leu Ala His Asn Glu Ile Arg Thr Val Ala
80 85 90
Ala Gly Ala Leu Ala Ser Leu Ser His Leu Lys Ser Leu Asp Leu
95 100 105
Ser His Asn Leu Ile Ser Asp Phe Ala Trp Ser Asp Leu His Asn
110 115 120
Leu Ser Ala Leu Gln Leu Leu Lys Met Asp Ser Asn Glu Leu Thr
125 130 135
Phe Ile Pro Arg Asp Ala Phe Arg Ser Leu Arg Ala Leu Arg Ser
140 145 150
Leu Gln Leu Asn His Asn Arg Leu His Thr Leu Ala Glu Gly Thr
155 160 165
Phe Thr Pro Leu Thr Ala Leu Ser His Leu Gln Ile Asn Glu Asn
170 175 180
Pro Phe Asp Cys Thr Cys Gly Ile Val Trp Leu Lys Thr Trp Ala
185 190 195
Leu Thr Thr Ala Val Ser Ile Pro Glu Gln Asp Asn Ile Ala Cys
200 205 210
16
CA 02372511 2002-05-14
Thr Ser Pro His Val Leu Lys Gly Thr Pro Leu Ser Arg Leu Pro
215 220 225
Pro Leu Pro Cys Ser Ala Pro Ser Val Gln Leu Ser Tyr Gln Pro
230 235 240
Ser Gln Asp Cly Ala Glu Leu Arg Pro Gly Phe Val Leu Ala Leu
245 250 255
His Cys Asp Val Asp Gly Gln Pro Ala Pro Gln Leu His Trp His
260 265 270
Ile Gln Ile Pro Ser Gly Ile Val Glu Ile Thr Ser Pro Asn Val
275 280 285
Gly Thr Asp Cly Arg Ala Leu Pro Gly Thr Pro Val Ala Ser Ser
290 295 300
Gln Pro Arg Phe Gin Ala Phe Ala Asn Gly Ser Leu Leu Ile Pro
305 310 315
Asp Phe Gly Lys Leu Glu Glu Gly Thr Tyr Ser Cys Leu Ala Thr
320 325 330
Asn Glu Leu Cly Ser Ala Glu Ser Ser Val Asp Val Ala Leu Ala
335 340 345
Thr Pro Gly Clu Gly Gly Glu Asp Thr Leu Gly Arg Arg Phe His
350 355 360
Gly Lys Ala Val Glu Gly Lys Gly Cys Tyr Thr Val Asp Asn Glu
365 370 375
Val Gln Pro Ser Gly Pro Glu Asp Asn Val Val. Ile Ile Tyr Leu
380 385 390
Ser Arg Ala Gly Asn Pro Glu Ala Ala Val Ala Glu Gly Val Pro
395 400 405
Gly Gln Leu Pro Pro Gly Leu Leu Leu Leu Gly Gln Ser Leu Leu
410 415 420
Leu Phe Phe Phe Leu Thr Ser Phe
425
<210> 18
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 18
gtggctggca cacaatgaga tc 22
<210> 19
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
17
CA 02372511 2002-05-14
<400> 19
ccaatgtgtg caagcggttg tg 22
<210> 20
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 20
tcaagagcct ggacctcagc cacaatctca tctctgactt tgcctggagc 50
<210> 21
<211> 2033
<212> DNA
<213> Homo Sapien
<400> 21
ccaggccggg aggcgacgcg cccagccgtc taaacgggaa cagccctggc 50
tgagggagct gcagcgcagc agagtatctg acggcgccag gttgcgtagg 100
tgcggcacga ggagttttcc cggcagcgag gaggtcctga gcagcatggc 150
ccggaggagc gccttccctg ccgccgcgct ctggctctgg agcatcctcc 200
tgtgcctgct ggcactgcgg gcggaggccg ggccgccgca ggaggagagc 250
ctgtacctat ggatcgatgc tcaccaggca agagtactca taggatttga 300
agaagatatc ctgattgttt cagaggggaa aatggcacct tttacacatg 350
atttcagaaa agcgcaacag agaatgccag ctattcctgt caatatccat 400
tccatgaatt ttacctggca agctgcaggg caggcagaat acttctatga 450
attcctgtcc ttgcgctccc tggataaagg catcatggca gatccaaccg 500
tcaatgtccc tctgctggga acagtgcctc acaaggcatc agttgttcaa 550
gttggtttcc catgtcttgg aaaacaggat ggggtggcag catttgaagt 600
ggatgtgatt gttatgaatt ctgaaggcaa caccattctc caaacacctc 650
aaaatgctat cttctttaaa acatgtcaac aagctgagtg cccaggcggg 700
tgccgaaatg gaggcttttg taatgaaaga cgcatctgcg agtgtcctga 750
tgggttccac ggacctcact gtgagaaagc cctttgtacc ccacgatgta 800
tgaatggtgg actttgtgtg actcctggtt tctgcatctg cccacctgga 850
ttctatggag tgaactgtga caaagcaaac tgctcaacca cctgctttaa 900
tggagggacc tgtttctacc ctggaaaatg tatttgccct ccaggactag 950
agggagagca gtgtgaaatc agcaaatgcc cacaaccctg tcgaaatgga 1000
ggtaaatgca ttggtaaaag caaatgtaag tgttccaaag gttaccaggg 1050
agacctctgt tcaaagcctg tctgcgagcc tggctgtggt gcacatggaa 1100
18
CA 02372511 2002-05-14
cctgccatga acccaacaaa tgccaatgtc aagaaggttg gcatggaaga 1150
cactgcaata aaaggtacga agccagcctc atacatgccc tgaggccagc 1200
aggcgcccag ctcaggcagc acacgccttc acttaaaaag gccgaggagc 1250
ggcgggatcc acctgaatcc aattacatct ggtgaactcc gacatctgaa 1300
acgttttaag ttacaccaag ttcatagcct ttgttaacct ttcatgtgtt 1350
gaatgttcaa ataatgttca ttacacttaa gaatactggc ctgaatttta 1400
ttagcttcat tataaatcac tgagctgata tttactcttc cttttaagtt 1450
ttctaagtac gtctgtagca tgatggtata gattttcttg tttcagtgct 1500
ttgggacaga ttttatatta tgtcaattga tcaggttaaa attttcagtg 1550
tgtagttggc agatattttc aaaattacaa tgcatttatg gtgtctgggg 1600
gcaggggaac atcagaaagg ttaaattggg caaaaatgcg taagtcacaa 1650
gaatttggat ggtgcagtta atgttgaagt tacagcattt cagattttat 1700
tgtcagatat ttagatgttt gttacatttt taaaaattgc tcttaatttt 1750
taaactctca atacaatata ttttgacctt accattattc cagagattca 1800
gtattaaaaa aaaaaaaatt acactgtggt agtggcattt aaacaatata 1850
atatattcta aacacaatga aatagggaat ataatgtatg aactttttgc 1900
attggcttga agcaatataa tatattgtaa acaaaacaca gctcttacct 1950
aataaacatt ttatactgtt tgtatgtata aaataaaggt gctgctttag 2000
ttttttggaa aaaaaaaaaa aaaaaaaaaa aaa 2033
<210> 22
<211> 379
<212> PRT
<213> Homo Sapien
<400> 22
Met Ala Arg Arg Ser Ala Phe Pro Ala Ala Ala Leu Trp Leu Trp
1 5 10 15
Ser Ile Leu Leu Cys Leu Leu Ala Leu Arg Ala Glu Ala Gly Pro
20 25 30
Pro Gln Glu Glu Ser Leu Tyr Leu Trp Ile Asp Ala His Gln Ala
35 40 45
Arg Val Leu Ile Gly Phe Glu Glu Asp Ile Leu Ile Val Ser Glu
50 55 60
Gly Lys Met Ala Pro Phe Thr His Asp Phe Arg Lys Ala Gln Gln
65 70 75
Arg Met Pro Ala Ile Pro Val Asn Ile His Ser Met Asn Phe Thr
80 85 90
Trp Gln Ala Ala Gly Gln Ala Glu Tyr Phe Tyr Glu Phe Leu Ser
19
CA 02372511 2002-05-14
95 100 105
Leu Arg Ser Leu Asp Lys Gly Ile Met Ala Asp Pro Thr Val Asn
110 115 120
Val Pro Leu Leu Gly Thr Val Pro His Lys Ala Ser Val Val Gln
125 130 135
Val Gly Phe Pro Cys Leu Gly Lys Gln Asp Gly Val Ala Ala Phe
140 145 150
Glu Val Asp Val Ile Val Met Asn Ser Glu Gly Asn Thr Ile Leu
155 160 165
Gln Thr Pro Gln Asn Ala Ile Phe Phe Lys Thr Cys Gln Gln Ala
170 175 180
Glu Cys Pro Gly Gly Cys Arg Asn Gly Gly Phe Cys Asn Glu Arg
185 190 195
Arg Ile Cys Glu Cys Pro Asp Gly Phe His Gly Pro His Cys Glu
200 205 210
Lys Ala Leu Cys Thr Pro Arg Cys Met Asn Gly Gly Leu Cys Val
215 220 225
Thr Pro Gly Phe Cys Ile Cys Pro Pro Gly Phe Tyr Gly Val Asn
230 235 240
Cys Asp Lys Ala Asn Cys Ser Thr Thr Cys Phe Asn Gly Gly Thr
245 250 255
Cys Phe Tyr Pro Gly Lys Cys Ile Cys Pro Pro Gly Leu Glu Gly
260 265 270
Glu Gln Cys Glu Ile Ser Lys Cys Pro Gln Pro Cys Arg Asn Gly
275 280 285
Gly Lys Cys Ile Gly Lys Ser Lys Cys Lys Cys Ser Lys Gly Tyr
290 295 300
Gln Gly Asp Leu Cys Ser Lys Pro Val Cys Glu Pro Gly Cys Gly
305 310 315
Ala His Gly Thr Cys His Glu Pro Asn Lys Cys Gln Cys Gln Glu
320 325 330
Gly Trp His Gly Arg His Cys Asn Lys Arg Tyr Glu Ala Ser Leu
335 340 345
Ile His Ala Leu Arg Pro Ala Gly Ala Gln Leu Arg Gln His Thr
350 355 360
Pro Ser Leu Lys Lys Ala Glu Glu Arg Arg Asp Pro Pro Glu Ser
365 370 375
Asn Tyr Ile Trp
<210> 23
<211> 783
<212> DNA
<213> Homo Sapien
CA 02372511 2002-05-14
<400> 23
agaacctcag aaatgtgagt tatttgggaa tggctgtttg taaatgtcct 50
tacgtaagcc aagaggaggt cttgacttgg ggtcccaggg gtaccgcaga 100
tcccagggac tggagcagca ctagcaagct ctggaggatg agccaggagt 150
ctggaattga ggctgagcca aagaccccag ggccgtctca gtctcataaa 200
aggggatcag gcaggaggag tttgggagaa acctgagaag ggcctgattt 250
gcagcatcat gatgggcctc tccttggcct ctgctgtgct cctggcctcc 300
ctcctgagtc tccaccttgg aactgccaca cgtgggagtg acatatccaa 350
gacctgctgc ttccaataca gccacaagcc ccttccctgg acctgggtgc 400
gaagctatga attcaccagt aacagctgct cccagcgggc tgtgatattc 450
actaccaaaa gaggcaagaa agtctgtacc catccaagga aaaaatgggt 500
gcaaaaatac atttctttac tgaaaactcc gaaacaattg tgactcagct 550
gaattttcat ccgaggacgc ttggaccccg ctcttggctc tgcagccctc 600
tggggagcct gcggaatctt ttctgaaggc tacatggacc cgctggggag 650
gagagggtgt ttcctcccag agttacttta ataaaggttg ttcatagagt 700
tgaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 750
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 783
<210> 24
<211> 94
<212> PRT
<213> Homo Sapien
<400> 24
Met Met Gly Leu Ser Leu Ala Ser Ala Val Leu Leu Ala Ser Leu
1 5 10 15
Leu Ser Leu His Leu Gly Thr Ala Thr Arg Gly Ser Asp Ile Ser
20 25 30
Lys Thr Cys Cys Phe Gln Tyr Ser His Lys Pro Leu Pro Trp Thr
35 40 45
Trp Val Arg Ser Tyr Glu Phe Thr Ser Asn Ser Cys Ser Gln Arg
50 55 60
Ala Val Ile Phe Thr Thr Lys Arg Gly Lys Lys Val Cys Thr His
65 70 75
Pro Arg Lys Lys Trp Val Gln Lys Tyr Ile Ser Leu Leu Lys Thr
80 85 90
Pro Lys Gln Leu
<210> 25
<211> 23
<212> DNA
21
CA 02372511 2002-05-14
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 25
ggatcaggca ggaggagttt ggg 23
<210> 26
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 26
ggatgggtac agactttctt gcc 23
<210> 27
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 27
atgatgggcc tctccttggc ctctgctgtg ctcctggcct ccctcctgag 50
<210> 28
<211> 3552
<212> DNA
<213> Homo Sapien
<400> 28
gcgagaacct ttgcacgcgc acaaactacg gggacgattt ctgattgatt 50
tttggcgctt tcgatccacc ctcctccctt ctcatgggac tttggggaca 100
aagcgtcccg accgcctcga gcgctcgagc agggcgctat ccaggagcca 150
ggacagcgtc gggaaccaga ccatggctcc tggaccccaa gatccttaag 200
ttcgtcgtct tcatcgtcgc ggttctgctg ccggtccggg ttgactctgc 250
caccatcccc cggcaggacg aagttcccca gcagacagtg gccccacagc 300
aacagaggcg cagcctcaag gaggaggagt gtccagcagg atctcataga 350
tcagaatata ctggagcctg taacccgtgc acagagggtg tggattacac 400
cattgcttcc aacaatttgc cttcttgcct gctatgtaca gtttgtaaat 450
caggtcaaac aaataaaagt tcctgtacca cgaccagaga caccgtgtgt 500
cagtgtgaaa aaggaagctt ccaggataaa aactcccctg agatgtgccg 550
gacgtgtaga acagggtgtc ccagagggat ggtcaaggtc agtaattgta 600
cgccccggag tgacatcaag tgcaaaaatg aatcagctgc cagttccact 650
gggaaaaccc cagcagcgga ggagacagtg accaccatcc tggggatgct 700
22
CA 02372511 2002-05-14
tgcctctccc tatcactacc ttatcatcat agtggtttta gtcatcattt 750
tagctgtggt tgtggttggc ttttcatgtc ggaagaaatt catttcttac 800
ctcaaaggca tctgctcagg tggtggagga ggtcccgaac gtgtgcacag 850
agtccttttc cggcggcgtt catgtccttc acgagttcct ggggcggagg 900
acaatgcccg caacgagacc ctgagtaaca gatacttgca gcccacccag 950
gtctctgagc aggaaatcca aggtcaggag ctggcagagc taacaggtgt 1000
gactgtagag tcgccagagg agccacagcg tctgctggaa caggcagaag 1050
ctgaagggtg tcagaggagg aggctgctgg ttccagtgaa tgacgctgac 1100
tccgctgaca tcagcacctt gctggatgcc tcggcaacac tggaagaagg 1150
acatgcaaag gaaacaattc aggaccaact ggtgggctcc gaaaagctct 1200
tttatgaaga agatgaggca ggctctgcta cgtcctgcct gtgaaagaat 1250
ctcttcagga aaccagagct tccctcattt accttttctc ctacaaaggg 1300
aagcagcctg gaagaaacag tccagtactt gacccatgcc ccaacaaact 1350
ctactatcca atatggggca gcttaccaat ggtcctagaa ctttgttaac 1400
gcacttggag taatttttat gaaatactgc gtgtgataag caaacgggag 1450
aaatttatat cagattcttg gctgcatagt tatacgattg tgtattaagg 1500
gtcgttttag gccacatgcg gtggctcatg cctgtaatcc cagcattttg 1550
ataggctgag gcaggtggat tgcttgagct cgggagtttg agaccagcct 1600
catcaacaca gtgaaactcc atctcaattt aaaaagaaaa aaagtggttt 1650
taggatgtca ttctttgcag ttcttcatca tgagacaagt ctttttttct 1700
gcttcttata ttgcaagctc catctctact ggtgtgtgca tttaatgaca 1750
tctaactaca gatgccgcac agccacaatg ctttgcctta tagtttttta 1800
actttagaac gggattatct tgttattacc tgtattttca gtttcggata 1850
tttttgactt aatgatgaga ttatcaagac gtagccctat gctaagtcat 1900
gagcatatgg acttacgagg gttcgactta gagttttgag ctttaagata 1950
ggattattgg ggcttacccc caccttaatt agagaaacat ttatattgct 2000
tactactgta ggctgtacat ctcttttccg atttttgtat aatgatgtaa 2050
acatggaaaa actttaggaa atgcacttat taggctgttt acatgggttg 2100
cctggataca aatcagcagt caaaaatgac taaaaatata actagtgacg 2150
gagggagaaa tcctccctct gtgggaggca cttactgcat tccagttctc 2200
cctcctgcgc cctgagactg gaccagggtt tgatggctgg cagcttctca 2250
aggggcagct tgtcttactt gttaatttta gaggtatata gccatattta 2300
23
CA 02372511 2002-05-14
tttataaata aatatttatt tatttattta taagtagatg tttacatatg 2350
cccaggattt tgaagagcct ggtatctttg ggaagccatg tgtctggttt 2400
gtcgtgctgg gacagtcatg ggactgcatc ttccgacttg tccacagcag 2450
atgaggacag tgagaattaa gttagatccg agactgcgaa gagcttctct 2500
ttcaagcgcc attacagttg aacgttagtg aatcttgagc ctcatttggg 2550
ctcagggcag agcaggtgtt tatctgcccc ggcatctgcc atggcatcaa 2600
gagggaagag tggacggtgc ttgggaatgg tgtgaaatgg ttgccgactc 2650
aggcatggat gggcccctct cgcttctggt ggtctgtgaa ctgagtccct 2700
gggatgcctt ttagggcaga gattcctgag ctgcgtttta gggtacagat 2750
tccctgtttg aggagcttgg cccctctgta agcatctgac tcatctcaga 2800
gatatcaatt cttaaacact gtgacaacgg gatctaaaat ggctgacaca 2850
tttgtccttg tgtcacgttc cattatttta tttaaaaacc tcagtaatcg 2900
ttttagcttc tttccagcaa actcttctcc acagtagccc agtcgtggta 2950
ggataaatta cggatatagt cattctaggg gtttcagtct tttccatctc 3000
aaggcattgt gtgttttgtt ccgggactgg tttggctggg acaaagttag 3050
aactgcctga agttcgcaca ttcagattgt tgtgtccatg gagttttagg 3100
aggggatggc ctttccggtc ttcggacttc catcctctcc cacttccatc 3150
tggcgtccca caccttgtcc cctgcacttc tggatgacac agggtgctgc 3200
tgcctcctag tctttgcctt tgctgggcct tctgtgcagg agacttggtc 3250
tcaaagctca gagagagcca gtccggtccc agctcctttg tcccttcctc 3300
agaggccttc cttgaagatg catctagact accagcctta tcagtgttta 3350
agcttattcc tttaacataa gcttcctgac aacatgaaat tgttggggtt 3400
ttttggcttt ggttgatttg tttaggtttt gctttatacc cgggccaaat 3450
agcacataac acctggttat atatgaaata ctcatatgtt tatgaccaaa 3500
ataaatatga aacctcatrt taaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3550
as 3552
<210> 29
<211> 386
<212> PRT
<213> Homo Sapien
<400> 29
Met Gly Leu Trp Gly Gln Ser Val Pro Thr Ala Ser Ser Ala Arg
1 5 10 15
Ala Gly Arg Tyr Pro Gly Ala Arg Thr Ala Ser Gly Thr Arg Pro
20 25 30
24
CA 02372511 2002-05-14
Trp Leu Leu Asp Pro Lys Ile Leu Lys Phe Val Val Phe Ile Val
35 40 45
Ala Val Leu Leu Pro Val Arg Val Asp Ser Ala Thr Ile Pro Arg
50 55 60
Gln Asp Glu Val Pro Gln Gln Thr Val Ala Pro Gln Gln Gln Arg
65 70 75
Arg Ser Leu Lys Glu Glu Glu Cys Pro Ala Gly Ser His Arg Ser
80 85 90
Glu Tyr Thr Gly Ala Cys Asn Pro Cys Thr Glu Gly Val Asp Tyr
95 100 105
Thr Ile Ala Ser Asn Asn Leu Pro Ser Cys Leu Leu Cys Thr Val
110 115 120
Cys Lys Ser Gly Gln Thr Asn Lys Ser Ser Cys Thr Thr Thr Arg
125 130 135
Asp Thr Val Cys Gln Cys Glu Lys Gly Ser Phe Gln Asp Lys Asn
140 145 150
Ser Pro Glu Net Cys Arg Thr Cys Arg Thr Gly Cys Pro Arg Gly
155 160 165
Met Val Lys Val Ser Asn Cys Thr Pro Arg Ser Asp Ile Lys Cys
