Note: Descriptions are shown in the official language in which they were submitted.
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PROTEASES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of proteases
and to the use of
these sequences in the diagnosis, treatment, and prevention of
gastrointestinal, cardiovascular,
autoimmune/inflarmnatory, cell proliferative, developmental, epithelial,
neurological, and
reproductive disorders, and in the assessment of the effects of exogenous
compounds on the
expression of nucleic acid and amino acid sequences of proteases.
BACKGROUND OF THE INVENTION
Proteases cleave proteins and peptides at the peptide bond that forms the
backbone of the
protein or peptide chain. Proteolysis is one of the most important and
frequent enzymatic reactions
that occurs both within and outside of cells. Proteolysis is responsible for
the activation and
maturation of nascent polypeptides, the degradation of misfolded and damaged
proteins, and the
controlled turnover of peptides within the cell. Proteases participate in
digestion, endocrine function,
and tissue remodeling during embryonic development, wound healing, and normal
growth. Proteases
can play a role in regulatory processes by affecting the half life of
regulatory proteins. Proteases are
involved in the etiology or progression of disease states such as
inflammation, angiogenesis, tumor
dispersion and metastasis, cardiovascular disease, neurological disease, and
bacterial, parasitic, and
viral infections.
Proteases can be categorized on the basis of where they cleave their
substrates.
Exopeptidases, which include aminopeptidases, dipeptidyl peptidases,
tripeptidases,
carboxypeptidases, peptidyl-di-peptidases, dipeptidases, and omega peptidases,
cleave residues at the
termini of their substrates. Endopeptidases, including serine proteases,
cysteine proteases, and
metalloproteases, cleave at residues within the peptide. Four principal
categories of mammalian
proteases have been identified based on active site structure, mechanism of
action, and overall three-
dimensional structure. (See Beynon, R.J. and J.S. Bond (1994) Proteolytic
Enzymes: A Practical
Approach, Oxford University Press, New York NY, pp. 1-5.)
Serine Proteases
The serine proteases (SPs) are a large, widespread family of proteolytic
enzymes that include
the digestive enzymes trypsin and chymotrypsin, components of the complement
and blood-clotting
cascades, and enzymes that control the degradation and turnover of
macromolecules within the cell
and in the extracellular matrix. Most of the more than 20 subfamilies can be
grouped into six clans,
each with a common ancestor. These six clans are hypothesized to have
descended from at least four
evolutionarily distinct ancestors. SPs are named for the presence of a serine
residue found in the
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active catalytic site of most families. The active site is defined by the
catalytic triad, a set of
conserved asparagine, histidine, and serine residues critical for catalysis.
These residues form a
charge relay network that facilitates substrate binding. Other residues
outside the active site form an
oxyanion hole that stabilizes the tetrahedral transition intermediate formed
during catalysis. SPs have
a wide range of substrates and can be subdivided into subfamilies on the basis
of their substrate
specificity. The main subfamilies are named for the residues) after which they
cleave: trypases
(after arginine or lysine), aspases (after aspartate), chymases (after
phenylalanine or leucine), metases
(methionine), and serases (after serine) (Rawlings, N.D, and A.J. Barrett
(1994) Methods Enzymol.
244:19-61).
Most mammalian serine proteases are synthesized as zymogens, inactive
precursors that are
activated by proteolysis. For example, trypsinogen is converted to its active
form, trypsin, by
enteropeptidase. Enteropeptidase is an intestinal protease that removes an N-
terminal fragment from
trypsinogen. The remaining active fragment is trypsin, which in turn activates
the precursors of the
other pancreatic enzymes. Likewise, proteolysis of prothrombin, the precursor
of thrombin, generates
three separate polypeptide fragments. The N-terminal fragment is released
while the other two
fragments, which comprise active thrombin, remain associated through disulfide
bonds.
The two largest SP subfamilies are the chymotrypsin (S1) and subtilisin (S8)
families. Some
members of the chymotrypsin family contain two structural domains unique to
this family. Kringle
domains are triple-looped, disulfide cross-linked domains found in varying
copy number. Kringles
are thought to play a role in binding mediators such as membranes, other
proteins or phospholipids,
and in the regulation of proteolytic activity (PROSITE PDOC00020). Apple
domains are 90 amino-
acid repeated domains, each containing six conserved cysteines. Three
disulfide bonds link the first
and sixth, second and fifth, and third and fourth cysteines (PROSITE
PDOC00376). Apple domains
are involved in protein-protein interactions. S 1 family members include
trypsin, chymotrypsin,
coagulation factors IX-III, complement factors B, C, and D, granzymes,
kallikrein, and tissue- and
urokinase-plasminogen activators. The subtilisin family has members found in
the eubacteria,
archaebacteria, eukaryotes, and viruses. Subtilisins include the proprotein-
processing endopeptidases
kexin and furin and the pituitary prohormone convertases PC1, PC2, PC3, PC6,
and PACE4
(Rawlings and Barrett, supra).
SPs have functions in many normal processes and some have been implicated in
the etiology
or treatment of disease. Enterokinase, the initiator of intestinal digestion,
is found in the intestinal
brush border, where it cleaves the acidic propeptide from trypsinogen to yield
active trypsin
(Kitamoto, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91:7588-7592).
Prolylcarboxypeptidase, a
lysosomal serine peptidase that cleaves peptides such as angiotensin II and
III and [des-Arg9]
bradykinin, shares sequence homology with members of both the serine
carboxypeptidase and
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prolylendopeptidase families (Tan, F. et al. (1993) J. Biol. Chem. 268:16631-
16638). The protease
neuropsin may influence synapse formation and neuronal connectivity in the
hippocampus in
response to neural signaling (Chen, Z.-L. et al. (1995) J. Neurosci. 15:5088-
5097). Tissue
plasminogen activator is useful for acute management of stroke (Zivin, J.A.
(1999) Neurology 53:14-
19) and myocardial infarction (Ross, A.M. (1999) Clin: Cardiol. 22:165-171).
Some receptors (PAR,
for proteinase-activated receptor), highly expressed throughout the digestive
tract, are activated by
proteolytic cleavage of an extracellular domain. The major agonists for PARs,
thrombin, trypsin, and
mast Bell tryptase, are released in allergy and inflammatory conditions.
Control of PAR activation by
proteases has been suggested as a promising therapeutic target (Vergnolle, N.
(2000) Aliment.
Pharmacol. Ther. 14:257-266; Rice, K.D. et al. (1998) Curr. Pharm. Des. 4:381-
396). Prostate-
specific antigen (PSA) is a kallikrein-like serine protease synthesized and
secreted exclusively by
epithelial Bells in the prostate gland. Serum PSA is elevated in prostate
cancer and is the most
sensitive physiological marker for monitoring cancer progression and response
to therapy. PSA can
also identify the prostate as the origin of a metastatic tumor (Brawer, M.K.
and P.H. Lange (1989)
Urology 33:11-16).
The signal peptidase is a specialized class of SP found in all prokaryotic and
eukaryotic cell
types that serves in the processing of signal peptides from certain proteins.
Signal peptides are
amino-terminal domains of a protein which direct the protein from its
ribosomal assembly site to a
particular cellular or extracellular location. Once the protein has been
exported, removal of the signal
sequence by a signal peptidase and posttranslational processing, e.g.,
glycosylation or
phosphorylation, activate the protein. Signal peptidases exist as mufti-
subunit complexes in both
yeast and mammals. The canine signal peptidase complex is composed of five
subunits, all
associated with the microsomal membrane and containing hydrophobic regions
that span the
membrane one or more times (Shelness, G.S. and G. Blobel (1990) J. Biol. Chem.
265:9512-9519).
Some of these subunits serve to fix the complex in its proper position on the
membrane while others
contain the actual catalytic activity.
Another family of proteases which have a serine in their active site are
dependent on the
hydrolysis of ATP for their activity. These proteases contain proteolytic core
domains and regulatory
ATPase domains which can be identified by the presence of the P-loop, an
ATP/GTP-binding motif
(PROSITE PDOC00803). Members of this family include the eukaryotic
mitochondrial matrix
proteases, Clp protease and the proteasome. Clp protease was originally found
in plant chloroplasts
but is believed to be widespread in both prokaryotic and eukaryotic cells. The
gene for early-onset
torsion dystonia encodes a protein related to Clp protease (Ozelius, L.J. et
al. (1998) Adv. Neurol.
78:93-105).
The proteasome is an intracellular protease complex found in some bacteria and
in all
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eukaryotic cells, and plays an important role in cellular physiology.
Proteasomes are associated with
the ubiquitin conjugation system (UCS), a major pathway for the degradation of
cellular proteins of
all types, including proteins that function to activate or repress cellular
processes such as transcription
and cell cycle progression (Ciechanover, A. (1994) Cell 79:13-21). In the UCS
pathway, proteins
targeted for degradation are conjugated to ubiquitin, a small heat stable
protein. The ubiquitinated
protein is then recognized and degraded by the proteasome. The resultant
ubiquitin-peptide complex
is hydrolyzed by a ubiquitin carboxyl terminal hydrolase, and free ubiquitin
is released for
reutilization by the UCS. Ubiquitin-proteasome systems are implicated in the
degradation of mitotic
cyclic kinases, oncoproteins, tumor suppressor genes (p53), cell surface
receptors associated with
signal transduction, transcriptional regulators, and mutated or damaged
proteins (Ciechanover, supra).
This pathway has been implicated in a number of diseases, including cystic
fibrosis, Angelman's
syndrome, and Liddle syndrome (reviewed in Schwartz, A.L. and A. Ciechanover
(1999} Annu. Rev.
Med. 50:57-74). A murine proto-oncogene, Unp, encodes a nuclear ubiquitin
protease whose
overexpression leads to oncogenic transformation of NIH3T3 cells. The human
homologue of this
gene is consistently elevated in small cell tumors and adenocarcinomas of the
lung (Gray, D.A.
(1995) Oncogene 10:2179-2183). Ubiquitin carboxyl terminal hydrolase is
involved in the
differentiation of a lymphoblastic leukemia cell line to a non-dividing mature
state (Maki, A. et al.
(1996) Differentiation 60:59-66). In neurons, ubiquitin carboxyl terminal
hydrolase (PGP 9.5)
expression is strong in the abnormal structures that occur in human
neurodegenerative diseases
(Lowe, J. et al. (1990) J. Pathol. 161:153-160). The proteasome is a large
(2000 kDa) multisubunit
complex composed of a central catalytic core containing a variety of proteases
arranged in four seven-
membered rings with the active sites facing inwards into the central cavity,
and terminal ATPase
subunits covering the outer port of the cavity and regulating substrate entry
(for review, see Schmidt,
M. et al. (1999) Curr. Opin. Chem. Biol. 3:584-591).
Cysteine Proteases
Cysteine proteases (CPs) are involved in diverse cellular processes ranging
from the
processing of precursor proteins to intracellular degradation. Nearly half of
the CPs known are
present only in viruses. CPs have a cysteine as the major catalytic residue at
the active site where
catalysis proceeds via a thioester intermediate and is facilitated by nearby
histidine and asparagine
residues. A glutamine residue is also important, as it helps to form an
oxyanion hole. Two important
CP families include the papain-like enzymes (C1) and the calpains (C2). Papain-
like family members
are generally lysosomal or secreted and therefore are synthesized with signal
peptides as well as
propeptides. Most members bear a conserved motif in the propeptide that may
have structural
significance (Karrer, K.M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:3063-
3067). Three-
dimensional structures of papain family members show a bilobed molecule with
the catalytic site
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located between the two lobes. Papains include cathepsins B, C, H, L, and S,
certain plant allergens
and dipeptidyl peptidase (for a review, see Rawlings, N.D. and A.J. Barrett
(1994) Methods Enzymol.
244:461-486).
Some CPs are expressed ubiquitously, while others are produced only by cells
of the immune
system. Of particular note, CPs are produced by monocytes, macrophages and
other cells which
migrate to sites of inflammation and secrete molecules involved in tissue
repair. Overabundance of
these repair molecules plays a role in certain disorders. In autoimmune
diseases such as rheumatoid
arthritis, secretion of the cysteine peptidase cathepsin C degrades collagen,
laminin, elastin and other
structural proteins found in the extracellular matrix of bones. Bone weakened
by such degradation is
also more susceptible to tumor invasion and metastasis. Cathepsin L expression
may also contribute
to the influx of mononuclear cells which exacerbates the destruction of the
rheumatoid synovium
(Keyszer, G.M. (1995) Arthritis Rheum. 38:976-984).
Calpains are calcium-dependent cytosolic endopeptidases which contain both an
N-terminal
catalytic domain and a C-terminal calcium-binding domain. Calpain is expressed
as a proenzyme
heterodimer consisting of a catalytic subunit unique to each isoform and a
regulatory subunit common
to different isoforms. Each subunit bears a calcium-binding EF-hand domain.
The regulatory subunit
also contains a hydrophobic glycine-rich domain that allows the enzyme to
associate with cell
membranes. Calpains are activated by increased intracellular calcium
concentration, which induces a
change in conformation and limited autolysis. The resultant active molecule
requires a lower calcium
concentration for its activity (Chan, S.L. and M.P. Mattson (1999) J.
Neurosci. Res. 58:167-190).
Calpain expression is predominantly neuronal, although it is present in other
tissues. Several chronic
neurodegenerative disorders, including ALS, Parkinson's disease and
Alzheimer's disease are
associated with increased calpain expression (Chan and Mattson, supra).
Calpain-mediated
breakdown of the cytoskeleton has been proposed to contribute to brain damage
resulting from head
injury (McCracken, E. et al. (1999) J. Neurotrauma 16:749-761). Calpain-3 is
predominantly
expressed in skeletal muscle, and is responsible for limb-girdle muscular
dystrophy type 2A (Minami,
N. et al. (1999) J. Neurol. Sci. 171:31-37).
Another family of thiol proteases is the caspases, which are involved in the
initiation and
execution phases of apoptosis. A pro-apoptotic signal can activate initiator
caspases that trigger a
proteolytic caspase cascade, leading to the hydrolysis of target proteins and
the classic apoptotic
death of the cell. Two active site residues, a cysteine and a histidine, have
been implicated in the
catalytic mechanism. Caspases acre among the most specific endopeptidases,
cleaving after aspartate
residues. Caspases are synthesized as inactive zymogens consisting of one
large (p20) and one small
(p10) subunit separated by a small spacer region, and a variable N-terminal
prodomain. This
prodomain interacts with cofactors that can positively or negatively affect
apoptosis. An activating
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signal causes autoproteolytic cleavage of a specific aspartate residue (D297
in the caspase-1
numbering convention) and removal of the spacer and prodomain, leaving a
p101p20 heterodimer.
Two of these heterodimers interact via their small subunits to form the
catalytically active tetramer.
The long prodomains of some caspase family members have been shown to promote
dimerization and
auto-processing of procaspases. Some caspases contain a "death effector
domain" in their prodomain
by which they can be recruited into self activating complexes with other
caspases and FADD protein
associated death receptors or the TNF receptor complex. In addition, two
dimers from different
caspase family members can associate, changing the substrate specificity of
the resultant tetramer.
Endogenous caspase inhibitors (inhibitor of apoptosis proteins, or IAPs) also
exist. All these
interactions have clear effects on the control of apoptosis (reviewed in Chan
and Mattson, supra;
Salveson, G.S. and V.M. Dixit (1999) Proc. Natl. Acad. Sci. USA 96:10964-
10967).
Caspases have been implicated in a number of diseases. Mice lacking some
caspases have
severe nervous system defects due to failed apoptosis in the neuroepithelium
and suffer early
lethality. Others show severe defects in the inflammatory response, as
caspases are responsible for
processing IL-lb and possibly other inflammatory cytokines (Chap and Mattson,
supra). Cowpox
virus and baculoviruses target caspases to avoid the death of their host cell
and promote successful
infection. In addition, increases in inappropriate apoptosis have been
reported in AIDS,
neurodegenerative diseases and ischemic injury, while a decrease in cell death
is associated with
cancer (Salveson and Dixit, supra; Thompson, C.B. (1995) Science 267:1456-
1462).
Aspartyl proteases
Aspartyl proteases (APs) include the lysosomal proteases cathepsins D and E,
as well as
chymosin, renin, and the gastric pepsins. Most retroviruses encode an AP,
usually as part of the Col
polyprotein. APs, also called acid proteases, are monomeric enzymes consisting
of two domains,
each domain containing one half of the active site with its own catalytic
aspartic acid residue. APs
are most active in the range of pH 2-3, at which one of the aspartate residues
is ionized and the other
neutral. The pepsin family of APs contains many secreted enzymes, and all are
likely to be
synthesized with signal peptides and propeptides. Most family members have
three disulfide loops,
the first ~5 residue loop following the first aspartate, the second 5-6
residue loop preceding the
second aspartate, and the third and largest loop occurring toward the C
terminus. Retropepsins, on
the other hand, are analogous to a single domain of pepsin, and become active
as homodimers with
each retropepsin monomer contributing one half of the active site.
Retropepsins are required for
processing the viral polyproteins.
APs have roles in various tissues, and some have been associated with disease.
Renin
mediates the first step in processing the hormone angiotensin, which is
responsible for regulating
electrolyte balance and blood pressure (reviewed in Crews, D.E. and S.R.
Williams (1999) Hum.
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Biol. 71:475-503). Abnormal regulation and expression of cathepsins are
evident in various
inflammatory disease states. Expression of cathepsin D is elevated in synovial
tissues from patients
with rheumatoid arthritis and osteoarthritis. The increased expression and
differential regulation of
the cathepsins are linked to the metastatic potential of a variety of cancers
(Chambers, A.F. et al.
(1993) Crit. Rev. Oncol. 4:95-114).
Metalloproteases
Metalloproteases require a metal ion for activity, usually manganese or zinc.
Examples of
manganese metalloenzymes include aminopeptidase P and human proline
dipeptidase (PEPD).
Aminopeptidase P can degrade bradykinin, a nonapeptide activated in a variety
of inflammatory
responses. Aminopeptidase P has been implicated in coronary
ischemia/reperfusion injury.
Administration of aminopeptidase P inhibitors has been shown to have a
cardioprotective effect in
rats (Ersahin, C. et al (1999) J. Cardiovasc. Pharmacol. 34:604-611).
Most zinc-dependent metalloproteases share a common sequence in the zinc-
binding domain.
The active site is made up of two histidines which act as zinc ligands and a
catalytic glutamic acid C
terminal to the first histidine. Proteins containing this signature sequence
are known as the
metzincins and include aminopeptidase N, angiotensin-converting enzyme,
neurolysin, the matrix
metalloproteases and the adamalysins (ADAMS). An alternate sequence is found
in the zinc
carboxypeptidases, in which all three conserved residues - two histidines and
a glutamic acid - are
involved in zinc binding.
A number of the neutral metalloendopeptidases, including angiotensin
converting enzyme and
the aminopeptidases, are involved in the metabolism of peptide hormones. High
aminopeptidase B
activity, for example, is found in the adrenal glands and neurohypophyses of
hypertensive rats (Prieto,
I. et al. (1998) Horm. Metab. Res. 30:246-248). Oligopeptidase M/neurolysin
can hydrolyze
bradykinin as well as neurotensin (Serizawa, A. et al. (1995) J. Biol. Chem
270:2092-2098).
Neurotensin is a vasoactive peptide that can act as a neurotransmitter in the
brain, where it has been
implicated in limiting food intake (Tritos, N.A. et al. (1999) Neuropeptides
33:339-349).
The matrix metalloproteases (MMPs) are a family of at least 23 enzymes that
can degrade
components of the extracellular matrix (ECM). They are Zn+2 endopeptidases
with an N-terminal
catalytic domain. Nearly all members of the family have a hinge peptide and C-
terminal domain
which can bind to substrate molecules in the ECM or to inhibitors produced by
the tissue (TIIVVIPs, for
tissue inhibitor of metalloprotease; Campbell, LL. et al. (1999) Trends
Neurosci. 22:285). The
presence of fibronectin-like repeats, transmembrane domains, or C-terminal
hemopexinase-like
domains can be used to separate MMPs into collagenase, gelatinase, stromelysin
and membrane-type
MMP subfamilies. In the inactive form, the Zn+z ion in the active site
interacts with a cysteine in the
pro-sequence. Activating factors disrupt the Zn+Z-cysteine interaction, or
"cysteine switch," exposing
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the active site. This partially activates the enzyme, which then cleaves off
its propeptide and becomes
fully active. MMPs are often activated by the serine proteases plasmin and
furin. MMPs are often
regulated by stoichiometric, noncovalent interactions with inhibitors; the
balance of protease to
inhibitor, then, is very important in tissue homeostasis (reviewed in Yong,
V.W. et al. (1998) Trends
Neurosci.21:75).
MMPs are implicated in a number of diseases including osteoarthritis
(Mitchell, P. et al.
(1996) J. Clin. Invest. 97:761), atherosclerotic plaque rupture (Sukhova, G.K.
et al. (1999)
Circulation 99:2503), aortic aneurysm (Schneiderman, J. et al. (1998) Am. J.
Path. 152:703),
non-healing wounds (Saarialho-Kere, U.K. et al. (1994) J. Clin. Invest.
94:79), bone resorption
(Blavier, L. and J.M. Delaisse (1995) J. Cell Sci. 108:3649), age-related
macular degeneration (Stem,
B. et al. (1998) Invest. Ophthalmol. Vis. Sci. 39:2194), emphysema (Finlay,
G.A. et al. (1997) Thorax
52:502), myocardial infarction (Rohde, L.E. et al. (1999) Circulation 99:3063)
and dilated
cardiomyopathy (Thomas, C.V. et al. (1998) Circulation 97:1708). MMP
inhibitors prevent
metastasis of mammary carcinoma and experimental tumors in rat, and Lewis lung
carcinoma,
hemangioma, and human ovarian carcinoma xenografts in mice (Eccles, S.A. et
al. (1996) Cancer
Res. 56:2815; Anderson et al. (1996) Cancer Res. 56:715-718; Volpert, O.V. et
al. (1996) J. Clin.
Invest. 98:671; Taraboletti, G. et al. (1995) J. NCI 87:293; Davies, B. et al.
(1993) Cancer Res.
53:2087). MMPs may be active in Alzheimer's disease. A number of MMPs are
implicated in
multiple sclerosis, and administration of MMP inhibitors can relieve some of
its symptoms (reviewed
in Yong, supra).
Another family of metalloproteases is the ADAMS, for A Disintegrin and
Metalloprotease
Domain, which they share with their close relatives the adamalysins, snake
venom metalloproteases
(SVMPs). ADAMS combine features of both cell surface adhesion molecules and
proteases,
containing a prodomain, a protease domain, a disintegrin domain, a cysteine
rich domain, an
epidermal growth factor repeat, a transmembrane domain, and a cytoplasmic
tail. The first three
domains listed above are also found in the SVMPs. The ADAMs possess four
potential functions:
proteolysis, adhesion, signaling and fusion. The ADAMs share the metzincin
zinc binding sequence
and are inhibited by some MMP antagonists such as TllVIP-1.
ADAMs are implicated in such processes as sperm-egg binding and fusion,
myoblast fusion,
and protein-ectodomain processing or shedding of cytokines, cytokine
receptors, adhesion proteins
and other extracellular protein domains (Schlondorff, J. and C.P. Blobel
(1999) J. Cell. Sci.
112:3603-3617). The Kuzbanian protein cleaves a substrate in the NOTCH pathway
(possibly
NOTCH itself), activating the program for lateral inhibition in Droso~hila
neural development. Two
ADAMS, TACE (ADAM 17) and ADAM 10, are proposed to have analogous roles in the
processing
of amyloid precursor protein in the brain (Schlondorff and Blobel, supra).
TALE has also been
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identified as the TNF activating enzyme (Black, R.A. et al. (1997) Nature
385:729). TNF is a
pleiotropic cytokine that is important in mobilizing host defenses in response
to infection or trauma,
but can cause severe damage in excess and is often overproduced in autoimmune
disease. TACE
cleaves membrane-bound pro-TNF to release a soluble form. Other ADAMs may be
involved in a
similar type of processing of other membrane-bound molecules.
The ADAMTS sub-family has all of the features of ADAM family metalloproteases
and
contain an additional thrombospondin domain (TS). The prototypic ADAMTS was
identified in
mouse, found to be expressed in heart and kidney and upregulated by
proinflaxnrnatory stimuli (Kuno,
K. et al. (1997) J. Biol. Chem. 272:556-562). To date eleven members are
recognized by the Human
Genome Organization (HUGO;
http://www.gene.ucl.ac.uk/users/hesterladamts.html#Approved).
Members of this family have the ability to degrade aggrecan, a high molecular
weight proteoglycan
which provides cartilage with important mechanical properties including
compressibility, and which
is lost during the development of arthritis. Enzymes which degrade aggrecan
are thus considered
attractive targets to prevent and slow the degradation of articular cartilage
(See, e.g., Tortorella, M.D.
(1999) Science 284:1664; Abbaszade, I. (1999) J. Biol. Chem. 274:23443). Other
members are
reported to have antiangiogenic potential (Kuno et al., supra) and/or
procollagen processing (Colige,
A. et al. (1997) Proc. Natl. Acad. Sei. USA 94:2374).
Protease inhibitors
Protease inhibitors and other regulators of protease activity control the
activity and effects of
proteases. Protease inhibitors have been shown to control pathogenesis in
animal models of
proteolytic disorders (Murphy, G. (1991) Agents Actions Suppl. 35:69-76). Low
levels of the
cystatins, low molecular weight inhibitors of the cysteine proteases,
correlate with malignant
progression of tumors (Catkins, C. et al. (1995) Biol. Biochem. Hoppe Seyler
376:71-80). Serpins are
inhibitors of nnammalian plasma serine proteases. Many serpins serve to
regulate the blood clotting
cascade and/or the complement cascade in mammals. Sp32 is a positive regulator
of the mammalian
acrosomal protease, acrosin, that binds the proenzyme, proaerosin, and thereby
aides in packaging the
enzyme into the acrosomal matrix (Baba, T. et al. (1994) J. Biol. Chem.
269:10133-10140). The
Kunitz family of serine protease inhibitors are characterized by one or more
"Kunitz domains"
containing a series of cysteine residues that are regularly spaced over
approximately 50 amino acid
residues and form three intrachain disulfide bonds. Members of this family
include aprotinin, tissue
factor pathway inhibitor (TFPI-1 and TFPI-2), inter-oc-trypsin inhibitor, and
bikunin. (Manor, C.W. et
al. (1997) J. Biol. Chem. 272:12202-12208.) Members of this family are potent
inhibitors (in the
nanomolar range) against serine proteases such as kallikrein and plasmin.
Aprotinin has clinical
utility in reduction of perioperative blood loss.
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The discovery of new proteases, and the polynucleotides encoding them,
satisfies a need in
the art by providing new compositions which are useful in the diagnosis,
prevention, and treatment of
gastrointestinal, cardiovascular, autoimmune/inflammatory, cell proliferative,
developmental,
epithelial, neurological, and reproductive disorders, and in the assessment of
the effects of exogenous
compounds on the expression of nucleic acid and amino acid sequences of
proteases.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, proteases, referred to
collectively as "PRTS"
and individually as "PRTS-l," "PRTS-2," "PRTS-3," "PRTS-4," "PRTS-5," "PRTS-
6," "PRTS-7,"
"PRTS-8," "PRTS-9," "PRTS-10," "PRTS-11," "PRTS-12," "PRTS-13," "PRTS-14," and
"PRTS-
15." In one aspect, the invention provides an isolated polypeptide selected
from the group consisting
of a) a polypeptide comprising an amino acid sequence selected from the group
consisting of SEQ ll~
N0:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence
at least 90%
identical to an amino acid sequence selected from the group consisting of SEQ
ID NO:1-15, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-15, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-15. In one
alternative, the
invention provides an isolated polypeptide comprising the amino acid sequence
of SEQ ID NO:1-15.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected
from the group consisting of a) a polypeptide comprising an amino acid
sequence selected from the
group consisting of SEQ ~ NO:1-15, b) a polypeptide comprising a naturally
occurring amino acid
sequence at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ
ID NO:1-15, c) a biologically active fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-15, and d) an immunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ m N0:1-15.