170 175 180
Lys Asn Glu Ser Ala Ala Ser Ser Thr Gly Lys Thr Pro Ala Ala
185 190 195
Glu Glu Thr Val Thr Thr Ile Leu Gly Met Leu Ala Ser Pro Tyr
200 205 210
His Tyr Leu Ile Ile Ile Val Val Leu Val Ile Ile Leu Ala Val
215 220 225
Val Val Val Gly Phe Ser Cys Arg Lys Lys Phe Ile Ser Tyr Leu
230 235 240
Lys Gly Ile Cys Ser Gly Gly Gly Gly Gly Pro Glu Arg Val His
245 250 255
Arg Val Leu Phe Arg Arg Arg Ser Cys Pro Ser Arg Val Pro Gly
260 265 270
Ala Glu Asp Asn Ala Arg Asn Glu Thr Leu Ser Asn Arg Tyr Leu
275 280 285
Gln Pro Thr Gln Val Ser Glu Gln Glu Ile Gln Gly Gln Glu Leu
290 295 300
Ala Glu Leu Thr Gly Val Thr Val Glu Ser Pro Glu Glu Pro Gln
305 310 315
Arg Leu Leu Glu Gln Ala Glu Ala Glu Gly Cys Gln Arg Arg Arg
320 325 330
Leu Leu Val Pro Val Asn Asp Ala Asp Ser Ala Asp Ile Ser Thr
335 340 345
CA 02372511 2002-05-14
Leu Leu Asp Ala Ser Ala Thr Leu Glu Glu Gly His Ala Lys Glu
350 355 360
Thr Ile Gln Asp Gln Leu Val Gly Ser Glu Lys Leu Phe Tyr Glu
365 370 375
Glu Asp Glu Ala Gly Ser Ala Thr Ser Cys Leu
380 385
<210> 30
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 30
cataaaagtt cctgcaccat gaccagagac acagtgtgtc agtgtaaaga 50
<210> 31
<211> 963
<212> DNA
<213> Homo Sapien
<400> 31
gcggcacctg gaagatgcgc ccattggctg gtggcctgct caaggtggtg 50
ttcgtggtct tcgcctcctt gtgtgcctgg tattcggggt acctgctcgc 100
agagctcatt ccagatgcac ccctgtccag tgctgcctat agcatccgca 150
gcatcgggga gaggcctgtc ctcaaagctc cagtccccaa aaggcaaaaa 200
tgtgaccact ggactccctg cccatctgac acctatgcct acaggttact 250
cagcggaggt ggcagaagca agtacgccaa aatctgcttt gaggataacc 300
tacttatggg agaacagctg ggaaatgttg ccagaggaat aaacattgcc 350
attgtcaact atgtaactgg gaatgtgaca gcaacacgat gttttgatat 400
gtatgaaggc gataactctg gaccgatgac aaagtttatt cagagtgctg 450
ctccaaaatc cctgctcttc atggtgacct atgacgacgg aagcacaaga 500
ctgaataacg atgccaagaa tgccatagaa gcacttggaa gtaaagaaat 550
caggaacatg aaattcaggt ctagctgggt atttattgca gcaaaaggct 600
tggaactccc ttccgaaatt cagagagaaa agatcaacca ctctgatgct 650
aagaacaaca gatattctgg ctggcctgca gagatccaga tagaaggctg 700
catacccaaa gaacgaagct gacactgcag ggtcctgagt aaatgtgttc 750
tgtataaaca aatgcagctg gaatcgctca agaatcttat ttttctaaat 800
ccaacagccc atatttgatg agtattttgg gtttgttgta aaccaatgaa 850
catttgctag ttgtatcaaa tcttggtacg cagtattttt ataccagtat 900
tttatgtagt gaagatgtca attagcagga aactaaaatg aatggaaatt 950
26
CA 02372511 2002-05-14
cttaaaaaaa aaa 963
<210> 32
<211> 235
<212> PRT
<213> Homo Sapien
<400> 32
Met Arg Pro Leu Ala Gly Gly Leu Leu Lys Val Val Phe Val Val
1 5 10 15
Phe Ala Ser Leu Cys Ala Trp Tyr Ser Gly Tyr Leu Leu Ala Glu
20 25 30
Leu Ile Pro Asp Ala Pro Leu Ser Ser Ala Ala Tyr Ser Ile Arg
35 40 45
Ser Ile Gly Glu Arg Pro Val Leu Lys Ala Pro Val Pro Lys Arg
50 55 60
Gln Lys Cys Asp His Trp Thr Pro Cys Pro Ser Asp Thr Tyr Ala
65 70 75
Tyr Arg Leu Leu Ser Gly Gly Gly Arg Ser Lys Tyr Ala Lys Ile
80 85 90
Cys Phe Glu Asp Asn Leu Leu Met Gly Glu Gln Leu Gly Asn Val
95 100 105
Ala Arg Gly Ile Asn Ile Ala Ile Val Asn Tyr Val Thr Gly Asn
110 115 120
Val Thr Ala Thr Arg Cys Phe Asp Met Tyr Glu Gly Asp Asn Ser
125 130 135
Gly Pro Met Thr Lys Phe Ile Gln Ser Ala Ala Pro Lys Ser Leu
140 145 150
Leu Phe Met Val Thr Tyr Asp Asp Gly Ser Thr Arg Leu Asn Asn
155 160 165
Asp Ala Lys Asn Ala Ile Glu Ala Leu Gly Ser Lys Glu Ile Arg
170 175 180
Asn Met Lys Phe Arg Ser Ser Trp Val Phe Ile Ala Ala Lys Gly
185 190 195
Leu Glu Leu Pro Ser Glu Ile Gln Arg Glu Lys Ile Asn His Ser
200 205 210
Asp Ala Lys Asn Asn Arg Tyr Ser Gly Trp Pro Ala Glu Ile Gln
215 220 225
Ile Glu Gly Cys Ile Pro Lys Glu Arg Ser
230 235
<210> 33
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
27
CA 02372511 2002-05-14
<400> 33
ggctggcctg cagagatc 18
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 34
aatgtgacca ctggactccc 20
<210> 35
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 35
aggcttggaa ctcccttc 18
<210> 36
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 36
aagattcttg agcgattcca gctg 24
<210> 37
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 37
aatccctgct cttcatggtg acctatgacg acggaagcac aagactg 47
<210> 38
<211> 1215
<212> DNA
<213> Homo Sapien
<400> 38
ccggggaggg gagggcccgt cccgcccctc cccgtctctc cccgcccctc 50
cccgtccctc ccgccgaagc tccgtcccgc ccgcgggccg gctccgccct 100
cacctcccgg ccgcggctgc cctctgcccg ggttgtccaa gatggagggc 150
gctccaccgg ggtcgctcgc cctccggctc ctgctgttcg tggcgctacc 200
cgcctccggc tggctgacga cgggcgcccc cgagccgccg ccgctgtccg 250
28
CA 02372511 2002-05-14
gagccccaca ggacggcatc agaattaatg taactacact gaaagatgat 300
ggggacatat ctaaacagca ggttgttctt aacataacct atgagagtgg 350
acaggtgtat gtaaatgact tacctgtaaa tagtggtgta acccgaataa 400
gctgtcagac tttgatagtg aagaatgaaa atcttgaaaa tttggaggaa 450
aaagaatatt ttggaattgt cagtgtaagg attttagttc atgagtggcc 500
tatgacatct ggttccagtt tgcaactaat tgtcattcaa gaagaggtag 550
tagagattga tggaaaacaa gttcagcaaa aggatgtcac tgaaattgat 600
attttagtta agaaccgggg agtactcaga cattcaaact ataccctccc 650
tttggaagaa agcatgctct actctatttc tcgagacagt gacattttat 700
ttacccttcc taacctctcc aaaaaagaaa gtgttagttc actgcaaacc 750
actagccagt atcttatcag gaatgtggaa accactgtag atgaagatgt 800
tttacctggc aagttacctg aaactcctct cagagcagag ccgccatctt 850
catataaggt aatgtgtcag tggatggaaa agtttagaaa agatctgtgt 900
aggttctgga gcaacgtttt cccagtattc tttcagtttt tgaacatcat 950
ggtggttgga attacaggag cagctgtggt aataaccatc ttaaaggtgt 1000
ttttcccagt ttctgaatac aaaggaattc ttcagttgga taaagtggac 1050
gtcatacctg tgacagctat caacttatat ccagatggtc cagagaaaag 1100
agctgaaaac cttgaagata aaacatgtat ttaaaacgcc atctcatatc 1150
atggactccg aagtagcctg ttgcctccaa atttgccact tgaatataat 1200
tttctttaaa tcgtt 1215
<210> 39
<211> 330
<212> PRT
<213> Homo Sapien
<400> 39
Met Glu Gly Ala Pro Pro Gly Ser Leu Ala Leu Arg Leu Leu Leu
1 5 10 15
Phe Val Ala Leu Pro Ala Ser Gly Trp Leu Thr Thr Gly Ala Pro
20 25 30
Glu Pro Pro Pro Leu Ser Gly Ala Pro Gln Asp Gly Ile Arg Ile
35 40 45
Asn Val Thr Thr Leu Lys Asp Asp Gly Asp Ile Ser Lys Gln Gln
50 55 60
Val Val Leu Asn Ile Thr Tyr Glu Ser Gly Gln Val Tyr Val Asn
65 70 75
Asp Leu Pro Val Asn Ser Gly Val Thr Arg Ile Ser Cys Gln Thr
80 85 90
29
CA 02372511 2002-05-14
Leu Ile Val Lys Asn Glu Asn Leu Glu Asn Leu Glu Glu Lys Glu
95 100 105
Tyr Phe Gly Ile Val Ser Val Arg Ile Leu Val His Glu Trp Pro
110 115 120
Met Thr Ser Gly Ser Ser Leu Gln Leu Ile Val Ile Gln Glu Glu
125 130 135
Val Val Glu Ile Asp Gly Lys Gln Val Gln Gln Lys Asp Val Thr
140 145 150
Glu Ile Asp Ile Leu Val Lys Asn Arg Gly Val Leu Arg His Ser
155 160 165
Asn Tyr Thr Leu Pro Leu Glu Glu Ser Met Leu Tyr Ser Ile Ser
170 175 180
Arg Asp Ser Asp Ile Leu Phe Thr Leu Pro Asn Leu Ser Lys Lys
185 190 195
Glu Ser Val .Ser Ser Leu Gln Thr Thr Ser Gln Tyr Leu Ile Arg
200 205 210
Asn Val Glu Thr Thr Val Asp Glu Asp Val Leu Pro Gly Lys Leu
215 220 225
Pro Glu Thr Pro Leu Arg Ala Glu Pro Pro Ser Ser Tyr Lys Val
230 235 240
Met Cys Gln Trp Met Glu Lys Phe Arg Lys Asp Leu Cys Arg Phe
245 250 255
Trp Ser Asn Val Phe Pro Val Phe Phe Gln Phe Leu Asn Ile Met
260 265 270
Val Val Gly Ile Thr Gly Ala Ala Val Val Ile Thr Ile Leu Lys
275 280 285
Val Phe Phe Pro Val Ser Glu Tyr Lys Gly Ile Leu Gln Leu Asp
290 295 300
Lys Val Asp Val Ile Pro Val Thr Ala Ile Asn Leu Tyr Pro Asp
305 310 315
Gly Pro Glu Lys Arg Ala Glu Asn Leu Glu Asp Lys Thr Cys Ile
320 325 330
<210> 40
<211> 2498
<212> DNA
<213> Homo Sapien
<400> 40
cgtctctgcg ttcgccatgc gtcccggggc gccagggcca ctctggcctc 50
tgccctgggg ggccctggct tgggccgtgg gcttcgtgag ctccatgggc 100
tcggggaacc c.cgcgcccgg tggtgtttgc tggctccagc agggccagga 150
ggccacctgc agcctggtgc tccagactga tgtcacccgg gccgagtgct 200
gtgcctccgg caacattgac accgcctggt ccaacctcac ccacccgggg 250
CA 02372511 2002-05-14
aacaagatca acctcctcgg cttcttgggc cttgtccact gccttccctg 300
caaagattcg tgcgacggcg tggagtgcgg cccgggcaag gcgtgccgca 350
tgctgggggg ccgcccgcgc tgcgagtgcg cgcccgactg ctcggggctc 400
ccggcgcggc tgcaggtctg cggctcagac ggcgccacct accgcgacga 450
gtgcgagctg cgcgccgcgc gctgccgcgg ccacccggac ctgagcgtca 500
tgtaccgggg ccgctgccgc aagtcctgtg agcacgtggt gtgcccgcgg 550
ccacagtcgt gcgtcgtgga ccagacgggc agcgcccact gcgtggtgtg 600
tcgagcggcg c.cctgccctg tgccctccag ccccggccag gagctttgcg 650
gcaacaacaa cgtcacctac atctcctcgt gccacatgcg ccaggccacc 700
tgcttcctgg gccgctccat cggcgtgcgc cacgcgggca gctgcgcagg 750
cacccctgag gagccgccag gtggtgagtc tgcagaagag gaagagaact 800
tcgtgtgagc ctgcaggaca ggcctgggcc tggtgcccga ggccccccat 850
catcccctgt tatttattgc cacagcagag tctaatttat atgccacgga 900
cactccttag a.gcccggatt cggaccactt ggggatccca gaacctccct 950
gacgatatcc tggaaggact gaggaaggga ggcctggggg ccggctggtg 1000
ggtgggatag acctgcgttc cggacactga gcgcctgatt tagggccctt 1050
ctctaggatg ccccagcccc taccctaaga cctattgccg gggaggattc 1100
cacacttccg ctcctttggg gataaaccta ttaattattg ctactatcaa 1150
gagggctggg cattctctgc tggtaattcc tgaagaggca tgactgcttt 1200
tctcagcccc aagcctctag tctgggtgtg tacggagggt ctagcctggg 1250
tgtgtacgga gggtctagcc tgggtgagta cggagggtct agcctgggtg 1300
agtacggagg gtctagcctg ggtgagtacg gagggtctag cctgggtgtg 1350
tatggaggat ctagcctggg tgagtatgga gggtctagcc tgggtgagta 1400
tggagggtct agcctgggtg tgtatggagg gtctagcctg ggtgagtatg 1450
gagggtctag cctgggtgtg tatggagggt ctagcctggg tgagtatgga 1500
gggtctagcc tgggtgtgta cggagggtct agtctgagtg cgtgtgggga 1550
cctcagaaca ctgtgacctt agcccagcaa gccaggccct tcatgaaggc 1600
caagaaggct gccaccattc cctgccagcc caagaactcc agcttcccca 1650
ctgcctctgt gtgccccttt gcgtcctgtg aaggccattg agaaatgccc 1700
agtgtgcccc ctgggaaagg gcacggcctg tgctcctgac acgggctgtg 1750
cttggccaca gaaccaccca gcgtctcccc tgctgctgtc cacgtcagtt 1800
catgaggcaa cgtcgcgtgg tctcagacgt ggagcagcca gcggcagctc 1850
31
CA 02372511 2002-05-14
agagcagggc actgtgtccg gcggagccaa gtccactctg ggggagctct 1900
ggcggggacc acgggccact gctcacccac tggccccgag gggggtgtag 1950
acgccaagac tcacgcatgt gtgacatccg gagtcctgga gccgggtgtc 2000
ccagtggcac cactaggtgc ctgctgcctc cacagtgggg ttcacaccca 2050
gggctccttg gtcccccaca acctgccccg gccaggcctg cagacccaga 2100
ctccagccag acctgcctca cccaccaatg cagccggggc tggcgacacc 2150
agccaggtgc tggtcttggg ccagttctcc cacgacggct caccctcccc 2200
tccatctgcg ttgatgctca gaatcgccta cctgtgcctg cgtgtaaacc 2250
acagcctcag accagctatg gggagaggac aacacggagg atatccagct 2300
tccccggtct ggggtgagga atgtggggag cttgggcatc ctcctccagc 2350
ctcctccagc ccccaggcag tgccttacct gtggtgccca gaaaagtgcc 2400
cctaggttgg tgggtctaca ggagcctcag ccaggcagcc caccccaccc 2450
tggggccctg cctcaccaag gaaataaaga ctcaagccat aaaaaaaa 2498
<210> 41
<211> 263
<212> PRT
<213> Homo Sapien
<400> 41
Met Arg Pro Gly Ala Pro Gly Pro Leu Trp Pro Leu Pro Trp Gly
1 5 10 15
Ala Leu Ala Trp Ala Val Gly Phe Val Ser Ser Met Gly Ser Gly
20 25 30
Asn Pro Ala Pro Gly Gly Val Cys Trp Leu Gln Gln Gly Gln Glu
35 40 45
Ala Thr Cys Ser Leu Val Leu Gln Thr Asp Val Thr Arg Ala Glu
50 55 60
Cys Cys Ala Ser Gly Asn Ile Asp Thr Ala Trp Ser Asn Leu Thr
65 70 75
His Pro Gly Asn Lys Ile Asn Leu Leu Gly Phe Leu Gly Leu Val
80 85 90
His Cys Leu Pro Cys Lys Asp Ser Cys Asp Gly Val Glu Cys Gly
95 100 105
Pro Gly Lys Ala Cys Arg Met Leu Gly Gly Arg Pro Arg Cys Glu
110 115 120
Cys Ala Pro Asp Cys Ser Gly Leu Pro Ala Arg Leu Gln Val Cys
125 130 135
Gly Ser Asp Gly Ala Thr Tyr Arg Asp Glu Cys Glu Leu Arg Ala
140 145 150
Ala Arg Cys Arg Gly His Pro Asp Leu Ser Val Met Tyr Arg Gly
32
CA 02372511 2002-05-14
155 160 165
Arg Cys Arg Lys Ser Cys Glu His Val Val Cys Pro Arg Pro Gln
170 175 180
Ser Cys Val Val Asp Gln Thr Gly Ser Ala His Cys Val Val Cys
185 190 195
Arg Ala Ala Pro Cys Pro Val Pro Ser Ser Pro Gly Gln Glu Leu
200 205 210
Cys Gly Asn Asn Asn Val Thr Tyr Ile Ser Ser Cys His Met Arg
215 220 225
Gln Ala Thr Cys Phe Leu Gly Arg Ser Ile Gly Val Arg His Ala
230 235 240
Gly Ser Cys Ala Gly Thr Pro Glu Glu Pro Pro Gly Gly Glu Ser
245 250 255
Ala Glu Glu Clu Glu Asn Phe Val
260
<210> 42
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 42
tcctgtgagc acgtggtgtg 20
<210> 43
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 43
gggtgggata gacctgcg 18
<210> 44
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 44
aaggccaaga aggctgcc 18
<210> 45
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
33
CA 02372511 2002-05-14
<400> 45
ccaggcctgc agacccag 18
<210> 46
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 46
cttcctcagt ccttccagga tatc 24
<210> 47
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 47
aagctggata tcctccgtgt tgtc 24
<210> 48
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 48
cctgaagagg catgactgct tttctca 27
<210> 49
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 49
ggggataaac ctattaatta ttgctac 27
<210> 50
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 50
aacgtcacct acatctcctc gtgccacatg cgccaggcca cctg 44
<210> 51
<211> 1690
<212> DNA
<213> Homo Sapien
34
CA 02372511 2002-05-14
<400> 51
tgcagagctt gtggaggcca tggggcgcgt cgtcgcggag ctcgtctcct 50
cgctgctggg gttgtggctg ttgctgtgca gctgcggatg ccccgagggc 100
gccgagctgc gtgctccgcc agataaaatc gcgattattg gagccggaat 150
tggtggcact tcagcagcct attacctgcg gcagaaattt gggaaagatg 200
tgaagataga cctgtttgaa agagaagagg tcgggggccg cctggctacc 250
atgatggtgc aggggcaaga atacgaggca ggaggttctg tcatccatcc 300
tttaaatctg cacatgaaac gttttgtcaa agacctgggt ctctctgctg 350
ttcaggcctc tggtggccta ctggggatat ataatggaga gactctggta 400
tttgaggaga gcaactggtt cataattaac gtgattaaat tagtttggcg 450
ctatggattt caatccctcc gtatgcacat gtgggtagag gacgtgttag 500
acaagttcat gaggatctac cgctaccagt ctcatgacta tgccttcagt 550
agtgtcgaaa aattacttca tgctctagga ggagatgact tccttggaat 600
gcttaatcga acacttcttg aaaccttgca aaaggccggc ttttctgaga 650
agttcctcaa taaaatgatt gctcctgtta tgagggtcaa ttatggccaa 700
agcacggaca tcaatgcctt tgtgggggcg gtgtcactgt cctgttctga 750
ttctggcctt tgggcagtag aaggtggcaa taaacttgtt tgctcagggc 800