In one alternative, the polynucleotide encodes a polypeptide selected from the
group consisting of
SEQ ll~ NO:1-15. In another alternative, the polynucleotide is selected from
the group consisting of
SEQ m N0:16-30.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-15, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ m NO:1-15, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ )D NO:1-15, and d) an immunogenic fragment of a polypeptide
having an amino
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acid sequence selected from the group consisting of SEQ ID NO:1-15. In one
alternative, the
invention provides a cell transformed with the recombinant polynucleotide. In
another alternative, the
invention provides a transgenic organism comprising the recombinant
polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID N0:1-15, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO: l-15, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ll~ NO:1-15, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ll~ NO:1-15. The
method comprises a)
culturing a cell under conditions suitable for expression of the polypeptide,
wherein said cell is
transformed with a recombinant polynucleotide comprising a promoter sequence
operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the polypeptide so
expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ ID N0:1-15, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ 1D N0:1-15, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-15, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-15.
The invention further provides an isolated polynucleotide selected from the
group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ >D N0:16-30, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ ~
N0:16-30, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
In one alternative, the
polynucleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
selected from the group
consisting of a) a polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:16-30, b) a polynucleotide comprising a naturally
occurring polynucleotide
sequence at least 90% identical to a polynucleotide sequence selected from the
group consisting of
SEQ m NO:16-30, c) a polynucleotide complementary to the polynucleotide of a),
d) a
polynucleotide complementary to the polynucleotide of b), and e) an RNA
equivalent of a)-d). The
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method comprises a) hybridizing the sample with a probe comprising at least 20
contiguous
nucleotides comprising a sequence complementary to said target polynucleotide
in the sample, and
which probe specifically hybridizes to said target polynucleotide, under
conditions whereby a
hybridization complex is formed between said probe and said target
polynucleotide or fragments
thereof, and b) detecting the presence or absence of said hybridization
complex, and optionally, if
present, the amount thereof. In one alternative, the probe comprises at least
60 contiguous
nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ m N0:16-30, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ >D
N0:16-30, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method
comprises a) amplifying said target polynucleotide or fragment thereof using
polymerase chain
reaction amplification, and b) detecting the presence or absence of said
amplified target
polynucleotide or fragment thereof, and, optionally, if present, the amount
thereof.
The invention further provides a composition comprising an effective amount of
a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-15, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ m N0:1-15, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ m NO:1-
15, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ )~ NO:1-15, and a pharmaceutically acceptable excipient. In
one embodiment, the
composition comprises an amino acid sequence selected from the group
consisting of SEQ >I7 NO: l-
15. The invention additionally provides a method of treating a disease or
condition associated with
decreased expression of functional PRTS, comprising administering to a patient
in need of such
treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ m N0:1-15, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ m NO:1-15, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ m NO:1-
15, and d) an
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immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-15. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional PRTS, comprising
administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID N0:1-15, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-15, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO: l-15,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-15. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
invention provides a composition comprising an antagonist compound identified
by the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with overexpression of functional
PRTS, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-15, b) a
polypeptide comprising a
naturally occurnng amino acid sequence at least 90% identical to an amino acid
sequence selected
from the group consisting of SEQ ~ NO:1-15, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ )D
N0:1-15, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-15. The method comprises a) combining the
polypeptide with at least
one test compound under suitable conditions, and b) detecting binding of the
polypeptide to the test
compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-15, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-15, c) a biologically active fragment
of a polypeptide
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having an amino acid sequence selected from the group consisting of SEQ >D
NO:1-15, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID N0:1-15. The method comprises a) combining the
polypeptide with at least
one test compound under conditions permissive for the activity of the
polypeptide, b) assessing the
activity of the polypeptide in the presence of the test compound, and c)
comparing the activity of the
polypeptide in the presence of the test compound with the activity of the
polypeptide in the absence
of the test compound, wherein a change in the activity of the polypeptide in
the presence of the test
compound is indicative of a compound that modulates the activity of the
polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
polynucleotide sequence selected from the group consisting of SEQ ID N0:16-30,
the method
comprising a) exposing a sample comprising the target polynucleotide to a
compound, and b)
detecting altered expression of the target polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
)D N0:16-30, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ m N0:16-30,
iii) a
polynucleotide having a sequence complementary to i), iv) a polynucleotide
complementary to the
polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization
occurs under conditions
whereby a specific hybridization complex is formed between said probe and a
target polynucleotide
in the biological sample, said target polynucleotide selected from the group
consisting of i) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:16-30, ii) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID N0:16-30,
iii) a polynucleotide complementary to the polynucleotide of i), iv) a
polynucleotide complementary
to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
Alternatively, the target
polynucleotide comprises a fragment of a polynucleotide sequence selected from
the group consisting
of i)-v) above; c) quantifying the amount of hybridization complex; and d)
comparing the amount of
hybridization complex in the treated biological sample with the amount of
hybridization complex in
an untreated biological sample, wherein a difference in the amount of
hybridization complex in the
treated biological sample is indicative of toxicity of the test compound.
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BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the presentinvention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptides of the invention. The probability scores for the
matches between each
polypeptide and its homalog(s) are also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for
analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide sequences of the invention, along with selected fragments of
the polynucleotide
sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which
will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing
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the cell lines, protocols, reagents and vectors which are reported in the
publications and which might
be used in connection with the invention. Nothing herein is to be construed as
an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"PRTS" refers to the amino acid sequences of substantially purified PRTS
obtained from any
species, particularly a mammalian species, including bovine, ovine, porcine,
marine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
PRTS. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of PRTS either by
directly interacting with
PRTS or by acting on components of the biological pathway in which PRTS
participates.
An "allelic variant" is an alternative form of the gene encoding PRTS. Allelic
variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more times
in a given sequence.
"Altered" nucleic acid sequences encoding PRTS include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as PRTS or a
polypeptide with at least one functional characteristic of PRTS. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding PRTS, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
PRTS. The encoded protein may also be "altered," and may contain deletions,
insertions, or
substitutions of amino acid residues which produce a silent change and result
in a functionally
equivalent PRTS. Deliberate amino acid substitutions may be made on the basis
of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the
residues, as long as the biological or immunological activity of PRTS is
retained. For example,
negatively charged amino acids may include aspartic acid and glutamic acid,
and positively charged
amino acids may include lysine and arginine. Amino acids with uncharged polar
side chains having
similar hydrophilicity values may include: asparagine and glutamine; and
serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
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The terms "amino acid" and'"amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid
sequence to the complete native amino acid sequence associated with the
recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of PRTS. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of PRTS either by
directly interacting with PRTS or by acting on components of the biological
pathway in which PRTS
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')Z, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind PRTS polypeptides can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide
used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived
from the translation of
RNA, or synthesized chemically, and can be conjugated to a carrier protein if
desired. Commonly
used carriers that are chemically coupled to peptides include bovine serum
albumin, thyroglobulin,
and keyhole limpet hemocyanin (KI,H). The coupled peptide is then used to
immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that
binds to a
specific molecular target. Aptamers are derived from an in vitro evolutionary
process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large
combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may
include
deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other
nucleotide-like molecules.
The nucleotide components of an aptamer may have modified sugar groups (e.g.,
the 2'-OH group of a
ribonucleotide may be replaced by 2'-F or 2'-NHZ), which may improve a desired
property, e.g.,
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resistance to nucleases or longer lifetime in blood. Aptamers may be
conjugated to other molecules,
e.g., a high molecular weight carrier to slow clearance of the aptamer from
the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by
photo-activation of a
cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-
13.)
The term "intramer" refers to an aptamer which is expressed in vivo. For
example, a vaccinia
virus-based RNA expression system has been used to express specific RNA
aptamers at high levels in
the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA
96:3606-3610).
The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or
other left-
handed nucleotide derivatives or nucleotide-like molecules. Aptamers
containing left-handed
nucleotides are resistant to degradation by naturally occurring enzymes, which
normally act on
substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic PRTS, or of
any oligopeptide thereof,
to induce a specific immune response in appropriate animals or cells and to
bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising
a given amino acid sequence" refer broadly to any composition containing the
given polynucleotide
or amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding PRTS or fragments of
PRTS may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
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associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl
sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit
(Applied
Biosystems, Foster City CA) in the 5' and/or the 3' direction, and
resequenced, or wluch has been
assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
Wn or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended and
assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of
the protein is conserved and not significantly changed by such substitutions.
The table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp ~ Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val lle, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
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The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which
retains at least one biological or immunological function of the natural
molecule. A derivative
polypeptide is one modified by glycosylation, pegylation, or any similar
process that retains at least
one biological or immunological function of the polypeptide from which it was
derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
exon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of PRTS or the polynucleotide encoding PRTS
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ m N0:16-30 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:16-30, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:16-30 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:16-30 from related polynucleotide sequences. The precise length of a
fragment of SEQ
m N0:16-30 and the region of SEQ m NO:16-30 to which the fragment corresponds
are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
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A fragment of SEQ ID NO:1-15 is encoded by a fragment of SEQ ID N0:16-30. A
fragment
of SEQ ID NO:1-15 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:1-15. For example, a fragment of SEQ ID NO:1-15 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID NO:1-15.
The precise length of
a fragment of SEQ ID NO: l-15 and the region of SEQ ID N0:1-15 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation
codon (e.g., methionine) followed by an open reading frame and a translation
termination codon. A
"full length" polynucleotide sequence encodes a "full length" polypeptide
sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between
two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise al banments of polynucleotide sequences, the default
parameters are set as
follows: I~tuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available
from several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
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The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: S and Extension Gap: 2 penalties
Gap x drop-off.' S0
Expect: l0
Word Size: 11
Filter: orz
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length
supported by the sequences shown herein, in the tables, figures, or Sequence
Listing, may be used to
describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "°lo identity," as applied to
polypeptide sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some
alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the charge
and hydrophobicity at the
site of substitution, thus preserving the structure (and therefore function)
of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
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residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 11 and Exterzsiorz Gap: 1 penalties
Gap x drop-off.' SO
Expect: l0
Word Size: 3
Filter: orz
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill
in the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
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Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1% (w/v) SDS, and about 100 ~tg/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al.
(1989) Molecular Clonin~Y A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring
Harbor Press,
Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present
invention include wash conditions of 68°C in the presence of about 0.2
x SSC and about 0.1% SDS,
for 1 hour. Alternatively, temperatures of about 65°C, 60°C,
55°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present
at about 0.1%.
Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents
include, for instance, sheared and denatured salmon sperm DNA at about 100-200
,ug/ml. Organic
solvent, such as formamide at a concentration of about 35-50% v/v, may also be
used under particular
circumstances, such as for RNA:DNA hybridizations. Useful variations on these
wash conditions
will be readily apparent to those of ordinary skill in the art. Hybridization,
particularly under high
stringency conditions, may be suggestive of evolutionary similarity between
the nucleotides. Such
similarity is strongly indicative of a similar role for the nucleotides and
their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amind acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of PRTS
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
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mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment
of PRTS which is useful in any of the antibody production methods disclosed
herein or known in the
art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of PRTS. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of PRTS.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an PRTS may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in
the art. These processes may occur synthetically or biochemically. Biochemical
modifications will
vary by cell type depending on the enzymatic milieu of PRTS.
"Probe" refers to nucleic acid sequences encoding PRTS, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
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DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2"d
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
primer selection program (available to the public from the Whitehead
InstitutelMTT Center for
Genome Research, Cambridge MA) allows the user to input a "mispriming
library," in which
sequences to avoid as primer binding sites are user-specified. Primer3 is
useful, in particular, for the
selection of oligonucleotides for microarrays. (The source code for the latter
two primer selection
programs may also be obtained from their respective sources and modified to
meet the user's specific
needs.) The PrimeGen program (available to the public from the UK Human Genome
Mapping
Project Resource Centre, Cambridge UI~) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
polynucleotide fragments identified by any of the above selection methods are
useful in hybridization
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technologies, for example, as PCR or sequencing primers, microarray elements,
or specific probes to
identify fully or partially complementary polynucleotides in a sample of
nucleic acids. Methods of
oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or ehromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing PRTS,
nucleic acids encoding PRTS, or fragments thereof may comprise a bodily fluid;
an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
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molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
comprising the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" or "expression profile" refers to the collective pattern
of gene
expression by a particular cell type or tissue under given conditions at a
given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or eukaryotic host Bell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or
viral infection, electroporation, heat shock, lipofection, and particle
bombardment. The term
"transformed cells" includes stably transformed cells in which the inserted
DNA is capable of
replication either as an autonomously replicating plasmid or as part of the
host chromosome, as well
as transiently transformed cells which express the inserted DNA or RNA for
limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, plants and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention
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into such organisms are widely known and provided in references such as
Sambrook et al. (1989),
su ra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an
"allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides will generally have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least
92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human proteases (PRTS), the
polynucleotides
encoding PRTS, and the use of these compositions for the diagnosis, treatment,
or prevention of
gastrointestinal, cardiovascular, autoimmune/inflammatory, cell proliferative,
developmental,
epithelial, neurological, and reproductive disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
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single Incyte project identification number (Incyte Project )D). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide ID) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ m NO:) and
an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID)
as shown.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2
show the
polypeptide sequence identification number (Polypeptide SEQ DJ NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide >D) for polypeptides of the
invention. Column 3
shows the GenBank identification number (GenBank ID NO:) of the nearest
GenBank homolog.
Column 4 shows the probability scores for the matches between each polypeptide
and its homolog(s).
Column 5 shows the annotation of the GenBank homolog(s) along with relevant
citations where
applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1
and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the
corresponding
Incyte polypeptide sequence number (Incyte Polypeptide ID) for each
polypeptide of the invention.
Column 3 shows the number of amino acid residues in each polypeptide. Column 4
shows potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the
MOTIFS program of the GCG sequence analysis software package (Genetics
Computer Group,
Madison WI). Column 6 shows amino acid residues comprising signature
sequences, domains, and
motifs. Column 7 shows analytical methods for protein structure/function
analysis and in some cases,
searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are proteases. For example,
SEQ ll~ N0:3 is 50%
identical to Xenopus ADAM 13 metalloprotease (GenBank ID g1916617) as
determined by the Basic
Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability
score is 2.1e-208,
which indicates the probability of obtaining the observed polypeptide sequence
alignment by chance.
SEQ ID N0:3 also contains a neutral zinc metalloprotease active site domain
and a disintegrin
domain, as determined by searching for statistically significant matches in
the hidden Markov model
(HMM)-based PFAM database of conserved protein family domains. (See Table 3.)
The presence of
these motifs is confirmed by BLM'S, MOTIFS, and PROF1LESCAN analyses,
providing further
corroborative evidence that SEQ ID N0:3 is a protease of the ADAM family. In
an alternate
example, SEQ ID N0:4 is 44% identical to human zinc metalloprotease ADAMTS7
(GenBank m
g5923788) as determined by the Basic Local Alignment Search Tool (BLAST). (See
Table 2.) The
BLAST probability score is 2.2e-143, which indicates the probability of
obtaining the observed
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polypeptide sequence alignment by chance. SEQ ID N0:4 also contains a
Reprolysin (M12B) family
zinc metalloprotease site and a Thrombospondin type 1 domain as determined by
searching for
statistically significant matches in the hidden Markov model (I-BVIM)-based
PFAM database of
conserved protein family domains. (See Table 3.) Data from BLIMPS and MOTIFS
analyses
provide further corroborative evidence that SEQ ID N0:4 is a metalloprotease
(note that the
"Thrombospondin type 1 domains" are found at the carboxy-terminal end, and are
characteristic of
the ADAMTS metalloprotease protein family). In an alternate example, SEQ ID
NO:S is 62%
identical to mouse distal intestinal serine protease (GenBank ID 85921501) as
determined by the
Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is
5.3e-99, which indicates the probability of obtaining the observed polypeptide
sequence alignment by
chance. SEQ ID N0:5 also contains a trypsin family serine protease active site
domain as determined
by searching for statistically significant matches in the hidden Markov model
(HMM)-based PFAM
database of conserved protein family domains. (See Table 3.) The presence of
this motif is
confirmed by BLM'S, MOTIFS, and PROFILESCAN analyses. BLIMPS analysis also
reveals the
presence of kringle and type I fibronectin domains. Together, these data
provide further
corroborative evidence that SEQ ID N0:5 is a trypsin family serine protease.
In an alternate example,
SEQ ID N0:8 is 45% identical to human membrane-type serine protease 1 (GenBank
117 86002714)
as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
The BLAST
probability score is 6.1e-69, which indicates the probability of obtaining the
observed polypeptide
sequence alignment by chance. SEQ 1D N0:8 also contains a trypsin domain as
determined by
searching for statistically significant matches in the hidden Markov model
(IllVIM)-based PFAM
database of conserved protein family domains. (See Table 3.) Data from BLIMPS,
MOTIFS, and
PROFIL,ESCAN analyses provide further corroborative evidence that SEQ ID N0:8
is a serine
protease. In an alternate example, SEQ ID N0:11 is 49% identical to mouse ADAM
4 protein
precursor (GenBank ID 8965014) as determined by the Basic Local Alignment
Search Tool
(BLAST). (See Table 2.) The BLAST probability score is 4.1e-117, which
indicates the probability
of obtaining the observed polypeptide sequence alignment by chance. SEQ ID
N0:11 also contains a
reprolysin family propeptide domain and a disintegrin domain as determined by
searching for
statistically significant matches in the hidden Markov model (HMM)-based PFAM
database of
conserved protein family domains. (See Table 3.) Data from BLllVIPS and
PROF1LESCAN analyses
provide further corroborative evidence that SEQ ID NO:11 is an ADAM family
metalloprotease. In
an alternate example, SEQ ll~ N0:12 is 42% identical to bovine enteropeptidase
(GenBank ID
8416132) as determined by the Basic Local Alignment Search Tool (BLAST). (See
Table 2.) The
BLAST probability score is 2.2e-47, which indicates the probability of
obtaining the observed
polypeptide sequence alignment by chance. SEQ ID N0:12 also contains a trypsin
domain as
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determined by searching for statistically significant matches in the hidden
Markov model (HMM)-
based PFAM database of conserved protein family domains. (See Table 3.) Data
from BLIIUVIPS,
MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that
SEQ m N0:12 is
a trypsin family serine protease. In an alternate example, SEQ ID N0:13 is 52%
identical from
residues 110 to 4g2 to Saccharomyces cerevisiae Maplp methionine
aminopeptidase (GenBank ID
g662342) as determined by the Basic Local Alignment Search Tool (BLAST), with
a probability
score of 1.6e-99. (See Table 2.) SEQ ID N0:13 also contains a metallopeptidase
family M24
domain as determined by searching for statistically significant matches in the
hidden Markov model
(I~ZM)-based PFAM database of conserved protein family domains. (See Table 3.)
Data from
BLI1VVIPS and PROFII,ESCAN analyses provide further corroborative evidence
that SEQ )D N0:13 is
a methionine aminopeptidase. In an alternate example, SEQ ID N0:15 is 36%
identical to Xenopus
epidermis-specific serine protease (GenBank ID g6009515) as determined by the
Basic Local
Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is
7.7e-52, which
indicates the probability of obtaining the observed polypeptide sequence
alignment by chance. SEQ
ID N0:15 also contains a trypsin family protease active site domain as
determined by searching for
statistically significant matches in the hidden Markov model (HNI1~~I)-based
PFAM database of
conserved protein family domains. (See Table 3.) The presence of this motif is
confirmed by
BLM'S, MOT1FS, and PROFILESCAN analyses. BLM'S analysis also reveals that SEQ
~
NO:15 contains a kringle domain, providing further corroborative evidence that
SEQ ID NO:15 is a
protease of the trypsin family. SEQ ID N0:2-3, SEQ ID N0:6-7, SEQ ID N0:9-10
and SEQ )D
N0:14 were analyzed and annotated in a similar manner. The algorithms and
parameters for the
analysis of SEQ ID NO: l-15 are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Columns 1 and 2 list the
polynucleotide sequence
identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte
polynucleotide
consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide
of the invention.
Column 3 shows the length of each polynucleotide sequence in basepairs. Column
4 lists fragments
of the polynucleotide sequences which are useful, for example, in
hybridization or amplification
technologies that identify SEQ ll~ N0:16-30 or that distinguish between SEQ ID
N0:16-30 and
related polynucleotide sequences. Column 5 shows identification numbers
corresponding to cDNA
sequences, coding sequences (exons) predicted from genomic DNA, and/or
sequence assemblages
comprised of both cDNA and genomic DNA. These sequences were used to assemble
the full length
polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the
nucleotide start (5')
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and stop (3') positions of the cDNA and/or genomic sequences in column 5
relative to their respective
full length sequences.
The identification numbers in Column 5 of Table 4 may refer specifically, for
example, to
Incyte cDNAs along with their corresponding cDNA libraries. For example,
7635792H1 is the
identification number of an Incyte cDNA sequence, and SINTD1E01 is the cDNA
library from which
it is derived. Incyte cDNAs for which cDNA libraries are not indicated were
derived from pooled
cDNA libraries (e.g., 55147856J1). Alternatively, the identification numbers
in column 5 may refer
to GenBank cDNAs or ESTs (e.g., g876900) Which contributed to the assembly of
the full length
polynucleotide sequences. In addition, the identification numbers in column 5
may identify
sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database
(i.e., those
sequences including the designation ".ENST"). Alternatively, the
identification numbers in column 5
may be derived from the NCBI RefSeq Nucleotide Sequence Records Database
(i.e., those sequences
including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence
Records (i.e., those
sequences including the designation "NP"). Alternatively, the identification
numbers in column 5
may refer to assemblages of both cDNA and Genscan-predicted exons brought
together by an "exon
stitching" algorithm. For example, FL 1~'.d~XXXX_Nl 1Vz_YYYYY_N3 IV4
represents a "stitched"
sequence in which X~XXX is the identification number of the cluster of
sequences to which the
algorithm was applied, and YYYYY is the number of the prediction generated by
the algorithm, and
N1,2, j.,a, if present, represent specific exons that may have been manually
edited during analysis (See
Example V). Alternatively, the identification numbers in column 5 may refer to
assemblages of
exons brought together by an "exon-stretching" algorithm. For example,
FLXXXX~X gAAAAA~BBBBB_1 lV is the identification number of a "stretched"
sequence, with
XXX~PI~X being the Incyte project identification number, gAAAAA being the
GenBank identification
number of the human genomic sequence to which the "exon-stretching" algorithm
was applied,
gBBBBB being the GenBank identification number or NCBI RefSeq identification
number of the
nearest GenBank protein homolog, and N refernng to specific exons (See Example
V). In instances
where a RefSeq sequence was used as a protein homolog for the "exon-
stretching" algorithm, a
RefSeq identifier (denoted by "NM," "NP,"' or "NT") may be used in place of
the GenBank identifier
(i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs
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GNN, GFG,Exon prediction from genomic sequences using,
for example,
ENST GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Banger Centre,
Cambridge, UK).
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences
(see Example V).
INCY Full length transcript and exon prediction
from mapping of EST
sequences to the genome. Genomic location
and EST composition
data are combined to predict the exons and
resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in
column 5 was obtained to confirm the final consensus polynucleotide sequence,
but the relevant
Incyte cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide
sequences which were assembled using Incyte eDNA sequences. The representative
cDNA library is
the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences which
were used to assemble and confirm the above polynucleotide sequences. The
tissues and vectors
which were used to construct the cDNA libraries shown in Table 5 are described
in Table 6.
The invention also encompasses PRTS variants. A preferred PRTS variant is one
which has
at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid
sequence identity to the PRTS amino acid sequence, and which contains at least
one functional or
structural characteristic of PRTS.
The invention also encompasses polynucleotides which encode PRTS. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:16-30, which encodes PRTS. The
polynucleotide
sequences of SEQ ID NO:16-30, as presented in the Sequence Listing, embrace
the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced
with uracil, and the
sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
PRTS. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at
least about 85 %, or even at least about 95 % polynucleotide sequence identity
to the polynucleotide
sequence encoding PRTS. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ 117
N0:16-30 which has at least about 70%, or alternatively at least about 85%, or
even at least about
95% polynucleotide sequence identity to a nucleic acid sequence selected from
the group consisting
34.
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of SEQ )D N0:16-30. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of PRTS.
In addition, or in the alternative, a polynucleotide variant of the invention
is a splice variant
of a polynucleotide sequence encoding PRTS. A splice variant may have portions
which have
significant sequence identity to the polynucleotide sequence encoding PRTS,
but will generally have
a greater or lesser number of polynucleotides due to additions or deletions of
blocks of sequence
arising from alternate splicing of exons during mRNA processing. A splice
variant may have less
than about 70%, or alternatively less than about 60%, or alternatively less
than about 50%
polynucleotide sequence identity to the polynucleotide sequence encoding PRTS
over its entire
length; however, portions of the splice variant will have at least about 70%,
or alternatively at least
about 85%, or alternatively at least about 95%, or alternatively 100%
polynucleotide sequence
identity to portions of the polynucleotide sequence encoding PRTS. Any one of
the splice variants
described above can encode an amino acid sequence which contains at least one
functional or
structural characteristic of PRTS.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
genetic code, a multitude of polynucleotide sequences encoding PRTS, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring PRTS, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode PRTS and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurnng PRTS under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding PRTS or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate at which
expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding PRTS and its derivatives without altering the encoded amino
acid sequences
include the production of RNA transcripts having more desirable properties,
such as a greater
half life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode PRTS
and
PRTS derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the
synthetic sequence may be inserted into any of the many available expression
vectors and cell
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systems using reagents well known in the art. Moreover, synthetic chemistry
may be used to
introduce mutations into a sequence encoding PRTS or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:16-30 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Bioloay, John Wiley ~ Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding PRTS may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unlalown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments
adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme
digestions and ligations may be used to insert an engineered double-stranded
sequence into a region
of unknown sequence before performing PCR. Other methods which may be used to
retrieve
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WO 02/38744 PCT/USO1/51034
unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991)
Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and
PROMOTERFINDER libraries
(Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need
to screen libraries
and is useful in finding intron/exon junctions. For all PCR-based methods,
primers may be designed
using commercially available software, such as OLIGO 4.06 primer analysis
software (National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in
length, to have a GC content of about 50% or more, and to anneal to the
template at temperatures of
about 6~°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode PRTS may be cloned in recombinant DNA molecules that direct
expression of PRTS,
or fragments or functional equivalents thereof, in appropriate host cells. Due
to the inherent
degeneracy of the genetic code, other DNA sequences which encode substantially
the same or a
functionally equivalent amino acid sequence may be produced and used to
express PRTS.
The nucleotide sequences of the present invention can be engineered using
methods generally
laiown in the art in order to alter PRTS-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
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The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of PRTS, such as its biological or enzymatic
activity or its ability to
bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding PRTS may be synthesized, in whole or
in part,
using chemical methods well known in the art. '(See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.)
Alternatively, PRTS itself or a fragment thereof may be synthesized using
chemical methods. For
example, peptide synthesis can be performed using various solution-phase or
solid-phase techniques.
(See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Pro cu
roes, WH Freeman, New
York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.)
Automated synthesis
may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the
amino acid sequence of PRTS, or any part thereof, may be altered during direct
synthesis and/or
combined with sequences from other proteins, or any part thereof, to produce a
variant polypeptide or
a polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, su ra, pp. 28-53.)
In order to express a biologically active PRTS, the nucleotide sequences
encoding PRTS or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which contains
the necessary elements for transcriptional and translational control of the
inserted coding sequence in
a suitable host. These elements include regulatory sequences, such as
enhancers, constitutive and
'i8
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inducible promoters, and 5' and 3' untranslated regions in the vector and in
polynucleotide sequences
encoding PRTS. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
PRTS. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding PRTS and its initiation codon and upstream regulatory
sequences are inserted
into the appropriate expression vector, no additional transcriptional or
translational control signals
may be needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted,
exogenous translational control signals including an in-frame ATG initiation
codon should be
provided by the vector. Exogenous translational elements and initiation codons
may be of various
origins, both natural and synthetic. The efficiency of expression may be
enhanced by the inclusion of
enhancers appropriate for the particular host cell system used. (See, e.g.,
Scharf, D. et al. (1994)
Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding PRTS and appropriate transcriptional and
translational control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in
vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel,
F.M. et al. (1995)
Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, ch. 9,
13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding PRTS. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV,
or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti
or pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, s, upra; Ausubel, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~v (1992) McGraw
Hill, New
York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and
Harnngton, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors
derived from retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993)
Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
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(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature
389:239-242.)
The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding PRTS. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding PRTS can be
achieved using a
multifunctional E. coli vector such as PBLLTESCRIPT (Stratagene, La Jolla CA)
or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding PRTS into the
vector's multiple
cloning site disrupts the lacZ gene, allowing a colorimetric screening
procedure for identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
in vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of PRTS are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of PRTS may be used.
For example, vectors
containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of PRTS. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, su ra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and
Scorer, C.A. et al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of PRTS. Transcription of
sequences
encoding PRTS may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Broglie, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.)
These constructs can be introduced into plant cells by direct DNA
transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of
Science and Technolo~y
(1992) McGraw Hill, New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding PRTS
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses PRTS in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
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Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression
of PRTS in cell lines is preferred. For example, sequences encoding PRTS can
be transformed into
cell lines using expression vectors which may contain viral origins of
replication and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media
before being switched to selective media. The purpose of the selectable marker
is to confer resistance
to a selective agent, and its presence allows growth and recovery of cells
which successfully express
the introduced sequences. Resistant clones of stably transformed cells may be
propagated using
tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk- and apr cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For example,
dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hvsD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech),13 glucuronidase and its substrate 13-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding PRTS is inserted within a marker gene sequence, transformed
cells containing
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sequences encoding PRTS can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding PRTS under the
control of a single
promoter. Expression of the marker gene in response to induction or selection
usually indicates
expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding PRTS
and that express
PRTS may be identified by a variety of procedures known to those of skill in
the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations,
PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane, solution, or
chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
Immunological methods for detecting and measuring the expression of PRTS using
either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on PRTS is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunology, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding PRTS
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding PRTS, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for
ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic
agents, as well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
Host cells transformed with nucleotide sequences encoding PRTS may be cultured
under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors
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containing polynucleotides which encode PRTS may be designed to contain signal
sequences which
direct secretion of PRTS through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, andlor activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding PRTS may be ligated to a heterologous sequence resulting in
translation of a
fusion protein in any of the aforementioned host systems. For example, a
chimeric PRTS protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of PRTS activity.
Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available
affinity matrices. Such moieties include, but are not limited to, glutathione
S-transferase (GST),
maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their
cognate fusion proteins on immobilized glutathione, maltose, phenylarsine
oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable
immunoaffinity
purification of fusion proteins using commercially available monoclonal and
polyclonal antibodies
that specifically recognize these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the PRTS encoding sequence and the
heterologous protein
sequence, so that PRTS may be cleaved away from the heterologous moiety
following purification.
Methods for fusion protein expression and purification are discussed in
Ausubel (1995, supra, ch. 10).
A variety of commercially available kits may also be used to facilitate
expression and purification of
fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled PRTS may
be achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
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PRTS of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to PRTS. At least one and up to a plurality of test
compounds may be screened
for specific binding to PRTS. Examples of test compounds include antibodies,
oligonucleotides,
proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
PRTS, e.g., a ligand or fragment thereof, a natural substrate, a structural or
functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which PRTS
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express PRTS,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or
E. coli. Cells expressing PRTS or cell membrane fractions which contain PRTS
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either PRTS or the
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example,
the assay may comprise the steps of combining at least one test compound with
PRTS, either in
solution or affixed to a solid support, and detecting the binding of PRTS to
the compound.
Alternatively, the assay may detect or measure binding of a test compound in
the presence of a
labeled competitor. Additionally, the assay may be carried out using cell-free
preparations, chemical
libraries, or natural product mixtures, and the test compounds) may be free in
solution or affixed to a
solid support.
PRTS of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of PRTS. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for PRTS
activity, wherein PRTS is combined with at least one test compound, and the
activity of PRTS in the
presence of a test compound is compared with the activity of PRTS in the
absence of the test
compound. A change in the activity of PRTS in the presence of the test
compound is indicative of a
compound that modulates the activity of PRTS. Alternatively, a test compound
is combined with an
in vitro or cell-free system comprising PRTS under conditions suitable for
PRTS activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of PRTS
may do so indirectly and need not come in direct contact with the test
compound. At least one and up
to a plurality of test compounds may be screened.
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In another embodiment, polynucleotides encoding PRTS or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem
(ES) cells. Such techniques are well known in the art and are useful for the
generation of animal
models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent
No. 5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker gene, e.g., the neomycin phosphotransferase
gene (neo; Capecchi, M.R.
(1989) Science 244:1288-1292). The vector integrates into the corresponding
region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using
the Cre-loxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding PRTS may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding PRTS can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a
region of a polynucleotide encoding PRTS is injected into animal ES cells, and
the injected sequence
integrates into the animal cell genome. Transformed cells are injected into
blastulae, and the
blastulae are implanted as described above. Transgenic progeny or inbred lines
are studied and
treated with potential pharmaceutical agents to obtain information on
treatment of a human disease.
Alternatively, a mammal inbred to overexpress PRTS, e.g., by secreting PRTS in
its milk, may also
serve as a convenient source of that protein (Janne, J. et al. (1998)
Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of PRTS and proteases. In addition, the expression of PRTS is
closely associated
with reproductive, normal and tumorous gastrointestinal, urogenital, bone
tumor, breast, brain, testis,
and adrenal tumor tissues, as well as with adherent mononuclear cells.
Therefore, PRTS appears to
play a role in gastrointestinal, cardiovascular, autoimmune/inflammatory, cell
proliferative,
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developmental, epithelial, neurological, and reproductive disorders. In the
treatment of disorders
associated with increased PRTS expression or activity, it is desirable to
decrease the expression 'or
activity of PRTS. In the treatment of disorders associated with decreased PRTS
expression or
activity, it is desirable to increase the expression or activity of PRTS.
Therefore, in one embodiment, PRTS or a fragment or derivative thereof may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of PRTS. Examples of such disorders include, but are not limited to,
a gastrointestinal
disorder, such as dysphagia, peptic esophagitis, esophageal spasm, esophageal
stricture, esophageal
carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia,
nausea, emesis,
gastroparesis, antral or pyloric edema, abdominal angina, pyrosis,
gastroenteritis, intestinal
obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis,
cholecystitis, cholestasis,
pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis,
hyperbilirubinemia, cirrhosis,
passive congestion of the liver, hepatoma, infectious colitis, ulcerative
colitis, ulcerative proctitis,
Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma,
colonic
obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea,
constipation, gastrointestinal
hemorrhage, acquired immunodeficiency syndrome (A1177S) enteropathy, jaundice,
hepatic
encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis,
Wilson's disease, alpha~-
antitrypsin deficiency, Reye's syndrome, primary sclerosing cholangitis, liver
infarction, portal vein
obstruction and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic
vein thrombosis, veno-
occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy,
intrahepatic cholestasis of
pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and
carcinomas; a
cardiovascular disorder, such as arteriovenous fistula, atherosclerosis,
hypertension, vasculitis,
Raynaud's disease, aneurysms, arterial dissections, varicose veins,
thrombophlebitis and
phlebothrombosis, vascular tumors, and complications of thrombolysis, balloon
angioplasty, vascular
c
replacement, and coronary artery bypass graft surgery, congestive heart
failure, ischemic heart
disease, angina pectoris, myocardial infarction, hypertensive heart disease,
degenerative valvular
heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic
valve, mitral annular
calcification, mitral valve prolapse, rheumatic fever and rheumatic heart
disease, infective
endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic
lupus erythematosus,
carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic
heart disease,
congenital heart disease, and complications of cardiac transplantation; an
autoimmune/inflammatory
disorder, such as acquired immunodeficiency syndrome (AIDS), Addison's
disease, adult respiratory
distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis,
atherosclerotic plaque rupture, autoimmune hemolytic anemia, autoimmune
thyroiditis, autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
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dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema,
episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema
nodosum, atrophic
gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Haslumoto's
thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis,
myasthenia gravis,
myocardial or pericardial inflammation, osteoarthritis, degradation of
articular cartilage, osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid
arthritis, scleroderma, Sjogren's
syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic
sclerosis, thrombocytopenic
purpura, ulcerative colitis, uveitis, Werner syndrome, complications of
cancer, hemodialysis, and
extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal,
and helminthic infections, and
trauma; a cell proliferative disorder such as actinic keratosis,
arteriosclerosis, atherosclerosis, bursitis,
cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis,
paroxysmal nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas, parathyroid,
penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and
uterus; a developmental
disorder, such as renal tubular acidosis, anemia, Cushing's syndrome,
achondroplastic dwa~sm,
Duchenne and Becker muscular dystrophy, bone resorption, epilepsy, gonadal
dysgenesis, WAGR
syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation), Smith-
Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial
dysplasia, hereditary
keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and
neurofibromatosis,
hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea
and cerebral palsy,
spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract,
age-related macular
degeneration, and sensorineural hearing loss; an epithelial disorder, such as
dyshidrotic eczema,
allergic contact dermatitis, keratosis pilaris, melasma, vitiligo, actinic
keratosis, basal cell carcinoma,
squamous cell carcinoma, seborrheic keratosis, folliculitis, herpes simplex,
herpes zoster, varicella,
candidiasis, dermatophytosis, scabies, insect bites, cherry angioma, keloid,
dermatofibroma,
acrochordons, urticaria, transient acantholytic dermatosis, xerosis, eczema,
atopic dermatitis, contact
dermatitis, hand eczema, nummular eczema, lichen simplex chronicus, asteatotic
eczema, stasis
dermatitis and stasis ulceration, seborrheic dermatitis, psoriasis, lichen
planus, pityriasis rosea,
impetigo, ecthyma, dermatophytosis, tinea versicolor, warts, acne vulgaris,
acne rosacea, pemphigus
vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, bullous pemphigoid,
herpes gestationis,
dermatitis herpetiformis, linear IgA disease, epidermolysis bullosa acquisita,
dermatomyositis, lupus
erythematosus, scleroderma and morphea, erythroderma, alopecia, figurate skin
lesions,
telangiectasias, hypopigmentation, hyperpigmentation, vesicles/bullae,
exanthems, cutaneous drug
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reactions, papulonodular skin lesions, chronic non-healing wounds,
photosensitivity diseases,
epidermolysis bullosa simplex, epidermolytic hyperkeratosis, epidermolytic and
nonepidermolytic
palmoplantar keratoderma, ichthyosis bullosa of Siemens, ichthyosis
exfoliativa, keratosis palmaris et
plantaris, keratosis palmoplantaris, palmoplantar keratoderma, keratosis
punctata, Meesmann's
corneal dystrophy, pachyonychia congenita, white sponge nevus, steatocystoma
multiplex, epidermal
nevi/epidermolytic hyperkeratosis type, monilethrix, trichothiodystrophy,
chronic
hepatitis/cryptogenic cirrhosis, and colorectal hyperplasia; a neurological
disorder, such as epilepsy,
ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's
disease, Pick's disease,
Huntington's disease, dementia, Parkinson's disease and other extrapyramidal
disorders, amyotrophic
lateral sclerosis and other motor neuron disorders, progressive neural
muscular atrophy, retinitis
pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating
diseases, bacterial and
viral meningitis, brain abscess, subdural empyema, epidural abscess,
suppurative intracranial
thrombophlebitis, myelitis and radiculitis, viral central nervous system
disease, prion diseases
including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker
syndrome, fatal
familial insomnia, nutritional and metabolic diseases of the nervous system,
neurofibromatosis,
tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental
retardation and other developmental disorders of the central nervous system
including Down
syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system
disorders, cranial nerve
disorders, spinal cord diseases, muscular dystrophy and other neuromuscular
disorders, peripheral
nervous system disorders, dermatomyositis and polymyositis, inherited,
metabolic, endocrine, and
toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders
including mood, anxiety,
and schizophrenic disorders, seasonal affective disorder (SAD), akathesia,
amnesia, catatonia,
diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses,
postherpetic neuralgia,
Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and familial
frontotemporal dementia; and a reproductive disorder, such as infertility,
including tubal disease,
ovulatory defects, and endometriosis, a disorder of prolactin production, a
disruption of the estrous
cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian
hyperstimulation
syndrome, an endometrial or ovarian tumor, a uterine fibroid, autoimmune
disorders, an ectopic
pregnancy, and teratogenesis; cancer of the breast, fibrocystic breast
disease, and galactorrhea; a
disruption of spermatogenesis, abnormal sperm physiology, cancer of the
testis, cancer of the
prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease,
impotence, carcinoma of the
male breast, and gynecomastia.
In another embodiment, a vector capable of expressing PRTS or a fragment or
derivative
thereof may be administered to a subject to treat or prevent a disorder
associated with decreased
expression or activity of PRTS including, but not limited to, those described
above.
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In a further embodiment, a composition comprising a substantially purified
PRTS in
conjunction with a suitable pharmaceutical carrier may be administered to a
subject to treat or prevent
a disorder associated with decreased expression or activity of PRTS including,
but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of PRTS
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of PRTS including, but not limited to, those listed above.
In a further embodiment, an antagonist of PRTS may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of PRTS.
Examples of such
disorders include, but are not limited to, those gastrointestinal,
cardiovascular,
autoimmune/inflammatory, cell proliferative, developmental, epithelial,
neurological, and
reproductive disorders described above. In one aspect, an antibody which
specifically binds PRTS
may be used directly as an antagonist or indirectly as a targeting or delivery
mechanism for bringing a
pharmaceutical agent to cells or tissues which express PRTS.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding PRTS may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of PRTS including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment or prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
An antagonist of PRTS may be produced using methods which are generally known
in the art.
In particular, purified PRTS may be used to produce antibodies or to screen
libraries of
pharmaceutical agents to identify those which specifically bind PRTS.
Antibodies to PRTS may also
~be generated using methods that are well known in the art. Such antibodies
may include, but are not
limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab
fragments, and
fragments produced by a Fab expression library. Neutralizing antibodies (i.e.,
those which inhibit
dimer formation) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans,
and others may be immunized by injection with PRTS or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral
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gels such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among
adjuvants used in
humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable.
It is preferred that the oligopeptides, peptides, or (fragments used to induce
antibodies to
PRTS have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein. Short stretches
of PRTS amino acids may be fused with those of another protein, such as KLH,
and antibodies to the
chimeric molecule may be produced.
Monoclonal antibodies to PRTS may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-hybridoma
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
hnmunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
PRTS-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g.,
Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunoglobulin libraries or panels of highly
specific binding reagents as
disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl.
Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for PRTS may also be
generated.
For example, such fragments include, but are not limited to, F(ab')2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
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Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Nwnerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
PRTS and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering PRTS epitopes is generally used, but a
competitive binding assay may
also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for PRTS. Affinity
is expressed as an
association constant, Ka, which is defined as the molar concentration of PRTS-
antibody complex
divided by the molar concentrations of free antigen and free antibody under
equilibrium conditions.
The Ka determined for a preparation of polyclonal antibodies, which are
heterogeneous in their
affinities for multiple PRTS epitopes, represents the average affinity, or
avidity, of the antibodies for
PRTS. The Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a
particular PRTS epitope, represents a true measure of affinity. High-affinity
antibody preparations
with Ka ranging from about 109 to 1012 Llmole are preferred for use in
immunoassays in which the
PRTS-antibody complex must withstand rigorous manipulations. Low-affinity
antibody preparations
with Ka ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of PRTS, preferably in active
form, from the
antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Ap rp oath, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies,
John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml,
preferably 5-10 mg specific antibody/ml, is generally employed in procedures
requiring precipitation
of PRTS-antibody complexes. Procedures for evaluating antibody specificity,
titer, and avidity, and
guidelines for antibody quality and usage in various applications, are
generally available. (See, e.g.,
Catty, supra, and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding PRTS, or
any fragment
or complement thereof, may be used for therapeutic purposes. In one aspect,
modifications of gene
expression can be achieved by designing complementary sequences or antisense
molecules (DNA,
RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of
the gene encoding
PRTS. Such technology is well known in the art, and antisense oligonucleotides
or larger fragments
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can be designed from various locations along the coding or control regions of
sequences encoding
PRTS. (See, e.g., Agrawal, S., ed. (1996) Antisense Thera ep utics, Humana
Press Inc., Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding PRTS may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeBciency (SCID)-X1 disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites
(e.g., against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
USA. 93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Trypanosoma cruzi). In the
case where a genetic deficiency in PRTS expression or regulation causes
disease, the expression of
PRTS from an appropriate population of transduced cells may alleviate the
clinical manifestations
caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
PRTS are treated by constructing mammalian expression vectors encoding PRTS
and introducing
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these vectors by mechanical means into PRTS-deficient cells. Mechanical
transfer technologies for
use with cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii)
ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv)
receptor-mediated gene
transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson
(1993) Annu. Rev.
Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H.
Recipon (1998) Curr.
Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of PRTS include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA),
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
PRTS may
be expressed using (i) a constitutively active promoter, (e.g., from
cytomegalovirus (CMV), Rous
sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ~3-actin genes),
(ii) an inducible
promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard
(1992) Proc. Natl.
Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769;
Rossi, F.M.V. and
H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in
the T-REX plasmid
(Invitrogen)); the ecdysone-inducible promoter (available in the plasmids
PVGRXR and PIIVD;
Invitrogen);,the FK506/rapamycin inducible promoter; or the RU486/mifepristone
inducible promoter
(Rossi, F.M.V. and H.M. Blau, su ra)), or (iii) a tissue-specific promoter or
the native promoter of the
endogenous gene encoding PRTS from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), ox by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of
these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to PRTS expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding PRTS under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirns vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
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receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant")
discloses a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by
reference. Propagation of retrovirus vectors, transduction of a population of
cells (e.g., CD4+ T-
cells), and the return of transduced cells to a patient are procedures well
known to persons skilled in
the art of gene therapy and have been well documented (Ranga, U. et al. (1997)
J. Virol. 71:7020-
7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)
Blood 89:2283-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding PRTS to cells which have one or more genetic
abnormalities with respect to
the expression of PRTS. The construction and packaging of adenovirus-based
vectors are well known
to those with ordinary skill in the art. Replication defective adenovirus
vectors have proven to be
versatile for importing genes encoding immunoregulatory proteins into intact
islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for
gene therapy"), hereby
incorporated by reference. For adenoviral vectors, see also Antinozzi, P.A. et
al. (1999) Annu. Rev.
Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature 18:389:239-242,
both incorporated by
reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding PRTS to target cells which have one or more genetic
abnormalities with
respect to the expression of PRTS. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing PRTS to cells of the central nervous
system, for which HSV has a
tropism. The construction and packaging of herpes-based vectors are well known
to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is hereby
incorporated by reference. U:S. Patent No. 5,804,413 teaches the use of
recombinant HSV d92 which
consists of a genome containing at least one exogenous gene to be transferred
to a cell under the
control of the appropriate promoter for purposes including human gene therapy.
Also taught by this
patent are the construction and use of recombinant HSV strains deleted for
ICP4, ICP27 and ICP22.
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For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and
Xu, H. et al. (1994)
Dev. Biol. 163:152-161, hereby incorporated by reference. The manipulation of
cloned heipesvirus
sequences, the generation of recombinant virus following the transfection of
multiple plasmids
containing different segments of the large herpesvirus genomes, the growth and
propagation of
herpesvirus, and the infection of cells with herpesvirus are techniques well
lrnown to those of
ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA vixus)
vector is used to
deliver polynucleotides encoding PRTS to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
PRTS into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
PRTS-coding RNAs and the synthesis of high levels of PRTS in vector transduced
cells. While
alphavirus infection is typically associated with cell lysis within a few
days, the ability to establish a
persistent infection in hamster normal kidney cells (BHK-21) with a variant of
Sindbis virus (SIN)
indicates that the lytic replication of alphaviruses can be altered to suit
the needs of the gene therapy
application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host
range of alphaviruses will
allow the introduction of PRTS into a variety of cell types. The specific
transduction of a subset of
cells in a population may require the sorting of cells prior to transduction.
The methods of
manipulating infectious cDNA clones of alphaviruses, performing alphavirus
cDNA and RNA
transfections, and performing alphavirus infections, are well known to those
with ordinary skill in the
art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions
-10 and +10 from the start site, may also be employed to inhibit gene
expression. Similarly,
inhibition can be achieved using triple helix base-pairing methodology. Triple
helix pairing is useful
because it causes inhibition of the ability of the double helix to open
sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using
triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et
al. (1994) in Huber, B.E.
and B.I. Carr, Molecular and Immunolo icg-Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163-
177.) A complementary sequence or antisense molecule may also be designed to
block translation of
mRNA by preventing the transcript from binding to ribosomes.
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Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding PRTS.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding PRTS. Such DNA sequences may be incorporated into a wide
variety of vectors
with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these
cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can be
introduced into cell lines,
cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine,
queosine, and wybutosine, as well as acetyl-, methyl-, thin-, and similarly
modified forms of adenine,
cytidine, guanine, thymine, and uridine which are not as easily recognized by
endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding PRTS. Compounds
which may be effective in altering expression of a specific polynucleotide may
include, but are not
limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming
oligonucleotides,
transcription factors and other polypeptide transcriptional regulators, and
non-macromolecular
chemical entities which are capable of interacting with specific
polynucleotide sequences. Effective
compounds may alter polynucleotide expression by acting as either inhibitors
or promoters of
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polynucleotide expression. Thus, in the treatment of disorders associated with
increased PRTS
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding PRTS may be therapeutically useful, and in the treatment of disorders
associated with
decreased PRTS expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding PRTS may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical andlor structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding PRTS is exposed to at least one test compound thus
obtained. The sample
may comprise, for example, an intact or permeabilized cell, or an in vitro
cell-free or reconstituted
biochemical system. Alterations in the expression of a polynucleotide encoding
PRTS are assayed by
any method commonly known in the art. Typically, the expression of a specific
nucleotide is
detected by hybridization with a probe having a nucleotide sequence
complementary to the sequence
of the polynucleotide encoding PRTS. The amount of hybridization may be
quantified, thus forming
the basis for a comparison of the expression of the polynucleotide both with
and without exposure to
one or more test compounds. Detection of a change in the expression of a
polynucleotide exposed to
a test compound indicates that the test compound is effective in altering the
expression of the
polynucleotide. A screen for a compound effective in altering expression of a
specific polynucleotide
can be carried out, for example, using a Schizosaccharomyces pombe gene
expression system
(Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000)
Nucleic Acids Res.
28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000)
Biochem. Biophys. Res.
Commun. 268:8-13). A particular embodiment of the present invention involves
screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide
nucleic acids, and modified oligonucleotides) for antisense activity against a
specific polynucleotide
sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
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using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition
which generally comprises an active ingredient formulated with a
pharmaceutically acceptable
excipient. Excipients may include, for example, sugars, starches, celluloses,
gums, and proteins.
Various formulations are commonly known and are thoroughly discussed in the
latest edition of
Remin~,ton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such
compositions may
consist of PRTS, antibodies to PRTS, and mimetics, agonists, antagonists, or
inhibitors of PRTS.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, infra-
arterial, intramedullary,
intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of
fast-acting formulations is well-known in the art. In the case of
macromolecules (e.g. larger peptides
and proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the
lung have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton,
J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage
of administration
without needle injection, and obviates the need fox potentially toxic
penetration enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising PRTS or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of
the macromolecule. Alternatively, PRTS or a fragment thereof may be joined to
a short cationic N-
terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs,
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monkeys, or pigs. An animal model may also be used to determine the
appropriate concentration
range and route of administration. Such information can then be used to
determine useful doses and
routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example PRTS
or fragments thereof, antibodies of PRTS, and agonists, antagonists or
inhibitors of PRTS, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDso (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting compositions may be administered every 3 to 4
days, every week,
or biweekly depending on the half life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about O.l ,ug to 100,000 ~.g, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind PRTS may be used for
the
diagnosis of disorders characterized by expression of PRTS, or in assays to
monitor patients being
treated with PRT5 or agonists, antagonists, or inhibitors of PRTS. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
for PRTS include methods which utilize the antibody and a label to detect PRTS
in human body
fluids or in extracts of cells or tissues. The antibodies may be used with or
without modification, and
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may be labeled by covalent or non-covalent attachment of a reporter molecule.
A wide variety of
reporter molecules, several of which are described above, are known in the art
and may be used.
A variety of protocols for measuring PRTS, including ELISAs, RIAs, and FAGS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
PRTS expression. Normal
or standard values for PRTS expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibodies to PRTS under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of FRTS
expressed in
subject, control, and disease samples from biopsied tissues are compared with
the standard values.
Deviation between standard and subject values establishes the parameters for
diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding PRTS may
be used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect
and quantify gene expression in biopsied tissues in which expression of PRTS
may be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
PRTS, and to monitor regulation of PRTS levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding PRTS or closely related
molecules may be used to
identify nucleic acid sequences which encode PRTS. The specificity of the
probe, whether it is made
from a highly specific region, e.g., the 5'regulatory region, or from a less
specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding PRTS, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the PRTS encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:16-30 or from
genomic sequences including promoters, enhancers, and introns of the PRTS
gene.