ttctgcaggc atccaaaagc aatcttatat ctggctcagt aatgtacatc 850
gaggagaaaa caaagaccaa gtacacagga aatccaacaa agatgtatga 900
agtggtctac caaattggaa ctgagactcg ttcagacttc tatgacatcg 950
tcttggtggc cactccgttg aatcgaaaaa tgtcgaatat tacttttctc 1000
aactttgatc ctccaattga ggaattccat caatattatc aacatatagt 1050
gacaacttta gttaaggggg aattgaatac atctatcttt agctctagac 1100
ccatagataa atttggcctt aatacagttt taaccactga taattcagat 1150
ttgttcatta acagtattgg gattgtgccc tctgtgagag aaaaggaaga 1200
tcctgagcca tcaacagatg gaacatatgt ttggaagatc ttttcccaag 1250
aaactcttac taaagcacaa attttaaagc tctttctgtc ctatgattat 1300
gctgtgaaga agccatggct tgcatatcct cactataagc ccccggagaa 1350
atgcccctct atcattctcc atgatcgact ttattacctc aatggcatag 1400
agtgtgcagc aagtgccatg gagatgagtg ccattgcagc ccacaacgct 1450
gcactccttg cctatcaccg ctggaacggg cacacagaca tgattgatca 1500
ggatggctta tatgagaaac ttaaaactga actatgaagt gacacactcc 1550
CA 02372511 2002-05-14
tttttcccct c.ctagttcca aatgactatc agtggcaaaa aagaacaaaa 1600
tctgagcaga gatgattttg aaccagatat tttgccatta tcattgttta 1650
ataaaagtaa tccctgctgg tcataggaaa aaaaaaaaaa 1690
<210> 52
<211> 505
<212> PRT
<213> Homo Sapien
<400> 52
Met Gly Arg Val Val Ala Glu Leu Val Ser Ser Leu Leu Gly Leu
1 5 10 15
Trp Leu Leu Leu Cys Ser Cys Gly Cys Pro Glu Gly Ala Glu Leu
20 25 30
Arg Ala Pro Pro Asp Lys Ile Ala Ile Ile Gly Ala Gly Ile Gly
35 40 45
Gly Thr Ser Ala Ala Tyr Tyr Leu Arg Gln Lys Phe Gly Lys Asp
50 55 60
Val Lys Ile Asp Leu Phe Glu Arg Glu Glu Val Gly Gly Arg Leu
65 70 75
Ala Thr Met Met Val Gln Gly Gln Glu Tyr Glu Ala Gly Gly Ser
80 85 90
Val Ile His Pro Leu Asn Leu His Met Lys Arg Phe Val Lys Asp
95 100 105
Leu Gly Leu Ser Ala Val Gln Ala Ser Gly Gly Leu Leu Gly Ile
110 115 120
Tyr Asn Gly Glu Thr Leu Val Phe Glu Glu Ser Asn Trp Phe Ile
125 130 135
Ile Asn Val Ile Lys Leu Val Trp Arg Tyr Gly Phe Gln Ser Leu
140 145 150
Arg Met His Met Trp Val Glu Asp Val Leu Asp Lys Phe Met Arg
155 160 165
Ile Tyr Arg Tyr Gln Ser His Asp Tyr Ala Phe Ser Ser Val Glu
170 175 180
Lys Leu Leu His Ala Leu Gly Gly Asp Asp Phe Leu Gly Met Leu
185 190 195
Asn Arg Thr Leu Leu Glu Thr Leu Gln Lys Ala Gly Phe Ser Glu
200 205 210
Lys Phe Leu Asn Glu Met Ile Ala Pro Val Met Arg Val Asn Tyr
215 220 225
Gly Gln Ser Thr Asp Ile Asn Ala Phe Val Gly Ala Val Ser Leu
230 235 240
Ser Cys Ser Asp Ser Gly Leu Trp Ala Val Glu Gly Gly Asn Lys
245 250 255
36
CA 02372511 2002-05-14
Leu Val Cys Ser Gly Leu Leu Gln Ala Ser Lys Ser Asn Leu Ile
260 265 270
Ser Gly Ser Val Met Tyr Ile Glu Glu Lys Thr Lys Thr Lys Tyr
275 280 285
Thr Gly Asn Pro Thr Lys Met Tyr Glu Val Val Tyr Gln Ile Gly
290 295 300
Thr Glu Thr Arg Ser Asp Phe Tyr Asp Ile Val Leu Val Ala Thr
305 310 315
Pro Leu Asn Arg Lys Met Ser Asn Ile Thr Phe Leu Asn Phe Asp
320 325 330
Pro Pro Ile jGlu Glu Phe His Gln Tyr Tyr Gln His Ile Val Thr
335 340 345
Thr Leu Val Lys Gly Glu Leu Asn Thr Ser Ile Phe Ser Ser Arg
350 355 360
Pro Ile Asp Lys Phe Gly Leu Asn Thr Val Leu Thr Thr Asp Asn
365 370 375
Ser Asp Leu Phe Ile Asn Ser Ile Gly Ile Val Pro Ser Val Arg
380 385 390
Glu Lys Glu Asp Pro Glu Pro Ser Thr Asp Gly Thr Tyr Val Trp
395 400 405
Lys Ile Phe Ser Gln Glu Thr Leu Thr Lys Ala Gln Ile Leu Lys
410 415 420
Leu Phe Leu Ser Tyr Asp Tyr Ala Val Lys Lys Pro Trp Leu Ala
425 430 435
Tyr Pro His Tyr Lys Pro Pro Glu Lys Cys Pro Ser Ile Ile Leu
440 445 450
His Asp Arg Leu Tyr Tyr Leu Asn Gly Ile Glu Cys Ala Ala Ser
455 460 465
Ala Met Glu Met Ser Ala Ile Ala Ala His Asn Ala Ala Leu Leu
470 475 480
Ala Tyr His Arg Trp Asn Gly His Thr Asp Met Ile Asp Gln Asp
485 490 495
Gly Leu Tyr Glu Lys Leu Lys Thr Glu Leu
500 505
<210> 53
<211> 728
<212> DNA
<213> Homo Sapien
<400> 53
catttccaac aagagcactg gccaagtcag cttcttctga gagagtctct 50
agaagacatg atgctacact cagctttggg tctctgcctc ttactcgtca 100
cagtttcttc caaccttgcc attgcaataa aaaaggaaaa gaggcctcct 150
37
CA 02372511 2002-05-14
cagacactct caagaggatg gggagatgac atcacttggg tacaaactta 200
tgaagaaggt ctcttttatg ctcaaaaaag taagaagcca ttaatggtta 250
ttcatcacct ggaggattgt caatactctc aagcactaaa gaaagtattt 300
gcccaaaatg aagaaataca agaaatggct cagaataagt tcatcatgct 350
aaaccttatg c.atgaaacca ctgataagaa tttatcacct gatgggcaat 400
atgtgcctag aatcatgttt gtagaccctt ctttaacagt tagagctgac 450
atagctggaa gatactctaa cagattgtac acatatgagc ctcgggattt 500
acccctattg atagaaaaca tgaagaaagc attaagactt attcagtcag 550
agctataaga gatgatggaa aaaagccttc acttcaaaga agtcaaattt 600
catgaagaaa acctctggca cattgacaaa tactaaatgt gaaagtatat 650
agattttgta atattactat ttagtttttt taatgtgttt gcaatagtct 700
tattaaaata attgtttttt aaatctga 728
<210> 54
<211> 166
<212> PRT
<213> Homo Sapien
<400> 54
Met Met Leu His Ser Ala Leu Gly Leu Cys Leu Leu Leu Val Thr
1 5 10 15
Val Ser Ser Asn Leu Ala Ile Ala Ile Lys Lys Glu Lys Arg Pro
20 25 30
Pro Gln Thr Leu Ser Arg Gly Trp Gly Asp Asp Ile Thr Trp Val
35 40 45
Gln Thr Tyr.Glu Glu Gly Leu Phe Tyr Ala Gln Lys Ser Lys Lys
50 55 60
Pro Leu Met Val Ile His His Leu Glu Asp Cys Gln Tyr Ser Gln
65 70 75
Ala Leu Lys Lys Val Phe Ala Gln Asn Glu Glu Ile Gln Glu Met
80 85 90
Ala Gln Asn Lys Phe Ile Met Leu Asn Leu Met His Glu Thr Thr
95 100 105
Asp Lys Asn Leu Ser Pro Asp Gly Gin Tyr Val Pro Arg Ile Met
110 115 120
Phe Val Asp Pro Ser Leu Thr Val Arg Ala Asp Ile Ala Gly Arg
125 130 135
Tyr Ser Asn Arg Leu Tyr Thr Tyr Glu Pro Arg Asp Leu Pro Leu
140 145 150
Leu Ile Glu Asn Met Lys Lys Ala Leu Arg Leu Ile Gln Ser Glu
155 160 165
38
CA 02372511 2002-05-14
Leu
<210> 55
<211> 537
<212> DNA
<213> Homo Sapien
<400> 55
taaaacagct acaatattcc agggccagtc acttgccatt tctcataaca 50
gcgtcagaga gaaagaactg actgaaacgt ttgagatgaa gaaagttctc 100
ctcctgatca cagccatctt ggcagtggct gttggtttcc cagtctctca 150
agaccaggaa caaaaaaaaa gaagtatcag tgacagcgat gaattagctt 200
cagggttttt tgtgttccct tacccatatc catttcgccc acttccacca 250
attccatttc caagatttcc atggtttaga cgtaattttc ctattccaat 300
acctgaatct gcccctacaa ctccccttcc tagcgaaaag taaacaagaa 350
ggataagtca cgataaacct ggtcacctga aattgaaatt gagccacttc 400
cttgaagaat caaaattcct gttaataaaa gaaaaacaaa tgtaattgaa 450
atagcacaca gcattctcta gtcaatatct ttagtgatct tctttaataa 500
acatgaaagc aaagattttg gtttcttaat ttccaca 537
<210> 56
<211> 85
<212> PRT
<213> Homo Sapien
<400> 56
Met Lys Lys Val Leu Leu Leu Ile Thr Ala Ile Leu Ala Val Ala
1 5 10 15
Val Gly Phe Pro Val Ser Gln Asp Gln Glu Arg Glu Lys Arg Ser
20 25 30
Ile Ser Asp Ser Asp Glu Leu Ala Ser Gly Phe Phe Val Phe Pro
35 40 45
Tyr Pro Tyr Pro Phe Arg Pro Leu Pro Pro Ile Pro Phe Pro Arg
50 55 60
Phe Pro Trp Phe Arg Arg Asn Phe Pro Ile Pro Ile Pro Glu Ser
65 70 75
Ala Pro Thr Thr Pro Leu Pro Ser Glu Lys
80 85
<210> 57
<211> 2997
<212> DNA
<213> Homo Sapien
<400> 57
cggacgcgtg ggcgggcgcg ccgggaggga ccggcggcgg catgggccgg 50
39
CA 02372511 2002-05-14
gggccctggg atgcgggccc gtctcgccgc ctgctgccgc tgttgctgct 100
gctcggcctg gcccgcggcg ccgcgggagc gccgggcccc gacggtttag 150
acgtctgtgc cacttgccat gaacatgcca catgccagca aagagaaggg 200
aagaagatct gtatttgcaa ctatggattt gtagggaacg ggaggactca 250
gtgtgttgat aaaaatgagt gccagtttgg agccactctt gtctgtggga 300
accacacatc ttgccacaac acccccgggg gcttctattg catttgcctg 350
gaaggatatc gagccacaaa caacaacaag acattcattc ccaacgatgg 400
caccttttgt acagacatag atgagtgtga agtttctggc ctgtgcaggc 450
atggagggcg atgcgtgaac actcatggga gctttgaatg ctactgtatg 500
gatggatact tgccaaggaa tggacctgaa cctttccacc cgaccaccga 550
tgccacatca tgcacagaaa tagactgtgg tacccctcct gaggttccag 600
atggctatat cataggaaat tatacgtcta gtctgggcag ccaggttcgt 650
tatgcttgca gagaaggatt cttcagtgtt ccagaagata cagtttcaag 700
ctgcacaggc ctgggcacat gggagtcccc aaaattacat tgccaagaga 750
tcaactgtgg caaccctcca gaaatgcggc acgccatctt ggtaggaaat 800
cacagctcca ggctgggcgg tgtggctcgc tatgtctgtc aagaggcctt 850
tgagagccct ggaggaaaga tcacttctgt ttgcacagag aaaggcacct 900
ggagagaaag tactttaaca tgcacagaaa ttctgacaaa gattaatgat 950
gtatcactgt ttaatgatac ctgtgtgaga tggcaaataa actcaagaag 1000
aataaacccc aagatctcat atgtgatatc cataaaagga caacggttgg 1050
accctatgga atcagttcgt gaggagacag tcaacttgac cacagacagc 1100
aggaccccag aagtgtgcct agccctgtac ccaggcacca actacaccgt 1150
gaacatctcc acagcacctc ccaggcgctc gatgccagcc gtcatcggtt 1200
tccagacagc tgaagttgat ctcttagaag atgatggaag tttcaatatt 1250
tcaatattta atgaaacttg tttgaaattg aacaggcgtt ctaggaaagt 1300
tggatcagaa cacatgtacc aatttaccgt tctgggtcag aggtggtatc 1350
tggctaactt ttctcatgca acatcgttta acttcacaac gagggaacaa 1400
gtgcctgtag tgtgtttgga tctgtaccct acgactgatt atacggtgaa 1450
tgtgaccctg ctgagatctc ctaagcggca ctcagtgcaa ataacaatag 1500
caactccccc agcagtaaaa cagaccatca gtaacatttc aggatttaat 1550
gaaacctgct tgagatggag aagcatcaag acagctgata tggaggagat 1600
gtatttattc cacatttggg gccagagatg gtatcagaag gaatttgccc 1650
CA 02372511 2002-05-14
aggaaatgac ctttaatatc agtagcagca gccgagatcc cgaggtgtgc 1700
ttggacctac gtccgggtac caactacaat gtcagtctcc gggctctgtc 1750
ttcggaactt cctgtggtca tctccctgac aacccagata acagagcctc 1800
ccctcccgga agtagaattt tttacggtgc acagaggacc tctaccacgc 1850
ctcagactga ggaaagccaa ggagaaaaat ggaccaatca gttcatatca 1900
ggtgttagtg cttcccctgg ccctccaaag cacattttct tgtgattctg 1950
aaggcgcttc ctccttcttt agcaacgcct ctgatgctga tggatacgtg 2000
gctgcagaac tactggccaa agatgttcca gatgatgcca tggagatacc 2050
tataggagac aggctgtact atggggaata ttataatgca cccttgaaaa 2100
gagggagtga ttactgcatt atattacgaa tcacaagtga atggaataag 2150
gtgagaagac actcctgtgc agtttgggct caggtgaaag attcgtcact 2200
catgctgctg cagatggcgg gtgttggact gggttccctg gctgttgtga 2250
tcattctcac attcctctcc ttctcagcgg tgtgatggca gatggacact 2300
gagtggggag gatgcactgc tgctgggcag gtgttctggc agcttctcag 2350
gtgcccgcac agaggctccg tgtgacttcc gtccagggag catgtgggcc 2400
tgcaactttc tccattccca gctgggcccc attcctggat ttaagatggt 2450
ggctatccct gaggagtcac cataaggaga aaactcagga attctgagtc 2500
ttccctgcta caggaccagt tctgtgcaat gaacttgaga ctcctgatgt 2550
acactgtgat attgaccgaa ggctacatac agatctgtga atcttggctg 2600
ggacttcctc tgagtgatgc ctgagggtca gctcctctag acattgactg 2650
caagagaatc tctgcaacct cctatataaa agcatttctg ttaattcatt 2700
cagaatccat tctttacaat atgcagtgag atgggcttaa gtttgggcta 2750
gagtttgact ttatgaagga ggtcattgaa aaagagaaca gtgacgtagg 2800
caaatgtttc aagcacttta gaaacagtac ttttcctata attagttgat 2850
atactaatga gaaaatatac tagcctggcc atgccaataa gtttcctgct 2900
gtgtctgtta ggcagcattg ctttgatgca atttctattg tcctatatat 2950
tcaaaagtaa tgtctacatt ccagtaaaaa tatcccgtaa ttaaaaa 2997
<210> 58
<211> 747
<212> PRT
<213> Homo Sapien
<400> 58
Met Gly Arg Gly Pro Trp Asp Ala Gly Pro Ser Arg Arg Leu Leu
1 5 10 15
41
CA 02372511 2002-05-14
Pro Leu Leu Leu Leu Leu Gly Leu Ala Arg Gly Ala Ala Gly Ala
20 25 30
Pro Gly Pro Asp Gly Leu Asp Val Cys Ala Thr Cys His Glu His
35 40 45
Ala Thr Cys Gln Gln Arg Glu Gly Lys Lys Ile Cys Ile Cys Asn
50 55 60
Tyr Gly Phe Val Gly Asn Gly Arg Thr Gln Cys Val Asp Lys Asn
65 70 75
Glu Cys Gln Phe Gly Ala Thr Leu Val Cys Gly Asn His Thr Ser
80 85 90
Cys His Asn Thr Pro Gly Gly Phe Tyr Cys Ile Cys Leu Glu Gly
95 100 105
Tyr Arg Ala Thr Asn Asn Asn Lys Thr Phe Ile Pro Asn Asp Gly
110 115 120
Thr Phe Cys Thr Asp Ile Asp Glu Cys Glu Val Ser Gly Leu Cys
125 130 135
Arg His Gly Gly Arg Cys Val Asn Thr His Gly Ser Phe Glu Cys
140 145 150
Tyr Cys Met Asp Gly Tyr Leu Pro Arg Asn Gly Pro Glu Pro Phe
155 160 165
His Pro Thr Thr Asp Ala Thr Ser Cys Thr Glu Ile Asp Cys Gly
170 175 180
Thr Pro Pro Glu Val Pro Asp Gly Tyr Ile Ile Gly Asn Tyr Thr
185 190 195
Ser Ser Leu Gly Ser Gln Val Arg Tyr Ala Cys Arg Glu Gly Phe
200 205 210
Phe Ser Val Pro Glu Asp Thr Val Ser Ser Cys Thr Gly Leu Gly
215 220 225
Thr Trp Glu Ser Pro Lys Leu His Cys Gln Glu Ile Asn Cys Gly
230 235 240
Asn Pro Pro Glu Met Arg His Ala Ile Leu Val Gly Asn His Ser
245 250 255
Ser Arg Leu Gly Gly Val Ala Arg Tyr Val Cys Gin Glu Gly Phe
260 265 270
Glu Ser Pro Gly Gly Lys Ile Thr Ser Val Cys Thr Glu Lys Gly
275 280 285
Thr Trp Arg Glu Ser Thr Leu Thr Cys Thr Glu Ile Leu Thr Lys
290 295 300
Ile Asn Asp Val Ser Leu Phe Asn Asp Thr Cys Val Arg Trp Gln
305 310 315
Ile Asn Ser Arg Arg Ile Asn Pro Lys Ile Ser Tyr Val Ile Ser
320 325 330
42
CA 02372511 2002-05-14
Ile Lys Gly Gln Arg Leu Asp Pro Met Glu Ser Val Arg Glu Glu
335 340 345
Thr Val Asn Leu Thr Thr Asp Ser Arg Thr Pro Glu Val Cys Leu
350 355 360
Ala Leu Tyr Pro Gly Thr Asn Tyr Thr Val Asn Ile Ser Thr Ala
365 370 375
Pro Pro Arg Arg Ser Met Pro Ala Val Ile Gly Phe Gln Thr Ala
380 385 390
Glu Val Asp Leu Leu Glu Asp Asp Gly Ser Phe Asn Ile Ser Ile
395 400 405
Phe Asn Glu Thr Cys Leu Lys Leu Asn Arg Arg Ser Arg Lys Val
410 415 420
Gly Ser Glu His Met Tyr Gln Phe Thr Val Leu Gly Gln Arg Trp
425 430 435
Tyr Leu Ala Asn Phe Ser His Ala Thr Ser Phe Asn Phe Thr Thr
440 445 450
Arg Glu Gln Val Pro Val Val Cys Leu Asp Leu Tyr Pro Thr Thr
455 460 465
Asp Tyr Thr Val Asn Val Thr Leu Leu Arg Ser Pro Lys Arg His
470 475 480
Ser Val Gln Ile Thr Ile Ala Thr Pro Pro Ala Val Lys Gln Thr
485 490 495
Ile Ser Asn Ile Ser Gly Phe Asn Glu Thr Cys Leu Arg Trp Arg
500 505 510
Ser Ile Lys Thr Ala Asp Met Glu Glu Met Tyr Leu Phe His Ile
515 520 525
Trp Gly Gln Arg Trp Tyr Gln Lys Glu Phe Ala Gln Glu Met Thr
530 535 540
Phe Asn Ile Ser Ser Ser Ser Arg Asp Pro Glu Val Cys Leu Asp
545 550 555
Leu Arg Pro Gly Thr Asn Tyr Asn Val Ser Leu Arg Ala Leu Ser
560 565 570
Ser Glu Leu Pro Val Val Ile Ser Leu Thr Thr Gln Ile Thr Glu
575 580 585
Pro Pro Leu Pro Glu Val Glu Phe Phe Thr Val His Arg Gly Pro
590 595 600
Leu Pro Arg Leu Arg Leu Arg Lys Ala Lys Glu Lys Asn Gly Pro
605 610 615
Ile Ser Ser Tyr Gln Val Leu Val Leu Pro Leu Ala Leu Gln Ser
620 625 630
Thr Phe Ser Cys Asp Ser Glu Gly Ala Ser Ser Phe Phe Ser Asn
635 640 645
43
CA 02372511 2002-05-14
Ala Ser Asp Ala Asp Gly Tyr Val Ala Ala Glu Leu Leu Ala Lys
650 655 660
Asp Val Pro Asp Asp Ala Met Glu Ile Pro Ile Gly Asp Arg Leu
665 670 675