Means for producing specific hybridization probes for DNAs encoding PRTS
include the
cloning of polynucleotide sequences encoding PRTS or PRTS derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 355,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
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Polynucleotide sequences encoding PRTS may be used for the diagnosis of
disorders
associated with expression of PRTS. Examples of such disorders include, but
are not limited to, a
gastrointestinal disorder, such as dysphagia, peptic esophagitis, esophageal
spasm, esophageal
stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric
carcinoma, anorexia, nausea,
emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis,
gastroenteritis, intestinal
obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis,
cholecystitis, cholestasis,
pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis,
hyperbilirubinemia, cirrhosis,
passive congestion of the liver, hepatoma, infectious colitis, ulcerative
colitis, ulcerative proctitis,
Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma,
colonic
obstruction, irntable bowel syndrome, short bowel syndrome, diarrhea,
constipation, gastrointestinal
hemorrhage, acquired immunodeficiency syndrome (A)DS) enteropathy, jaundice,
hepatic
encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis,
Wilson's disease, alphal-
antitrypsin deficiency, Reye's syndrome, primary sclerosing cholangitis, liver
infarction, portal vein
obstruction and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic
vein thrombosis, veno-
occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy,
intrahepatic cholestasis of
pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and
carcinomas; a
cardiovascular disorder, such as arteriovenous fistula, atherosclerosis,
hypertension, vasculitis,
Raynaud's disease, aneurysms, arterial dissections, varicose veins,
thrombophlebitis and
phlebothrombosis, vascular tumors, and complications of thrombolysis, balloon
angioplasty, vascular
replacement, and coronary artery bypass graft surgery, congestive heart
failure, ischemic heart
disease, angina pectoris, myocardial infarction, hypertensive heart disease,
degenerative valvular
heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic
valve, mitral annular
calcification, mitral valve prolapse, rheumatic fever and rheumatic heart
disease, infective
endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic
lupus erythematosus,
carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic
heart disease,
congenital heart disease, and complications of cardiac transplantation; an
autoimmune/inflammatory
disorder, such as acquired immunodeficiency syndrome (A)DS), Addison's
disease, adult respiratory
distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis,
atherosclerotic plaque rupture, autoimmune hemolytic anemia, autoimmune
thyroiditis, autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema,
episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema
nodosum, atrophic
gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's
thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis,
myasthenia gravis,
myocardial or pericardial inflammation, osteoarthritis, degradation of
articular cartilage, osteoporosis,
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pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid
arthritis, scleroderma, Sjogren's
syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic
sclerosis, thrombocytopenic
purpura, ulcerative colitis, uveitis, Werner syndrome, complications of
cancer, hemodialysis, and
extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal,
and helminthic infections, and
trauma; a cell proliferative disorder such as actinic keratosis,
arteriosclerosis, atherosclerosis, bursitis,
cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis,
paroxysmal nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas, parathyroid,
penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and
uterus; a developmental
disorder, such as renal tubular acidosis, anemia, Cushing's syndrome,
achondroplastic dwarfism,
Duchenne and Becker muscular dystrophy, bone resorption, epilepsy, gonadal
dysgenesis, WAGR
syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation), Smith-
Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial
dysplasia, hereditary
keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and
neurofibromatosis,
hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea
and cerebral palsy,
spins bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract,
age-related macular
degeneration, and sensorineural hearing loss; an epithelial disorder, such as
dyshidrotic eczema,
allergic contact dermatitis, keratosis pilaris, melasma, vitiligo, actinic
keratosis, basal cell carcinoma,
squamous cell carcinoma, seborrheic keratosis, folliculitis, herpes simplex,
herpes zoster, varicella,
candidiasis, dermatophytosis, scabies, insect bites, cherry angioma, keloid,
dermatofibroma,
acrochordons, urticaria, transient acantholytic dermatosis, xerosis, eczema,
atopic dermatitis, contact
dermatitis, hand eczema, nummular eczema, lichen simplex chronicus, asteatotic
eczema, stasis
dermatitis and stasis ulceration, seborrheic dermatitis, psoriasis, lichen
planus, pityriasis roses,
impetigo, ecthyma, dermatophytosis, tines versicolor, warts, acne vulgaris,
acne rosacea, pemphigus
vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, bullous pemphigoid,
herpes gestationis,
dermatitis herpetiformis, linear IgA disease, epidermolysis bullosa acquisita,
dermatomyositis, lupus
erythematosus, scleroderma and morphea, erythroderma, alopecia, figurate skin
lesions,
telangiectasias, hypopigmentation, hyperpigmentation, vesicleslbullae,
exanthems, cutaneous drug
reactions, papulonodular skin lesions, chronic non-healing wounds,
photosensitivity diseases,
epidermolysis bullosa simplex, epidermolytic hyperkeratosis, epidermolytic and
nonepidermolytic
palmoplantar keratoderma, ichthyosis bullosa of Siemens, ichthyosis
exfoliativa, keratosis palmaris et
plantaris, keratosis palmoplantaris, palmoplantar keratoderma, keratosis
punctata, Meesmann's
corneal dystrophy, pachyonychia congenita, white sponge nevus, steatocystoma
multiplex, epidermal
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nevi/epidermolytic hyperkeratosis type, monilethrix, trichothiodystrophy,
chronic
hepatitis/cryptogenic cirrhosis, and colorectal hypezplasia; a neurological
disorder, such as epilepsy,
ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's
disease, Pick's disease,
Huntington's disease, dementia, Parkinson's disease and other extrapyramidal
disorders, amyotrophic
lateral sclerosis and other motor neuron disorders, progressive neural
muscular atrophy, retinitis
pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating
diseases, bacterial and
viral meningitis, brain abscess, subdural empyema, epidural abscess,
suppurative intracranial
thrombophlebitis, myelitis and radiculitis, viral central nervous system
disease, prion diseases
including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker
syndrome, fatal
familial insomnia, nutritional and metabolic diseases of the nervous system,
neurofibromatosis,
tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental
retardation and other developmental disorders of the central nervous system
including Down
syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system
disorders, cranial nerve
disorders, spinal cord diseases, muscular dystrophy and other neuromuscular
disorders, peripheral
nervous system disorders, dermatomyositis and polymyositis, inherited,
metabolic, endocrine, and
toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders
including mood, anxiety,
and schizophrenic disorders, seasonal affective disorder (SAD), akathesia,
amnesia, catatonia,
diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses,
postherpetic neuralgia,
Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and familial
frontotemporal dementia; and a reproductive disorder, such as infertility,
including tubal disease,
ovulatory defects, and endometriosis, a disorder of prolactin production, a
disruption of the estrous
cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian
hyperstimulation
syndrome, an endometrial or ovarian tumor, a uterine fibroid, autoimmune
disorders, an ectopic
pregnancy, and teratogenesis; cancer of the breast, fibrocystic breast
disease, and galactorrhea; a
disruption of spermatogenesis, abnormal sperm physiology, cancer of the
testis, cancer of the
prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease,
impotence, carcinoma of the
male breast, and gynecomastia. The polynucleotide sequences encoding PRTS may
be used in
Southern or northern analysis, dot blot, or other membrane-based technologies;
in PCR technologies;
in dipstick, pin, and multiformat ELISA-like assays; and in microarrays
utilizing fluids or tissues
from patients to detect altered PRTS expression. Such qualitative or
quantitative methods are well
known in the art.
In a particular aspect, the nucleotide sequences encoding PRTS may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding PRTS may be labeled by standard methods and added to a
fluid or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a
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suitable incubation period, the sample is washed and the signal is quantified
and compared with a
standard value. If the amount of signal in the patient sample is significantly
altered in comparison to
a control sample then the presence of altered levels of nucleotide sequences
encoding PRTS in the
sample indicates the presence of the associated disorder. Such assays may also
be used to evaluate
the efficacy of a particular therapeutic treatment regimen in animal studies,
in clinical trials, or to
monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of PRTS,
a normal or standard profile for expression is established. This may be
accomplished by combining
body fluids or cell extracts taken from normal subjects, either animal or
human, with a sequence, or a
fragment thereof, encoding PRTS, under conditions suitable for hybridization
or amplification.
Standard hybridization may be quantified by comparing the values obtained from
normal subjects
with values from an experiment in which a known amount of a substantially
purified polynucleotide
is used. Standard values obtained in this manner may be compared with values
obtained from
samples from patients who are symptomatic for a disorder. Deviation from
standard values is used to
establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding PRTS
may involve the use of PCR. These oligomers may be chemically synthesized,
generated
enzymatically, or produced in vitro. Oligomers will preferably contain a
fragment of a polynucleotide
encoding PRTS, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
PRTS, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding PRTS may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
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substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic
disease in humans. Methods of SNP detection include, but are not limited to,
single-stranded
conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In
SSCP,
oligonucleotide primers derived from the polynucleotide sequences encoding
PRTS are used to
amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived,
for example,
from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
SNPs in the DNA cause
differences in the secondary and tertiary structures of PCR products in single-
stranded form, and
these differences are detectable using gel electrophoresis in non-denaturing
gels. In fSCCP, the
oligonucleotide primers are fluorescently labeled, which allows detection of
the amplimers in high-
throughput equipment such as DNA sequencing machines. Additionally, sequence
database analysis
methods, termed in silico SNP (isSNP), are capable of identifying
polymorphisms by comparing the
sequence of individual overlapping DNA fragments which assemble into a common
consensus
sequence. These computer-based methods filter out sequence variations due to
laboratory preparation
of DNA and sequencing errors using statistical models and automated analyses
of DNA sequence
chromatograms. In the alternative, SNPs may be detected and characterized by
mass spectrometry
using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San
Diego CA).
Methods which may also be used to quantify the expression of PRTS include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. >inmunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives
rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as.a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and
effective treatment regimen for that patient. For example, therapeutic agents
which are highly
effective and display the fewest side effects may be selected for a,patient
based on his/her
pharmacogenomic profile.
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In another embodiment, PRTS, fragments of PRTS, or antibodies specific for
PRTS may be
used as elements on a microarray. The microarray may be used to monitor or
measure protein-protein
interactions, drug-target interactions, and gene expression profiles, as
described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines,
biopsies, or other biological samples. The transcript image may thus reflect
gene expression in vivo,
as in the case of a tissue or biopsy sample, or in vitro, as in the case of a
cell line.
Transcript images which profile the expression of the polynucleotides of the
present
invention may also be used in conjunction with in vitro model systems and
preclinical evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental'
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and
toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson
(2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference
herein). If a test
compound has a signature similar to that of a compound with known toxicity, it
is likely to share
those toxic properties. These fingerprints or signatures are most useful and
refined when they contain
expression information from a large number of genes and gene families.
Ideally, a genome-wide
measurement of expression provides the highest quality signature. Even genes
whose expression is
not altered by any tested compounds are important as well, as the levels of
expression of these genes
are used to normalize the rest of the expression data. The normalization
procedure is useful for
comparison of expression data after treatment with different compounds. While
the assignment of
gene function to elements of a toxicant signature aids in interpretation of
toxicity mechanisms,
knowledge of gene function is not necessary for the statistical matching of
signatures which leads to
prediction of toxicity. (See, for example, Press Release 00-02 from the
National Institute of
Environmental Health Sciences, released February 29, 2000, available at
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http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
treated biological sample are hybridized with one or more probes specific to
the polynucleotides of
the present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a
proteome can be subjected individually to further analysis. Proteome
expression patterns, or profiles,
are analyzed by quantifying the number of expressed proteins and their
relative abundance under
given conditions and at a given time. A profile of a cell's proteome may thus
be generated by
separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the
separation is achieved using two-dimensional gel electrophoresis, in which
proteins from a sample are
separated by isoelectric focusing in the first dimension, and then according
to molecular weight by
sodium dodecyl sulfate slab gel electrophoresis.in the second dimension
(Steiner and Anderson,
supra). The proteins are visualized in the gel as discrete and uniquely
positioned spots, typically by
staining the gel with an agent such as Coomassie Blue or silver or fluorescent
stains. The optical
density of each protein spot is generally proportional to the level of the
protein in the sample. The
optical densities of equivalently positioned protein spots from different
samples, for example, from
biological samples either treated or untreated with a test compound or
therapeutic agent, are
compared to identify any changes in protein spot density related to the
treatment. The proteins in the
spots are partially sequenced using, for example, standard methods employing
chemical or enzymatic
cleavage followed by mass spectrometry. The identity of the protein in a spot
may be determined by
comparing its partial sequence, preferably of at least 5 contiguous amino acid
residues, to the
polypeptide sequences of the present invention. In some cases, further
sequence data may be
obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for PRTS
to quantify the
levels of PRTS expression. In one embodiment, the antibodies are used as
elements on a microarray,
and protein expression levels are quantified by exposing the microarray to the
sample and detecting
the levels of protein bound to each array element (Lueking, A. et al. (1999)
Anal. Biochem. 270:103-
111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be
performed by a
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variety of methods known in the art, for example, by reacting the proteins in
the sample with a thiol-
or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at each
array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to
rapid degradation of mRNA, so proteomic profiling may be more reliable and
informative in such
cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological
sample. A difference in the amount of protein between the two samples is
indicative of a toxic
response to the test compound in the treated sample. Individual proteins are
identified by sequencing
the amino acid residues of the individual proteins and comparing these partial
sequences to the
polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological' sample are
incubated with antibodies specific to the polypeptides of the present
invention. The amount of
protein recognized by the antibodies is quantified. The amount of protein in
the treated biological
sample is compared with the amount in an untreated biological sample. A
difference in the amount of
protein between the two samples is indicative of a toxic response to the test
compound in the treated
sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are
well known and thoroughly described in DNA Microarrays: A Practical Approach,
M. Schena, ed.
(1999) Oxford University Press, London, hereby expressly incorporated by
reference.
In another embodiment of the invention, nucleic acid sequences encoding PRTS
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
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coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
of a mufti-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs),
bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g., Haxrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop
genetic linkage maps, for example, which correlate the inheritance of a
disease state with the
inheritance of a particular chromosome region or restriction fragment length
polymorphism (RFLP).
(See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding PRTS on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
Tn situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another marninalian
species, such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to 11q22-23,
any sequences mapping to that area may represent associated or regulatory
genes for further
investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The
nucleotide sequence of
the instant invention may also be used to detect differences in the
chromosomal location due to
translocation, inversion, etc., among normal, carrier, or affected
individuals.
In another embodiment of the invention, PRTS, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between PRTS and the agent being tested may be measured.
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Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with PRTS, or
fragments thereof,
and washed. Bound PRTS is then detected by methods well known in the art.
Purified PRTS can
also be coated directly onto plates for use in the aforementioned drug
screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture the peptide
and immobilize it on a
solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing
antibodies capable of binding PRTS specifically compete with a test compound
for binding PRTS. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with PRTS.
In additional embodiments, the nucleotide sequences which encode PRTS may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
properties of nucleotide sequences that axe currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are,
therefore, to be construed as merely illustrative, and not limitative of the
remainder of the disclosure
in any way whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below
including U.S. Ser. No. 60/241,573, U.S. Ser. No. 60/243,643, U.S. Ser. No.
60/245,256, U.S. Ser.
No. 60/248,395, U.S. Ser. No. 60/249,826, U.S. Ser. No. 60/252,303, U.S. Ser.
No. 60/250,981, are
expressly incorporated by reference herein.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database
(Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues
were homogenized
and lysed in guanidinium isothiocyanate, while others were homogenized and
lysed in phenol or in a
suitable mixture of denaturants, such as TRIZOL (Life Technologies), a
monophasic solution of
phenol and guanidine isothiocyanate. The resulting lysates were centrifuged
over CsCI cushions or
extracted with chloroform. RNA was precipitated from the lysates with either
isopropanol or sodium
acetate and ethanol, or by other routine methods.
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Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, su ra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBI~-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid
(Invitrogen),
PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), or
pINCY (Incyte
Genomics), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli
cells including XLl-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DH10B,
or
ElectroMAX DHlOB from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP 96
plasmid purification kit from QIAGEN. Following precipitation, plasmids were
resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
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using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example 1I were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-
200 thermal
cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension
using the techniques
disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sa
iens,
Rattus norve i~ cus, Mus musculus, Caenorhabditis ele,g~anS, Saccharomyces
cerevisiae,
Schizosaccharomyces bombe, and Candida albicans (Incyte Genomics, Palo Alto
CA); and hidden
Markov model (HMM)-based protein family databases such as PFAM. (HMM is a
probabilistic
approach which analyzes consensus primary structures of gene families. See,
for example, Eddy,
S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The queries were performed
using programs based
on BLAST, FASTA, BLIMPS, and IIMMER. The Incyte cDNA sequences were assembled
to
produce full length polynucleotide sequences. Alternatively, GenBank cDNAs,
GenBank ESTs,
stitched sequences, stretched sequences, or Genscan-predicted coding sequences
(see Examples IV
and V) were used to extend Incyte cDNA assemblages to full length. Assembly
was performed using
programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened
for open
reading frames using programs based on GeneMark, BLAST, and FASTA. The full
length
polynucleotide sequences were translated to derive the corresponding full
length polypeptide
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sequences. Alternatively, a polypeptide of the invention may begin at any of
the methionine residues
of the full length translated polypeptide. Full length polypeptide sequences
were subsequently
analyzed by querying against databases such as the GenBank protein databases
(genpept), SwissProt,
the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden
Markov
model (ITVIM)-based protein family databases such as PFAM. Full length
polynucleotide sequences
are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering,
South San
Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide
sequence
alignments are generated using default parameters specified by the CLUSTAL
algorithm as
incorporated into the MEGALIGN multisequence alignment program (DNASTAR),
which also
calculates the percent identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and
threshold parameters. The first column of Table 7 shows the tools, programs,
and algorithms used,
the second column provides brief descriptions thereof, the third column
presents appropriate
references, all of which are incorporated by reference herein in their
entirety, and the fourth column
presents, where applicable, the scores, probability values, and other
parameters used to evaluate the
strength of a match between two sequences (the higher the score or the lower
the probability value,
the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide
and polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ
ID N0:16-30. Fragments from about 20 to about 4000 nucleotides which are
useful in hybridization
and amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative proteases were initially identified by running the Genscan gene
identification
program against public genomic sequence databases (e.g., gbpri and gbhtg).
Genscan is a general-
purpose gene identification program which analyzes genomic DNA sequences from
a variety of
organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and
Burge, C. and S. Karlin
(1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates
predicted exons to form an
assembled cDNA sequence extending from a methionine to a stop codon. The
output of Genscan is a
FASTA database of polynucleotide and polypeptide sequences. The maximum range
of sequence for
Genscan to analyze ~at once was set to 30 kb. To determine which of these
Genscan predicted cDNA
sequences encode proteases, the encoded polypeptides were analyzed by querying
against PFAM
models for proteases. Potential proteases were also identified by homology to
Incyte cDNA
sequences that had been annotated as proteases. These selected Genscan-
predicted sequences were
then compared by BLAST analysis to the genpept and gbpri public databases.
Where necessary, the
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Genscan-predicted sequences were then edited by comparison to the top BLAST
hit from genpept to
correct errors in the sequence predicted by Genscan, such as extra or omitted
exons. BLAST analysis
was also used to fmd any Incyte cDNA or public cDNA coverage of the Genscan-
predicted
sequences, thus providing evidence for transcription. When Incyte cDNA
coverage was available,
this information was used to correct or confirm the Genscan predicted
sequence. Full length
polynucleotide sequences were obtained by assembling Genscan-predicted coding
sequences with
Incyte cDNA sequences and/or public cDNA sequences using the assembly process
described in
Example IIC. Alternatively, full length polynucleotide sequences were derived
entirely from edited or
unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
III were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster Were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
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analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example 1V. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as probes to
search for
homologous genomic sequences from the public human genome databases. Partial
DNA sequences
were therefore "stretched" or extended by the addition of homologous genomic
sequences. The
resultant stretched sequences were examined to determine whether it contained
a complete gene.
VI. Chromosomal Mapping of PRTS Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:16-30 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:16-30 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ 1D NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM
distances are based on genetic markers mapped by Genethon which provide
boundaries for radiation
hybrid markers whose sequences were included in each of the clusters. Human
genome maps and
other resources available to the public, such as the NCBI "GeneMap'99" World
Wide Web site
(http://www.ncbi.nlin.nih.govlgenemap/), can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel
(1995) supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
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much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the
computer search can be modified to determine whether any particular match is
categorized as exact or
similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding PRTS are analyzed with
respect to the
tissue sources from which they were derived. For example, some full length
sequences are
assembled, at least in part, with overlapping Incyte cDNA sequences (see
Example )~. Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each
human tissue is
classified into one of the following organ/tissue categories: cardiovascular
system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female;
genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal
system; nervous
system; pancreas; respiratory system; sense organs; skin; stomatognathic
system; unclassified/mixed;
or urinary tract. The number of libraries in each category is counted and
divided by the total number
of libraries across all categories. Similarly, each human tissue is classified
into one of the following
disease/condition categories: cancer, cell line, developmental, inflammation,
neurological, trauma,
cardiovascular, pooled, and other, and the number of libraries in each
category is counted and divided
by the total number of libraries across all categories. The resulting
percentages reflect the tissue- and
disease-specific expression of cDNA encoding PRTS. cDNA sequences and cDNA
library/tissue
information are found in the L1FESEQ GOLD database (Incyte Genomics, Palo Alto
CA).
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VIII. Extension of PRTS Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mgz+, (NH4)ZS04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step l: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SKI- were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ,u1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~l of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ,u1 to 10 ,u1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
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Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37°C in
384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase ,
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted
with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5'regulatory sequences using the above procedure along with
oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ DD N0:16-30 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ~cCi of
~Y 32P~ adenosine- triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
Ase I, Bgl II, Eco RT, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
X. Microarrays
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The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra.),
mechanical nucrospotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena (1999),
supra). Suggested substrates include silicon, silica, glass slides, glass
chips, and silicon wafers.
Alternatively, a procedure analogous to a dot or slot blot may also be used to
arrange and link
elements to the surface of a substrate using thermal, UV, chemical, or
mechanical bonding
procedures. A typical array may be produced using available methods and
machines well known to
those of ordinary skill in the art and may contain any appropriate number of
elements. (See, e.g.,
Schena, M. et al. (1995) Science 270:467-470; Shalom D. et al. (1996) Genome
Res. 6:639-645;
Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample axe conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
2,0 complementarity and the relative abundance of each polynucleotide which
hybridizes to an element
on the microarray may be assessed. In one embodiment, microarray preparation
and usage is
described in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)~ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)~
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/~,1 oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/~tl RNase inhibitor, 500 ~,M dATP, 500 ,uM
dGTP, 500 ~M dTTP, 40
,uM dCTP, 40 ~.M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium
hydroxide and
incubated for 20 minutes at 85° C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
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using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and
resuspended in 14 ~,15X SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
~.g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water,
and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides
are cured in a
110°C oven.
Array elements are applied to the coated glass substrate using a procedure
described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~.1 of the array
element DNA, at an average
concentration of 100 ng/~,1, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in
0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 ~,1 of sample mixture consisting of 0.2 ~,g
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered
with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just
slightly larger than a microscope slide. The chamber is kept at 100% humidity
internally by the
addition of 140 p1 of 5X SSC in a corner of the chamber. The chamber
containing the arrays is
incubated for about 6.5 hours at 60° C. The arrays are washed for 10
min at 45° C in a first wash
buffer (1X SSC, 0.1% SDS), three times for 10 minutes each at 45°C in a
second wash buffer (0.1X
SSC), and dried.
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Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are
differentially expressed, the calibration is done by labeling samples of the
calibrating cDNA with the
two fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A!D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstallc
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each
spot is centered in each element of the grid. The fluorescence signal within
each element is then
integrated to obtain a numerical value corresponding to the average intensity
of the signal. The
software used for signal analysis is the GEMTOOLS gene expression analysis
program (Incyte).
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XI. Complementary Polynucleotides
Sequences complementary to the PRTS-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring PRTS. Although
use of oligonucleotides
comprising from about 15 to 30 base pairs is described, essentially the same
procedure is used with
smaller or with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO
4.06 software (National Biosciences) and the coding sequence of PRTS. To
inhibit transcription, a
complementary oligonucleotide is designed from the most unique 5' sequence and
used to prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is
designed to prevent ribosomal binding to the PRTS-encoding transcript.
XII. Expression of PRTS
Expression and purification of PRTS is achieved using bacterial or virus-based
expression
systems. For expression of PRTS in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express PRTS upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of PRTS in eulcaryotic cells is
achieved by infecting insect
or mammalian cell lines with recombinant Autographica californica nuclear
polyhedrosis virus
(AcMNPV), commonly lmown as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding PRTS by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera fruginerda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.)
In most expression systems, PRTS is synthesized as a fusion protein with,
e.g., glutathione S-
transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting
rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-
kilodalton enzyme from Schistosoma j~onicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
PRTS at specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity
purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman
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Kodak). 6-His, a stretch of six consecutive histidine residues, enables
purification on metal-chelate
resins (QIAGEN). Methods for protein expression and purification are discussed
in Ausubel (1995,
supra, ch. 10 and 16). Purified PRTS obtained by these methods can be used
directly in the assays
shown in Examples XVI, XVII, XVIII, and XlX where applicable.
XIII. Functional Assays
PRTS function is assessed by expressing the sequences encoding PRTS at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA),
both of which
contain the cytomegalovirus promoter. 5-10 ,ug of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ,ug of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
The influence of PRTS on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding PRTS and either CD64 or CD64-GFP.
CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind to
conserved regions of human
immunoglobulin G (IgG). Transfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success
NY). mRNA can be purified from the cells using methods well known by those of
skill in the art.
Expression of mRNA encoding PRTS and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
XIV. Production of PRTS Specific Antibodies
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PRTS substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the PRTS amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, su__Pra,, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-KL,H complex in complete Freund's adjuvant. Resulting antisera
are tested for
antipeptide and anti-PRTS activity by, for example, binding the peptide or
PRTS to a substrate,
blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat
anti-rabbit IgG.
XV. Purification of Naturally Occurring PRTS Using Specific Antibodies
Naturally occurring or recombinant PRTS is substantially purified by
immunoaffinity
chromatography using antibodies specific fox PRTS. An immunoaffinity column is
constructed by
covalently coupling anti-PRTS antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing PRTS are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of PRTS (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/PRTS binding (e.g., a buffer of pH 2 to pH 3, or a high concentration
of a chaotrope, such as
urea or thiocyanate ion), and PRTS is collected.
XVI. Identification of Molecules Which Interact with PRTS
PRTS, or biologically active fragments thereof, are labeled with'z5I Bolton-
Hunter reagent.
(See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a mufti-well plate are incubated with the
labeled PRTS, washed,
and any wells with labeled PRTS complex are assayed. Data obtained using
different concentrations
of PRTS are used to calculate values for the number, affinity, and association
of PRTS with the
candidate molecules.
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Alternatively, molecules interacting with PRTS are analyzed using the yeast
two-hybrid
system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or
using commercially
available kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
PRTS may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
XVII. Demonstration of PRTS Activity
Protease activity is measured by the hydrolysis of appropriate synthetic
peptide substrates
conjugated with various chromogenic molecules in which the degree of
hydrolysis is quantified by
spectrophotometric (or.fluorometric) absorption of the released chromophore
(Beynon, R.J. and J.S.
Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford University
Press, New York NY,
pp.25-55). Peptide substrates are designed according to the category of
protease activity as
endopeptidase (serine, cysteine, aspartic proteases, or metalloproteases),
aminopeptidase (leucine
aminopeptidase), or carboxypeptidase (carboxypeptidases A and B, procollagen C-
proteinase).
Commonly used chromogens are 2-naphthylamine, 4-nitroaniline, and furylacrylic
acid. Assays are .
performed at ambient temperature and contain an aliquot of the enzyme and the
appropriate substrate
in a suitable buffer. Reactions are carried out in an optical cuvette, and the
increaseldecrease in
absorbents of the chromogen released during hydrolysis of the peptide
substrate is measured. The
change in absorbents is proportional to the enzyme activity in the assay.
An alternate assay for ubiquitin hydrolase activity measures the hydrolysis of
a ubiquitin
precursor. The assay is performed at ambient temperature and contains an
aliquot of PRTS and the
appropriate substrate in a suitable buffer. Chemically synthesized human
ubiquitin-valine may be
used as substrate. Cleavage of the C-terminal valine residue from the
substrate is monitored by
capillary electrophoresis (Franklin, K. et al. (1997) Anal. Biochem. 247:305-
309).
In the alternative, an assay for protease activity takes advantage of
fluorescence resonance
energy transfer (FRET) that occurs when one donor and one acceptor fluorophore
with an appropriate
spectral overlap are in close proximity. A flexible peptide linker containing
a cleavage site specific
for PRTS is fused between a red-shifted variant (RSGFP4) and a blue variant
(BFPS) of Green
Fluorescent Protein. This fusion protein has spectral properties that suggest
energy transfer is
occurring from BFPS to RSGFP4. When the fusion protein is incubated with PRTS,
the substrate is
cleaved; and the two fluorescent proteins dissociate. This is accompanied by a
marked decrease in
energy transfer which is quantified by comparing the emission spectra before
and after the addition of
PRTS (Mitre, R.D. et al. (1996) Gene 173:13-17). This assay can also be
performed in living cells.
In this case the fluorescent substrate protein is expressed constitutively in
cells and PRTS is
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introduced on an inducible vector so that FRET can be monitored in the
presence and absence of
PRTS {Sagot, I. et al. (1999) FEBS Lett. 447:53-57).
XVIII. Identification of PRTS Substrates
Phage display libraries can be used to identify optimal substrate sequences
for PRTS. A
random hexamer followed by a linker and a known antibody epitope is cloned as
an N-terminal
extension of gene III in a filamentous phage library. Gene III codes for a
coat protein, and the epitope
will be displayed on the surface of each phage particle. The library is
incubated with PRTS under
proteolytic conditions so that the epitope will be removed if the hexamer
codes for a PRTS cleavage
site. An antibody that recognizes the epitope is added along with immobilized
protein A. Uncleaved
phage, which still bear the epitope, are removed by centrifugation. Phage in
the supernatant are then
amplified and undergo several more rounds of screening. Individual phage
clones are then isolated
and sequenced. Reaction kinetics for these peptide substrates can be studied
using an assay in
Example XVII, and an optimal cleavage sequence can be derived (Ke, S.H. et al.