Tyr Tyr Gly Glu Tyr Tyr Asn Ala Pro Leu Lys Arg Gly Ser Asp
680 685 690
Tyr Cys Ile Ile Leu Arg Ile Thr Ser Glu Trp Asn Lys Val Arg
695 700 705
Arg His Ser Cys Ala Val Trp Ala Gln Val Lys Asp Ser Ser Leu
710 715 720
Met Leu Leu Gln Met Ala Gly Val Gly Leu Gly Ser Leu Ala Val
725 730 735
Val Ile Ile Leu Thr Phe Leu Ser Phe Ser Ala Val
740 745
<210> 59
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 59
ccacttgcca tgaacatgcc ac 22
<210> 60
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 60
cctcttgaca gacatagcga gccac 25
<210> 61
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 61
cactcttgtc tgtgggaacc acacatcttg ccacaactgt ggc 43
<210> 62
<211> 2015
<212> DNA
<213> Homo Sapien
<400> 62
ggaaaaggta cccgcgagag acagccagca gttctgtgga gcagcggtgg 50
ccggctagga tgggctgtct ctggggtctg gctctgcccc ttttcttctt 100
44
CA 02372511 2002-05-14
ctgctgggag gttggggtct ctgggagctc tgcaggcccc agcacccgca 150
gagcagacac t.gcgatgaca acggacgaca cagaagtgcc cgctatgact 200
ctagcaccgg g.ccacgccgc tctggaaact caaacgctga gcgctgagac 250
ctcttctagg gcctcaaccc cagccggccc cattccagaa gcagagacca 300
ggggagccaa gagaatttcc cctgcaagag agaccaggag tttcacaaaa 350
acatctccca acttcatggt gctgatcgcc acctccgtgg agacatcagc 400
cgccagtggc agccccgagg gagctggaat gaccacagtt cagaccatca 450
caggcagtga tcccgaggaa gccatctttg acaccctttg caccgatgac 500
agctctgaag aggcaaagac actcacaatg gacatattga cattggctca 550
cacctccaca gaagctaagg gcctgtcctc agagagcagt gcctcttccg 600
acggccccca tccagtcatc accccgtcac gggcctcaga gagcagcgcc 650
tcttccgacg gcccccatcc agtcatcacc ccgtcacggg cctcagagag 700
cagcgcctct tccgacggcc cccatccagt catcaccccg tcatggtccc 750
cgggatctga tgtcactctc ctcgctgaag ccctggtgac tgtcacaaac 800
atcgaggtta ttaattgcag catcacagaa atagaaacaa caacttccag 850
catccctggg gcctcagaca tagatctcat ccccacggaa ggggtgaagg 900
cctcgtccac ctccgatcca ccagctctgc ctgactccac tgaagcaaaa 950
ccacacatca ctgaggtcac agcctctgcc gagaccctgt ccacagccgg 1000
caccacagag tcagctgcac ctcatgccac ggttgggacc ccactcccca 1050
ctaacagcgc cacagaaaga gaagtgacag cacccggggc cacgaccctc 1100
agtggagctc tggtcacagt tagcaggaat cccctggaag aaacctcagc 1150
cctctctgtt gagacaccaa gttacgtcaa agtctcagga gcagctccgg 1200
tctccataga ggctgggtca gcagtgggca aaacaacttc ctttgctggg 1250
agctctgctt cctcctacag cccctcggaa gccgccctca agaacttcac 1300
cccttcagag acaccgacca tggacatcgc aaccaagggg cccttcccca 1350
ccagcaggga c-cctcttcct tctgtccctc cgactacaac caacagcagc 1400
cgagggacga a.cagcacctt agccaagatc acaacctcag cgaagaccac 1450
gatgaagccc caacagccac gcccacgact gcccggacga ggccgaccac 1500
agacgtgagt g.caggtgaaa atggaggttt cctcctcctg cggctgagtg 1550
tggcttcccc ggaagacctc actgacccca gagtggcaga aaggctgatg 1600
cagcagctcc accgggaact ccacgcccac gcgcctcact tccaggtctc 1650
cttactgcgt gtcaggagag gctaacggac atcagctgca gccaggcatg 1700
CA 02372511 2002-05-14
tcccgtatgc caaaagaggg tgctgcccct agcctgggcc cccaccgaca 1750
gactgcagct gcgttactgt gctgagaggt acccagaagg ttcccatgaa 1800
gggcagcatg tccaagcccc taaccccaga tgtggcaaca ggaccctcgc 1850
tcacatccac cggagtgtat gtatggggag gggcttcacc tgttcccaga 1900
ggtgtccttg gactcacctt ggcacatgtt ctgtgtttca gtaaagagag 1950
acctgatcac ccatctgtgt gcttccatcc tgcattaaaa ttcactcagt 2000
gtggcccaaa aaaaa 2015
<210> 63
<211> 482
<212> PRT
<213> Homo Sapien
<400> 63
Met Gly Cys Leu Trp Gly Leu Ala Leu Pro Leu Phe Phe Phe Cys
1 5 10 15
Trp Glu Val Gly Val Ser Gly Ser Ser Ala Gly Pro Ser Thr Arg
20 25 30
Arg Ala Asp Thr Ala Met Thr Thr Asp Asp Thr Glu Val Pro Ala
35 40 45
Met Thr Leu Ala Pro Gly His Ala Ala Leu Glu Thr Gln Thr Leu
50 55 60
Ser Ala Glu Thr Ser Ser Arg Ala Ser Thr Pro Ala Gly Pro Ile
65 70 75
Pro Glu Ala Glu Thr Arg Gly Ala Lys Arg Ile Ser Pro Ala Arg
80 85 90
Glu Thr Arg Ser Phe Thr Lys Thr Ser Pro Asn Phe Met Val Leu
95 100 105
Ile Ala Thr Ser Val Glu Thr Ser Ala Ala Ser Gly Ser Pro Glu
110 115 120
Gly Ala Gly Met Thr Thr Val Gln Thr Ile Thr Gly Ser Asp Pro
125 130 135
Glu Glu Ala Ile Phe Asp Thr Leu Cys Thr Asp Asp Ser Ser Glu
140 145 150
Glu Ala Lys Thr Leu Thr Met Asp Ile Leu Thr Leu Ala His Thr
155 160 165
Ser Thr Glu Ala Lys Gly Leu Ser Ser Glu Ser Ser Ala Ser Ser
170 175 180
Asp Gly Pro His Pro Val Ile Thr Pro Ser Arg Ala Ser Glu Ser
185 190 195
Ser Ala Ser Ser Asp Gly Pro His Pro Val Ile Thr Pro Ser Arg
200 205 210
Ala Ser Glu Ser Ser Ala Ser Ser Asp Gly Pro His Pro Val Ile
46
CA 02372511 2002-05-14
215 220 225
Thr Pro Ser Trp Ser Pro Gly Ser Asp Val Thr Leu Leu Ala Glu
230 235 240
Ala Leu Val Thr Val Thr Asn Ile Glu Val Ile Asn Cys Ser Ile
245 250 255
Thr Glu Ile Glu Thr Thr Thr Ser Ser Ile Pro Gly Ala Ser Asp
260 265 270
Ile Asp Leu Ile Pro Thr Glu Gly Val Lys Ala Ser Ser Thr Ser
275 280 285
Asp Pro Pro Ala Leu Pro Asp Ser Thr Glu Ala Lys Pro His Ile
290 295 300
Thr Glu Val Thr Ala Ser Ala Glu Thr Leu Ser Thr Ala Gly Thr
305 310 315
Thr Glu Ser Ala Ala Pro His Ala Thr Val Gly Thr Pro Leu Pro
320 325 330
Thr Asn Ser Ala Thr Glu Arg Glu Val Thr Ala Pro Gly Ala Thr
335 340 345
Thr Leu Ser.Gly Ala Leu Val Thr Val Ser Arg Asn Pro Leu Glu
350 355 360
Glu Thr Ser Ala Leu Ser Val Glu Thr Pro Ser Tyr Val Lys Val
365 370 375
Ser Gly Ala Ala Pro Val Ser Ile Glu Ala Gly Ser Ala Val Gly
380 385 390
Lys Thr Thr Ser Phe Ala Gly Ser Ser Ala Ser Ser Tyr Ser Pro
395 400 405
Ser Glu Ala Ala Leu Lys Asn Phe Thr Pro Ser Glu Thr Pro Thr
410 415 420
Met Asp Ile Ala Thr Lys Gly Pro Phe Pro Thr Ser Arg Asp Pro
425 430 435
Leu Pro Ser Val Pro Pro Thr Thr Thr Asn Ser Ser Arg Gly Thr
440 445 450
Asn Ser Thr Leu Ala Lys Ile Thr Thr Ser Ala Lys Thr Thr Met
455 460 465
Lys Pro Gin Gin Pro Arg Pro Arg Leu Pro Gly Arg Gly Arg Pro
470 475 480
Gln Thr
<210> 64
<211> 1252
<212> DNA
<213> Homo Sapien
<400> 64
gcctctgaat tgttgggcag tctggcagtg gagctctccc cggtctgaca 50
47
CA 02372511 2002-05-14
gccactccag aggccatgct tcgtttcttg ccagatttgg ctttcagctt 100
cctgttaatt ctggctttgg gccaggcagt ccaatttcaa gaatatgtct 150
ttctccaatt tctgggctta gataaggcgc cttcacccca gaagttccaa 200
cctgtgcctt atatcttgaa gaaaattttc caggatcgcg aggcagcagc 250
gaccactggg gtctcccgag acttatgcta cgtaaaggag ctgggcgtcc 300
gcgggaatgt acttcgcttt ctcccagacc aaggtttctt tctttaccca 350
aagaaaattt cccaagcttc ctcctgcctg cagaagctcc tctactttaa 400
cctgtctgcc atcaaagaaa gggaacagtt gacattggcc cagctgggcc 450
tggacttggg gcccaattct tactataacc tgggaccaga gctggaactg 500
gctctgttcc t.ggttcagga gcctcatgtg tggggccaga ccacccctaa 550
gccaggtaaa atgtttgtgt tgcggtcagt cccatggcca caaggtgctg 600
ttcacttcaa c.ctgctggat gtagctaagg attggaatga caacccccgg 650
aaaaatttcg ggttattcct ggagatactg gtcaaagaag atagagactc 700
aggggtgaat tttcagcctg aagacacctg tgccagacta agatgctccc 750
ttcatgcttc cctgctggtg gtgactctca accctgatca gtgccaccct 800
tctcggaaaa ggagagcagc catccctgtc cccaagcttt cttgtaagaa 850
cctctgccac cgtcaccagc tattcattaa cttccgggac ctgggttggc 900
acaagtggat cattgccccc aaggggttca tggcaaatta ctgccatgga 950
gagtgtccct t.ctcactgac catctctctc aacagctcca attatgcttt 1000
catgcaagcc ctgatgcatg ccgttgaccc agagatcccc caggctgtgt 1050
gtatccccac caagctgtct cccatttcca tgctctacca ggacaataat 1100
gacaatgtca ttctacgaca ttatgaagac atggtagtcg atgaatgtgg 1150
gtgtgggtag gatgtcagaa atgggaatag aaggagtgtt cttagggtaa 1200
atcttttaat aaaactacct atctggttta tgaccactta gatcgaaatg 1250
tc 1252
<210> 65
<211> 364
<212> PRT
<213> Homo Sapien
<400> 65
Met Leu Arg Phe Leu Pro Asp Leu Ala Phe Ser Phe Leu Leu Ile
1 5 10 15
Leu Ala Leu Gly Gln Ala Val Gln Phe Gln Glu Tyr Val Phe Leu
20 25 30
Gln Phe Leu Gly Leu Asp Lys Ala Pro Ser Pro Gln Lys Phe Gln
48
CA 02372511 2002-05-14
35 40 45
Pro Val Pro Tyr Ile Leu Lys Lys Ile Phe Gln Asp Arg Glu Ala
50 55 60
Ala Ala Thr Thr Gly Val Ser Arg Asp Leu Cys Tyr Val Lys Glu
65 70 75
Leu Gly Val Arg Gly Asn Val Leu Arg Phe Leu Pro Asp Gln Gly
80 85 90
Phe Phe Leu Tyr Pro Lys Lys Ile Ser Gln Ala Ser Ser Cys Leu
95 100 105
Gln Lys Leu Leu Tyr Phe Asn Leu Ser Ala Ile Lys Glu Arg Glu
110 115 120
Gln Leu Thr Leu Ala Gln Leu Gly Leu Asp Leu Gly Pro Asn Ser
125 130 135
Tyr Tyr Asn Leu Gly Pro Glu Leu Glu Leu Ala Leu Phe Leu Val
140 145 150
Gln Glu Pro His Val Trp Gly Gln Thr Thr Pro Lys Pro Gly Lys
155 160 165
Met Phe Val Leu Arg Ser Val Pro Trp Pro Gln Gly Ala Val His
170 175 180
Phe Asn Leu Leu Asp Val Ala Lys Asp Trp Asn Asp Asn Pro Arg
185 190 195
Lys Asn Phe Gly Leu Phe Leu Glu Ile Leu Val Lys Glu Asp Arg
200 205 210
Asp Ser Gly Val Asn Phe Gin Pro Glu Asp Thr Cys Ala Arg Leu
215 220 225
Arg Cys Ser Leu His Ala Ser Leu Leu Val Val Thr Leu Asn Pro
230 235 240
Asp Gln Cys His Pro Ser Arg Lys Arg Arg Ala Ala Ile Pro Val
245 250 255
Pro Lys Leu Ser Cys Lys Asn Leu Cys His Arg His Gln Leu Phe
260 265 270
Ile Asn Phe Arg Asp Leu Gly Trp His Lys Trp Ile Ile Ala Pro
275 280 285
Lys Gly Phe Net Ala Asn Tyr Cys His Gly Glu Cys Pro Phe Ser
290 295 300
Leu Thr Ile Ser Leu Asn Ser Ser Asn Tyr Ala Phe Met Gln Ala
305 310 315
Leu Met His Ala Val Asp Pro Glu Ile Pro Gln Ala Val Cys Ile
320 325 330
Pro Thr Lys Leu Ser Pro Ile Ser Met Leu Tyr Gln Asp Asn Asn
335 340 345
Asp Asn Val Ile Leu Arg His Tyr Glu Asp Met Val Val Asp Glu
49
CA 02372511 2002-05-14
350 355 360
Cys Gly Cys Gly
<210> 66
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 66
gtctgacagc cactccagag 20
<210> 67
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 67
tctccaattt ctgggcttag ataaggcgcc ttcaccccag aagttcc 47
<210> 68
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 68
gtcccaggtt atagtaagaa ttgg 24
<210> 69
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 69
gtgttgcggt cagtcccatg 20
<210> 70
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 70
gctgtctccc atttccatgc 20
<210> 71
<211> 24
<212> DNA
CA 02372511 2002-05-14
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 71
cgactaccat gtcttcataa tgtc 24
<210> 72
<211> 2849
<212> DNA
<213> Homo Sapien
<400> 72
cactttctcc ctctcttcct ttactttcga gaaaccgcgc ttccgcttct 50
ggtcgcagag acctcggaga ccgcgccggg gagacggagg tgctgtgggt 100
gggggggacc tgtggctgct cgtaccgccc cccaccctcc tcttctgcac 150
tgccgtcctc cggaagacct tttcccctgc tctgtttcct tcaccgagtc 200
tgtgcatcgc cccggacctg gccgggagga ggcttggccg gcgggagatg 250
ctctaggggc ggcgcgggag gagcggccgg cgggacggag ggcccggcag 300
gaagatgggc tcccgtggac agggactctt gctggcgtac tgcctgctcc 350
ttgcctttgc ctctggcctg gtcctgagtc gtgtgcccca tgtccagggg 400
gaacagcagg agtgggaggg gactgaggag ctgccgtcgc ctccggacca 450
tgccgagagg gctgaagaac aacatgaaaa atacaggccc agtcaggacc 500
aggggctccc tgcttcccgg tgcttgcgct gctgtgaccc cggtacctcc 550
atgtacccgg cgaccgccgt gccccagatc aacatcacta tcttgaaagg 600
ggagaagggt gaccgcggag atcgaggcct ccaagggaaa tatggcaaaa 650
caggctcagc aaggaccagg ggccacactg gacccaaagg gcagaagggc 700
tccatggggg cccctgggga gcggtgcaag agccactacg ccgccttttc 750
ggtgggccgg aagaagccca tgcacagcaa ccactactac cagacggtga 800
tcttcgacac ggagttcgtg aacctctacg accacttcaa catgttcacc 850
ggcaagttct actgctacgt gcccggcctc tacttcttca gcctcaacgt 900
gcacacctgg aaccagaagg agacctacct gcacatcatg aagaacgagg 950
aggaggtggt gatcttgttc gcgcaggtgg gcgaccgcag catcatgcaa 1000
agccagagcc tgatgctgga gctgcgagag caggaccagg tgtgggtacg 1050
cctctacaag ggcgaacgtg agaacgccat cttcagcgag gagctggaca 1100
cctacatcac cttcagtggc tacctggtca agcacgccac cgagccctag 1150
ctggccggcc acctcctttc ctctcgccac cttccacccc tgcgctgtgc 1200
tgaccccacc gcctcttccc cgatccctgg actccgactc cctggctttg 1250
51
CA 02372511 2002-05-14
gcattcagtg agacgccctg cacacacaga aagccaaagc gatcggtgct 1300
cccagatccc gcagcctctg gagagagctg acggcagatg aaatcaccag 1350
ggcggggcac ccgcgagaac cctctgggac cttccgcggc cctctctgca 1400
cacatcctca agtgaccccg cacggcgaga cgcgggtggc ggcagggcgt 1450
cccagggtgc ggcaccgcgg ctccagtcct tggaaataat taggcaaatt 1500
ctaaaggtct caaaaggagc aaagtaaacc gtggaggaca aagaaaaggg 1550
ttgttatttt tgtctttcca gccagcctgc tggctcccaa gagagaggcc 1600
ttttcagttg agactctgct taagagaaga tccaaagtta aagctctggg 1650
gtcaggggag gggccggggg caggaaacta cctctggctt aattctttta 1700
agccacgtag gaactttctt gagggatagg tggaccctga catccctgtg 1750
gccttgccca agggctctgc tggtctttct gagtcacagc tgcgaggtga 1800
tgggggctgg ggccccaggc gtcagcctcc cagagggaca gctgagcccc 1850
ctgccttggc tccaggttgg tagaagcagc cgaagggctc ctgacagtgg 1900
ccagggaccc ctgggtcccc caggcctgca gatgtttcta tgaggggcag 1950
agctccttgg tacatccatg tgtggctctg ctccacccct gtgccacccc 2000
agagccctgg ggggtggtct ccatgcctgc caccctggca tcggctttct 2050
gtgccgcctc ccacacaaat cagccccaga aggccccggg gccttggctt 2100
ctgtttttta taaaacacct caagcagcac tgcagtctcc catctcctcg 2150
tgggctaagc atcaccgctt ccacgtgtgt tgtgttggtt ggcagcaagg 2200
ctgatccaga ccccttctgc ccccactgcc ctcatccagg cctctgacca 2250
gtagcctgag aggggctttt tctaggcttc agagcagggg agagctggaa 2300
ggggctagaa agctcccgct tgtctgtttc tcaggctcct gtgagcctca 2350
gtcctgagac cagagtcaag aggaagtaca cgtcccaatc acccgtgtca 2400
ggattcactc tcaggagctg ggtggcagga gaggcaatag cccctgtggc 2450
aattgcagga ccagctggag cagggttgcg gtgtctccac ggtgctctcg 2500
ccctgcccat ggccacccca gactctgatc tccaggaacc ccatagcccc 2550
tctccacctc accccatgtt gatgcccagg gtcactcttg ctacccgctg 2600
ggcccccaaa cccccgctgc ctctcttcct tccccccatc ccccacctgg 2650
ttttgactaa tcctgcttcc ctctctgggc ctggctgccg ggatctgggg 2700
tccctaagtc cctctcttta aagaacttct gcgggtcaga ctctgaagcc 2750
gagttgctgt gggcgtgccc ggaagcagag cgccacactc gctgcttaag 2800
ctcccccagc tctttccaga aaacattaaa ctcagaattg tgttttcaa 2849
52
CA 02372511 2002-05-14
<210> 73
<211> 281
<212> PRT
<213> Homo Sapien
<400> 73
Met Gly Ser Arg Gly Gln Gly Leu Leu Leu Ala Tyr Cys Leu Leu
1 5 10 15
Leu Ala Phe Ala Ser Gly Leu Val Leu Ser Arg Val Pro His Val
20 25 30
Gln Gly Glu .Gln Gln Glu Trp Glu Gly Thr Glu Glu Leu Pro Ser
35 40 45
Pro Pro Asp His Ala Glu Arg Ala Glu Glu Gln His Glu Lys Tyr
50 55 60
Arg Pro Ser Gin Asp Gln Gly Leu Pro Ala Ser Arg Cys Leu Arg
65 70 75
Cys Cys Asp Pro Gly Thr Ser Met Tyr Pro Ala Thr Ala Val Pro
80 85 90