(1997) J. Biol.
Chem. 272:16603-16609).
To screen for in vivo PRTS substrates, this method can be expanded to screen a
cDNA
expression library displayed on the surface of phage particles (T7SELECT 10-3
Phage display vector,
Novagen, Madison Wn or yeast cells (pYDl yeast display vector kit, Invitrogen,
Carlsbad CA). In
this case, entire cDNAs are fused between Gene III and the appropriate
epitope.
XIX. Identification of PRTS Inhibitors
Compounds to be tested are arrayed in the wells of a mufti-well plate in
varying
concentrations along with an appropriate buffer and substrate, as described in
the assays in Example
XVII. PRTS activity is measured for each well and the ability of each compound
to inhibit PRTS
activity can be determined, as well as the dose-response kinetics. This assay
could also be used to
identify molecules which enhance PRTS activity.
In the alternative, phage display libraries can be used to screen for peptide
PRTS inhibitors.
Candidates are found among peptides which bind tightly to a protease. In this
case, mufti-well plate
wells are coated with PRTS and incubated with a random peptide phage display
library or a cyclic
peptide library (Koivunen, E. et al. (1999) Nat. Biotechnol. 17:768-774).
Unbound phage are washed
away and selected phage amplified and rescreened for several more rounds.
Candidates axe tested for
PRTS inhibitory activity using an assay described in Example XV1I.
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the
invention. Although the invention has been described in connection with
certain embodiments, it
should be understood that the invention as claimed should not be unduly
limited to such specific
86
86
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
embodiments. Indeed, various modifications of the described modes for carrying
out the invention
which are obvious to those skilled in molecular biology or related fields are
intended to be within the
scope of the following claims.
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Table 5
PolynucleotideIncyte ProjectRepresentative Library
SEQ m:
m NO:
16 6926819CB S1NTDIE01
1
17 7473526CB1 ESOGTME01
18 7478443CB UTRSTME01
1
19 3533147CB1 CONUTUTOl
20 7483438CB1 K)DNTUTOl
21 7246467CB TESTNOT03
1
22 7997881CB1 BRSTNOT07
23 7484378CB1 BONMTUE02
24 7473143CB PONSAZTO1
1
25 4382838CB1 BRAENOKOl
26 6717888CB1 TESTNOT17
27 7472044CB ADRETUT05
1
28 7477384CB MPHGNOT03
1
30 7077175CB BON5TUT01
1
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p ~ c w o
d P.~ U can E-~ E-
118
lls
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
<110> INCYTE GENOMICS, INC.
LEE, Ernestine A.
HAFALIA, April
YUE, Henry
LAL, Preeti G.
YAO, Monique G.
LU, Yan
WALIA, Narinder K.
WARREN, Bridget A.
LU, Dyung Aina M.
BAUGHN, Mariah R.
DELEGEANE, Angelo M.
BURFORD, Neil
BOROWSKY, Mark L.
LEE, Sally
XU, Yuming
GRIFFIN, Jennifer A.
KALLICK, Deborah A.
GANDHI, Ameena R.
ARVIZU, Chandra
ISON, Craig H.
TANG, Y. Tom
AZIMZAI, Yalda
ELLIOTT, Vicki S.
SWARNAKAR, Anita
RAMKUMAR, Jayalaxini
NGUYEN, Danniel B.
TRIBOULEY, Catherine M.
L0, Terence P.
AU-YOUNG, Janice
THANGAVELU, Kavitha
KEARNEY, Liam
<120> PROTEASES
<130> PI-0263 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/241,573; 60/243,643; 60/245,256; 60!248,395; 60!249,826
60/252,303; 60/250,981
<151> 2000-10-18; 2000-10-25; 2000-11-02; 2000-11-13; 2000-11-16
2000-11-20; 2000-12-01
<160> 32
<170> PERL Program
<210> 1
<211> 334
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6926819CD1
<400> 1
Met Asn Pro Ser Leu Leu Leu A1a Ala Phe Phe Leu Gly Ile Ala
1 5 . 10 15
Ser Ala A1a Leu Thr Arg Asp His Ser Leu Asp Ala Gln Trp Thr
20 25 30
1/39
CA 02425829 2003-04-08
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Lys Trp Lys Ala Lys His Lys Arg Leu Tyr Gly Met Asn Arg Asn
35 40 45
His Trp Ile Arg Val Leu Trp Glu Lys Asp Val Lys Met Ile Glu
50 55 60
Gln His Asn Gln Glu Tyr Ser Gln Gly Lys His Ser Phe Thr Met
65 70 75
Ala Met Asn Ala Phe Gly Asp Met Val Ser Glu Glu Phe Arg Gln
80 85 90
Val Met Asn G1y Phe G1n Tyr Gln Lys His Arg Lys Gly Lys Gln
95 100 105
Phe Gln Glu Arg Leu Leu Leu Glu Ile Pro Thr Ser Val Asp Trp
110 115 120
Arg G1u Lys Gly Tyr Met Thr Pro Val Lys Asp Gln Gln Gly Gln
125 130 135
Cys Gly Ser Cys Trp Ala Phe Ser A1a Thr Gly Ala Leu Glu G1y
140 145 150
Gln Met Phe Trp Lys Thr Gly Lys Leu Ile Ser Leu Asn Glu Gln
155 160 165 ff
Asn Leu Val Asp Cys Ser Gly Pro G1n Gly Asn Glu Gly Cys Asn
170 175 180
Gly Asp Phe Met Asp Asn Pro Phe Arg Tyr Val Gln Glu Asn Gly
185 190 195
Gly Leu Asp Ser Glu Ala Ser Tyr Pro Tyr Glu Gly Lys Val Lys
200 205 210
Thr Cys Arg Tyr Asn Pro Lys Tyr Ser Ala A1a Asn Asp Thr Gly
215 220 225
Phe Val Asp I1e Pro Ser Arg Glu Lys Asp Leu Ala Lys Ala Val
230 235 240
Ala Thr Val Gly Pro Ile Ser Val A1a Val G1y Ala Ser His Val
245 250 ' 255
Phe Phe Gln Phe Tyr Lys Lys Gly Ile Tyr Phe Glu Pro Arg Cys
260 265 270
Asp Pro Glu Gly Leu Asp His Ala Met Leu Val Val Gly Tyr Ser
275 280 285
Tyr Glu Gly Ala Asp Ser Asp Asn Asn Lys Tyr Trp Leu Va1 Lys
290 295 300
Asn Ser Trp Gly Lys Asn Trp G1y Met Asp Gly Tyr Ile Lys Met
305 310 315
Ala Lys Asp Arg Arg Asn Asn Cys Gly Ile Ala Thr Ala Ala Ser
320 325 330
Tyr Pro Thr Val
<210> 2
<211> 511
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473526CD1
<400> 2
Met Ser Leu Trp Pro Pro Phe Arg Cys Arg Trp Lys Leu Ala Pro
1 5 10 15
Arg Tyr Ser Arg Arg Ala Ser Pro Gln Gln Pro Gln Gln Asp Phe
20 25 30
Glu Ala Leu Leu Ala Glu Cys Leu Arg Asn Gly Cys Leu Phe Glu
35 40 45
Asp Thr Ser Phe Pro Ala Thr Leu Ser Ser I1e Gly Ser Gly Ser
50 55 60
Leu Leu Gln Lys Leu Pro Pro Arg Leu Gln Trp Lys Arg Pro Pro
65 70 75
2/39
CA 02425829 2003-04-08
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Glu Leu His Ser Asn Pro Gln Phe Tyr Phe Ala Lys Ala Lys Arg
80 85 90
Leu Asp Leu Cys Gln G1y Ile Val Gly Asp Cys Trp Phe Leu Ala
95 100 105
Ala Leu Gln Ala Leu Ala Leu His Gln Asp Ile Leu Ser Arg Va1
110 115 120
Val Pro Leu Asn Gln Ser Phe Thr Glu Lys Tyr Ala Gly Ile Phe
125 130 135
Arg Phe Trp Phe Trp His Tyr Gly Asn Trp Val Pro Val Val T1e
140 145 150
Asp Asp Arg Leu Pro Val Asn Glu Ala Gly Gln Leu Val Phe Val
155 160 165
Ser Ser Thr Tyr Lys Asn Leu Phe Trp Gly Ala Leu Leu Glu Lys
170 175 180
Ala Tyr Ala Lys Leu Ser Gly Ser Tyr Glu Asp Leu Gln Ser Gly
185 190 195
Gln Val Ser Glu Ala Leu Val Asp Phe Thr Gly Gly Val Thr Met
200 205 210
Thr Ile Asn Leu Ala Glu Ala His Gly Asn Leu Trp Asp Ile Leu
215 220 225
Ile Glu Ala Thr Tyr Asn Arg Thr Leu Ile Gly Cys Gln Thr His
230 235 240
Ser Gly Glu Lys Ile Leu Glu Asn Gly Leu Val Glu Gly His Ala
245 250 255
Tyr Thr Leu Thr Gly Ile Arg Lys Val Thr Cys Lys His Arg Pro
260 265 270
Glu Tyr Leu Val Lys Leu Arg Asn Pro Trp Gly Lys Val Glu Trp
275 280 285
Lys Gly Asp Trp Ser Asp Ser Ser Ser Lys Trp Glu Leu Leu Ser
290 295 300
Pro Lys Glu Lys Ile Leu Leu Leu Arg Lys Asp Asn Asp G1y Glu
305 310 315
Phe Trp Met Thr Leu G1n Asp Phe Lys Thr His Phe Val Leu Leu
320 325 330
Val Ile Cys Lys Leu Thr Pro Gly Leu Leu Ser Gln G1u Ala Ala
335 340 345
Gln Lys Trp Thr Tyr Thr Met Arg Glu Gly Arg Trp Glu Lys Arg
350 355 360
Ser Thr Ala G1y Gly Gln Arg Gln Leu Leu Gln Asp Thr Phe Trp
365 370 375
Lys Asn Pro Gln Phe Leu Leu Ser Val Trp Arg Pro Glu Glu Gly
380 385 390
Arg Arg Ser Leu Arg Pro Cys Ser Val Leu Val Ser Leu Leu G1n
395 400 405
Lys Pro Arg His Arg Cys Arg Lys Arg Lys Pro Leu Leu Ala Ile
410 415 420
Gly Phe Tyr Leu Tyr Arg Met Asn Lys Tyr His Asp Asp Gln Arg
425 430 435
Arg Leu Pro Pro Glu Phe Phe Gln Arg Asn Thr Pro Leu Ser G1n
440 445 450
Pro Asp Arg Phe Leu Lys Glu Lys Glu Va1 Ser Gln Glu Leu Cys
455 460 465
Leu Glu Pro Gly Thr Tyr Leu Ile Val Pro Ala Tyr Trp Arg Pro
470 475 480
Thr Arg Ser Gln Ser Ser Ser Ser Gly Ser Ser Pro G1y Ser Thr
485 490 495
Ser Phe Met Lys Leu Ala Ala Ile Leu Val Ser Ser Ser Gln Arg
500 505 510
Arg
<210> 3
<211> 812
3/39
CA 02425829 2003-04-08
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<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7478443CD1
<400> 3
Met Gly Trp Arg Pro Arg Arg Ala Arg Gly Thr Pro Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Leu Leu Leu Trp Pro Va1 Pro Gly Ala Gly Val
20 25 30
Leu Gln Gly His Ile Pro Gly Gln Pro Val Thr Pro His Trp Val
35 40 45
Leu Asp Gly Gln Pro Trp Arg Thr Val Ser Leu Glu Glu Pro Val
50 55 60
Ser Lys Pro Asp Met Gly Leu Val Ala Leu Glu Ala Glu Gly Gln
65 70 75
Glu Leu Leu Leu Glu Leu Glu Lys Asn His Arg Leu Leu Ala Pro
80 85 90
Gly Tyr Ile Glu Thr His Tyr Gly Pro Asp Gly Gln Pro Val Val
95 100 105
Leu Ala Pro Asn His Thr Asp His Cys His Tyr Gln Gly Arg Val
110 115 120
Arg Gly Phe Pro Asp Ser Trp Val Val Leu Cys Thr Cys Ser G1y
125 130 135
Met Ser Gly Leu Ile Thr Leu Ser Arg Asn Ala Ser Tyr Tyr Leu
140 145 150
Arg Pro Trp Pro Pro Arg Gly Ser Lys Asp Phe Ser Thr His Glu
155 160 165
Ile Phe Arg Met Glu Gln Leu Leu Thr Trp Lys Gly Thr Cys Gly
170 175 180
His Arg Asp Pro Gly Asn Lys Ala Gly Met Thr Ser Leu Pro Gly
185 190 195
Gly Pro Gln Ser Arg Gly Arg Arg Glu Ala Arg Arg Thr Arg Lys
200 205 210
Tyr Leu G1u Leu Tyr Ile Val Ala Asp His Thr Leu Phe Leu Thr
215 220 225
Arg His Arg Asn Leu Asn His Thr Lys Gln Arg Leu Leu Glu Val
230 235 240
Ala Asn Tyr Val Asp Gln Leu Leu Arg Thr Leu Asp Ile Gln Val
245 250 255
Ala Leu Thr Gly Leu Glu Val Trp Thr Glu Arg Asp Arg Ser Arg
260 265 270
Va1 Thr Gln Asp Ala Asn Ala Thr Leu Trp Ala Phe Leu Gln Trp
275 280 285
Arg Arg Gly Leu Trp A1a Gln Arg Pro His Asp Ser Ala Gln Leu
290 295 300
Leu Thr Gly Arg Ala Phe Gln Gly Ala Thr Val Gly Leu Ala Pro
305 310 315
Val Glu Gly Met Cys Arg A1a G1u Ser Ser Gly Gly Val Ser Thr
320 325 330
Asp His Ser Glu Leu Pro I1e Gly Ala Ala Ala Thr Met Ala His
335 340 345
Glu Ile Gly His Ser Leu Gly Leu Ser His Asp Pro Asp Gly Cys
350 355 360
Cys Val Glu Ala Ala Ala Glu Ser Gly Gly Cys Val Met A1a Ala
365 370 375
Ala Thr Gly His Pro Phe Pro Arg Val Phe Ser Ala Cys Ser Arg
380 385 390
Arg Gln Leu Arg A1a Phe Phe Arg Lys Gly Gly Gly Ala Cys Leu
395 400 405
Ser Asn Ala Pro Asp Pro Gly Leu Pro Va1 Pro Pro Ala Leu Cys
4/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
410 415 420
G1y Asn Gly Phe Val Glu Ala Gly Glu Glu Cys Asp Cys Gly Pro
425 430 435
Gly Gln Glu Cys Arg Asp Leu Cys Cys Phe A1a His Asn Cys Ser
440 445 450
Leu Arg Pro Gly Ala Gln Cys Ala His Gly Asp Cys Cys Val Arg
455 460 465
Cys Leu Leu Lys Pro Ala G1y Ala Leu Cys Arg Gln Ala Met Gly
470 475 480
Asp Cys Asp Leu Pro Glu Phe Cys Thr Gly Thr Ser Ser His Cys
485 490 495
Pro Pro Asp Val Tyr Leu Leu Asp Gly Ser Pro Cys Ala Arg Gly
500 505 510
Ser Gly Tyr Cys Trp Asp Gly Ala Cys Pro Thr Leu Glu Gln Gln
515 520 525
Cys Gln Gln Leu Trp Gly Pro Gly Ser His Pro Ala Pro Glu Ala
530 535 540
Cys Phe Gln Va1 Val Asn Ser Ala G1y Asp Ala His Gly Asn Cys
545 550 555
Gly Gln Asp Ser Glu Gly His Phe Leu Pro Cys Ala Gly Arg Asp
560 565 570
Ala Leu Cys G1y Lys Leu Gln Cys Gln Gly Gly Lys Pro Ser Leu
575 580 585
Leu Ala Pro His Met Val Pro Val Asp Ser Thr Val His Leu Asp
590 595 600
Gly Gln Glu Val Thr Cys Arg Gly Ala Leu Ala Leu Pro Ser Ala
605 610 615
Gln Leu Asp Leu Leu Gly Leu Gly Leu Val Glu Pro Gly Thr Gln
620 625 630
Cys G1y Pro Arg Met Val Cys Gln Ser Arg Arg Cys Arg Lys Asn
635 640 645
Ala Phe Gln Glu Leu Gln Arg Cys Leu Thr A1a Cys His Ser His
650 655 660
Gly Val Cys Asn Ser Asn His Asn Cys His Cys Ala Pro Gly Trp
665 670 675
Ala Pro Pro Phe Cys Asp Lys Pro Gly Phe Gly Gly Ser Met Asp
680 685 690
Ser G1y Pro Val Gln Ala Glu Asn His Asp Thr Phe Leu Leu Ala
695 700 705
Met Leu Leu Ser Val Leu Leu Pro Leu Leu Pro Gly Ala Gly Leu
710 715 720
Ala Trp Cys Cys Tyr Arg Leu Pro Gly Ala His Leu Gln Arg Cys
725 730 735
Ser Trp Gly Cys Arg Arg Asp Pro Ala Cys Ser Gly Pro Lys Asp
740 745 750
Gly Pro His Arg Asp His Pro Leu Gly Gly Val His Pro Met Glu
755 760 765
Leu Gly Pro Thr Ala Thr Gly Gln Pro Trp Pro Leu Asp Pro Glu
770 775 780
Asn Ser His Glu Pro Ser Ser His Pro Glu Lys Pro Leu Pro Ala
785 790 795
Val Ser Pro Asp Pro Gln Asp Gln Val Gln Met Pro Arg Ser Cys
800 805 810
Leu Trp
<210> 4
<211> 1236
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
5/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
<223> Incyte ID No: 3533147CD1
<400> 4
Met Thr Gly Thr Gly Gly Arg Lys Pro Thr Gly Asp Lys Gln Glu
1 5 10 15
Val His Pro Trp Glu Lys Gln Glu Val Arg Glu Gln Thr Glu Ser
20 25 30
Pro Gln Glu Leu Thr Arg Ser Pro Gln Gly Thr Asp Arg Asn Asp
35 40 45
Thr Val Thr Ile Tyr Thr Asp Thr Gln Ser Arg Lys Ala Gly Ala
50 55 ' 60
Ser Arg Lys Ile Arg Asn Met Leu Asn Ile Tyr Leu Val Trp Leu
65 70 75
Val Lys Ile Asn Gln Ile Ile Ile Asn Val Phe Tyr Gln Asn Pro
80 85 90
Glu Pro Thr Ile Trp Asn Ser Ala Phe Ile Va1 Asp Ile Thr Ala
95 100 105
Ile Va1 Pro Thr Ala Leu Phe Pro Phe Asn Va1 Ala Lys Pro Lys
110 115 120
Met Leu Val Glu Asn Leu G1n Glu Gly Asp Phe Arg Glu Leu Arg
125 130 135
Gly Asn Ser His His Cys Leu Thr Lys Lys Gly Leu Gly Asn A1a
140 145 150
Pro Pro Gly Leu Gln Phe Thr Leu Tyr Lys Cys Leu Asp Ser Ser
155 160 165
Arg Thr Ala Gln Pro His Ala Gly Leu His Tyr Val Asp Ile Asn
170 175 180
Ser Gly Met Ile Arg Thr Glu Glu Ala Asp Tyr Phe Leu Arg Pro
185 190 195
Leu Pro Ser His Leu Ser Trp Lys Leu Gly Arg Ala Ala Gln Gly
200 205 210
Ser Ser Pro Ser His Val Leu Tyr Lys Arg Ser Thr G1u Pro His
215 220 225
Ala Pro Gly A1a Ser Glu Val Leu Val Thr Ser Arg Thr Trp Glu
230 235 240
Leu Ala His G1n Pro Leu His Ser Ser Asp Leu Arg Leu Gly Leu
245 250 255
Pro Gln Lys G1n His Phe Cys Gly Arg Arg Lys Lys Tyr Met Pro
260 265 270
Gln Pro Pro Lys Glu Asp Leu Phe Ile Leu Pro Asp Glu Tyr Lys
275 280 285
Ser Cys Leu Arg His Lys Arg Ser Leu Leu Arg Ser His Arg Asn
290 295 300
Glu Glu Leu Asn Val Glu Thr Leu Val Val Val Asp Lys Lys Met
305 310 315
Met G1n Asn His Gly His Glu Asn Ile Thr Thr Tyr Val Leu Thr
320 325 330
Ile Leu Asn Met Val Ser Ala Leu Phe Lys Asp Gly Thr Ile Gly
335 340 345
G1y Asn Ile Asn Ile A1a Ile Val Gly Leu I1e Leu Leu Glu Asp
350 355 360
Glu G1n Pro Gly Leu Va1 Ile Ser His His Ala Asp His Thr Leu
365 370 375
Ser Ser Phe Cys Gln Trp Gln Ser Gly Leu Met Gly Lys Asp Gly
380 385. 390
Thr Arg His Asp His Ala Ile Leu Leu Thr Gly Leu Asp Ile Cys
395 400 405
Ser Trp Lys Asn Glu Pro Cys Asp Thr Leu Gly Phe Ala Pro Ile
410 415 420
Ser Gly Met Cys Ser Lys Tyr Arg Ser Cys Thr Ile Asn Glu Asp
425 430 435
Thr Gly Leu Gly Leu Ala Phe Thr I1e Ala His Glu Ser Gly His
440 445 450
6/39
CA 02425829 2003-04-08
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Asn Phe Gly Met Ile His Asp Gly Glu Gly Asn Met Cys Lys Lys
455 460 465
Ser Glu Gly Asn Ile Met Ser Pro Thr Leu Ala Gly Arg Asn Gly
470 475 480
Val Phe Ser Trp Ser Pro Cys Ser Arg Gln Tyr Leu His Lys Phe
485 490 495
Leu Ser Thr Ala Gln Ala Ile Cys Leu Ala Asp Gln Pro Lys Pro
500 505 510
Val Lys G1u Tyr Lys Tyr Pro Glu Lys Leu Pro Gly Glu Leu Tyr
515 520 525
Asp Ala Asn Thr Gln Cys Lys Trp G1n Phe Gly Glu Lys A1a Lys
530 535 540
Leu Cys Met Leu Asp Phe Lys Lys Asp Ile Cys Lys Ala Leu Trp
545 550 555
Cys His Arg Ile Gly Arg Lys Cys Glu Thr Lys Phe Met Pro Ala
560 565 570
Ala Glu Gly Thr Ile Cys Gly His Asp Met Trp Cys Arg Gly Gly
575 580 585
Gln Cys Val Lys Tyr Gly Asp Glu G1y Pro Lys Pro Thr His Gly
590 595 600
His Trp Ser Asp Trp Ser Ser Trp Ser Pro Cys Ser Arg Thr Cys
605 610 615
Gly Gly Gly Val Ser His Arg Ser Arg Leu Cys Thr Asn Pro Lys
620 625 630
Pro Ser His Gly Gly Lys Phe Cys Glu G1y Ser Thr Arg Thr Leu
635 640 645
Lys Leu Cys Asn Ser Gln Lys Cys Pro Arg Asp Ser Val Asp Phe
650 655 660
Arg Ala Ala Gln Cys Ala Glu His Asn Ser Arg Arg Phe Arg Gly
665 670 675
Arg His Tyr Lys Trp Lys Pro Tyr Thr Gln Val Glu Asp Gln Asp
680 685 690
Leu Cys Lys Leu Tyr Cys Ile Ala Glu Gly Phe Asp Phe Phe Phe
695 700 705
Ser Leu Ser Asn Lys Val Lys Asp Gly Thr Pro Cys Ser Glu Asp
710 715 720
Ser Arg Asn Val Cys Ile Asp Gly Ile Cys G1u Arg Val Gly Cys
725 730 735
Asp Asn Val Leu Gly Ser Asp Ala Val Glu Asp Val Cys Gly Val
740 745 750
Cys Asn Gly Asn Asn Ser Ala Cys Thr Ile His Arg Gly Leu Tyr
755 760 765
Thr Lys His His His Thr Asn Gln Tyr Tyr His Met Val Thr Ile
770 775 780
Pro Ser Gly Ala Arg Ser Ile Arg Ile Tyr Glu Met Asn Val Ser
785 790 795
Thr Ser Tyr Ile Ser Val Arg Asn Ala Leu Arg Arg Tyr Tyr Leu
800 805 810
Asn Gly His Trp Thr Val Asp Trp Pro Gly Arg Tyr Lys Phe Ser
815 820 825
G1y Thr Thr Phe Asp Tyr Arg Arg Ser Tyr Asn Glu Pro Glu Asn
830 ~ 835 840
Leu Ile Ala Thr Gly Pro Thr Asn Glu Thr Leu Ile Val Glu Leu
845 850 855
Leu Phe Gln Gly Arg Asn Pro Gly Va1 Ala Trp Glu Tyr Ser Met
860 865 870
Pro Arg Leu Gly Thr Glu Lys Gln Pro Pro Ala Gln Pro Ser Tyr
875 880 885
Thr Trp Ala Ile Val Arg Ser Glu Cys Ser Val Ser Cys Gly Gly
890 895 900
Gly Gln Met Thr Val Arg Glu Gly Cys Tyr Arg Asp Leu Lys Phe
905 910 915
Gln Val Asn Met Ser Phe Cys Asn Pro Lys Thr Arg Pro Val Thr
7/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
920 ' 925 930
Gly Leu Val Pro Cys Lys Val Ser Ala Cys Pro Pro Ser Trp Ser
935 940 945
Val Gly Asn Trp Ser Ala Cys Ser Arg Thr Cys Gly Gly Gly Ala
950 955 960
Gln Ser Arg Pro Va1 Gln Cys Thr Arg Arg Val His Tyr Asp Ser
965 970 975
Glu Pro Val Pro Ala Gly Leu Cys Pro Gln Leu Val Pro Pro Ala
980 985 990
Gly Arg Pro Ala Thr Leu Arg Ala Ala His Leu His Gly Ala Pro
995 1000 1005
Gly Pro Gly Gln Ser Ala His Thr Pro Val Gly Arg Val Glu G1u
1010 1015 1020
Arg Ala Va1 Ala Cys Lys Ser Thr Asn Pro Ser Ala Arg Ala Gln
1025 1030 1035
Leu Leu Pro Asp Ala Val Cys Thr Ser Glu Pro Lys Pro Arg Met
1040 1045 1050
His Glu Ala Cys Leu Leu Gln Arg Cys His Lys Pro Lys Lys Leu
1055 1060 1065
Gln Trp Leu Val Ser A1a Trp Ser G1n Cys Ser Val Thr Cys Glu
1070 1075 1080
Arg Gly Thr Gln Lys Arg Phe Leu Lys Cys Ala Glu Lys Tyr Val
1085 1090 1095
Ser Gly Lys Tyr Arg Glu Leu Ala Ser Lys Lys Cys Ser His Leu
1100 1105 1110
Pro Lys Pro Ser Leu Glu Leu Glu Arg Ala Cys Ala Pro Leu Pro
1115 1120 1125
Cys Pro Arg His Pro Pro Phe Ala Ala Ala Gly Pro Ser Arg Gly
1130 1135 1140
Ser Trp Phe Ala Ser Pro Trp Ser Gln Cys Thr Ala Ser Cys Gly
1145 1150 1155
Gly Gly Val Gln Thr Arg Ser Val Gln Cys Leu Ala Gly Gly Arg
1160 X165 1170
Pro Ala Ser Gly Cys Leu Leu His Gln Lys Pro Ser Ala Ser Leu
1175 1180 1185
Ala Cys Asn Thr His Phe Cys Pro Ile Ala Glu Lys Lys Asp Ala
1190 1195 1200
Phe Cys Lys Asp Tyr Phe His Trp Cys Tyr Leu Val Pro Gln His
1205 1210 1215
Gly Met Cys Ser His Lys Phe Tyr Gly Lys Gln Cys Cys Lys Thr
1220 1225 1230
Cys Ser Lys Ser Asn Leu
1235
<210> 5
<211> 304
<212> PRT
<21.3> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7483438CD1
<400> 5
Met Gly Leu Arg Ala Gly Pro Ile Leu Leu Leu Leu Leu Trp Leu
1 5 10 15
Leu Pro Gly A1a His Trp Asp Val Leu Pro her G1u Cys Gly His
20 25 30
Ser Lys Glu Ala Gly Arg Ile Val Gly Gly Gln Asp Thr Gln Glu
35 40 45
Gly Arg Trp Pro Trp Gln Val Gly Leu Trp Leu Thr Ser Val G1y
50 55 60
His Val Cys Gly Gly Ser Leu Ile His Pro Arg Trp Val Leu Thr
8/39
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WO 02/38744 PCT/USO1/51034
65 70 75
Ala Ala His Cys Phe Leu Arg Ser Glu Asp Pro Gly Leu Tyr His
80 85 90
Val Lys Val Gly Gly Leu Thr Pro Ser Leu Ser G1u Pro His Ser
95 100 105
Ala Leu Val Ala Val Arg Arg Leu Leu Val His Ser Ser Tyr His
110 115 120
Gly Thr Thr Thr Ser G1y Asp Ile Ala Leu Met G1u Leu Asp Ser
125 130 135
Pro Leu Gln A1a Ser Gln Phe Ser Pro I1e Cys Leu Pro G1y Pro
140 145 150
Gln Thr Pro Leu Ala I1e G1y Thr Val Cys Trp Val Asn Gly Leu
155 160 165
Gly Glu Val A1a Val Pro Leu Leu Asp Ser Asn Met Cys G1u Leu
170 175 180
Met Tyr His Leu G1y Glu Pro Ser Leu Ala Gly Gln Arg Leu Ile
185 190 195
Gln Asp Asp Met Leu Cys A1a Gly Ser Val Gln Gly Lys Lys Asp
200 205 210
Ser Cys Gln G1y Asp Ser Gly Gly Pro Leu Val Cys Pro Ile Asn
215 220 225
Asp Thr Trp Ile Gln Ala Gly Ile Val Ser Trp Gly Phe Gly Cys
230 235 240
Ala Arg Pro Phe Arg Pro Gly Val Tyr Thr Gln Val Leu Ser Tyr
245 250 255
Thr Asp Trp Ile Gln Arg Thr Leu Ala Glu Ser His Ser Gly Met
260 265 270
Ser Gly Ala Arg Pro Gly Ala Pro Gly Ser His Ser Gly Thr Ser
275 280 285
Arg Ser His Pro Val Leu Leu Leu Glu Leu Leu Thr Val Cys Leu
290 295 300
Leu Gly Ser Leu
<210> 6
<211> 980
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7246467CD1
<400> 6
Met Ser Pro Leu Lys Ile His Gly Pro Ile Arg I1e Arg Ser Met
1 5 10 15
Gln Thr Gly Ile Thr Lys Trp Lys Glu Gly Ser Phe Glu Ile Val
20 25 30
Glu Lys Glu Asn Lys Val Ser Leu Val Val His Tyr Asn Thr Gly
35 40 45
Gly Ile Pro Arg I1e Phe Gln Leu Ser His Asn Ile Lys Asn Val
50 55 60
Val Leu Arg Pro Ser Gly Ala Lys Gln Ser Arg Leu Met Leu Thr
65 70 75
Leu Gln Asp Asn Ser Phe Leu Ser Ile Asp Lys Val Pro Ser Lys
80 85 90
Asp Ala Glu Glu Met Arg Leu Phe Leu Asp Ala Va1 His Gln Asn
95 100 105
Arg Leu Pro Ala Ala Met Lys Pro Ser G1n Gly Ser Gly Ser Phe
110 115 120
Gly Ala Ile Leu Gly Ser Arg Thr Ser Gln Lys Glu Thr Ser Arg
125 130 135
Gln Leu Ser Tyr Ser Asp Asn Gln Ala Ser Ala Lys Arg Gly Ser
9/39
CA 02425829 2003-04-08
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140 145 150
Leu G1u Thr Lys Asp Asp Ile Pro Phe Arg Lys Val Leu G1y Asn
155 160 165
Pro Gly Arg Gly Ser Ile Lys Thr Val Ala Gly Ser Gly Ile Ala
170 175 180
Arg Thr Ile Pro Ser Leu Thr Ser Thr Ser Thr Pro Leu Arg Ser
185 190 195
G1y Leu Leu Glu Asn Arg Thr Glu Lys Arg Lys Arg Met Ile Ser
200 205 210
Thr Gly Ser Glu Leu Asn Glu Asp Tyr Pro Lys Glu Asn Asp Ser
215 220 225
Ser Ser Asn Asn Lys Ala Met Thr Asp Pro Ser Arg Lys Tyr Leu
230 235 240
Thr Ser Ser Arg Glu Lys Gln Leu Ser Leu Lys Gln Ser Glu Glu
245 250 255
Asn Arg Thr Ser Gly Gly Leu Leu Pro Leu Gln Ser Ser Ser Phe
260 265 270
Tyr Gly Ser Arg Ala Gly Ser Lys Glu His Ser Ser Gly Gly Thr
275 280 285
Asn Leu Asp Arg Thr Asn Val Ser Ser Gln Thr Pro Ser Ala Lys
290 295 300
Arg Ser Leu Gly Phe Leu Pro Gln Pro Val Pro Leu Ser Val Lys
305 310 315
Lys Leu Arg Cys Asn Gln Asp Tyr Thr G1y Trp Asn Lys Pro Arg
320 325 330
Val Pro Leu Ser Ser His Gln Gln Gln Gln Leu Gln Gly Phe Ser
335 340 345
Asn Leu Gly Asn Thr Cys Tyr Met Asn A1a Ile Leu Gln Ser Leu
350 355 360
Phe Ser Leu Gln Ser Phe Ala Asn Asp Leu Leu Lys Gln Gly Ile
365 370 375
Pro Trp Lys Lys Ile Pro Leu Asn Ala Leu Ile Arg Arg Phe Ala
380 385 390
His Leu Leu Val Lys Lys Asp Ile Cys Asn Ser Glu Thr Lys Lys
395 400 405
Asp Leu Leu Lys Lys Val Lys Asn Ala Ile Ser Ala Thr Ala Glu
410 415 420
Arg Phe Ser Gly Tyr Met Gln Asn Asp Ala His G1u Phe Leu Ser
425 430 435
Gln Cys Leu Asp Gln Leu Lys Glu Asp Met Glu Lys Leu Asn Lys
440 445 450
Thr Trp Lys Thr Glu Pro Val Ser Gly Glu Glu Asn Ser Pro Asp
455 460 465
Ile Ser Ala Thr Arg Ala Tyr Thr Cys Pro Val I1e Thr Asn Leu
470 475 480
Glu Phe Glu Val Gln His Ser Ile Ile Cys Lys Ala Cys Gly Glu
485 490 495
Ile Ile Pro Lys Arg Glu Gln Phe Asn Asp Leu Ser Ile Asp Leu
500 505 510
Pro Arg Arg Lys Lys Pro Leu Pro Pro Arg Ser Ile G1n Asp Ser
515 520 525
Leu Asp Leu Phe Phe Arg Ala Glu Glu Leu Glu Tyr Ser Cys Glu
530 535 540
Lys Cys Gly Gly Lys Cys Ala Leu Val Arg His Lys Phe Asn Arg
545 550 555
Leu Pro Arg Val Leu Ile Leu His Leu Lys Arg Tyr Ser Phe Asn
560 565 570
Val Ala Leu Ser Leu Asn Asn Lys Ile Gly Gln Gln Val I1e Ile
575 580 585
Pro Arg Tyr Leu Thr Leu Ser Ser His Cys Thr Glu Asn Thr Lys
590 595 600
Pro Pro Phe Thr Leu Gly Trp Ser Ala His Met Ala Met Ser Arg
605 610 615
10/39
CA 02425829 2003-04-08
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Pro Leu Lys Ala Ser Gln Met Va1 Asn Ser Cys Ile Thr Ser Pro
620 625 630
Ser Thr Pro Ser Lys Lys Phe Thr Phe Lys Ser Lys Ser Ser Leu
635 640 645
Ala Leu Cys Leu Asp Ser Asp Ser G1u Asp Glu Leu Lys Arg Ser
650 655 660
Val Ala Leu Ser Gln Arg Leu Cys Glu Met Leu G1y Asn Glu Gln
665 670 675
Gln G1n Glu Asp Leu Glu Lys Asp Ser Lys Leu Cys Pro I1e Glu
680 685 690
Pro Asp Lys Ser Glu Leu Glu Asn Ser Gly Phe Asp Arg Met Ser
695 700 705
G1u Glu Glu Leu Leu Ala Ala Va1 Leu Glu Ile Ser Lys Arg Asp
710 715 720
Ala Ser Pro Ser Leu Ser His Glu Asp Asp Asp Lys Pro Thr Ser
725 730 735
Ser Pro Asp Thr Gly Phe Ala Glu Asp Asp Ile Gln Glu Met Pro
740 745 750
Glu Asn Pro Asp Thr Met Glu Thr Glu Lys Pro Lys Thr Ile Thr
755 760 765
Glu Leu Asp Pro Ala Ser Phe Thr Glu Ile Thr Lys Asp Cys Asp
770 775 780
Glu Asn Lys G1u Asn Lys Thr Pro Glu Gly Ser Gln Gly Glu Val
785 790 795
Asp Trp Leu Gln Gln Tyr Asp Met Glu Arg Glu Arg Glu Glu Gln
800 805 810
Glu Leu Gln Gln Ala Leu Ala Gln Ser Leu G1n Glu Gln Glu Ala
815 820 825
Trp Glu Gln Lys Glu Asp Asp Asp Leu Lys Arg Ala Thr Glu Leu
830 835 840
Ser Leu Gln Glu Phe Asn Asn Ser Phe Val Asp Ala Leu Gly Ser
845 850 855
Asp Glu Asp Ser Gly Asn Glu Asp Val Phe Asp Met Glu Tyr Thr
860 865 870
Glu Ala Glu Ala Glu Glu Leu Lys Arg Asn Ala G1u Thr Gly Asn
875 880 885
Leu Pro His Ser Tyr Arg Leu Ile Ser Va1 Val Ser His Ile Gly
890 895 900
Ser Thr Ser Ser Ser G1y His Tyr Ile Ser Asp Val Tyr Asp Ile
905 910 915
Lys Lys Gln Ala Trp Phe Thr Tyr Asn Asp Leu Glu Val Ser Lys
920 925 930
I1e Gln Glu Ala Ala Val Gln Ser Asp Arg Asp Arg Ser Gly Tyr
935 940 945
Ile Phe Phe Tyr Met His Lys Glu Ile Phe Asp Glu Leu Leu Glu
950 955 960
Thr Glu Lys Asn Ser Gln Ser Leu Ser Thr Glu Val Gly Lys Thr
965 970 975
Thr Arg Gln Ala Ser
980
<210> 7
<211> 1251
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7997881CD1
<400> 7
Met Thr Ile Val Asp Lys Ala Ser Glu Ser Ser Asp Pro Ser Ala
1 5 10 15
11/39
CA 02425829 2003-04-08
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Tyr Gln Asn Gln Pro Gly Ser Ser Glu Ala Val Ser Pro Gly Asp
20 25 30
Met Asp Ala Gly Ser Ala Ser Trp Gly Ala Val Ser Ser Leu Asn
35 40 45
Asp Va1 Ser Asn His Thr Leu Ser Leu Gly Pro Val Pro Gly Ala
50 55 60
Val Va1 Tyr Ser Ser Ser Ser Val Pro Asp Lys Ser Lys Pro Ser
65 70 75
Pro Gln Lys Asp G1n Ala Leu Gly Asp Gly Ile Ala Pro Pro Gz,n
80 85 ~ ~0
Lys Val Leu Phe Pro Ser Glu Lys Ile Cys Leu Lys Trp Gln Gln
95 100 105
Thr His Arg Val Gly Ala Gly Leu Gln Asn Leu Gly Asn Thr Cys
110 115 120
Phe Ala Asn Ala Ala Leu Gln Cys Leu Thr Tyr Thr Pro Pro Leu
125 130 135
Ala Asn Tyr Met Leu Ser His Glu His Ser Lys Thr Cys His Ala
140 145 150
Glu Gly Phe Cys Met Met Cys Thr Met Gln Ala His Ile Thr G1n
155 160 165
Ala Leu Ser Asn Pro G1y Asp Val I1e Lys Pro Met Phe Val Ile
170 175 180
Asn Glu Met Arg Arg Ile Ala Arg His Phe Arg Phe Gly Asn Gln
185 190 195
Glu Asp Ala His Glu Phe Leu Gln Tyr Thr Val Asp Ala Met Gln
200 205 210
Lys Ala Cys Leu Asn G1y Ser Asn Lys Leu Asp Arg His Thr G1n
215 220 225
Ala Thr Thr Leu Val Cys Gln Ile Phe Gly Gly Tyr Leu Arg Ser
230 235 240
Arg Val Lys Cys Leu Asn Cys Lys Gly Val Ser Asp Thr Phe Asp
245 250 255
Pro Tyr Leu Asp Ile Thr Leu Glu Ile Lys Ala Ala Gln Ser Val
260 265 270
Asn Lys Ala Leu Glu Gln Phe Val Lys Pro Glu Gln Leu Asp Gly
275 280 285
Glu Asn Ser Tyr Lys Cys Ser Lys Cys Lys Lys Met Va1 Pro Ala
290 295 300
Ser Lys Arg Phe Thr Ile His Arg Ser Ser Asn Val Leu Thr Leu
305 310 315
Ser Leu Lys Arg Phe Ala Asn Phe Thr Gly G1y Lys I1e Ala Lys
320 325 330
Asp Val Lys Tyr Pro Glu Tyr Leu Asp Ile Arg Pro Tyr Met Ser
335 340 345
Gln Pro Asn Gly Glu Pro I1e Val Tyr Val Leu Tyr Ala Val Leu
350 355 360
Val His Thr Gly Phe Asn Cys His Ala Gly His Tyr Phe Cys Tyr
365 370 375
Ile Lys Ala Ser Asn Gly Leu Trp Tyr G1n Met Asn Asp Ser Ile
380 385 390
Val Ser Thr Ser Asp Ile Arg Ser Val Leu Ser Gln Gln Ala Tyr
395 400 405
Val Leu Phe Tyr Ile Arg Ser His Asp Va1 Lys Asn Gly Gly Glu
410 415 420
Leu Thr His Pro Thr His Ser Pro Gly Gln Ser Ser Pro Arg Pro
425 430 435
Val Ile Ser Gln Arg Val Val Thr Asn Lys Gln Ala Ala Pro Gly
440 445 450
Phe Ile Gly Pro Gln Leu Pro Ser His Met Ile Lys Asn Pro Pro
455 460 465
His Leu Asn Gly Thr Gly Pro Leu Lys Asp Thr Pro Ser Ser Ser
470 475 480
Met Ser Ser Pro Asn Gly Asn Ser Ser Val Asn Arg Ala Ser Pro
12/39
CA 02425829 2003-04-08
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485 490 495
Val Asn Ala Ser Ala Ser Val Gln Asn Trp Ser Val Asn Arg Ser
500 505 510
Ser Val Ile Pro Glu His Pro Lys Lys Gln Lys Ile Thr Ile Ser
515 520 525
Ile His Asn Lys Leu Pro Val Arg Gln Cys Gln Ser Gln Pro Asn
530 535 540
Leu His Ser Asn Ser Leu Glu Asn Pro Thr Lys Pro Val Pro Ser
545 550 555
Ser Thr Ile Thr Asn Ser Ala Va1 Gln Ser Thr Ser Asn Ala Ser
560 565 570
Thr Met Ser Val Ser Ser Lys Val Thr Lys Pro Ile Pro Arg Ser
575 580 585
Glu Ser Cys Ser Gln Pro Val Met Asn Gly Lys Ser Lys Leu Asn
590 595 600
Ser Ser Val Leu Val Pro Tyr Gly Ala Glu Ser Ser G1u Asp Ser
605 610 615
Asp Glu Glu Ser Lys Gly Leu Gly Lys Glu Asn Gly Ile Gly Thr
620 625 630
I1e Val Ser Ser His Ser Pro Gly Gln Asp Ala G1u Asp Glu Glu
635 640 645
A1a Thr Pro His Glu Leu Gln Glu Pro Met Thr Leu Asn Gly Ala
650 655 660
Asn Ser Ala Asp Ser Asp Ser Asp Pro Lys G1u Asn Gly Leu Ala
665 670 675
Pro Asp Gly Ala Ser Cys Gln Gly Gln Pro Ala Leu His Ser Glu
680 685 690
Asn Pro Phe Ala Lys Ala Asn Gly Leu Pro Gly Lys Leu Met Pro
695 700 705
Ala Pro Leu Leu Ser Leu Pro Glu Asp Lys Ile Leu Glu Thr Phe
710 715 720
Arg Leu Ser Asn Lys Leu Lys Gly Ser Thr Asp Glu Met Ser Ala
725 730 735
Pro Gly Ala Glu Arg Gly Pro Pro Glu Asp Arg Asp Ala Glu Pro
740 745 750
Gln Pro Gly Ser Pro Ala Ala Glu Ser Leu Glu Glu Pro Asp Ala
755 760 765
Ala Ala Gly Leu Ser Ser Thr Lys Lys Ala Pro Pro Pro Arg Asp
770 775 780
Pro Gly Thr Pro A1a Thr Lys Glu Gly Ala Trp Glu A1a Met Ala
785 790 795
Val Ala Pro Glu Glu Pro Pro Pro Ser Ala Gly Glu Asp Ile Va1
800 805 810
Gly Asp Thr Ala Pro Pro Asp Leu Cys Asp Pro Gly Ser Leu Thr
815 820 825
Gly Asp Ala Ser Pro Leu Ser Gln Asp Ala Lys Gly Met Ile Ala
830 835 840
Glu Gly Pro Arg Asp Ser Ala Leu Ala Glu Ala Pro Glu Gly Leu
845 850 855
Ser Pro Ala Pro Pro Ala Arg Ser Glu G1u Pro Cys Glu Gln Pro
860 865 870
Leu Leu Val His Pro Ser Gly Asp His Ala Arg Asp Ala Gln Asp
875 880 885
Pro Ser Gln Ser Leu Gly Ala Pro Glu Ala Ala Glu Arg Pro Pro
890 895 900
Ala Pro Val Leu Asp Met Ala Pro Ala Gly His Pro Glu Gly Asp
905 910 915
Ala G1u Pro Ser Pro G1y Glu Arg Val Glu Asp Ala Ala Ala Pro
920 925 930
Lys A1a Pro Gly Pro Ser Pro Ala Lys Glu Lys Ile G1y Ser Leu
935 940 945
Arg Lys Val Asp Arg Gly His Tyr Arg Ser Arg Arg Glu Arg Ser
950 955 960
13/39
CA 02425829 2003-04-08
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Ser Ser Gly Glu Pro Ala Arg Glu Ser Arg Ser Lys Thr Glu Gly
965 970 975
His Arg His Arg Arg Arg Arg Thr Cys Pro Arg Glu Arg Asp Arg
980 985 990
Gln Asp Arg His Ala Pro Glu His His Pro Gly His Gly Asp Arg
995 1000 1005
Leu Ser Pro Gly Glu Arg Arg Ser Leu Gly Arg Cys Ser His His
1010 1015 1020
His Ser Arg His Arg Ser Gly Val Glu Leu Asp Trp Val Arg His
1025 x.030 1035
His Tyr Thr Glu Gly Glu Arg Gly Trp Gly Arg Glu Lys Phe Tyr
1040 1045 1050
Pro Asp Arg Pro Arg Trp Asp Arg Cys Arg Tyr Tyr His Asp Arg
1055 1060 1065
Tyr Ala Leu Tyr Ala Ala Arg Asp Trp Lys Pro Phe His Gly Gly
1070 1075 1080
Arg Glu His Glu Arg Ala Gly Leu His Glu Arg Pro His Lys Asp
1085 1090 1095
His Asn Arg Gly Arg Arg Gly Cys Glu Pro Ala Arg Glu Arg Glu
1100 1105 1110
Arg His Arg Pro Ser Ser Pro Arg Ala Gly Ala Pro His Ala Leu
1115 1120 1125
Ala Pro His Pro Asp Arg Phe Ser His Asp Arg Thr Ala Leu Val
1130 1135 1140
Ala Gly Asp Asn Cys Asn Leu Ser Asp Arg Phe His Glu His Glu
1145 1150 1155
Asn Gly Lys Ser Arg Lys Arg Arg His Asp Ser Val Glu Asn Ser
1160 1165 1170
Asp Ser His Val Glu Lys Lys A1a Arg Arg Ser Glu Gln Lys Asp
1175 1180 1185
Pro Leu Glu Glu Pro Lys A1a Lys Lys His Lys Lys Ser Lys Lys
1190 1195 1200
Lys Lys Lys Ser Lys Asp Lys His Arg Asp Arg Asp Ser Arg His
1205 1210 1215
Gln Gln Asp Ser Asp Leu Ser A1a Ala Cys Ser Asp Ala Asp Leu
1220 1225 1230
His Arg His Lys Lys Lys Glu G1u Glu Lys Glu Glu Thr Phe Lys
1235 1240 1245
Lys Ile Arg Gly Leu Cys
1250
<210> 8
<211> 1128
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7484378CD1
<400> 8
Met Glu Pro Thr Val Ala Asp Val His Leu Val Pro Arg Thr Thr
1 5 10 15
Lys Glu Val Pro Ala Leu Asp Ala Ala Cys Cys Arg Ala Ala Ser
20 25 30
Ile Gly Val Val Ala Thr Ser Leu Val Val Leu Thr Leu Gly Val
35 40 45
Leu Leu Gly Gly Met Asn Asn Ser Arg His Ala Ala Leu Arg Ala
50 5r5 60
Ala Thr Leu Pro Gly Lys Val Tyr Ser Val Thr Pro Glu Ala Ser
65 70 75
Lys Thr Thr Asn Pro Pro Glu Gly Arg Asn Ser Glu His Ile Arg
80 85 90
14/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
Thr Ser Ala Arg Thr Asn Ser Gly His Thr I1e Phe Lys Lys Cys
95 100 105
Asn Thr Gln Pro Phe Leu Ser Thr Gln G1y Phe His Val Asp His
110 115 120
Thr Ala Glu Leu Arg Gly I1e Arg Trp Thr Ser Ser Leu Arg Arg
125 130 135
Glu Thr Ser Asp Tyr His Arg Thr Leu Thr Pro Thr Leu G1u Ala
140 145 150
Leu Leu His Phe Leu Leu Arg Pro Leu Gln Thr Leu Ser Leu Gly
155 160 165
Leu Glu Glu Glu Leu Leu Gln Arg Gly Ile Arg Ala Arg Leu Arg
170 175 180
Glu His Gly Ile Ser Leu Ala Ala Tyr Gly Thr Ile Val Ser Ala
185 190 195
Glu Leu Thr Gly Arg His Lys Gly Pro Leu Ala Glu Arg Asp Phe
200 205 210
Lys Ser Gly Arg Cys Pro Gly Asn Ser Phe Ser Cys Gly Asn Ser
215 . 220 225
Gln Cys Val Thr Lys Val Asn Pro Glu Cys Asp Asp Gln Glu Asp
230 235 240
Cys Ser Asp Gly Ser Asp Glu Ala His Cys Glu Cys Gly Leu Gln
245 250 255
Pro Ala Trp Arg Met Ala Gly Arg I1e Val Gly Gly Met Glu Ala
260 265 ~ 270
Ser Pro Gly Glu Phe Pro Trp Gln A1a Ser Leu Arg Glu Asn Lys
275 280 285
Glu His Phe Cys Gly Ala Ala Ile Ile Asn Ala Arg Trp Leu Val
290 295 300
Ser Ala A1a His Cys Phe Asn Glu Phe Gln Asp Pro Thr Lys Trp
305 310 315
Val Ala Tyr Val Gly Ala Thr Tyr Leu Ser Gly Ser Glu Ala Ser
320 325 330
Thr Val Arg Ala Gln Val Val Gln Ile Val Lys His Pro Leu Tyr
335 340 345
Asn Ala Asp Thr Ala Asp Phe Asp Val Ala Val Leu Glu Leu Thr
350 355 360
Ser Pro Leu Pro Phe Gly Arg His I1e Gln Pro Val Cys Leu Pro
365 370 375
Ala Ala Thr His Ile Phe Pro Pro Ser Lys Lys Cys Leu Ile Ser
380 385 390
Gly Trp Gly Tyr Leu Lys Glu Asp Phe Arg Lys His Leu Pro Arg
395 400 405
Pro Ala Met Val Lys Pro Glu Val Leu Gln Lys Ala Thr Val Glu
410 415 420
Leu Leu Asp Gln Ala Leu Cys Ala Ser Leu Tyr G1y His Ser Leu
425 430 435
Thr Asp Arg Met Val Cys Ala Gly Tyr Leu Asp Gly Lys Val Asp
440 445 450
Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys G1u Glu Pro
455 460 465
Ser Gly Arg Phe Phe Leu Ala Gly Ile Val Ser Trp Gly Ile Gly
470 475 480
Cys Ala Glu Ala Arg Arg Pro Gly Val Tyr Ala Arg Val Thr Arg
485 490 495
Leu Arg Asp Trp Ile Leu Glu Ala Thr Thr Lys Ala Ser Met Pro
500 505 510
Leu Ala Pro Thr Met Ala Pro Ala Pro Ala Ala Pro Ser Thr Ala
515 520 525
Trp Pro Thr Ser Pro Glu Ser Pro Val Val Ser Thr Pro Thr Lys
530 535 540
Ser Met Gln Ala Leu Ser Thr Val Pro Leu Asp Trp Val Thr Val
545 550 555
Pro Lys Leu G1n Glu Cys Gly Ala Arg Pro Ala Met Glu Lys Pro
15/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
560 565 570
Thr Arg Val Val Gly Gly Phe Gly Ala Ala Ser Gly Glu Val Pro
575 580 585
Trp G1n Val Ser Leu Lys Glu Gly Ser Arg His Phe Cys Gly Ala
590 595 600
Thr Val Val Gly Asp Arg Trp Leu Leu Ser Ala A1a His Cys Phe
605 610 615
Asn His Thr Lys Val Glu Gln Val Arg Ala His Leu Gly Thr Ala
620 625 630
Ser Leu Leu Gly Leu Gly Gly Ser Pro Val Lys Ile Gly Leu Arg
635 640 645
Arg Val Val Leu His Pro Leu Tyr Asn Pro Gly Ile Leu Asp Phe
650 655 660
Asp Leu Ala Val Leu Glu Leu Ala Ser Pro Leu Ala Phe Asn Lys
665 670 675
Tyr Ile Gln Pro Val Cys Leu Pro Leu Ala Ile G1n Lys Phe Pro
680 685 690
Val G1y Arg Lys Cys Met Ile Ser Gly Trp Gly Asn Thr Gln Glu
695 700 705
Gly Asn Ala Thr Lys Pro Glu Leu Leu Gln Lys Ala Ser Val Gly
710 715 720
Tle Ile Asp Gln Lys Thr Cys Ser Val Leu Tyr Asn Phe Ser Leu
725 730 735
Thr Asp Arg Met Ile Cys Ala Gly Phe Leu Glu Gly Lys Val Asp
740 745 750
Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Ala Cys Glu Glu Ala
755 760 765
Pro Gly Val Phe Tyr Leu Ala Gly Ile Val Ser Trp Gly Ile Gly
770 775 780
Cys A1a Gln Val Lys Lys Pro Gly Val Tyr Thr Arg Ile Thr Arg
785 790 795
Leu Lys Gly Trp Ile Leu Glu Ile Met Ser Ser Gln Pro Leu Pro
800 805 810
Met Ser Pro Pro Ser Thr Thr Arg Met Leu Ala Thr Thr Ser Pro