Gln Ile Asn Ile Thr Ile Leu Lys Gly Glu Lys Gly Asp Arg Gly
95 100 105
Asp Arg Gly Leu Gin Gly Lys Tyr Gly Lys Thr Gly Ser Ala Gly
110 115 120
Ala Arg Gly His Thr Gly Pro Lys Gly Gln Lys Gly Ser Met Gly
125 130 135
Ala Pro Gly Glu Arg Cys Lys Ser His Tyr Ala Ala Phe Ser Val
140 145 150
Gly Arg Lys Lys Pro Met His Ser Asn His Tyr Tyr Gln Thr Val
155 160 165
Ile Phe Asp Thr Glu Phe Val Asn Leu Tyr Asp His Phe Asn Met
170 175 180
Phe Thr Gly Lys Phe Tyr Cys Tyr Val Pro Gly Leu Tyr Phe Phe
185 190 195
Ser Leu Asn Val His Thr Trp Asn Gln Lys Glu Thr Tyr Leu His
200 205 210
Ile Met Lys Asn Glu Glu Glu Val Val Ile Leu Phe Ala Gln Val
215 220 225
Gly Asp Arg Ser Ile Met Gln Ser Gln Ser Leu Met Leu Glu Leu
230 235 240
Arg Glu Gln Asp Gln Val Trp Val Arg Leu Tyr Lys Gly Glu Arg
245 250 255
Glu Asn Ala Ile Phe Ser Glu Glu Leu Asp Thr Tyr Ile Thr Phe
260 265 270
Ser Gly Tyr Leu Val Lys His Ala Thr Glu Pro
275 280
53
CA 02372511 2002-05-14
<210> 74
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 74
tacaggccca gtcaggacca gggg 24
<210> 75
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 75
ctgaagaagt agaggccggg cacg 24
<210> 76
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 76
cccggtgctt gcgctgctgt gaccccggta cctccatgta cccgg 45
<210> 77
<211> 1042
<212> DNA
<213> Homo Sapien
<400> 77
gaattcggca cgagggaaga agagaaagaa aatctccggg gctgctggga 50
gcatataaag aagccctgtg gccttgctgg ttttaccatc cagaccagag 100
tcaggccaca gacggacatg gctgctcaag gctggtccat gctcctgctg 150
gctgtcctta acctaggcat cttcgtccgt ccctgtgaca ctcaagagct 200
acgatgtctg tgtattcagg aacactctga attcattcct ctcaaactca 250
ttaaaaatat aatggtgata ttcgagacca tttactgcaa cagaaaggaa 300
gtgatagcag tcccaaaaaa tgggagtatg atttgtttgg atcctgatgc 350
tccatgggtg aaggctactg ttggcccaat tactaacagg ttcctacctg 400
aggacctcaa acaaaaggaa tttccaccgg caatgaagct tctgtatagt 450
gttgagcatg aaaagcctct atatctttca tttgggagac ctgagaacaa 500
gagaatattt ccctttccaa ttcgggagac ctctagacac tttgctgatt 550
tagctcacaa cagtgatagg aattttctac gggactccag tgaagtcagc 600
54
CA 02372511 2002-05-14
ttgacaggca gtgatgccta aaagccactc atgaggcaaa gagtttcaag 650
gaagctctcc tcctggagtt ttggcgttct cattcttata ctctattccc 700
gcgttagtct ggtgtatgga tctatgagct ctcttttaat attttattat 750
aaatgtttta tttacttaac ttcctagtga atgttcacag gtgactgctc 800
ccccatcccc atttcttgat attacatata atggcatcat ataccccttt 850
attgactgac aaactactca gattgcttaa cattttgtgc ttcaaagtct 900
tatcccactc cactatgggc tgttacagag tgcatctcgg tgtagagcaa 950
ggctccttgt cttcagtgcc ccagggtgaa atacttcttt gaaaaatttt 1000
cattcatcag aaaatctgaa ataaaaatat gtcttaattg ag 1042
<210> 78
<211> 167
<212> PRT
<213> Homo Sapien
<400> 78
Met Ala Ala Gln Gly Trp Ser Met Leu Leu Leu Ala Val Leu Asn
1 5 10 15
Leu Gly Ile Phe Val Arg Pro Cys Asp Thr Gln Glu Leu Arg Cys
20 25 30
Leu Cys Ile Gin Glu His Ser Glu Phe Ile Pro Leu Lys Leu Ile
35 40 45
Lys Asn Ile Met Val Ile Phe Glu Thr Ile Tyr Cys Asn Arg Lys
50 55 60
Glu Val Ile Ala Val Pro Lys Asn Gly Ser Met Ile Cys Leu Asp
65 70 75
Pro Asp Ala Pro Trp Val Lys Ala Thr Val Gly Pro Ile Thr Asn
80 85 90
Arg Phe Leu Pro Glu Asp Leu Lys Gln Lys Glu Phe Pro Pro Ala
95 100 105
Met Lys Leu Leu Tyr Ser Val Glu His Glu Lys Pro Leu Tyr Leu
110 115 120
Ser Phe Gly Arg Pro Glu Asn Lys Arg Ile Phe Pro Phe Pro Ile
125 130 135
Arg Glu Thr Ser Arg His Phe Ala Asp Leu Ala His Asn Ser Asp
140 145 150
Arg Asn Phe Leu Arg Asp Ser Ser Glu Val Ser Leu Thr Gly Ser
155 160 165
Asp Ala
<210> 79
<211> 798
<212> DNA
CA 02372511 2002-05-14
<213> Homo Sapien
<220>
<221> unsure
<222> 794
<223> unknown base
<400> 79
cagacatggc tcagtcactg gctctgagcc tccttatcct ggttctggcc 50
tttggcatcc ccaggaccca aggcagtgat ggaggggctc aggactgttg 100
cctcaagtac agccaaagga agattcccgc caaggttgtc cgcagctacc 150
ggaagcagga accaagctta ggctgctcca tcccagctat cctgttcttg 200
ccccgcaagc gctctcaggc agagctatgt gcagacccaa aggagctctg 250
ggtgcagcag ctgatgcagc atctggacaa gacaccatcc ccacagaaac 300
cagcccaggg ctgcaggaag gacagggggg cctccaagac tggcaagaaa 350
ggaaagggct ccaaaggctg caagaggact gagcggtcac agacccctaa 400
agggccatag cccagtgagc agcctggagc cctggagacc ccaccagcct 450
caccagcgct tgaagcctga acccaagatg caagaaggag gctatgctca 500
ggggccctgg agcagccacc ccatgctggc cttgccacac tctttatcct 550
gctttaacca ccccatctgc attcccagct ctaccctgca tggctgagct 600
gcccacagca ggccaggtcc agagagaccg aggagggaga gtctcccagg 650
gagcatgaga ggaggcagca ggactgtccc cttgaaggag aatcatcagg 700
agcctggacc tgatacggct ccccagtaca ccccacctct tccttgtaaa 750
tatgatttat acctaactga ataaaaagct gttctgtctt cccnccca 798
<210> 80
<211> 134
<212> PRT
<213> Homo Sapien
<400> 80
Met Ala Gln Ser Leu Ala Leu Ser Leu Leu Ile Leu Val Leu Ala
1 5 10 15
Phe Gly Ile Pro Arg Thr Gln Gly Ser Asp Gly Gly Ala Gln Asp
20 25 30
Cys Cys Leu Lys Tyr Ser Gln Arg Lys Ile Pro Ala Lys Val Val
35 40 45
Arg Ser Tyr Arg Lys Gln Glu Pro Ser Leu Gly Cys Ser Ile Pro
50 55 60
Ala Ile Leu Phe Leu Pro Arg Lys Arg Ser Gln Ala Glu Leu Cys
65 70 75
Ala Asp Pro Lys Glu Leu Trp Val Gln Gln Leu Met Gln His Leu
80 85 90
56
CA 02372511 2002-05-14
Asp Lys Thr Pro Ser Pro Gln Lys Pro Ala Gln Gly Cys Arg Lys
95 100 105
Asp Arg Gly Ala Ser Lys Thr Gly Lys Lys Gly Lys Gly Ser Lys
110 115 120
Gly Cys Lys Arg Thr Glu Arg Ser Gln Thr Pro Lys Gly Pro
125 130
<210> 81
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 81
agacatggct cagtcactgg 20
<210> 82
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 82
gacccctaaa gggccatag 19
<210> 83
<211> 924
<212> DNA
<213> Homo Sapien
<400> 83
aaggagcagc ccgcaagcac caagtgagag gcatgaagtt acagtgtgtt 50
tccctttggc tcctgggtac aatactgata ttgtgctcag tagacaacca 100
cggtctcagg agatgtctga tttccacaga catgcaccat atagaagaga 150
gtttccaaga aatcaaaaga gccatccaag ctaaggacac cttcccaaat 200
gtcactatcc tgtccacatt ggagactctg cagatcatta agcccttaga 250
tgtgtgctgc gtgaccaaga acctcctggc gttctacgtg gacagggtgt 300
tcaaggatca tcaggagcca aaccccaaaa tcttgagaaa aatcagcagc 350
attgccaact ctttcctcta catgcagaaa actctgcggc aatgtcagga 400
acagaggcag tgtcactgca ggcaggaagc caccaatgcc accagagtca 450
tccatgacaa ctatgatcag ctggaggtcc acgctgctgc cattaaatcc 500
ctgggagagc tcgacgtctt tctagcctgg attaataaga atcatgaagt 550
aatgttctca gcttgatgac aaggaacctg tatagtgatc cagggatgaa 600
caccccctgt gcggtttact gtgggagaca gcccaccttg aaggggaagg 650
57
CA 02372511 2002-05-14
agatggggaa ggccccttgc agctgaaagt cccactggct ggcctcaggc 700
tgtcttattc cgcttgaaaa taggcaaaaa gtctactgtg gtatttgtaa 750
taaactctat ctgctgaaag ggcctgcagg ccatcctggg agtaaagggc 800
tgccttccca t.ctaatttat tgtaaagtca tatagtccat gtctgtgatg 850
tgagccaagt gatatcctgt agtacacatt gtactgagtg gtttttctga 900
ataaattcca tattttacct atga 924
<210> 84
<211> 177
<212> PRT
<213> Homo Sapien
<400> 84
Met Lys Leu Gin Cys Val Ser Leu Trp Leu Leu Gly Thr Ile Leu
1 5 10 15
Ile Leu Cys Ser Val Asp Asn His Gly Leu Arg Arg Cys Leu Ile
20 25 30
Ser Thr Asp Met His His Ile Glu Glu Ser Phe Gln Glu Ile Lys
35 40 45
Arg Ala Ile.Gln Ala Lys Asp Thr Phe Pro Asn Val Thr Ile Leu
50 55 60
Ser Thr Leu .Glu Thr Leu Gln Ile Ile Lys Pro Leu Asp Val Cys
65 70 75
Cys Val Thr Lys Asn Leu Leu Ala Phe Tyr Val Asp Arg Val Phe
80 85 90
Lys Asp HisGln Glu Pro Asn Pro Lys Ile Leu Arg Lys Ile Ser
95 100 105
Ser Ile Ala Asn Ser Phe Leu Tyr Met Gln Lys Thr Leu Arg Gln
110 115 120
Cys Gln Glu Gin Arg Gln Cys His Cys Arg Gln Glu Ala Thr Asn
125 130 135
Ala Thr Arg Val Ile His Asp Asn Tyr Asp Gln Leu Glu Val His
140 145 150
Ala Ala Ala Ile Lys Ser Leu Gly Glu Leu Asp Val Phe Leu Ala
155 160 165
Trp Ile Asn Lys Asn His Glu Val Met Phe Ser Ala
170 175
<210> 85
<211> 2137
<212> DNA
<213> Homo Sapien
<400> 85
gctcccagcc aagaacctcg gggccgctgc gcggtgggga ggagttcccc 50
gaaacccggc cgctaagcga ggcctcctcc tcccgcagat ccgaacggcc 100
58
CA 02372511 2002-05-14
tgggcggggt caccccggct gggacaagaa gccgccgcct gcctgcccgg 150
gcccggggag g-gggctgggg ctggggccgg aggcggggtg tgagtgggtg 200
tgtgcggggg gcggaggctt gatgcaatcc cgataagaaa tgctcgggtg 250
tcttgggcac ctacccgtgg ggcccgtaag gcgctactat ataaggctgc 300
cggcccggag ccgccgcgcc gtcagagcag gagcgctgcg tccaggatct 350
agggccacga ccatcccaac ccggcactca cagccccgca gcgcatcccg 400
gtcgccgccc agcctcccgc acccccatcg ccggagctgc gccgagagcc 450
ccagggaggt gccatgcgga gcgggtgtgt ggtggtccac gtatggatcc 500
tggccggcct ctggctggcc gtggccgggc gccccctcgc cttctcggac 550
gcggggcccc a.cgtgcacta cggctggggc gaccccatcc gcctgcggca 600
cctgtacacc tccggccccc acgggctctc cagctgcttc ctgcgcatcc 650
gtgccgacgg cgtcgtggac tgcgcgcggg gccagagcgc gcacagtttg 700
ctggagatca aggcagtcgc tctgcggacc gtggccatca agggcgtgca 750
cagcgtgcgg tacctctgca tgggcgccga cggcaagatg caggggctgc 800
ttcagtactc ggaggaagac tgtgctttcg aggaggagat ccgcccagat 850
ggctacaatg tgtaccgatc cgagaagcac cgcctcccgg tctccctgag 900
cagtgccaaa cagcggcagc tgtacaagaa cagaggcttt cttccactct 950
ctcatttcct gcccatgctg cccatggtcc cagaggagcc tgaggacctc 1000
aggggccact tggaatctga catgttctct tcgcccctgg agaccgacag 1050
catggaccca tttgggcttg tcaccggact ggaggccgtg aggagtccca 1100
gctttgagaa gtaactgaga ccatgcccgg gcctcttcac tgctgccagg 1150
ggctgtggta c.ctgcagcgt gggggacgtg cttctacaag aacagtcctg 1200
agtccacgtt ctgtttagct ttaggaagaa acatctagaa gttgtacata 1250
ttcagagttt tccattggca gtgccagttt ctagccaata gacttgtctg 1300
atcataacat tgtaagcctg tagcttgccc agctgctgcc tgggccccca 1350
ttctgctccc tcgaggttgc tggacaagct gctgcactgt ctcagttctg 1400
cttgaatacc tccatcgatg gggaactcac ttcctttgga aaaattctta 1450
tgtcaagctg aaattctcta attttttctc atcacttccc caggagcagc 1500
cagaagacag gcagtagttt taatttcagg aacaggtgat ccactctgta 1550
aaacagcagg taaatttcac tcaaccccat gtgggaattg atctatatct 1600
ctacttccag ggaccatttg cccttcccaa atccctccag gccagaactg 1650
actggagcag gcatggccca ccaggcttca ggagtagggg aagcctggag 1700
59
CA 02372511 2002-05-14
ccccactcca gccctgggac aacttgagaa ttccccctga ggccagttct 1750
gtcatggatg ctgtcctgag aataacttgc tgtcccggtg tcacctgctt 1800
ccatctccca gcccaccagc cctctgccca cctcacatgc ctccccatgg 1850
attggggcct cccaggcccc ccaccttatg tcaacctgca cttcttgttc 1900
aaaaatcagg aaaagaaaag atttgaagac cccaagtctt gtcaataact 1950
tgctgtgtgg aagcagcggg ggaagaccta gaaccctttc cccagcactt 2000
ggttttccaa catgatattt atgagtaatt tattttgata tgtacatctc 2050
ttattttctt acattattta tgcccccaaa ttatatttat gtatgtaagt 2100
gaggtttgtt ttgtatatta aaatggagtt tgtttgt 2137
<210> 86
<211> 216
<212> PRT
<213> Homo Sapien
<400> 86
Met Arg Ser Gly Cys Val Val Val His Val Trp Ile Leu Ala Gly
1 5 10 15
Leu Trp Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala
20 25 30
Gly Pro His Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg
35 40 45
His Leu Tyr Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu
50 55 60
Arg Ile Arg Ala Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser
65 70 75
Ala His Ser Leu Leu Glu Ile Lys Ala Val Ala Leu Arg Thr Val
80 85 90
Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu Cys Met Gly Ala
95 100 105
Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu Glu Asp Cys
110 115 120
Ala Phe Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Arg
125 130 135
Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys Gln
140 145 150
Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe
155 160 165
Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg
170 175 180
Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp
185 190 195
CA 02372511 2002-05-14
Ser Met Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg
200 205 210
Ser Pro Ser Phe Glu Lys
215
<210> 87
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 87
atccgcccag atggctacaa tgtgta 26
<210> 88
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 88
gcctcccggt ctccctgagc agtgccaaac agcggcagtg to 42
<210> 89
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 89
ccagtccggt gacaagccca as 22
<210> 90
<211> 1857
<212> DNA
<213> Homo Sapien
<400> 90
gtctgttccc aggagtcctt cggcggctgt tgtgtcagtg gcctgatcgc 50
gatggggaca aaggcgcaag tcgagaggaa actgttgtgc ctcttcatat 100
tggcgatcct gttgtgctcc ctggcattgg gcagtgttac agtgcactct 150
tctgaacctg aagtcagaat tcctgagaat aatcctgtga agttgtcctg 200
tgcctactcg ggcttttctt ctccccgtgt ggagtggaag tttgaccaag 250
gagacaccac cagactcgtt tgctataata acaagatcac agcttcctat 300
gaggaccggg tgaccttctt gccaactggt atcaccttca agtccgtgac 350
acgggaagac actgggacat acacttgtat ggtctctgag gaaggcggca 400
acagctatgg ggaggtcaag gtcaagctca tcgtgcttgt gcctccatcc 450
61
CA 02372511 2002-05-14
aagcctacag ttaacatccc ctcctctgcc accattggga accgggcagt 500
gctgacatgc tcagaacaag atggttcccc accttctgaa tacacctggt 550
tcaaagatgg gatagtgatg cctacgaatc ccaaaagcac ccgtgccttc 600
agcaactctt cctatgtcct gaatcccaca acaggagagc tggtctttga 650
tcccctgtca gcctctgata ctggagaata cagctgtgag gcacggaatg 700
ggtatgggac acccatgact tcaaatgctg tgcgcatgga agctgtggag 750
cggaatgtgg gggtcatcgt ggcagccgtc cttgtaaccc tgattctcct 800
gggaatcttg gtttttggca tctggtttgc ctatagccga ggccactttg 850
acagaacaaa gaaagggact tcgagtaaga aggtgattta cagccagcct 900
agtgcccgaa gtgaaggaga attcaaacag acctcgtcat tcctggtgtg 950
agcctggtcg gctcaccgcc tatcatctgc atttgcctta ctcaggtgct 1000
accggactct ggcccctgat gtctgtagtt tcacaggatg ccttatttgt 1050
cttctacacc ccacagggcc ccctacttct tcggatgtgt ttttaataat 1100
gtcagctatg tgccccatcc tccttcatgc cccccctccc tttcctacca 1150
ctgctgagtg gcctggaact tgtttaaagt gtttattccc catttctttg 1200
agggatcagg aaggaatcct gggtatgcca ttgacttccc ttctaagtag 1250
acagcaaaaa tggcgggggt cgcaggaatc tgcactcaac tgcccacctg 1300
gctggcaggg atctttgaat aggtatcttg agcttggttc tgggctcttt 1350
ccttgtgtac tgacgaccag ggccagctgt tctagagcgg gaattagagg 1400
ctagagcggc tgaaatggtt gtttggtgat gacactgggg tccttccatc 1450
tctggggccc actctcttct gtcttcccat gggaagtgcc actgggatcc 1500
ctctgccctg tcctcctgaa tacaagctga ctgacattga ctgtgtctgt 1550
ggaaaatggg agctcttgtt gtggagagca tagtaaattt tcagagaact 1600
tgaagccaaa aggatttaaa accgctgctc taaagaaaag aaaactggag 1650