815 820 825
Arg Thr Thr Ala Gly Leu Thr Val Pro Gly Ala Thr Pro Ser Arg
830 835 840
Pro Thr Pro Gly Ala A1a Ser Arg Va1 Thr Gly Gln Pro Ala Asn
845 850 855
Ser Thr Leu Ser Ala Val Ser Thr Thr Ala Arg Gly Gln Thr Pro
860 865 870
Phe Pro Asp A1a Pro Glu Ala Thr Thr His Thr Gln Leu Pro Asp
875 880 885
Cys Gly Leu Ala Pro Ala Ala Leu Thr Arg Ile Val Gly Gly Ser
890 895 900
Ala Ala Gly Arg Gly Glu Trp Pro Trp Gln Val Ser Leu Trp Leu
905 910 915
Arg Arg Arg Glu His Arg Cys Gly Ala Val Leu Val Ala Glu Arg
920 925 930
Trp Leu Leu Ser Ala Ala His Cys Phe Asp Va1 Tyr Gly Asp Pro
935 940 945
Lys Gln Trp Ala Ala Phe Leu Gly Thr Pro Phe Leu Ser Gly Ala
950 955 960
Glu Gly Gln Leu Glu Arg Val Ala Arg Ile Tyr Lys His Pro Phe
965 970 975
Tyr Asn Leu Tyr Thr Leu Asp Tyr Asp Val Ala Leu Leu Glu Leu
980 985 990
Ala Gly Pro Val Arg Arg Ser Arg Leu Val Arg Pro Ile Cys Leu
995 1000 1005
Pro Glu Pro Ala Pro Arg Pro Pro Asp Gly Thr Arg Cys Val Ile
1010 1015 1020
Thr Gly Trp Gly Ser Val Arg Glu G1y Gly Ser Met Ala Arg Gln
1025 1030 1035
16/39
CA 02425829 2003-04-08
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Leu Gln Lys Ala Ala Val Arg Leu Leu Ser Glu Gln Thr Cys Arg
1040 1045 1050
Arg Phe Tyr Pro Val Gln I1e Ser Ser Arg Met Leu Cys Ala Gly
1055 1060 1065
Phe Pro Gln Gly Gly Val Asp Ser Cys Ser Gly Asp Ala Gly Gly
1070 1075 1080
Pro Leu Ala Cys Arg Glu Pro Ser Gly Arg Trp Val Leu Thr Gly
1085 1090 1095
Val Thr Ser Trp Gly Tyr Gly Cys Gly Arg Pro His Phe Pro Gly
1100 1105 1110
Val Tyr Thr Arg Val Ala Ala Val Arg Gly Trp Ile Gly Gln His
1115 1120 1125
Ile Gln Glu
<210> 9
<211> 462
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473143CD1
<400> 9
Met Ile Pro Phe Thr Glu Leu Gly Gly Arg Gln Gln Lys Arg Arg
1 5 10 15
Glu Trp Val Gly Gly His Arg Glu His Pro Lys Gly Val Met Gly
20 25 30
Leu Ala His Arg Gly Met Ala Gly Leu Asp His Asp Val Val Ser
35 40 45
Asn Gln Cys Thr Ser Gly Lys Ser Pro Lys Ser Glu Arg Gly Ala
50 55 60
Glu Ala Leu Ala Arg Arg Leu Lys Gly Gly Arg Glu Arg Ala Gly
65 70 75
Ala Gly Lys Glu Tyr Gly Ile Val Gly Gly Ser Ser Gly His Cys
80 85 90
Cys Ser Lys Cys Gly Pro Thr Glu Gly Ile Ile Thr Ser Pro Gly
95 100 105
Ser Met Val Gly Arg Gln Ser Leu Gln Leu His Pro Gly Val Asp
110 115 120
Leu Asn Leu His Leu Arg Gln Ile Pro Gln Val Met Arg Val His
125 130 135
Ser Gln Asn Cys Thr Phe Gln Leu His Gly Pro Asn Gly Thr Val
140 145 150
G1u Ser Pro Gly Phe Pro Tyr Gly Tyr Pro Asn Tyr Ala Asn Cys
155 160 165
Thr Trp Thr Ile Thr Ala Glu Glu Gln His Arg Ile Gln Leu Val
170 175 180
Phe G1n Ser Phe Ala Leu Glu Glu Asp Phe Asp Val Leu Ser Val
185 190 195
Phe Asp G1y Pro Pro Gln Pro Glu Asn Leu Arg Thr Arg Leu Thr
200 205 210
Gly Phe G1n Leu Pro Ala Thr Ile Val Ser Ala Ala Thr Thr Leu
215 220 - 225
Ser Leu Arg Leu Ile Ser Asp Tyr Ala Val Ser Ala Gln Gly Phe
230 235 240
His Ala Thr Tyr Glu Val Leu Pro Ser His Thr Cys Gly Asn Pro
245 250 255
Gly Arg Leu Pro Asn Gly Ile Gln Gln Gly Ser Thr Phe Asn Leu
260 265 270
Gly Asp Lys Val Arg Tyr Ser Cys Asn Leu Gly Phe Phe Leu Glu
275 280 285
17/39
CA 02425829 2003-04-08
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Gly His Ala Val Leu Thr Cys His Ala Gly Ser Glu Asn Ser Ala
290 295 300
Thr Trp Asp Phe Pro Leu Pro Ser Cys Arg Ala Asp Asp Ala Cys
305 310 315
Gly Gly Thr Leu Arg Gly Gln Ser Gly Ile Ile Ser Ser Pro His
320 325 330
Phe Pro Ser Glu Tyr His Asn Asn Ala Asp Cys Thr Trp Thr I1e
335 340 345
Leu Ala Glu Leu Gly Asp Thr Ile Ala Leu Val Phe Ile Asp Phe
350 355 360
Gln Leu Glu Asp Gly Tyr Asp Phe Leu Glu Va1 Thr Gly Thr Glu
365 370 375
Gly Ser Ser Leu Trp Phe Thr Gly Ala Ser Leu Pro Ala Pro Val
380 385 390
Ile Ser Ser Lys Asn Trp Leu Arg Leu His Phe Thr Ser Asp Gly
395 400 405
Asn His Arg Gln Arg Gly Phe Ser Ala Gln Tyr Gln Val Lys Lys
410 415 420
Gln Ile Glu Leu Lys Ser Arg Gly Val Lys Leu Met Pro Ser Lys
425 430 435
Asp Asn Ser Gln Lys Thr Ser Val Cys Phe His Leu Thr Pro Arg
440 445 450
Ala Cys Leu Ser Leu Ser Ser Leu Leu Pro Cys Val
455 460
<210> 10
<211> 659
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4382838CD1
<400> 10
Met Leu Trp Ser Glu Arg Val Arg Pro Ser Tyr Ser Cys I1e Ala
1 5 10 15
Asn Asn Asn Val Gly Asn Pro Ala Lys Lys Ser Thr Asn Ile Ile
20 25 30
Val Arg Ala Leu Lys Lys Gly Arg Phe Trp Ile Thr Pro Asp Pro
35 40 45
Tyr His Lys Asp Asp Asn Ile Gln Ile Gly Arg Glu Val Lys Ile
50 55 60
Ser Cys Gln Val Glu Ala Val Pro Ser Glu Glu Val Thr Phe Ser
65 70 75
Trp Phe Lys Asn Gly Arg Pro Leu Arg Ser Ser Glu Arg Met Val
80 85 90
Ile Thr Gln Thr Asp Pro Asp Val Ser Pro Gly Thr Thr Asn Leu
95 100 105
Asp Ile Ile Asp Leu Lys Phe Thr Asp Phe Gly Thr Tyr Thr Cys
110 115 120
Val Ala Ser Leu Lys Gly Gly Gly Ile Ser Asp Ile Ser Ile Asp
125 130 135
Val Asn Ile Ser Ser Ser Thr Val Pro Pro Asn Leu Thr Val Pro
140 145 150
Gln Glu Lys Ser Pro Leu Val Thr Arg Glu Gly Asp Thr Ile Glu
155 160 165
Leu Gln Cys Gln Val Thr Gly Lys Pro Lys Pro Ile Ile Leu Trp
170 175 180
Ser Arg Ala Asp Lys Glu Val Ala Met Pro Asp Gly Ser Met Gln
185 190 195
Met Glu Ser Tyr Asp Gly Thr Leu Arg Ile Val Asn Val Ser Arg
200 205 210
18/39
CA 02425829 2003-04-08
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Glu Met Ser Gly Met Tyr Arg Cys Gln Thr Ser Gln Tyr Asn Gly
215 220 225
Phe Asn Val Lys Pro Arg Glu Ala Leu Val Gln Leu Ile Val Gln
230 235 240
Tyr Pro Pro Ala Val Glu Pro Ala Phe Leu Glu Ile Arg Gln Gly
245 250 255
Gln Asp Arg Ser Val Thr Met Ser Cys Arg Val Leu Arg Ala Tyr
260 265 270
Pro Ile Arg Val Leu Thr Tyr Glu Trp Arg Leu Gly Asn Lys Leu
275 280 285
Leu Arg Thr Gly Gln Phe Asp Ser Gln Glu Tyr Thr Glu Tyr Ala
290 295 300
Val Lys Ser Leu Ser Asn Glu Asn Tyr Gly Val Tyr Asn Cys Ser
305 310 315
Ile Ile Asn Glu Ala Gly Ala Gly Arg Cys Ser Phe Leu Val Thr
320 325 330
G1y Lys Ala Tyr Ala Pro Glu Phe Tyr Tyr Asp Thr Tyr Asn Pro
335 340 345
Val Trp Gln Asn Arg His Arg Val Tyr Ser Tyr Ser Leu Gln Trp
350 355 360
Thr G1n Met Asn Pro Asp Ala Val Asp Arg Ile Val Ala Tyr Arg
365 370 375
Leu Gly Ile Arg Gln Ala Gly G1n Gln Arg Trp Trp Glu Gln Glu
380 385 390
Ile Lys Ile Asn Gly Asn Ile G1n Lys Gly Glu Leu Ile Thr Tyr
395 400 405
Asn Leu Thr Glu Leu Ile Lys Pro Glu Ala Tyr Glu Val Arg Leu
410 415 420
Thr Pro Leu Thr Lys Phe Gly G1u Gly Asp Ser Thr Ile Arg Val
425 430 435
Ile Lys Tyr Ser Ala Pro Val Asn Pro His Leu Arg Glu Phe His
440 445 450
Arg Gly Phe Glu Asp Gly Asn Ile Cys Leu Phe Thr Gln Asp Asp
455 460 465
Thr Asp Asn Phe Asp Trp Thr Lys Gln Ser Thr Ala Thr Arg Asn
470 475 480
Thr Lys Tyr Thr Pro Asn Thr Gly Pro Asn Ala Asp Arg Ser Gly
485 490 495
Ser Lys Glu Gly Phe Tyr Met Tyr Ile Glu Thr Ser Arg Pro Arg
500 505 510
Leu Glu Gly Glu Lys Ala Arg Leu Pro Ser Pro Val Phe Ser I1e
515 520 525
Ala Pro Lys Asn Pro Tyr Gly Pro Thr Asn Thr Ala Tyr Cys Phe
530 535 540
Ser Phe Phe Tyr His Met Tyr Gly Gln His Ile Gly Val Leu Asn
545 550 555
Val Tyr Leu Arg Leu Lys Gly Gln Thr Thr Ile G1u Asn Pro Leu
560 565 570
Trp Ser Ser Ser Gly Asn Lys Gly Gln Arg Trp Asn Glu Ala His
575 580 585
Val Asn Ile Tyr Pro Ile Thr Ser Phe G1n Leu Ile Phe Glu Gly
590 595 600
Ile Arg Gly Pro Gly Ile Glu G1y Asp Ile Ala Ile Asp Asp Va1
605 610 615
Ser Ile Ala G1u Gly Glu Cys A1a Lys Gln Asp Leu Ala Thr Lys
620 625 630
Asn Ser Val Asp Gly Ala Val G1y Ile Leu Val His Ile Trp Leu
635 640 645
Phe Pro Ile Ile Val Leu Ile Ser Ile Leu Ser Pro Arg Arg
650 655
<210> 11
<211> 626
19/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6717888CD1
<400> 11
Met Gly Pro Ala Trp Val Gln Asp Pro Leu Thr Gly Ala Leu Trp
1 5 10 15
Leu Pro Val Leu Trp Ala Leu Leu Ser G1n Val~Tyr Cys Phe His
' 20 25 30
Asp Pro Pro Gly Trp Arg Phe Thr Ser Ser Glu Ile Val Ile Pro
35 40 45
Arg Lys Val Pro His Arg Arg Gly Gly Val Glu Met Pro Asp Gln
50 55 60
Leu Ser Tyr Ser Met His Phe Arg Gly Gln Arg His Val Ile His
65 70 75
Met Lys Leu Lys Lys Asn Met Met Pro Arg His Leu Pro Val Phe
80 85 90
Thr Asn Asn Asp Gln Gly Ala Met Gln Glu Asn Tyr Pro Phe Va1
95 100 105
Pro Arg Asp Cys Tyr Tyr Asp Cys Tyr Leu Glu Gly Val Pro Gly
110 115 120
Ser Val Ala Thr Leu Asp Thr Cys Arg Gly Gly Leu Arg Gly Met
125 130 135
Leu Gln Val Asp Asp Leu Thr Tyr Glu Ile Lys Pro Leu Glu Ala
140 145 150
Phe Ser Lys Phe Glu Tyr Val Val Ser Leu Leu Val Ser Glu Glu
155 160 165
Arg Pro Gly Glu Val Ser Arg Cys Lys Thr Glu Gly Glu Glu Ile
170 175 180
Asp Gln Glu Ser Glu Lys Val Lys Leu Ala Glu Thr Pro Arg Glu
185 190 195
Gly His Val Tyr Leu Trp Arg His His Arg Lys Asn Leu Lys Leu
200 205 210
His Tyr Thr Val Thr Asn Gly Leu Phe Met Gln Asn Pro Asn Met
215 220 225
Ser His Ile I1e Glu Asn Val Val Ile Ile Asn Ser Ile Ile His
230 235 240
Thr Ile Phe Lys Pro Val Tyr Leu Asn Val Tyr Val Arg Val Leu
245 250 255
Cys Ile Trp Asn Asp Met Asp Ile Val Met Tyr Asn Met Pro Ala
260 265 270
Asp Leu Va1 Val Gly Glu Phe Gly Ser Trp Lys Tyr Tyr Glu Trp
275 280 285
Phe Ser Gln Ile Pro His Asp Thr Ser Val Val Phe Thr Ser Asn
290 295 300
Arg Leu Gly Asn Thr Pro Arg Cys Gly Asp Lys Ile Lys Asn Gln
305 310 315
Arg G1u Glu Cys Asp Cys Gly Ser Leu Lys Asp Cys Ala Ser Asp
320 325 330
Arg Cys Cys Glu Thr Ser Cys Thr Leu Ser Leu Gly Ser Val Cys
335 340 345
Asn Thr Gly Leu Cys Cys His Lys Cys Lys Tyr Ala Ala Pro Gly
350 355 360
Val Val Cys Arg Asp Leu Gly Gly Ile Cys Asp Leu Pro Glu Tyr
365 370 375
Cys Asp G1y Lys Lys Glu Glu Cys Pro Asn Asp Ile Tyr Ile Gln
380 385' 390
Asp Gly Thr Pro Cys Ser Ala Val Ser Val Cys Ile Arg Gly Asn
395 400 405
Cys Ser Asp Arg Asp Met Gln Cys Gln Ala Leu Phe Gly Tyr Gln
20/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
410 415 420
Val Lys Asp Gly Ser Pro Ala Cys Tyr Arg Lys Leu Asn Arg Ile
425 430 435
Gly Asn Arg Phe Gly Asn Cys Gly Val Ile Leu Arg Arg Gly Gly
440 445 450
Ser Arg Pro Phe Pro Cys Glu Glu Asp Asp Val Phe Cys Gly Met
455 460 465
Leu His Cys Ser Arg Val Ser His I1e Pro Gly Gly Gly Glu His
470 475 480
Thr Thr ~Phe Cys Asn Ile Leu Val His Asp Ile Lys G1u Glu Lys
485 490 495
Cys Phe Gly Tyr Glu Ala His Gln Gly Thr Asp Leu Pro Glu Met
500 505 510
Gly Leu Val Val Asp Gly Ala Thr Cys Gly Pro Gly Ser Tyr Cys
515 520 525
Leu Lys Arg Asn Cys Thr Phe Tyr Gln Asp Leu His Phe Glu Cys
530 535 540
Asp Leu Lys Thr Cys Asn Tyr Lys Gly Val Cys Asn Asn Lys Lys
545 550 555
His Cys His Cys Leu His Glu Trp Gln Pro Pro Thr Cys Glu Leu
560 565 570
Arg Gly Lys Gly Gly Ser Ile Asp Ser Gly Pro Leu Pro Asp Lys
575 580 585
Gln Tyr Arg Ile Ala Gly Ser Ile Leu Val Asn Thr Asn Arg Ala
590 595 600
Leu Val Leu Ile Cys Ile Arg Tyr Ile Leu Phe Val Val Ser Leu
605 610 615
Leu Phe G1y Gly Phe Ser Gln Ala Ile Gln Cys
620 625
<210> 12
<211> 557
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472044CD1
<400> 12
Met Leu Leu Ala Val Leu Leu Leu Leu Pro Leu Pro Ser Ser Trp
1 5 10 15
Phe Ala His Gly His Pro Leu Tyr Thr Arg Leu Pro Pro Ser Ala
20 25 30
Leu Gln Val Phe Thr Leu Leu Leu Gly Ala Glu Thr Val Leu Gly
35 40 45
Arg Asn Leu Asp Tyr Val Cys Glu Gly Pro Cys Gly Glu Arg Arg
50 55 60
Pro Ser Thr Ala Asn Val Thr Arg Ala His Gly Arg I1e Val Gly
65 70 75
Gly Ser Ala Ala Pro Pro Gly Ala Trp Pro Trp Leu Val Arg Leu
80 85 90
Gln Leu G1y Gly Gln Pro Leu Cys Gly Gly Val Leu Val Ala Ala
95 100 105
Ser Trp Val Leu Thr Ala Ala His Cys Phe Val Gly Cys Arg Ser
110 115 120
Thr Arg Ser Ala Pro Asn Glu Leu Leu Trp Thr Val Thr Leu Ala
125 130 135
Glu Gly Ser Arg Gly Glu Gln Ala Glu Glu Val Pro Val Asn Arg
140 145 150
Ile Leu Pro His Pro Lys Phe Asp Pro Arg Thr Phe His Asn Asp
155 160 165
Leu Ala Leu Val Gln Leu Trp Thr Pro Val Ser Pro Gly Gly Ser
21/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
170 175 180
Ala Arg Pro Val Cys Leu Pro Gln Glu Pro Gln Glu Pro Pro Ala
185 190 195
Gly Thr Ala Cys Ala Ile Ala Gly Trp Gly Ala.Leu Phe Glu Asp
200 205 210
Gly Pro Glu Ala Glu Ala Val Arg Glu Ala Arg Val Pro Leu Leu
215 220 225
Ser Thr Asp Thr Cys Arg Arg Ala Leu Gly Pro Gly Leu Arg Pro
230 235 240
Ser Thr Met Leu Cys A1a Gly Tyr Leu Ala Gly G1y Val Asp Ser
245 250 255
Cys Gln G1y Asp Ser Gly Gly Pro Leu Thr Cys Ser Glu Pro Gly
260 265 270
Pro Arg Pro Arg Glu Val Leu Phe Gly Val Thr Ser Trp Gly Asp
275 280 285
Gly Cys Gly Glu Pro Gly Lys Pro Gly Val Tyr Thr Arg Val Ala
290 295 300
Val Phe Lys Asp Trp Leu Gln Glu Gln Met Ser Ala Ser Ser Ser
305 310 315
Ser Arg Glu Pro Ser Cys Arg Glu Leu Leu Ala Trp Asp Pro Pro
320 325 330
Gln Glu Leu Gln Ala Asp Ala Ala Arg Leu Cys Ala Phe Tyr Ala
335 340 345
Arg Leu Cys Pro Gly Ser Gln Gly Ala Cys Ala Arg Leu Ala His
350 355 360
Gln Gln Cys Leu Gln Arg Arg Arg Arg Cys Glu Leu Arg Ser Leu
365 370 375
Ala His Thr Leu Leu Gly Leu Leu Arg Asn Ala Gln Glu Leu Leu
380 385 390
Gly Pro Arg Pro Gly Leu Arg Arg Leu A1a Pro A1a Leu Ala Leu
395 400 405
Pro Ala Pro Ala Leu Arg Glu Ser Pro Leu His Pro Ala Arg Glu
410 415 420
Leu Arg Leu His Ser Gly Cys Pro Gly Leu Glu Pro Leu Arg Gln
425 430 435
Lys Leu Ala Ala Leu Gln Gly Ala His Ala Trp Ile Leu Gln Val
440 445 450
Pro Ser Glu His Leu Ala Met Asn Phe His Glu Val Leu Ala Asp
455 460 465
Leu Gly Ser Lys Thr Leu Thr Gly Leu Phe Arg Ala Trp Val Arg
470 475 480
Ala Gly Leu G1y Gly Arg His Val Ala Phe Ser Gly Leu Val Gly
485 490 495
Leu Glu Pro Ala Thr Leu Ala Arg Ser Leu Pro Arg Leu Leu Val
500 505 510
Gln Ala Leu Gln Ala Phe Arg Val Ala Ala Leu Ala Glu Gly Glu
515 520 525
Pro Glu Gly Pro Trp Met Asp Val Gly Gln Gly Pro Gly Leu G1u
530 535 540
Arg Lys Gly His His Pro Leu Asn Pro G1n Val Pro Pro Ala Arg
545 550 555
Gln Pro
<210> 13
<211> 494
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477384CD1
22/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
<400> 13
Met Gly Gly Pro Cys Arg Ala Pro Leu Gln Pro Gln Cys A1a Arg
1 5 10 15
Arg Arg Glu Ala Trp A1a Arg Arg His Arg Arg Arg Gly Ala Gly
20 25 30
Arg Arg Arg Arg Gly Gly A1a Pro A1a Ala Arg Ala Gly Arg Gly
35 40 45
Arg Gly Arg Gly Arg Gly Ala Leu Arg Gly Pro Gly Arg Pro Trp
50 55 60
Ala Pro Pro Pro Pro Ala Pro Arg Pro Ala A1a Gly Pro Ala Pro
65 70 75
Pro Pro Thr Arg Ser Leu Ser Pro Pro Leu Arg Pro Ala Val Pro
80 85 90
Pro Ser Arg Arg Arg Leu Phe Leu Gly Glu Ala Leu Phe Gln Arg
95 100 105
Ala Gly Ser Met Ala Ala Val Glu Thr Arg Val Cys Glu Thr Asp
110 115 120
Gly Cys Ser Ser Glu Ala Lys Leu Gln Cys Pro Thr Cys Ile Lys
125 130 135
Leu Gly Tle Gln Gly Ser Tyr Phe Cys Ser Gln Glu Cys Phe Lys
140 145 150
Gly Ser Trp Ala Thr His Lys Leu Leu His Lys Lys Ala Lys Asp
155 160 165
Glu Lys Ala Lys Arg Glu Val Ser Ser Trp Thr Val Glu Gly Asp
170 175 180
Ile Asn Thr Asp Pro Trp Ala Gly Tyr Arg Tyr Thr Gly Lys Leu
185 290 195
Arg Pro His Tyr Pro Leu Met Pro Thr Arg Pro Val Pro Ser Tyr
200 205 210
Ile Gln Arg Pro Asp Tyr Ala Asp His Pro Leu Gly Met Ser Glu
215 220 225
Ser Glu Gln Ala Leu Lys Gly Thr Ser Gln I1e Lys Leu Leu Ser
230 235 240
Ser Glu Asp Ile Glu Gly Met Arg Leu Val Cys Arg Leu Ala Arg
245 250 255
Glu Val Leu Asp Val Ala Ala G1y Met Ile Lys Pro Gly Val Thr
260 265 270
Thr G1u Glu Ile Asp His Ala Val His Leu Ala Cys Ile Ala Arg
275 280 285
Asn Cys Tyr Pro Ser Pro Leu Asn Tyr Tyr Asn Phe Pro Lys Ser
290 295 300
Cys Cys Thr Ser Val Asn Glu Val Ile Cys His Gly Ile Pro Asp
305 310 315
Arg Arg Pro Leu Gln Glu Gly Asp Ile Val Asn Val Asp Ile Thr
320 325 330
Leu Tyr Arg Asn Gly Tyr His Gly Asp Leu Asn Glu Thr Phe Phe
335 340 345
Val G1y Glu Val Asp Asp Gly Ala Arg Lys Leu Val Gln Thr Thr
350 355 360
Tyr Glu Cys Leu Met Gln Ala Ile Asp Ala Val Lys Pro Gly Val
365 370 375
Arg Tyr Arg Glu Leu Gly Asn Ile Ile Gln Lys His Ala Gln Ala
380 385 390
Asn Gly Phe Ser Val Val Arg Ser Tyr Cys Gly His Gly Ile His
395 400 405
Lys Leu Phe His Thr Ala Pro Asn Val Pro His Tyr Ala Lys Asn
410 415 420
Lys Ala Val Gly Val Met Lys Ser Gly His Va1 Phe Thr Ile Glu
425 430 435
Pro Met Ile Cys Glu Gly Gly Trp Gln Asp Glu Thr Trp Pro Asp
440 445 450
Gly Trp Thr Ala Val Thr Arg Asp Gly Lys Arg Ser Ala Gln Phe
455 460 465
23/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
Glu His Thr Leu Leu Val Thr Asp Thr Gly Cys Glu I1e Leu Thr
470 475 480
Arg Arg Leu Asp Ser Ala Arg Pro His Phe Met Ser Gln Phe
485 490
<210> 14
<211> 593
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7077175CD1
<400> 15
Met Asn Val Leu Lys Leu Asp Thr Leu Val Val A1a Gln Leu Trp
1 5 10 15
Arg Tyr Glu Asn Ala Lys Pro Thr Gly Glu Leu Gly Glu Pro Tyr
20 25 30
Glu Ala Gly Ile Asn Cys Ser Gly Ser Gly Ala Glu Glu Lys Glu
35 40 45
Asp Arg Arg Met Ala Ile Ile Trp Ala Val Pro Ser Thr Ser Val
50 55 60
Ser Trp Glu Gln Thr Ser Arg Lys Thr Gln Ile Arg Lys Lys Arg
65 70 75
Pro Ala Pro Arg Cys Lys Gln Leu Gly Thr Arg Gln Arg Val Leu
80 85 90
Pro Val Val Lys Pro Glu Val Leu Gln Lys Ala Thr Va1 G1u Leu
95 100 105
Leu Asp Gln Ala Leu Cys Ala Ser Leu Tyr Gly His Ser Leu Thr
110 115 120
Asp Arg Met Val Cys Ala Gly Tyr Leu Asp Gly Lys Val Asp Ser
125 130 135
Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Glu Glu Pro Ser
140 145 150
Gly Arg Phe Phe Leu Ala Gly Ile Val Ser Trp Gly Ile Gly Cys
155 160 165
Ala Glu Ala Arg Arg Pro Gly Val Tyr Ala Arg Val Thr Arg Leu
170 175 180
Arg Asp Trp Ile Leu Glu Ala Thr Thr Lys Ala Ser Met Pro Leu
185 190 195
Ala Pro Thr Met Ala Pro Ala Pro Ala Ala Pro Ser Thr Ala Trp
200 205 210
Pro Thr Ser Pro Glu Ser Pro Val Val Ser Thr Pro Thr Lys Ser
215 220 225
Met Gln Ala Leu Ser Thr Val Pro Leu Asp Trp Va1 Thr Val Pro
230 235 240
Lys Leu Gln Glu Cys Gly Ala Arg Pro Ala Met Glu Lys Pro Thr
245 250 255
Arg Val Val Gly Gly Phe G1y Ala A1a Ser Gly Glu Va1 Pro Trp
260 265 270
Gln Val Ser Leu Lys Glu Gly Ser Arg His Phe Cys Gly Ala Thr
275 280 285
Val Ala Gly Asp Arg Trp Leu Leu Ser Ala Ala His Cys Phe Asn
290 295 300
His Thr Lys Val Glu Gln Va1 Arg Ala His Leu Gly Thr Ala Ser
305 310 315
Leu Leu Gly Leu Gly Gly Ser Pro Val Lys Ile Gly Leu Arg Arg
320 325 330
Val Val Leu His Pro Leu Tyr Asn Pro Gly Ile Leu Asp Phe Asp
335 340 345
Leu Ala Val Leu Glu Leu Ala Ser Pro Leu Ala Phe Asn Lys Tyr
350 355 360
24/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