gctgggcgca gtggctcacg cctgtaatcc cagaggctga ggcaggcgga 1700
tcacctgagg tcgggagttc gggatcagcc tgaccaacat ggagaaaccc 1750
tactggaaat acaaagttag ccaggcatgg tggtgcatgc ctgtagtccc 1800
agctgctcag gagcctggca acaagagcaa aactccagct caaaaaaaaa 1850
aaaaaaa 1857
<210> 91
<211> 299
<212> PRT
<213> Homo Sapien
62
CA 02372511 2002-05-14
<400> 91
Met Gly Thr Lys Ala Gln Val Glu Arg Lys Leu Leu Cys Leu Phe
1 5 10 15
Ile Leu Ala Ile Leu Leu Cys Ser Leu Ala Leu Gly Ser Val Thr
20 25 30
Val His Ser Ser Glu Pro Glu Val Arg Ile Pro Glu Asn Asn Pro
35 40 45
Val Lys Leu.Ser Cys Ala Tyr Ser Gly Phe Ser Ser Pro Arg Val
50 55 60
Glu Trp Lys Phe Asp Gln Gly Asp Thr Thr Arg Leu Val Cys Tyr
65 70 75
Asn Asn Lys Ile Thr Ala Ser Tyr Glu Asp Arg Val Thr Phe Leu
80 85 90
Pro Thr Gly Ile Thr Phe Lys Ser Val Thr Arg Glu Asp Thr Gly
95 100 105
Thr Tyr Thr Cys Met Val Ser Glu Glu Gly Gly Asn Ser Tyr Gly
110 115 120
Glu Val Lys 'Val Lys Leu Ile Val Leu Val Pro Pro Ser Lys Pro
125 130 135
Thr Val Asn Ile Pro Ser Ser Ala Thr Ile Gly Asn Arg Ala Val
140 145 150
Leu Thr Cys Ser Glu Gln Asp Gly Ser Pro Pro Ser Glu Tyr Thr
155 160 165
Trp Phe Lys Asp Gly Ile Val Met Pro Thr Asn Pro Lys Ser Thr
170 175 180
Arg Ala Phe.Ser Asn Ser Ser Tyr Val Leu Asn Pro Thr Thr Gly
185 190 195
Glu Leu Val Phe Asp Pro Leu Ser Ala Ser Asp Thr Gly Glu Tyr
200 205 210
Ser Cys Glu Ala Arg Asn Gly Tyr Gly Thr Pro Met Thr Ser Asn
215 220 225
Ala Val Arg Met Glu Ala Val Glu Arg Asn Val Gly Val Ile Val
230 235 240
Ala Ala Val Leu Val Thr Leu Ile Leu Leu Gly Ile Leu Val Phe
245 250 255
Gly Ile Trp Phe Ala Tyr Ser Arg Gly His Phe Asp Arg Thr Lys
260 265 270
Lys Gly Thr Ser Ser Lys Lys Val Ile Tyr Ser Gln Pro Ser Ala
275 280 285
Arg Ser Glu Gly Glu Phe Lys Gln Thr Ser Ser Phe Leu Val
290 295
<210> 92
<211> 24
63
CA 02372511 2002-05-14
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 92
tcgcggagct gtgttctgtt tccc 24
<210> 93
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 93
tgatcgcgat ggggacaaag gcgcaagctc gagaggaaac tgttgtgcct 50
<210> 94
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 94
acacctggtt caaagatggg 20
<210> 95
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 95
taggaagagt tgctgaaggc acgg 24
<210> 96
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 96
ttgccttact caggtgctac 20
<210> 97
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 97
actcagcagt ggtaggaaag 20
64
CA 02372511 2002-05-14
<210> 98
<211> 1200
<212> DNA
<213> Homo Sapien
<400> 98
cccacgcgtc cgaacctctc cagcgatggg agccgcccgc ctgctgccca 50
acctcactct gtgcttacag ctgctgattc tctgctgtca aactcagtac 100
gtgagggacc agggcgccat gaccgaccag ctgagcaggc ggcagatccg 150
cgagtaccaa ctctacagca ggaccagtgg caagcacgtg caggtcaccg 200
ggcgtcgcat ctccgccacc gccgaggacg gcaacaagtt tgccaagctc 250
atagtggaga cggacacgtt tggcagccgg gttcgcatca aaggggctga 300
gagtgagaag tacatctgta tgaacaagag gggcaagctc atcgggaagc 350
ccagcgggaa gagcaaagac tgcgtgttca cggagatcgt gctggagaac 400
aactatacgg ccttccagaa cgcccggcac gagggctggt tcatggcctt 450
cacgcggcag gggcggcccc gccaggcttc ccgcagccgc cagaaccagc 500
gcgaggccca cttcatcaag cgcctctacc aaggccagct gcccttcccc 550
aaccacgccg agaagcagaa gcagttcgag tttgtgggct ccgcccccac 600
ccgccggacc aagcgcacac ggcggcccca gcccctcacg tagtctggga 650
ggcagggggc agcagcccct gggccgcctc cccacccctt tcccttctta 700
atccaaggac tgggctgggg tggcgggagg ggagccagat ccccgaggga 750
ggaccctgag ggccgcgaag catccgagcc cccagctggg aaggggcagg 800
ccggtgcccc aggggcggct ggcacagtgc ccccttcccg gacgggtggc 850
aggccctgga gaggaactga gtgtcaccct gatctcaggc caccagcctc 900
tgccggcctc ccagccgggc tcctgaagcc cgctgaaagg tcagcgactg 950
aaggccttgc agacaaccgt ctggaggtgg ctgtcctcaa aatctgcttc 1000
tcggatctcc ctcagtctgc ccccagcccc caaactcctc ctgactagac 1050
tgtaggaagg gacttttgtt tgtttgtttg tttcaggaaa aaagaaaggg 1100
agagagagga aaatagaggg ttgtccactc ctcacattcc acgacccagg 1150
cctgcacccc acccccaact cccagccccg gaataaaacc attttcctgc 1200
<210> 99
<211> 205
<212> PRT
<213> Homo Sapien
<400> 99
Met Gly Ala Ala Arg Leu Leu Pro Asn Leu Thr Leu Cys Leu Gln
1 5 10 15
CA 02372511 2002-05-14
Leu Leu Ile Leu Cys Cys Gln Thr Gln Tyr Val Arg Asp Gln Gly
20 25 30
Ala Met Thr Asp Gln Leu Ser Arg Arg Gln Ile Arg Glu Tyr Gln
35 40 45
Leu Tyr Ser Arg Thr Ser Gly Lys His Val Gln Val Thr Gly Arg
50 55 60
Arg Ile Ser Ala Thr Ala Glu Asp Gly Asn Lys Phe Ala Lys Leu
65 70 75
Ile Val Glu Thr Asp Thr Phe Gly Ser Arg Val Arg Ile Lys Gly
80 85 90
Ala Glu Ser flu Lys Tyr Ile Cys Met Asn Lys Arg Gly Lys Leu
95 100 105
Ile Gly Lys Pro Ser Gly Lys Ser Lys Asp Cys Val Phe Thr Glu
110 115 120
Ile Val Leu k3lu Asn Asn Tyr Thr Ala Phe Gln Asn Ala Arg His
125 130 135
Glu Gly Trp Phe Met Ala Phe Thr Arg Gln Gly Arg Pro Arg Gln
140 145 150
Ala Ser Arg Ser Arg Gln Asn Gln Arg Glu Ala His Phe Ile Lys
155 160 165
Arg Leu Tyr Cln Gly Gln Leu Pro Phe Pro Asn His Ala Glu Lys
170 175 180
Gln Lys Gln Phe Glu Phe Val Gly Ser Ala Pro Thr Arg Arg Thr
185 190 195
Lys Arg Thr Arg Arg Pro Gln Pro Leu Thr
200 205
<210> 100
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 100
cagtacgtga gggaccaggg cgccatga 28
<210> 101
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 101
ccggtgacct gcacgtgctt gcca 24
<210> 102
<211> 41
66
CA 02372511 2002-05-14
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<220>
<221> unsure
<222> 21
<223> unknown base
<400> 102
gcggatctgc cgcctgctca nctggtcggt catggcgccc t 41
<210> 103
<211> 1679
<212> DNA
<213> Homo Sapien
<400> 103
gttgtgtcct tcagcaaaac agtggattta aatctccttg cacaagcttg 50
agagcaacac aatctatcag gaaagaaaga aagaaaaaaa ccgaacctga 100
caaaaaagaa gaaaaagaag aagaaaaaaa atcatgaaaa ccatccagcc 150
aaaaatgcac aattctatct cttgggcaat cttcacgggg ctggctgctc 200
tgtgtctctt ccaaggagtg cccgtgcgca gcggagatgc caccttcccc 250
aaagctatgg acaacgtgac ggtccggcag ggggagagcg ccaccctcag 300
gtgcactatt gacaaccggg tcacccgggt ggcctggcta aaccgcagca 350
ccatcctcta tgctgggaat gacaagtggt gcctggatcc tcgcgtggtc 400
cttctgagca acacccaaac gcagtacagc atcgagatcc agaacgtgga 450
tgtgtatgac gagggccctt acacctgctc ggtgcagaca gacaaccacc 500
caaagacctc tagggtccac ctcattgtgc aagtatctcc caaaattgta 550
gagatttctt cagatatctc cattaatgaa gggaacaata ttagcctcac 600
ctgcatagca actggtagac cagagcctac ggttacttgg agacacatct 650
ctcccaaagc ggttggcttt gtgagtgaag acgaatactt ggaaattcag 700
ggcatcaccc gggagcagtc aggggactac gagtgcagtg cctccaatga 750
cgtggccgcg cccgtggtac ggagagtaaa ggtcaccgtg aactatccac 800
catacatttc agaagccaag ggtacaggtg tccccgtggg acaaaagggg 850
acactgcagt gtgaagcctc agcagtcccc tcagcagaat tccagtggta 900
caaggatgac aaaagactga ttgaaggaaa gaaaggggtg aaagtggaaa 950
acagaccttt cctctcaaaa ctcatcttct tcaatgtctc tgaacatgac 1000
tatgggaact acacttgcgt ggcctccaac aagctgggcc acaccaatgc 1050
cagcatcatg ctatttggtc caggcgccgt cagcgaggtg agcaacggca 1100
67
CA 02372511 2002-05-14
cgtcgaggag g-gcaggctgc gtctggctgc tgcctcttct ggtcttgcac 1150
ctgcttctca aattttgatg tgagtgccac ttccccaccc gggaaaggct 1200
gccgccacca ccaccaccaa cacaacagca atggcaacac cgacagcaac 1250
caatcagata tatacaaatg aaattagaag aaacacagcc tcatgggaca 1300
gaaatttgag ggaggggaac aaagaatact ttggggggaa aagagtttta 1350
aaaaagaaat tgaaaattgc cttgcagata tttaggtaca atggagtttt 1400
cttttcccaa acgggaagaa cacagcacac ccggcttgga cccactgcaa 1450
gctgcatcgt gcaacctctt tggtgccagt gtgggcaagg gctcagcctc 1500
tctgcccaca gagtgccccc acgtggaaca ttctggagct ggccatccca 1550
aattcaatca gtccatagag acgaacagaa tgagaccttc cggcccaagc 1600
gtggcgctgc gggcactttg gtagactgtg ccaccacggc gtgtgttgtg 1650
aaacgtgaaa taaaaagagc aaaaaaaaa 1679
<210> 104
<211> 344
<212> PRT
<213> Homo Sapien
<400> 104
Met Lys Thr Ile Gln Pro Lys Met His Asn Ser Ile Ser Trp Ala
1 5 10 15
Ile Phe Thr fly Leu Ala Ala Leu Cys Leu Phe Gln Gly Val Pro
20 25 30
Val Arg Ser Gly Asp Ala Thr Phe Pro Lys Ala Met Asp Asn Val
35 40 45
Thr Val Arg Gin Gly Glu Ser Ala Thr Leu Arg Cys Thr Ile Asp
50 55 60
Asn Arg Val Thr Arg Val Ala Trp Leu Asn Arg Ser Thr Ile Leu
65 70 75
Tyr Ala Gly Asn Asp Lys Trp Cys Leu Asp Pro Arg Val Val Leu
80 85 90
Leu Ser Asn Thr Gln Thr Gln Tyr Ser Ile Glu Ile Gln Asn Val
95 100 105
Asp Val Tyr Asp Glu Gly Pro Tyr Thr Cys Ser Val Gln Thr Asp
110 115 120
Asn His Pro Lys Thr Ser Arg Val His Leu Ile Val Gln Val Ser
125 130 135
Pro Lys Ile Val Glu Ile Ser Ser Asp Ile Ser Ile Asn Glu Gly
140 145 150
Asn Asn Ile Ser Leu Thr Cys Ile Ala Thr Gly Arg Pro Glu Pro
155 160 165
68
CA 02372511 2002-05-14
Thr Val Thr Trp Arg His Ile Ser Pro Lys Ala Val Gly Phe Val
170 175 180
Ser Glu Asp Glu Tyr Leu Glu Ile Gin Gly Ile Thr Arg Glu Gln
185 190 195
Ser Gly Asp Tyr Glu Cys Ser Ala Ser Asn Asp Val Ala Ala Pro
200 205 210
Val Val Arg Arg Val Lys Val Thr Val Asn Tyr Pro Pro Tyr Ile
215 220 225
Ser Glu Ala Lys Gly Thr Gly Val Pro Val Gly Gln Lys Gly Thr
230 235 240
Leu Gln Cys Glu Ala Ser Ala Val Pro Ser Ala Glu Phe Gln Trp
245 250 255
Tyr Lys Asp Asp Lys Arg Leu Ile Glu Gly Lys Lys Gly Val Lys
260 265 270
Val Glu Asn Arg Pro Phe Leu Ser Lys Leu Ile Phe Phe Asn Val
275 280 285
Ser Glu His Asp Tyr Gly Asn Tyr Thr Cys Val Ala Ser Asn Lys
290 295 300
Leu Gly His Thr Asn Ala Ser Ile Met Leu Phe Gly Pro Gly Ala
305 310 315
Val Ser Glu Val Ser Asn Gly Thr Ser Arg Arg Ala Gly Cys Val
320 325 330
Trp Leu Leu Pro Leu Leu Val Leu His Leu Leu Leu Lys Phe
335 340
<210> 105
<211> 1734
<212> DNA
<213> Homo Sapien
<400> 105
gtggactctg agaagcccag gcagttgagg acaggagaga gaaggctgca 50
gacccagagg gagggaggac agggagtcgg aaggaggagg acagaggagg 100
gcacagagac gcagagcaag ggcggcaagg aggagaccct ggtgggagga 150
agacactctg gagagagagg gggctgggca gagatgaagt tccaggggcc 200
cctggcctgc ctcctgctgg ccctctgcct gggcagtggg gaggctggcc 250
ccctgcagag cggagaggaa agcactggga caaatattgg ggaggccctt 300
gaacatggcc tgggagacgc cctgagcgaa ggggtgggaa aggccattgg 350
caaagaggcc ggaggggcag ctggctctaa agtcagtgag gcccttggcc 400
aagggaccag agaagcagtt ggcactggag tcaggcaggt tccaggcttt 450
ggcgcagcag atgctttggg caacagggtc ggggaagcag cccatgctct 500
gggaaacact gggcacgaga ttggcagaca ggcagaagat gtcattcgac 550
69
CA 02372511 2002-05-14
acggagcaga tgctgtccgc ggctcctggc agggggtgcc tggccacagt 600
ggtgcttggg aaacttctgg aggccatggc atctttggct ctcaaggtgg 650
ccttggaggc cagggccagg gcaatcctgg aggtctgggg actccgtggg 700
tccacggata ccccggaaac tcagcaggca gctttggaat gaatcctcag 750
ggagctccct ggggtcaagg aggcaatgga gggccaccaa actttgggac 800
caacactcag ggagctgtgg cccagcctgg ctatggttca gtgagagcca 850
gcaaccagaa tgaagggtgc acgaatcccc caccatctgg ctcaggtgga 900
ggctccagca actctggggg aggcagcggc tcacagtcgg gcagcagtgg 950
cagtggcagc aatggtgaca acaacaatgg cagcagcagt ggtggcagca 1000
gcagtggcag cagcagtggc agcagcagtg gcggcagcag tggcggcagc 1050
agtggtggca gcagtggcaa cagtggtggc agcagaggtg acagcggcag 1100
tgagtcctcc tggggatcca gcaccggctc ctcctccggc aaccacggtg 1150
ggagcggcgg aggaaatgga cataaacccg ggtgtgaaaa gccagggaat 1200
gaagcccgcg ggagcgggga atctgggatt cagggcttca gaggacaggg 1250
agtttccagc aacatgaggg aaataagcaa agagggcaat cgcctccttg 1300
gaggctctgg agacaattat cgggggcaag ggtcgagctg gggcagtgga 1350
ggaggtgacg ctgttggtgg agtcaatact gtgaactctg agacgtctcc 1400
tgggatgttt aactttgaca ctttctggaa gaattttaaa tccaagctgg 1450
gtttcatcaa ctgggatgcc ataaacaagg accagagaag ctctcgcatc 1500
ccgtgacctc cagacaagga gccaccagat tggatgggag cccccacact 1550
ccctccttaa aacaccaccc tctcatcact aatctcagcc cttgcccttg 1600
aaataaacct tagctgcccc acaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1650
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1700
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 1734
<210> 106
<211> 440
<212> PRT
<213> Homo Sapien
<400> 106
Met Lys Phe Cln Gly Pro Leu Ala Cys Leu Leu Leu Ala Leu Cys
1 5 10 15
Leu Gly Ser Gly Glu Ala Gly Pro Leu Gln Ser Gly Glu Glu Ser
20 25 30
Thr Gly Thr Asn Ile Gly Glu Ala Leu Gly His Gly Leu Gly Asp
35 40 45
CA 02372511 2002-05-14
Ala Leu Ser Glu Gly Val Gly Lys Ala Ile Gly Lys Glu Ala Gly
50 55 60
Gly Ala Ala Gly Ser Lys Val Ser Glu Ala Leu Gly Gln Gly Thr
65 70 75
Arg Glu Ala Val Gly Thr Gly Val Arg Gln Val Pro Gly Phe Gly
80 85 90
Ala Ala Asp Ala Leu Gly Asn Arg Val Gly Glu Ala Ala His Ala
95 100 105
Leu Gly Asn Thr Gly His Glu Ile Gly Arg Gln Ala Glu Asp Val
110 115 120
Ile Arg His Gly Ala Asp Ala Val Arg Gly Ser Trp Gln Gly Val
125 130 135
Pro Gly His Ser Gly Ala Trp Glu Thr Ser Gly Gly His Gly Ile
140 145 150
Phe Gly Ser Gln Gly Gly Leu Gly Gly Gln Gly Gln Gly Asn Pro
155 160 165
Gly Gly Leu Gly Thr Pro Trp Val His Gly Tyr Pro Gly Asn Ser
170 175 180
Ala Gly Ser Phe Gly Met Asn Pro Gln Gly Ala Pro Trp Gly Gln
185 190 195
Gly Gly Asn Gly Gly Pro Pro Asn Phe Gly Thr Asn Thr Gln Gly
200 205 210
Ala Val Ala Gln Pro Gly Tyr Gly Ser Val Arg Ala Ser Asn Gln
215 220 225
Asn Glu Gly Cys Thr Asn Pro Pro Pro Ser Gly Ser Gly Gly Gly
230 235 240
Ser Ser Asn Ser Gly Gly Gly Ser Gly Ser Gln Ser Gly Ser Ser
245 250 255
Gly Ser Gly Ser Asn Gly Asp Asn Asn Asn Gly Ser Ser Ser Gly
260 265 270
Gly Ser Ser Ser Gly Ser Ser Ser Gly Ser Ser Ser Gly Gly Ser
275 280 285
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Asn Ser Gly Gly Ser
290 295 300
Arg Gly Asp Ser Gly Ser Glu Ser Ser Trp Gly Ser Ser Thr Gly
305 310 315
Ser Ser Ser Gly Asn His Gly Gly Ser Gly Gly Gly Asn Gly His
320 325 330
Lys Pro Gly Cys Glu Lys Pro Gly Asn Glu Ala Arg Gly Ser Gly
335 340 345
Glu Ser Gly Ile Gln Gly Phe Arg Gly Gln Gly Val Ser Ser Asn
350 355 360
71
CA 02372511 2002-05-14
Met Arg Glu Ile Ser Lys Glu Gly Asn Arg Leu Leu Gly Gly Ser
365 370 375
Gly Asp Asn Tyr Arg Gly Gln Gly Ser Ser Trp Gly Ser Gly Gly
380 385 390
Gly Asp Ala Val Gly Gly Val Asn Thr Val Asn Ser Glu Thr Ser
395 400 405
Pro Gly Met Phe Asn Phe Asp Thr Phe Trp Lys Asn Phe Lys Ser
410 415 420
Lys Leu Gly Phe Ile Asn Trp Asp Ala Ile Asn Lys Asp Gln Arg
425 430 435
Ser Ser Arg Ile Pro
440
<210> 107
<211> 918
<212> DNA
<213> Homo Sapien
<400> 107
agccaggcag cacatcacag cgggaggagc tgtcccaggt ggcccagctc 50
agcaatggca atgggggtcc ccagagtcat tctgctctgc ctctttgggg 100
ctgcgctctg cctgacaggg tcccaagccc tgcagtgcta cagctttgag 150
cacacctact ttggcccctt tgacctcagg gccatgaagc tgcccagcat 200
ctcctgtcct catgagtgct ttgaggctat cctgtctctg gacaccgggt 250
atcgcgcgcc ggtgaccctg gtgcggaagg gctgctggac cgggcctcct 300
gcgggccaga cgcaatcgaa cccggacgcg ctgccgccag actactcggt 350
ggtgcgcggc tgcacaactg acaaatgcaa cgcccacctc atgactcatg 400
acgccctccc caacctgagc caagcacccg acccgccgac gctcagcggc 450
gccgagtgct acgcctgtat cggggtccac caggatgact gcgctatcgg 500
caggtcccga cgagtccagt gtcaccagga ccagaccgcc tgcttccagg 550
gcagtggcag aatgacagtt ggcaatttct cagtccctgt gtacatcaga 600
acctgccacc ggccctcctg caccaccgag ggcaccacca gcccctggac 650
agccatcgac ctccagggct cctgctgtga ggggtacctc tgcaacagga 700
aatccatgac ccagcccttc accagtgctt cagccaccac ccctccccga 750
gcactacagg tcctggccct gctcctccca gtcctcctgc tggtggggct 800
ctcagcatag agcccccctc caggatgctg gggacagggc tcacacacct 850
cattcttgct gcttcagccc ctatcacata gctcactgga aaatgatgtt 900
aaagtaagaa ttgcaaaa 918
<210> 108
72
CA 02372511 2002-05-14
<211> 251
<212> PRT
<213> Homo Sapien
<400> 108
Met Ala Met Gly Val Pro Arg Val Ile Leu Leu Cys Leu Phe Gly
1 5 10 15
Ala Ala Leu Cys Leu Thr Gly Ser Gln Ala Leu Gln Cys Tyr Ser
20 25 30
Phe Glu His Thr Tyr Phe Gly Pro Phe Asp Leu Arg Ala Met Lys
35 40 45
Leu Pro Ser Ile Ser Cys Pro His Glu Cys Phe Glu Ala Ile Leu
50 55 60
Ser Leu Asp Thr Gly Tyr Arg Ala Pro Val Thr Leu Val Arg Lys
65 70 75
Gly Cys Trp Thr Gly Pro Pro Ala Gly Gln Thr Gln Ser Asn Pro
80 85 90
Asp Ala Leu Pro Pro Asp Tyr Ser Val Val Arg Gly Cys Thr Thr
95 100 105
Asp Lys Cys Asn Ala His Leu Met Thr His Asp Ala Leu Pro Asn
110 115 120
Leu Ser Gln Ala Pro Asp Pro Pro Thr Leu Ser Gly Ala Glu Cys
125 130 135
Tyr Ala Cys Ile Gly Val His Gln Asp Asp Cys Ala Ile Gly Arg
140 145 150
Ser Arg Arg Val Gln Cys His Gln Asp Gln Thr Ala Cys Phe Gin
155 160 165
Gly Ser Gly Arg Met Thr Val Gly Asn Phe Ser Val Pro Val Tyr
170 175 180
Ile Arg Thr Cys His Arg Pro Ser Cys Thr Thr Glu Gly Thr Thr
185 190 195
Ser Pro Trp Thr Ala Ile Asp Leu Gln Gly Ser Cys Cys Glu Gly
200 205 210
Tyr Leu Cys Asn Arg Lys Ser Met Thr Gln Pro Phe Thr Ser Ala
215 220 225
Ser Ala Thr Thr Pro Pro Arg Ala Leu Gln Val Leu Ala Leu Leu
230 235 240
Leu Pro Val Leu Leu Leu Val Gly Leu Ser Ala
245 250
<210> 109
<211> 1813
<212> DNA
<213> Homo Sapien
<400> 109
ggagccgccc tgggtgtcag cggctcggct cccgcgcacg ctccggccgt 50
73
CA 02372511 2002-05-14
cgcgcagcct c-ggcacctgc aggtccgtgc gtcccgcggc tggcgcccct 100
gactccgtcc cggccaggga gggccatgat ttccctcccg gggcccctgg 150
tgaccaactt gctgcggttt ttgttcctgg ggctgagtgc cctcgcgccc 200
ccctcgcggg cccagctgca actgcacttg cccgccaacc ggttgcaggc 250
ggtggaggga ggggaagtgg tgcttccagc gtggtacacc ttgcacgggg 300
aggtgtcttc atcccagcca tgggaggtgc cctttgtgat gtggttcttc 350
aaacagaaag aaaaggagga tcaggtgttg tcctacatca atggggtcac 400
aacaagcaaa cctggagtat ccttggtcta ctccatgccc tcccggaacc 450
tgtccctgcg gctggagggt ctccaggaga aagactctgg cccctacagc 500
tgctccgtga atgtgcaaga caaacaaggc aaatctaggg gccacagcat 550
caaaacctta gaactcaatg tactggttcc tccagctcct ccatcctgcc 600
gtttccaggg tgtcgcccat gtgggggcaa acgtgaccct gagctgccag 650
tctccaagga gtaagcccgc tgtccaatac cagtgggatc ggcagcttcc 700
atccttccag actttctttg caccagcatt agatgtcatc cgtgggtctt 750
taagcctcac caacctttcg tcttccatgg ctggagtcta tgtctgcaag 800
gcccacaatg aggtgggcac tgcccaatgt aatgtgacgc tggaagtgag 850
cacagggcct ggagctgcag tggttcctgg agctgttgtg ggtaccctgg 900
ttggactggg gttgctggct gggctggtcc tcttgtacca ccgccggggc 950
aaggccctgg aggagccagc caatgatatc aaggaggatg ccattgctcc 1000
ccggaccctg ccctggccca agagctcaga cacaatctcc aagaatggga 1050
ccctttcctc tgtcacctcc gcacgagccc tccggccacc ccatggccct 1100
cccaggcctg gtgcattgac ccccacgccc agtctctcca gccaggccct 1150
gccctcacca agactgccca cgacagatgg ggcccaccct caaccaatat 1200
cccccatccc tggtggggtt tcttcctctg gcttgagccg catgggtgct 1250
gtgcctgtga tggtgcctgc ccagagtcaa gctggctctc tggtatgatg 1300
accccaccac tcattggcta aaggatttgg ggtctctcct tcctataagg 1350
gtcacctcta gcacagaggc ctgagtcatg ggaaagagtc acactcctga 1400
cccttagtac tctgccccca cctctcttta ctgtgggaaa accatctcag 1450
taagacctaa gtgtccagga gacagaagga gaagaggaag tggatctgga 1500
attgggagga gcctccaccc acccctgact cctccttatg aagccagctg 1550
ctgaaattag ctactcacca agagtgaggg gcagagactt ccagtcactg 1600
agtctcccag gcccccttga tctgtacccc acccctatct aacaccaccc 1650
74
CA 02372511 2002-05-14
ttggctccca ctccagctcc ctgtattgat ataacctgtc aggctggctt 1700
ggttaggttt tactggggca gaggataggg aatctcttat taaaactaac 1750
atgaaatatg t.gttgttttc atttgcaaat ttaaataaag atacataatg 1800
tttgtatgaa aaa 1813
<210> 110
<211> 390
<212> PRT
<213> Homo Sapien
<400> 110
Met Ile Ser Leu Pro Gly Pro Leu Val Thr Asn Leu Leu Arg Phe
1 5 10 15
Leu Phe Leu Gly Leu Ser Ala Leu Ala Pro Pro Ser Arg Ala Gln
20 25 30
Leu Gln Leu His Leu Pro Ala Asn Arg Leu Gln Ala Val Glu Gly
35 40 45
Gly Glu Val Val Leu Pro Ala Trp Tyr Thr Leu His Gly Glu Val
50 55 60
Ser Ser Ser Gln Pro Trp Glu Val Pro Phe Val Met Trp Phe Phe
65 70 75
Lys Gln Lys Glu Lys Glu Asp Gln Val Leu Ser Tyr Ile Asn Gly
80 85 90
Val Thr Thr Ser Lys Pro Gly Val Ser Leu Val Tyr Ser Met Pro
95 100 105
Ser Arg Asn Leu Ser Leu Arg Leu Glu Gly Leu Gln Glu Lys Asp
110 115 120
Ser Gly Pro Tyr Ser Cys Ser Val Asn Val Gln Asp Lys Gln Gly
125 130 135
Lys Ser Arg Gly His Ser Ile Lys Thr Leu G1u Leu Asn Val Leu
140 145 150
Val Pro Pro Ala Pro Pro Ser Cys Arg Leu Gln Gly Val Pro His
155 160 165
Val Gly Ala Asn Val Thr Leu Ser Cys Gln Ser Pro Arg Ser Lys
170 175 180
Pro Ala Val Gin Tyr Gln Trp Asp Arg Gln Leu Pro Ser Phe Gln
185 190 195
Thr Phe Phe Ala Pro Ala Leu Asp Val Ile Arg Gly Ser Leu Ser
200 205 210
Leu Thr Asn Leu Ser Ser Ser Met Ala Gly Val Tyr Val Cys Lys
215 220 225
Ala His Asn Glu Val Gly Thr Ala Gln Cys Asn Val Thr Leu Glu
230 235 240
Val Ser Thr Gly Pro Gly Ala Ala Val Val Ala Gly Ala Val Val
CA 02372511 2002-05-14
245 250 255
Gly Thr Leu Val Gly Leu Gly Leu Leu Ala Gly Leu Val Leu Leu
260 265 270
Tyr His Arg Arg Gly Lys Ala Leu Glu Glu Pro Ala Asn Asp Ile
275 280 285
Lys Glu Asp Ala Ile Ala Pro Arg Thr Leu Pro Trp Pro Lys Ser
290 295 300
Ser Asp Thr Ile Ser Lys Asn Gly Thr Leu Ser Ser Val Thr Ser
305 310 315
Ala Arg Ala Leu Arg Pro Pro His Gly Pro Pro Arg Pro Gly Ala
320 325 330
Leu Thr Pro Thr Pro Ser Leu Ser Ser Gln Ala Leu Pro Ser Pro
335 340 345
Arg Leu Pro Thr Thr Asp Gly Ala His Pro Gln Pro Ile Ser Pro
350 355 360
Ile Pro Gly Gly Val Ser Ser Ser Gly Leu Ser Arg Met Gly Ala
365 370 375
Val Pro Val Met Val Pro Ala Gln Ser Gln Ala Gly Ser Leu Val
380 385 390
<210> 111
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 111
agggtctcca ggagaaagac tc 22
<210> 112
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 112
attgtgggcc ttgcagacat agac 24
<210> 113
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 113
ggccacagca tcaaaacctt agaactcaat gtactggttc ctccagctcc 50
<210> 114
76
CA 02372511 2002-05-14
<211> 2479
<212> DNA
<213> Homo Sapien
<400> 114
acttgccatc acctgttgcc agtgtggaaa aattctccct gttgaatttt 50
ttgcacatgg aggacagcag caaagagggc aacacaggct gataagacca 100
gagacagcag ggagattatt ttaccatacg ccctcaggac gttccctcta 150
gctggagttc tggacttcaa cagaacccca tccagtcatt ttgattttgc 200
tgtttatttt ttttttcttt ttctttttcc caccacattg tattttattt 250
ccgtacttca gaaatgggcc tacagaccac aaagtggccc agccatgggg 300
cttttttcct gaagtcttgg cttatcattt ccctggggct ctactcacag 350
gtgtccaaac tcctggcctg ccctagtgtg tgccgctgcg acaggaactt 400
tgtctactgt aatgagcgaa gcttgacctc agtgcctctt gggatcccgg 450
agggcgtaac cgtactctac ctccacaaca accaaattaa taatgctgga 500
tttcctgcag aactgcacaa tgtacagtcg gtgcacacgg tctacctgta 550
tggcaaccaa ctggacgaat tccccatgaa ccttcccaag aatgtcagag 600
ttctccattt gcaggaaaac aatattcaga ccatttcacg ggctgctctt 650
gcccagctct tgaagcttga agagctgcac ctggatgaca actccatatc 700
cacagtgggg gtggaagacg gggccttccg ggaggctatt agcctcaaat 750
tgttgttttt gtctaagaat cacctgagca gtgtgcctgt tgggcttcct 800
gtggacttgc aagagctgag agtggatgaa aatcgaattg ctgtcatatc 850
cgacatggcc ttccagaatc tcacgagctt ggagcgtctt attgtggacg 900
ggaacctcct gaccaacaag ggtatcgccg agggcacctt cagccatctc 950
accaagctca aggaattttc aattgtacgt aattcgctgt cccaccctcc 1000
tcccgatctc ccaggtacgc atctgatcag gctctatttg caggacaacc 1050
agataaacca cattcctttg acagccttct caaatctgcg taagctggaa 1100
cggctggata tatccaacaa ccaactgcgg atgctgactc aaggggtttt 1150
tgataatctc tccaacctga agcagctcac tgctcggaat aacccttggt 1200
tttgtgactg cagtattaaa tgggtcacag aatggctcaa atatatccct 1250
tcatctctca acgtgcgggg tttcatgtgc caaggtcctg aacaagtccg 1300
ggggatggcc gtcagggaat taaatatgaa tcttttgtcc tgtcccacca 1350
cgacccccgg catccctctc ttcaccccag ccccaagtac agcttctccg 1400
accactcagc ctcccaccct ctctattcca aaccctagca gaagctacac 1450
77
CA 02372511 2002-05-14
gcctccaact cctaccacat cgaaacttcc cacgattcct gactgggatg 1500
gcagagaaag agtgacccca cctatttctg aacggatcca gctctctatc 1550
cattttgtga atgatacttc cattcaagtc agctggctct ctctcttcac 1600
cgtgatggca tacaaactca catgggtgaa aatgggccac agtttagtag 1650
ggggcatcgt tcagaagcgc atagtcagcg gtgagaagca acacctgagc 1700
ctggttaact tagagccccg atccacctat cggatttgtt tagtgccact 1750
ggatgctttt aactaccgcg cggtagaaga caccatttgt tcagaggcca 1800
ccacccatgc ctcctatctg aacaacggca gcaacacagc gtccagccat 1850
gagcagacga cgtcccacag catgggcccc ccctttctgc tggcgggctt 1900
gatcgggggc gcggtgatat ttgtgctggt ggtcttgctc agcgtctttt 1950
gctggcatat gcacaaaaag gggcgctaca cctcccagaa gtggaaatac 2000
aaccggggcc ggcggaaaga tgattattgc gaggcaggca ccaagaagga 2050
caactccatc ctggagatga cagaaaccag ttttcagatc gtctccttaa 2100
ataacgatca actccttaaa ggagatttca gactgcagcc catttacacc 2150
ccaaatgggg gcattaatta cacagactgc catatcccca acaacatgcg 2200
atactgcaac aacagcctgc cagacctgga gcactgccat acgtgacagc 2250
cagaggccca gcgttatcaa ggcggacaat tagactcttg agaacacact 2300
cgtgtgtgca cataaagaca cgcagattac atttgataaa tgttacacag 2350
atgcatttgt gcatttgaat actctgtaat ttatacggtg tactatataa 2400
tgggatttaa aaaaagtgct atcttttcta tttcaagtta attacaaaca 2450
gttttgtaac tctttgcttt ttaaatctt 2479
<210> 115
<211> 660
<212> PRT
<213> Homo Sapien
<400> 115
Met Gly Leu Gln Thr Thr Lys Trp Pro Ser His Gly Ala Phe Phe
1 5 10 15
Leu Lys Ser Trp Leu Ile Ile Ser Leu Gly Leu Tyr Ser Gln Val
20 25 30
Ser Lys Leu Leu Ala Cys Pro Ser Val Cys Arg Cys Asp Arg Asn
35 40 45
Phe Val Tyr Cys Asn Glu Arg Ser Leu Thr Ser Val Pro Leu Gly
50 55 60
Ile Pro Glu.Gly Val Thr Val Leu Tyr Leu His Asn Asn Gln Ile
65 70 75
78
CA 02372511 2002-05-14
Asn Asn Ala Gly Phe Pro Ala Glu Leu His Asn Val Gln Ser Val
80 85 90
His Thr Val Tyr Leu Tyr Gly Asn Gln Leu Asp Glu Phe Pro Met
95 100 105
Asn Leu Pro Lys Asn Val Arg Val Leu His Leu Gln Glu Asn Asn
110 115 120
Ile Gln Thr Ile Ser Arg Ala Ala Leu Ala Gin Leu Leu Lys Leu
125 130 135
Glu Glu Leu His Leu Asp Asp Asn Ser Ile Ser Thr Val Gly Val
140 145 150
Glu Asp Gly Ala Phe Arg Glu Ala Ile Ser Leu Lys Leu Leu Phe
155 160 165
Leu Ser Lys Asn His Leu Ser Ser Val Pro Val Gly Leu Pro Val
170 175 180
Asp Leu Gln Glu Leu Arg Val Asp Glu Asn Arg Ile Ala Val Ile
185 190 195
Ser Asp Met Ala Phe Gln Asn Leu Thr Ser Leu Glu Arg Leu Ile
200 205 210
Val Asp Gly Asn Leu Leu Thr Asn Lys Gly Ile Ala Glu Gly Thr
215 220 225
Phe Ser His Leu Thr Lys Leu Lys Glu Phe Ser Ile Val Arg Asn
230 235 240
Ser Leu Ser His Pro Pro Pro Asp Leu Pro Gly Thr His Leu Ile
245 250 255
Arg Leu Tyr Leu Gln Asp Asn Gln Ile Asn His Ile Pro Leu Thr
260 265 270
Ala Phe Ser Asn Leu Arg Lys Leu Glu Arg Leu Asp Ile Ser Asn
275 280 285
Asn Gln Leu Arg Met Leu Thr Gln Gly Val Phe Asp Asn Leu Ser
290 295 300
Asn Leu Lys Gln Leu Thr Ala Arg Asn Asn Pro Trp Phe Cys Asp
305 310 315
Cys Ser Ile Lys Trp Val Thr Glu Trp Leu Lys Tyr Ile Pro Ser
320 325 330
Ser Leu Asn Val Arg Gly Phe Met Cys Gln Gly Pro Glu Gln Val
335 340 345
Arg Gly Met Ala Val Arg Glu Leu Asn Met Asn Leu Leu Ser Cys
350 355 360
Pro Thr Thr Thr Pro Gly Leu Pro Leu Phe Thr Pro Ala Pro Ser
365 370 375
Thr Ala Ser Pro Thr Thr Gln Pro Pro Thr Leu Ser Ile Pro Asn
380 385 390
79
CA 02372511 2002-05-14
Pro Ser Arg Ser Tyr Thr Pro Pro Thr Pro Thr Thr Ser Lys Leu
395 400 405
Pro Thr Ile Pro Asp Trp Asp Gly Arg Glu Arg Val Thr Pro Pro
410 415 420
Ile Ser Glu Arg Ile Gln Leu Ser Ile His Phe Val Asn Asp Thr
425 430 435
Ser Ile Gln Val Ser Trp Leu Ser Leu Phe Thr Val Met Ala Tyr
440 445 450
Lys Leu Thr Trp Val Lys Met Gly His Ser Leu Val Gly Gly Ile
455 460 465
Val Gln Glu Arg Ile Val Ser Gly Glu Lys Gln His Leu Ser Leu
470 475 480
Val Asn Leu Clu Pro Arg Ser Thr Tyr Arg Ile Cys Leu Val Pro
485 490 495
Leu Asp Ala Phe Asn Tyr Arg Ala Val Glu Asp Thr Ile Cys Ser
500 505 510
Glu Ala Thr Thr His Ala Ser Tyr Leu Asn Asn Gly Ser Asn Thr
515 520 525
Ala Ser Ser His Glu Gln Thr Thr Ser His Ser Met Gly Ser Pro
530 535 540
Phe Leu Leu Ala Gly Leu Ile Gly Gly Ala Val Ile Phe Val Leu
545 550 555
Val Val Leu Leu Ser Val Phe Cys Trp His Met His Lys Lys Gly
560 565 570
Arg Tyr Thr Ser Gln Lys Trp Lys Tyr Asn Arg Gly Arg Arg Lys
575 580 585
Asp Asp Tyr Cys Glu Ala Gly Thr Lys Lys Asp Asn Ser Ile Leu
590 595 600
Glu Met Thr Glu Thr Ser Phe Gln Ile Val Ser Leu Asn Asn Asp
605 610 615
Gln Leu Leu Lys Gly Asp Phe Arg Leu Gln Pro Ile Tyr Thr Pro
620 625 630
Asn Gly Gly Ile Asn Tyr Thr Asp Cys His Ile Pro Asn Asn Met
635 640 645
Arg Tyr Cys Asn Ser Ser Val Pro Asp Leu Glu His Cys His Thr
650 655 660
<210> 116
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 116
CA 02372511 2002-05-14
cggtctacct gtatggcaac c 21
<210> 117
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 117
gcaggacaac cagataaacc ac 22
<210> 118
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 118
acgcagattt gagaaggctg tc 22
<210> 119
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 119
ttcacgggct gctcttgccc agctcttgaa gcttgaagag ctgcac 46
<210> 120
<211> 2857
<212> DNA
<213> Homo Sapien
<400> 120
tgaagagtaa tagttggaat caaaagagtc aacgcaatga actgttattt 50
actgctgcgt tttatgttgg gaattcctct cctatggcct tgtcttggag 100
caacagaaaa ctctcaaaca aagaaagtca agcagccagt gcgatctcat 150
ttgagagtga agcgtggctg ggtgtggaac caattttttg taccagagga 200
aatgaatacg actagtcatc acatcggcca gctaagatct gatttagaca 250
atggaaacaa ttctttccag tacaagcttt tgggagctgg agctggaagt 300
acttttatca ttgatgaaag aacaggtgac atatatgcca tacagaagct 350
tgatagagag gagcgatccc tctacatctt aagagcccag gtaatagaca 400
tcgctactgg aagggctgtg gaacctgagt ctgagtttgt catcaaagtt 450
tcggatatca atgacaatga accaaaattc ctagatgaac cttatgaggc 500
cattgtacca gagatgtctc cagaaggaac attagttatc caggtgacag 550
81
CA 02372511 2002-05-14
caagtgatgc tgacgatccc tcaagtggta ataatgctcg tctcctctac 600
agcttacttc aaggccagcc atatttttct gttgaaccaa caacaggagt 650
cataagaata tcttctaaaa tggatagaga actgcaagat gagtattggg 700
taatcattca agccaaggac atgattggtc agccaggagc gttgtctgga 750
acaacaagtg tattaattaa actttcagat gttaatgaca ataagcctat 800
atttaaagaa agtttatacc gcttgactgt ctctgaatct gcacccactg 850
ggacttctat aggaacaatc atggcatatg ataatgacat aggagagaat 900
gcagaaatgg attacagcat tgaagaggat gattcgcaaa catttgacat 950
tattactaat catgaaactc aagaaggaat agttatatta aaaaagaaag 1000
tggattttga gcaccagaac cactacggta ttagagcaaa agttaaaaac 1050
catcatgttc ctgagcagct catgaagtac cacactgagg cttccaccac 1100
tttcattaag atccaggtgg aagatgttga tgagcctcct cttttcctcc 1150
ttccatatta tgtatttgaa gtttttgaag aaaccccaca gggatcattt 1200
gtaggcgtgg tgtctgccac agacccagac aataggaaat ctcctatcag 1250
gtattctatt actaggagca aagtgttcaa tatcaatgat aatggtacaa 1300
tcactacaag taactcactg gatcgtgaaa tcagtgcttg gtacaaccta 1350
agtattacag ccacagaaaa atacaatata gaacagatct cttcgatccc 1400
actgtatgtg caagttctta acatcaatga tcatgctcct gagttctctc 1450
aatactatga gacttatgtt tgtgaaaatg caggctctgg tcaggtaatt 1500
cagactatca gtgcagtgga tagagatgaa tccatagaag agcaccattt 1550
ttactttaat ctatctgtag aagacactaa caattcaagt tttacaatca 1600
tagataatca agataacaca gctgtcattt tgactaatag aactggtttt 1650
aaccttcaag aagaacctgt cttctacatc tccatcttaa ttgccgacaa 1700
tggaatcccg tcacttacaa gtacaaacac ccttaccatc catgtctgtg 1750
actgtggtga cagtgggagc acacagacct gccagtacca ggagcttgtg 1800
ctttccatgg gattcaagac agaagttatc attgctattc tcatttgcat 1850
tatgatcata tttgggttta tttttttgac tttgggttta aaacaacgga 1900
gaaaacagat tctatttcct gagaaaagtg aagatttcag agagaatata 1950
ttccaatatg atgatgaagg gggtggagaa gaagatacag aggcctttga 2000
tatagcagag ctgaggagta gtaccataat gcgggaacgc aagactcgga 2050
aaaccacaag cgctgagatc aggagcctat acaggcagtc tttgcaagtt 2100
ggccccgaca gtgccatatt caggaaattc attctggaaa agctcgaaga 2150
82
CA 02372511 2002-05-14
agctaatact gatccgtgtg cccctccttt tgattccctc cagacctacg 2200
cttttgaggg aacagggtca ttagctggat ccctgagctc cttagaatca 2250
gcagtctctg atcaggatga aagctatgat taccttaatg agttgggacc 2300
tcgctttaaa agattagcat gcatgtttgg ttctgcagtg cagtcaaata 2350
attagggctt tttaccatca aaatttttaa aagtgctaat gtgtattcga 2400
acccaatggt agtcttaaag agttttgtgc cctggctcta tggcggggaa 2450
agccctagtc tatggagttt tctgatttcc ctggagtaaa tactccatgg 2500
ttattttaag ctacctacat gctgtcattg aacagagatg tggggagaaa 2550
tgtaaacaat cagctcacag gcatcaatac aaccagattt gaagtaaaat 2600
aatgtaggaa gatattaaaa gtagatgaga ggacacaaga tgtagtcgat 2650
ccttatgcga ttatatcatt atttacttag gaaagagtaa aaataccaaa 2700
cgagaaaatt taaaggagca aaaatttgca agtcaaatag aaatgtacaa 2750
atcgagataa catttacatt tctatcatat tgacatgaaa attgaaaatg 2800
tatagtcaga gaaattttca tgaattattc catgaagtat tgtttccttt 2850
atttaaa 2857
<210> 121
<211> 772
<212> PRT
<213> Homo Sapien
<400> 121
Met Asn Cys Tyr Leu Leu Leu Arg Phe Net Leu Gly Ile Pro Leu
1 5 10 15
Leu Trp Pro Cys Leu Gly Ala Thr Glu Asn Ser Gln Thr Lys Lys
20 25 30
Val Lys Gln Pro Val Arg Ser His Leu Arg Val Lys Arg Gly Trp
35 40 45
Val Trp Asn ln Phe Phe Val Pro Glu Glu Met Asn Thr Thr Ser
50 55 60
His His Ile Gly Gln Leu Arg Ser Asp Leu Asp Asn Gly Asn Asn
65 70 75
Ser Phe Gln Tyr Lys Leu Leu Gly Ala Gly Ala Gly Ser Thr Phe
80 85 90
Ile Ile Asp Glu Arg Thr Gly Asp Ile Tyr Ala Ile Gln Lys Leu
95 100 105
Asp Arg Glu Glu Arg Ser Leu Tyr Ile Leu Arg Ala Gln Val Ile
110 115 120
Asp Ile Ala Thr Gly Arg Ala Val Glu Pro Glu Ser Glu Phe Val
125 130 135
83
CA 02372511 2002-05-14
Ile Lys Val Ser Asp Ile Asn Asp Asn Glu Pro Lys Phe Leu Asp
140 145 150
Glu Pro Tyr Glu Ala Ile Val Pro Glu Met Ser Pro Glu Gly Thr
155 160 165
Leu Val Ile Cln Val Thr Ala Ser Asp Ala Asp Asp Pro Ser Ser
170 175 180
Gly Asn Asn Ala Arg Leu Leu Tyr Ser Leu Leu Gln Gly Gln Pro
185 190 195
Tyr Phe Ser Val Glu Pro Thr Thr Gly Val Ile Arg Ile Ser Ser
200 205 210
Lys Met Asp Arg Glu Leu Gln Asp Glu Tyr Trp Val Ile Ile Gln
215 220 225
Ala Lys Asp Met Ile Gly Gln Pro Gly Ala Leu Ser Gly Thr Thr
230 235 240
Ser Val Leu Ile Lys Leu Ser Asp Val Asn Asp Asn Lys Pro Ile
245 250 255
Phe Lys Glu Ser Leu Tyr Arg Leu Thr Val Ser Glu Ser Ala Pro
260 265 270
Thr Gly Thr.Ser Ile Gly Thr Ile Met Ala Tyr Asp Asn Asp Ile
275 280 285
Gly Glu Asn Ala Glu Met Asp Tyr Ser Ile Glu Glu Asp Asp Ser
290 295 300
Gln Thr Phe Asp Ile Ile Thr Asn His Glu Thr Gln Glu Gly Ile
305 310 315
Val Ile Leu Lys Lys Lys Val Asp Phe Glu His Gln Asn His Tyr
320 325 330
Gly Ile Arg Ala Lys Val Lys Asn His His Val Pro Glu Gln Leu
335 340 345
Met Lys Tyr His Thr Glu Ala Ser Thr Thr Phe Ile Lys Ile Gln
350 355 360
Val Glu Asp Val Asp Glu Pro Pro Leu Phe Leu Leu Pro Tyr Tyr
365 370 375
Val Phe Glu Val Phe Glu Glu Thr Pro Gin Gly Ser Phe Val Gly
380 385 390
Val Val Ser Ala Thr Asp Pro Asp Asn Arg Lys Ser Pro Ile Arg
395 400 405
Tyr Ser Ile Thr Arg Ser Lys Val Phe Asn Ile Asn Asp Asn Gly
410 415 420
Thr Ile Thr Thr Ser Asn Ser Leu Asp Arg Glu Ile Ser Ala Trp
425 430 435
Tyr Asn Leu Ser Ile Thr Ala Thr Glu Lys Tyr Asn Ile Glu Gln
440 445 450
84
CA 02372511 2002-05-14
Ile Ser Ser Ile Pro Leu Tyr Val Gln Val Leu Asn Ile Asn Asp
455 460 465
His Ala Pro Glu Phe Ser Gln Tyr Tyr Glu Thr Tyr Val Cys Glu
470 475 480
Asn Ala Gly Ser Gly Gln Val Ile Gln Thr Ile Ser Ala Val Asp
485 490 495
Arg Asp Glu Ser Ile Glu Glu His His Phe Tyr Phe Asn Leu Ser
500 505 510
Val Glu Asp Thr Asn Asn Ser Ser Phe Thr Ile Ile Asp Asn Gln
515 520 525
Asp Asn Thr Ala Val Ile Leu Thr Asn Arg Thr Gly Phe Asn Leu
530 535 540
Gln Glu Glu Pro Val Phe Tyr Ile Ser Ile Leu Ile Ala Asp Asn
545 550 555
Gly Ile Pro Ser Leu Thr Ser Thr Asn Thr Leu Thr Ile His Val
560 565 570
Cys Asp Cys Gly Asp Ser Gly Ser Thr Gln Thr Cys Gln Tyr Gln
575 580 585
Glu Leu Val Leu Ser Met Gly Phe Lys Thr Glu Val Ile Ile Ala
590 595 600
Ile Leu Ile Cys Ile Met Ile Ile Phe Gly Phe Ile Phe Leu Thr
605 610 615
Leu Gly Leu Lys Gln Arg Arg Lys Gln Ile Leu Phe Pro Glu Lys
620 625 630
Ser Glu Asp Phe Arg Glu Asn Ile Phe Gln Tyr Asp Asp Glu Gly
635 640 645
Gly Gly Glu Glu Asp Thr Glu Ala Phe Asp Ile Ala Glu Leu Arg
650 655 660
Ser Ser Thr Ile Met Arg Glu Arg Lys Thr Arg Lys Thr Thr Ser
665 670 675
Ala Glu Ile Arg Ser Leu Tyr Arg Gln Ser Leu Gln Val Gly Pro
680 685 690
Asp Ser Ala Ile Phe Arg Lys Phe Ile Leu Glu Lys Leu Glu Glu
695 700 705
Ala Asn Thr Asp Pro Cys Ala Pro Pro Phe Asp Ser Leu Gln Thr
710 715 720
Tyr Ala Phe Glu Gly Thr Gly Ser Leu Ala Gly Ser Leu Ser Ser
725 730 735
Leu Glu Ser Ala Val Ser Asp Gln Asp Glu Ser Tyr Asp Tyr Leu
740 745 750
Asn Glu Leu Gly Pro Arg Phe Lys Arg Leu Ala Cys Met Phe Gly
755 760 765
CA 02372511 2002-05-14
Ser Ala Val Gln Ser Asn Asn
770
<210> 122
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 122
cttgactgtc tctgaatctg caccc 25
<210> 123
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 123
aagtggtgga agcctccagt gtgg 24
<210> 124
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide probe
<400> 124
ccactacggt attagagcaa aagttaaaaa ccatcatggt tcctggagca 50
gc 52
<210> 125
<211> 1152
<212> DNA
<213> Homo Sapien
<400> 125
cttcagaaca ggttctcctt ccccagtcac cagttgctcg agttagaatt 50
gtctgcaatg gccgccctgc agaaatctgt gagctctttc cttatgggga 100
ccctggccac cagctgcctc cttctcttgg ccctcttggt acagggagga 150
gcagctgcgc ccatcatctc ccactgcagg cttgacaagt ccaacttcca 200
gcagccctat atcaccaacc gcaccttcat gctggctaag gaggctagct 250
tggctgataa caacacagac gttcgtctca ttggggagaa actgttccac 300
ggagtcagta tgagtgagcg ctgctatctg atgaagcagg tgctgaactt 350
cacccttgaa gaagtgctgt tccctcaatc tgataggttc cagccttata 400
tgcaggaggt ggtgcccttc ctggccaggc tcagcaacag gctaagcaca 450
tgtcatattg aaggtgatga cctgcatatc cagaggaatg tgcaaaagct 500
86
CA 02372511 2002-05-14
gaaggacaca gtgaaaaagc ttggagagag tggagagatc aaagcaattg 550
gagaactgga tttgctgttt atgtctctga gaaatgcctg catttgacca 600
gagcaaagct gaaaaatgaa taactaaccc cctttccctg ctagaaataa 650
caattagatg ccccaaagcg atttttttta accaaaagga agatgggaag 700
ccaaactcca tcatgatggg tggattccaa atgaacccct gcgttagtta 750
caaaggaaac caatgccact tttgtttata agaccagaag gtagactttc 800
taagcataga tatttattga taacatttca ttgtaactgg tgttctatac 850
acagaaaaca atttattttt taaataattg tctttttcca taaaaaagat 900
tactttccat tcctttaggg gaaaaaaccc ctaaatagct tcatttttcc 950
ataatcagta ctttatattt ataaatgtat ttattattat tataagatgg 1000
cattttattt atatcatttt attaatatgg atttatttat agaaacatca 1050
ttcgatattg ctacttgagt gtaaggctaa tattgatatt tatgacaata 1100
attatagagc tataacatgt ttatttgacc tcaataaaca cttggatatc 1150
cc 1152
<210> 126
<211> 179
<212> PRT
<213> Homo Sapien
<400> 126
Met Ala Ala Leu Gln Lys Ser Val Ser Ser Phe Leu Met Gly Thr
1 5 10 15
Leu Ala Thr Ser Cys Leu Leu Leu Leu Ala Leu Leu Val Gln Gly
20 25 30
Gly Ala Ala Ala Pro Ile Ser Ser His Cys Arg Leu Asp Lys Ser
35 40 45
Asn Phe Gln Gln Pro Tyr Ile Thr Asn Arg Thr Phe Met Leu Ala
50 55 60
Lys Glu Ala Ser Leu Ala Asp Asn Asn Thr Asp Val Arg Leu Ile
65 70 75
Gly Glu Lys Leu Phe His Gly Val Ser Met Ser Glu Arg Cys Tyr
80 85 90
Leu Met Lys Gin Val Leu Asn Phe Thr Leu Glu Glu Val Leu Phe
95 100 105
Pro Gln Ser Asp Arg Phe Gln Pro Tyr Met Gln Glu Val Val Pro
110 115 120
Phe Leu Ala Arg Leu Ser Asn Arg Leu Ser Thr Cys His Ile Glu
125 130 135
Gly Asp Asp Leu His Ile Gln Arg Asn Val Gln Lys Leu Lys Asp
140 145 150
87
CA 02372511 2002-05-14
Thr Val Lys Lys Leu Gly Glu Ser Gly Glu Ile Lys Ala Ile Gly
155 160 165
Glu Leu Asp Leu Leu Phe Met Ser Leu Arg Asn Ala Cys Ile
170 175
<210> 127
<211> 2557
<212> DNA
<213> Homo Sapien
<400> 127
gccctaacct t.cccagggct cagctctttg gagctgccca ttcctccggc 50
tgcgagaaag gacgcgcgcc ctgcgtcggg cgaagaaaag aagcaaaact 100
tgtcgggagg gtttcgtcat caacctcctt cccgcaaacc taaacctcct 150
gccggggcca tccctagaca gaggaaagtt cctgcagagc cgaccagccc 200
tagtggatct ggggcaggca gcggcgctgg ctgtggaatt agatctgttt 250
tgaacccagt ggagcgcatc gctggggctc ggaagtcacc gtccgcgggc 300
accgggttgg cgctgcccga gtggaaccga cagtttgcga gcctcggctg 350
caagtggcct ctcctccccg cggttgttgt tcagtgtcgg gtgagggctg 400
cgagtgtggc aagttgcaaa gagagcctca gaggtccgaa gagcgctgcg 450
ctcctactcg cgttcgcttc ttcctcttct cggttcccta ctgtgaaatc 500
gcagcgacat ttacaaaggc ctccgggtcc taccgagacc gatccgcagc 550
gtttggcccg gtcgtgccta ttgcatcggg agcccccgag caccggcgaa 600
atggcgaggt tcccgaaggc cgacctggcc gctgcaggag ttatgttact 650
ttgccacttc ttcacggacc agtttcagtt cgccgatggg aaacccggag 700
accaaatcct tgattggcag tatggagtta ctcaggcctt ccctcacaca 750
gaggaggagg tggaagttga ttcacacgcg tacagccaca ggtggaaaag 800
aaacttggac tttctcaagg cggtagacac gaaccgagca agcgtcggcc 850
aagactctcc tgagcccaga agcttcacag acctgctgct ggatgatggg 900
caggacaata acactcagat cgaggaggat acagaccaca attactatat 950
atctcgaata tatggtccat ctgattctgc cagccgggat ttatgggtga 1000
acatagacca aatggaaaaa gataaagtga agattcatgg aatattgtcc 1050
aatactcatc ggcaagctgc aagagtgaat ctgtccttcg attttccatt 1100
ttatggccac ttcctacgtg aaatcactgt ggcaaccggg ggtttcatat 1150
acactggaga a.gtcgtacat cgaatgctaa cagccacaca gtacatagca 1200
cctttaatgg caaatttcga tcccagtgta tccagaaatt caactgtcag 1250
atattttgat aatggcacag cacttgtggt ccagtgggac catgtacatc 1300
88
CA 02372511 2002-05-14
tccaggataa ttataacctg ggaagcttca cattccaggc aaccctgctc 1350
atggatggac gaatcatctt tggatacaaa gaaattcctg tcttggtcac 1400
acagataagt tcaaccaatc atccagtgaa agtcggactg tccgatgcat 1450
ttgtcgttgt ccacaggatc caacaaattc ccaatgttcg aagaagaaca 1500
atttatgaat accaccgagt agagctacaa atgtcaaaaa ttaccaacat 1550
ttcggctgtg gagatgaccc cattacccac atgcctccag tttaacagat 1600
gtggcccctg tgtatcttct cagattggct tcaactgcag ttggtgtagt 1650
aaacttcaaa gatgttccag tggatttgat cgtcatcggc aggactgggt 1700
ggacagtgga tgccctgaag agtcaaaaga gaagatgtgt gagaatacag 1750
aaccagtgga aacttcttct cgaaccacca caaccgtagg agcgacaacc 1800
acccagttca gggtcctaac taccaccaga agagcagtga cttctcagtt 1850
tcccaccagc ctccctacag aagatgatac caagatagca ctacatctaa 1900
aagataatgg agcttctaca gatgacagtg cagctgagaa gaaaggggga 1950
accctccacg ctggcctcat cattggaatc ctcatcctgg tcctcattgt 2000
agccacagcc attcttgtga cagtctatat gtatcaccac ccaacatcag 2050
cagccagcat cttctttatt gagagacgcc caagcagatg gcctgcgatg 2100
aagtttagaa gaggctctgg acatcctgcc tatgctgaag ttgaaccagt 2150
tggagagaaa gaaggcttta ttgtatcaga gcagtgctaa aatttctagg 2200
acagaacaac accagtactg gtttacaggt gttaagacta aaattttgcc 2250
tataccttta agacaaacaa acaaacacac acacaaacaa gctctaagct 2300
gctgtagcct gaagaagaca agatttctgg acaagctcag cccaggaaac 2350
aaagggtaaa caaaaaacta aaacttatac aagataccat ttacactgaa 2400
catagaattc cctagtggaa tgtcatctat agttcactcg gaacatctcc 2450
cgtggactta t.ctgaagtat gacaagatta taatgctttt ggcttaggtg 2500
cagggttgca aagggatcag aaaaaaaaaa tcataataaa gctttagttc 2550
atgaggg 2557
<210> 128
<211> 529
<212> PRT
<213> Homo Sapien
<400> 128
Met Ala Arg Phe Pro Lys Ala Asp Leu Ala Ala Ala Gly Val Met
1 5 10 15
Leu Leu Cys His Phe Phe Thr Asp Gln Phe Gln Phe Ala Asp Gly
20 25 30
89
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
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