Ile Gln Pro Val Cys Leu Pro Leu A1a Ile Gln Lys Phe Pro Val
365 370 375
Gly Arg Lys Cys Met Ile Ser Gly Trp Gly Asn Thr Gln Glu Gly
380 385 390
Asn Ala Thr Lys Pro Glu Leu Leu G1n Lys Ala Ser Val Gly Ile
395 400 405
Ile Asp Gln Lys Thr Cys Ser Val Leu Tyr Asn Phe Ser Leu Thr
410 415 420
Asp Arg Met Ile Cys Ala G1y Phe Leu Glu Gly Lys Val Asp Ser
425 430 435
Cys Gln Gly Asp Ser Gly Gly Pro Leu Ala Cys Glu Glu Ala Pro
440 445 450
G1y Val Phe Tyr Leu Ala Gly Ile Val Ser Trp Gly Ile Gly Cys
455 460 465
Ala Gln Val Lys Lys Pro Gly Val Tyr Thr Arg Ile Thr Arg Leu
470 475 480
Lys Gly Trp Ile Leu Glu I1e Met Ser Ser Gln Pro Leu Pro Met
485 490 495
Ser Pro Pro Ser Thr Thr Arg Met Leu Ala Thr Thr Ser Pro Arg
500 505 510
Thr Thr Ala Gly Leu Thr Val Pro Gly Ala Thr Pro Ser Arg Pro
515 520 525
Thr Pro Gly Ala Ala Ser Arg Val Thr Gly Gln Pro Ala Asn Ser
530 535 540
Thr Leu Ser Ala Val Ser Thr Thr Ala Arg Gly Gln Thr Pro Phe
545 550 555
Pro Asp Ala Pro Glu Ala Thr Thr His Thr Gln Leu Pro Gly Thr
560 565 570
Gly Arg Asp Gly Gly Ile Pro Gly Ser Gly Gly Ser His Val Asn
575 580 585
Gln Pro Gly Leu Pro Asn Lys Thr
590
<210> 15
<211> 319
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7480124CD1
<400> 16
Met Gly Pro Leu Gly Pro Ser Ala Leu Gly Leu Leu Leu Leu Leu
1 5 10 15
Leu Val Val Ala Pro Pro Arg Val Ala Ala Leu Val His Arg Gln
20 25 30
Pro Glu Asn Gln Gly Ile Ser Leu Thr Gly Ser Val Ala Cys Gly
35 40 45
Arg Pro Ser Met Glu Gly Lys Ile Leu Gly Gly Val Pro Ala Pro
50 55 60
Glu Arg Lys Trp Pro Trp Gln Val Ser Val His Tyr Ala Gly Leu
65 70 75
His Val Cys Gly Gly Ser Ile Leu Asn Glu Tyr Trp Va1 Leu Ser
80 85 90
Ala Ala His Cys Phe His Arg Asp Lys Asn Ile Lys Ile Tyr Asp
95 100 105
Met Tyr Val Gly Leu Va1 Asn Leu Arg Val Ala Gly Asn His Thr
110 115 120
Gln Trp Tyr Gly Val Asn Arg Val Ile Leu His Pro Thr Tyr Gly
125 130 135
Met Tyr His Pro Ile Gly Gly Asp Val Ala Leu Val Gln Leu Lys
140 145 150
25/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
Thr Arg Ile Val Phe Ser Glu Ser Val Leu Pro Val Cys Leu Ala
155 160 165
Thr Pro G1u Val Asn Leu Thr Ser Ala Asn Cys Trp Ala Thr Gly
170 175 180
Trp Gly Leu Val Ser Lys Gln Gly G1u Thr Ser Asp Glu Leu Gln
185 190 195
Glu Val Gln Leu Pro Leu Ile Leu G1u Pro Trp Cys His Leu Leu
200 205 210
Tyr Gly His Met Ser Tyr Ile Met Pro Asp Met Leu Cys Ala Gly
215 220 225
Asp Ile Leu Asn Ala Lys Thr Val Cys Glu Gly Asp Ser Gly Gly
230 235 240
Pro Leu Val Cys G1u Phe Asn Arg Ser Trp Leu Gln Ile Gly Ile
245 250 255
Val Ser Trp Gly Arg Gly Cys Ser Asn Pro Leu Tyr Pro Gly Va1
260 265 270
Tyr Ala Ser Val Ser Tyr Phe Ser Lys Trp Ile Cys Asp Asn Ile
275 280 285
Glu Ile Thr Pro Thr Pro Ala Gln Pro Ala Pro Ala Leu Ser Pro
290 295 300
Ala Leu Gly Pro Thr Leu Ser Val Leu Met Ala Met Leu Ala Gly
305 310 315
Trp Ser Val Leu
<210> 16
<211> 2406
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6926819CB1
<400> 17
gttaagctga aaatgcacac agggctcctg taaatttctt ttcataaacc acccgcccag 60
ggcattaaat agggtactta gttgatccga accctccagg gagacctccg acccttctct 120
tCgtagCCCC CagCtCCCCt cccccggttc cactgaggca aggggactga gctgctccac 1.80
atgccaggag tcagcacgcc ggaaggcccc gcccagcggc tggcgcagcc aatcgcagag 240
cgggcaagtg gtgggggcgg gcctgcctgg gcggcaaggg ggcagcgggg tctaggggct 300
ttacaggtca attagctgct ttcgggcggc cttaggcgac aggagactcc tggacccagc 360
acctgcccac tgtgcctgtc cacctgtggc tacagcagCt gagaccccag tgggctaaag 420
attggacagg ggcccaccag ggacccagca agtccttcag ctctgtgagt gagggatttt 480
ccggagtgcc aggccgcagt attcccaggg ccgtggggtg ggacagggag gctcgacccc 540
ggcaaatcag gcagaggcgc cccttgctcc ctgcaacatc gcccacgtcc tggggccaca 600
gtgagcatga gcggagggcg ggagcaagag ccaggggacc tggcctgggt ccccagccca 660
aagcctggga agctgcctac ccacccctgt gtgggcgcgg acactgggga ctctggcttc 720
cggtggttcg gccacctgat tcagtttatg ctctgtgagg ggagctggag tgttggcagg 780
actggcccac ctgcaggact gcaggactgc gggaacggcg gtagatgggt gctctccttc 840
ecagtttgtc ctgggaagac attcaataac tgtttcatta caaggggcat ttggaaaaca 900
tacttcacct tctgttgtgt attagccaag aacaaggtgt gatgtgactt cccaattatt 960
ggggatccct ttgtcccttc ttgaaattag atgtcttcat tcttgaggtt ttgcctggat 1020
gacctcagca caattggtac aaaacctggg ccaatggttt cctagtttcc cggttgttgc 1080
cttaagcttc tcgcccatca ggtaccttcc tgtccttgtt catagcctgt catcatcatt 1140
ccagaaaact gtttcaactc ctacagctgt ggacaggctg cttttcattt tggtgggtcc 1200
ctccaatacc tCCaCttgCC CtgtttttCt ccagccacat ccttggcctc ttccacagtc 1260
cttaggtaaa tgcttggaag aataatttaa atatttttat tctaccatgg tggccctagt 1320
ttctcagggg gtagtaaaat ggctttttag gatcggtcta atcagatcct catttctttt 1380
cccttcctag atttttgaaa catgaatcct tcactcctcc tggctgcctt tttcctggga 1440
attgcctcag ctgctctaac acgtgaccac agtttagacg cacaatggac caagtggaag 1500
gcaaagcaca agagattata tggcatgaac aggaaccact ggattagagt cctctgggag 1560
aaggacgtga agatgattga gcagcacaat caggaataca gccaagggaa acacagcttc 1620
acaatggcca tgaacgcctt tggagacatg gtaagtgaag aattcaggca ggtgatgaat 1680
26/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
ggttttcaat accagaagca caggaagggg aaacagttcc aggaacgcct gcttcttgag 1740
atccccacat ctgtggactg gagagagaaa ggctacatga ctcctgtgaa ggatcagcag 1800
ggtcagtgtg gctcttgttg ggcttttagt gcaactggtg ctctggaagg gcagatgttc 1860
tggaaaacag gcaaacttat ctcactgaat gagcagaatc tggtagactg ctctgggcct 1920
caaggcaatg agggctgcaa tggtgact~tc atggataatc ccttccggta tgttcaggag 1980
aacggaggcc tggactctga ggcatcctat ccatatgaag gaaaggttaa aacctgtagg 2040
tacaatccca agtattctgc tgctaatgac actggttttg tggacatccc ttcacgggag 2100
aaggacctgg cgaaggcagt ggcaactgtg gggcccatct ctgttgctgt tggtgcaagc 2160
catgtcttct tccagttcta taaaaaagga atttattttg agccacgctg tgaccctgaa 2220
ggcctggatc atgctatgct ggtggttggc tacagctatg aaggagcaga ctcagataac 2280
aataaatatt ggctggtgaa gaacagctgg ggtaaaaact ggggcatgga tggctacata 2340
aagatggcca aagaccggag gaacaactgt ggaattgcca cagcagccag ctaccccact 2400
gtgtga 2406
<210> 17
<211> 1967
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473526CB1
<400> 18
cggacgcgtg ggcggacgcg tgggtgccca ggcgcttaaa gaagcaaaat ctcttgtgca 60
ggagcagcag agactcctca ggaagactca ctggactgta cccaccacct gccatgtctc.120
tgtggccacc tttccgatgc agatggaagc tggcgccaag gtactctagg agggcgtctc 180
cacagcaacc ccaacaggac tttgaggccc tgctggcaga gtgcctgagg aatggctgcc 240
tctttgaaga caccagcttc ccggccaccc tgagctccat cggcagtggc tccctgctgc 300
agaagctgcc accccgcctg cagtggaaga ggcccccgga gctgcacagc aatccccagt 360
tttattttgc caaggccaaa aggctggatc tgtgccaggg gatagtagga gactgctggt 420
tcttggctgc tttgcaagct ctggccttgc accaggacat cctgagccgg gttgttcccc 480
tgaatcagag tttcactgag aagtatgctg gcatcttccg gttctggttc tggcactatg 540
ggaactgggt tcctgtggtg atcgatgacc gtctgcctgt gaatgaggct ggccagctgg 600
tctttgtctc ctccacctat aagaacttgt tctggggagc acttctggaa aaggcctatg 660
ccaagctctc tggttcctat gaagacttgc agtcaggaca ggtgtctgaa gcccttgtag 720
acttcactgg aggggtgaca atgaccatca acctggcaga agcccatggc aacctctggg 780
acatcctcat cgaagccacc tacaacagaa ccctcattgg ctgccagacc cactcagggg 840
agaagattct ggagaatggg ctggtggaag gccatgccta tactctcaca ggaatcagga 900
aggtgacctg caaacataga cctgaatatc tcgtcaagct acggaacccc tggggaaagg 960
tggaatggaa aggagactgg agtgacagtt caagtaaatg ggagctgctg agccccaagg 1020
agaagattct gcttctgagg aaagacaatg acggagaatt ctggatgacg ctgcaggact 1080
ttaaaacaca tttcgtgctc ctggttatct gtaaactgac cccaggcctg ttgagccagg 1140
aggcggccca gaagtggacg tacaccatgc gggaggggag atgggagaag cggagcacag 1200
ctggtggcca gaggcagttg ctgcaggaca cattttggaa gaacccgcag ttcctgctgt 1260
ctgtctggag gcccgaggag ggcaggagat ccctgaggcc ctgcagcgtg ctggtgtccc 1320
tgctccagaa gcccaggcac aggtgccgca agcggaagcc tctcctcgcc attggcttct 1380
acctctatag gatgaacaag taccatgatg accagaggag actgccccct gagttcttcc 1440
agagaaacac tcctctgagc cagcctgata ggtttctcaa ggagaaagaa gtgagtcagg 1500
agctgtgtct ggaaccaggg acgtacctca tcgtgcctgc atattggagg cccaccagaa 1560
gtcagagttc gtcctcaggg tcttctccag gaagcacatc ttttatgaaa ttggcagcaa 1620
ttctggtgtc gtcttctcaa aggagataga agaccaaaat gaaaggcagg atgaattctt 1680
caccaaattc ttttgaaaag catccagaga ttaatgcagt tcaacttcag aacctcctga 1740
accagatgac ctggtcaagt ctggggagca gacagccctt tctttagcct ggaagcctgc 1800
aggggatcct ggccttactg accttaatgc atcaggtact atgagcatcc caggaatcag 1860
gcacctgttg gaaggagtga agtctctcag aaggtctcca caagcaacac cgtgggtcag 1920
gaactgaact ggagcaatgg acgtgcagaa ggagcagaac acgccag 1967
<210> 18
<211> 3446
<212> DNA
<213> Homo Sapiens
27/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
<220>
<221> misc_feature
<223> Incyte ID No: 7478443CB1
<400> 19
tgcctagagg ccgaggagct cacagctatg ggctggaggc cccggagagc tcgggggacc 60
ccgttgctgc tgctgctact actgctgctg ctctggccag tgccaggcgc cggggtgctt 120
caaggacata tccctgggca gccagtcacc ccgcactggg tcctggatgg acaaccctgg 180
cgcaccgtca gcctggagga gccggtctcg aagccagaca tggggctggt ggccctggag 240
gctgaaggcc aggagctcct gcttgagctg gagaagaacc acaggctgct ggccccagga 300
tacatagaaa cccactacgg cccagatggg cagccagtgg tgctggcccc caaccacacg 360
gatcattgcc actaccaagg gcgagtaagg ggcttccccg actcctgggt agtcctctgc 420
acctgctctg ggatgagtgg cctgatcacc ctcagcagga atgccagcta ttatctgcgt 480
ccctggccac cccggggctc caaggacttc tcaacccacg agatctttcg gatggagcag 540
ctgctcacct ggaaaggaac ctgtggccac agggatcctg ggaacaaagc gggcatgacc 600
agccttcctg gtggtcccca gagcaggggc aggcgagaag cgcgcaggac ccggaagtac 660
ctggaactgt acattgtggc agaccacacc ctgttcttga ctcggcaccg aaacttgaac 720
cacaccaaac agcgtctcct ggaagtcgcc aactacgtgg accagcttct caggactctg 780
gacattcagg tggcgctgac cggcctggag gtgtggaccg agcgggaccg cagccgcgtc 840
acgcaggacg ccaacgccac gctctgggcc ttcctgcagt ggcgccgggg gctgtgggcg 900
cagcggcccc acgactccgc gcagctgctc acgggccgcg ccttccaggg cgccacagtg 960
ggcctggcgc ccgtcgaggg catgtgccgc gccgagagct cgggaggcgt gagcacggac 1020
cactcggagc tccccatcgg cgccgcagcc accatggccc atgagatcgg ccacagcctc 1080
ggcctcagcc acgaccccga cggctgctgc gtggaggctg cggccgagtc cggaggctgc 1140
gtcatggctg cggccaccgg gcacccgttt ccgcgcgtgt tcagcgcctg cagccgccgc 1200
cagctgcgcg ccttcttccg caaggggggc ggcgcttgcc tctccaatgc cccggacccc 1260
ggactcccgg tgccgccggc gctctgcggg aacggcttcg tggaagcggg cgaggagtgt 1320
gactgcggcc ctggccagga gtgccgcgac ctctgctgct ttgctcacaa ctgctcgctg 1380
cgcccggggg cccagtgcgc ccacggggac tgctgcgtgc gctgcctgct gaagccggct 1440
ggagcgctgt gccgccaggc catgggtgac tgtgacctcc ctgagttttg cacgggcacc 1500
tcctcccact gtcccccaga cgtttaccta ctggacggct caccctgtgc caggggcagt 1560
ggctactgct gggatggcgc atgtcccacg ctggagcagc agtgccagca gctctggggg 1620
cctggctccc acccagctcc cgaggcctgt ttccaggtgg tgaactctgc gggagatgct 1680
catggaaact gcggccagga cagcgagggc cacttcctgc cctgtgcagg gagggatgcc 1740
ctgtgtggga agctgcagtg ccagggtgga aagcccagcc tgctcgcacc gcacatggtg 1800
ccagtggact ctaccgttca cctagatggc caggaagtga cttgtcgggg agccttggca 1860
ctccccagtg cccagctgga cctgcttggc ctgggcctgg tagagccagg cacccagtgt 1920
ggacctagaa tggtgtgcca gagcaggcgc tgcaggaaga atgccttcca ggagcttcag 1980
cgctgcctga ctgcctgcca cagccacggg gtttgcaata gcaaccataa ctgccactgt 2040
gctccaggct gggctccacc cttctgtgac aagccaggct ttggtggcag catggacagt 2100
ggccctgtgc aggctgaaaa ccatgacacc ttcctgctgg ccatgctcct cagcgtcctg 2160
ctgcctctgc tcccaggggc cggcctggcc tggtgttgct accgactccc aggagcccat 2220
ctgcagcgat gcagctgggg ctgcagaagg gaccctgcgt gcagtggccc caaagatggc 2280
ccacacaggg accaccccct gggcggcgtt caccccatgg agttgggccc cacagccact 2340
ggacagccct ggcccctgga ccctgagaac tctcatgagc ccagcagcca ccctgagaag 2400
cctctgccag cagtctcgcc tgacccccaa gatcaagtcc agatgccaag atcctgcctc 2460
tggtgagagg tagctcctaa aatgaacaga tttaaagaca ggtggccact gacagccact 2520
ccaggaactt gaactgcagg ggcagagcca gtgaatcacc ggacctccag cacctgcagg 2580
cagcttggaa gtttcttccc cgagtggagc ttcgacccac ccactccagg aacccagagc 2640
cacattagaa gttcctgagg gctggagaac actgctgggc acactctcca gctcaataaa 2700
ccatcagtcc cagaagcaaa ggtcacacag cccctgacct ccctcaccag tggaggctgg 2760
gtagtgctgg ccatcccaaa agggctctgt cctgggagtc tggtgtgtct cctacatgca 2820
atttccacgg acccagctct gtggagggca tgactgctgg ccagaagcta gtggtcctgg 2880
ggccctatgg ttcgactgag tccacactcc cctggagcct ggctggcctc tgcaaacaaa 2940
cataattttg gggaccttcc~ttcctgtttc ttcccaccct gtcttctccc ctaggtggtt 3000
cctgagcccc cacccccaat cccagtgcta cacctgaggt tctggagctc agaatctgac 3060
agcctctccc ccattctgtg tgtgtcgggg ggacagaggg aaccatttaa gaaaagatac 3120
caaagtagaa gtcaaaagaa agacatgttg gctataggcg tggtggctca tgcctataat 3180
cccagcactt tgggaagccg gggtaggagg atcaccagag gccagcaggt ccacaccagc 3240
ctgggcaaca cagcaagaca ccgcatctac agaaaaattt taaaattagc tgggcgtggt 3300
ggtgtgtacc tgtaggccta gctgctcagg aggctgaagc aggaggatca cttgagcctg 3360
agttcaacac tgcagtgagc tatggtggca ccactgcact ccagcctggg tgacagagca 3420
agaccctgtc tctaaaataa atttta 3446
28/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
<210> 19
<211> 4888
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3533147CB1
<400> 20
atgacaggaa caggaggcag gaagcccact ggggacaaac aggaagtcca cccctgggaa 60
aaacaggaag tgagggaaca gacagaaagt ccacaggagc tgacaagaag tccacagggg 120
acagacagga atgatacagt gaccatctat actgacaccc aaagccgaaa ggctggcgct 180
tctcgtaaaa tcagaaacat gctcaacatt taccttgttt ggttagttaa gataaaccag 240
ataataatca atgtctttta tcaaaatcca gaaccaacta tctggaattc tgcatttatt 300
gtggacataa cagcaatagt tccaacagca ttatttccat ttaatgtggc caagccaaaa 360
atgctcgtgg agaatttaca ggaaggtgac ttcagggagc ttcgtggtaa cagccaccac 420
tgcctgacca aaaagggtct aggaaatgct cctccaggcc tgcagttcac actgtacaaa 480
tgtctggact catccaggac agcccagccc catgcagggc ttcactacgt ggacattaat 540
tcaggcatga tacgaacaga agaggcagat tacttcctaa ggccacttcc ttcacacctc 600
tcatggaaac tcggcagagc tgcccaaggc agctcgccat cccacgtact gtacaagaga 660
tccacagagc cccatgctcc tggggccagt gaggtcctgg tgacctcaag gacatgggag 720
ctggcacatc aacccctgca cagcagcgac cttcgcctgg gactgccaca aaagcagcat 780
ttctgtggaa gacgcaagaa atacatgccc cagcctccca aggaagacct cttcatcttg 840
ccagatgagt ataagtcttg cttacggcat aagcgctctc ttctgaggtc ccatagaaat 900
gaagaactga acgtggagac cttggtggtg gtcgacaaaa agatgatgca aaaccatggc 960
catgaaaata tcaccaccta cgtgctcacg atactcaaca tggtatctgc tttattcaaa 1020
gatggaacaa taggaggaaa catcaacatt gcaattgtag gtctgattct tctagaagat 1080
gaacagccag gactggtgat aagtcaccac gcagaccaca ccttaagtag cttctgccag 1140
tggcagtctg gattgatggg gaaagatggg actcgtcatg accacgccat cttactgact 1200
ggtctggata tatgttcctg gaagaatgag ccctgtgaca ctttgggatt tgcacccata 1260
agtggaatgt gtagtaaata tcgcagctgc acgattaatg aagatacagg tcttggactg 1320
gccttcacca ttgcccatga gtctggacac aactttggca tgattcatga tggagaaggg 1380
aacatgtgta aaaagtccga gggcaacatc atgtccccta cattggcagg acgcaatgga 1440
gtcttctcct ggtcaccctg cagccgccag tatctacaca aatttctaag caccgctcaa 1500
gctatctgcc ttgctgatca gccaaagcct gtgaaggaat acaagtatcc tgagaaattg 1560
ccaggagaat tatatgatgc aaacacacag tgcaagtggc agttcggaga gaaagccaag 1620
ctctgcatgc tggactttaa aaaggacatc tgtaaagccc tgtggtgcca tcgtattgga 1680
aggaaatgtg agactaaatt tatgccagca gcagaaggca caatttgtgg gcatgacatg 1740
tggtgccggg gaggacagtg tgtgaaatat ggtgatgaag gccccaagcc cacccatggc 1800
cactggtcgg actggtcttc ttggtcccca tgctccagga cctgcggagg gggagtatct 1860
cataggagtc gcctctgcac caaccccaag ccatcgcatg gagggaagtt ctgtgagggc 1920
tccactcgca ctctgaagct ctgcaacagt cagaaatgtc cccgggacag tgttgacttc 1980
cgtgctgctc agtgtgccga gcacaacagc agacgattca gagggcggca ctacaagtgg 2040
aagccttaca ctcaagtaga agatcaggac ttatgcaaac tctactgtat cgcagaagga 2100
tttgatttct tcttttcttt gtcaaataaa gtcaaagatg ggactccatg ctcggaggat 2160
agccgtaatg tttgtataga tgggatatgt gagagagttg gatgtgacaa tgtccttgga 2220
tctgatgctg ttgaagacgt ctgtggggtg tgtaacggga ataactcagc ctgcacgatt 2280
cacaggggtc tctacaccaa gcaccaccac accaaccagt attatcacat ggtcaccatt 2340
ccttctggag cccggagtat ccgcatctat gaaatgaacg tctctacctc ctacatttct 2400
gtgcgcaatg ccctcagaag gtactacctg aatgggcact ggaccgtgga ctggcccggc 2460
cggtacaaat tttcgggcac tactttcgac tacagacggt cctataatga gcccgagaac 2520
ttaatcgcta ctggaccaac caacgagaca ctgattgtgg agctgctgtt tcagggaagg 2580
aacccgggtg ttgcctggga atactccatg cctcgcttgg ggaccgagaa gcagccccct 2640
gcccagccca gctacacttg ggccatcgtg cgctctgagt gctccgtgtc ctgcggaggg 2700
ggacagatga ccgtgagaga gggctgctac agagacctga agtttcaagt aaatatgtcc 2760
ttctgcaatc ccaagacacg acctgtcacg gggctggtgc cttgcaaagt atctgcctgt 2820
cctcccagct ggtccgtggg gaactggagt gcctgcagtc ggacgtgtgg cgggggtgcc 2880
cagagccgcc ccgtgcagtg cacacggcgg gtgcactatg actcggagcc agtcccggca 2940
ggcctgtgcc ctcagctggt ccctccagca ggcaggcctg caactctcag agctgcccac 3000
ctgcatggag cgccgggccc tgggcagagt gctcacacac ctgtgggaag ggtggaggaa 3060
cgggcagtgg cctgtaagag caccaacccc tcggccagag cgcagctgct gcccgacgct 3120
gtctgcacct ccgagcccaa gcccaggatg catgaagcct gtctgcttca gcgctgccac 3180
29/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
aagcccaaga agctgcagtg gctggtgtcc gcctggtccc agtgctctgt gacatgtgaa 3240
agaggaacac agaaaagatt cttaaaatgt gctgaaaagt atgtttctgg aaagtatcga 3300
gagctggcct caaagaagtg ctcacatttg ccgaagccca gcctggagct ggaacgtgcc 3360
tgcgccccgc ttccatgccc caggcacccc ccatttgctg ctgcgggacc ctcgaggggc 3420
agctggtttg cctcaccctg gtctcagtgc acggccagct gtgggggagg cgttcagacg 3480
aggtccgtgc agtgcctggc tgggggccgg ccggcctcag gctgcctcct gcaccagaag 3540
ccttcggcct ccctggcctg caacactcac ttctgcccca ttgcagagaa gaaagatgcc 3600
ttctgcaaag actacttcca ctggtgctac ctggtacccc agcacgggat gtgcagccac 3660
aagttctacg gcaagcagtg ctgcaagact tgctctaagt ccaacttgtg agttgggacc 3720
gctctccgta gcagagaaag tgcctgcgtg gcacagaaat ttcccacaaa tgagctgtgc 3780
aatctacgtc ggaatacatc caaggaagag caaagccaaa agaagaaaac cgtgttaggc 3840
tctttgacca ggagtgtatg tatgtggttc actgtgagcc tgggtgcaga cctgtgtccc 3900
catgcacaca gtgtctcctg tcaggctgaa atgtggcacc ctggcagaca gagctgtggc 3960
tcgtgaggca gaaggcaggc accacaacgg gagaggcagc actcacccct gcctgttgca 4020
gctaaatcaa gtcaaaaaga caggcgaggc tgaacttgct aaatgtctgg tgccttagaa 4080
aaagaaggaa aggccatgaa ataaggaaaa catacaaaat atgtaccccc tagttcacca 4140
gcctcccctc ccactaggag ggcccctcga gccatcagga gtgaccaact tcctgggtgg 4200
aggtcagggg agctccagga ggctgcccag gctcctcctc ctcctcccca gcggccgagc 4260
atctcttacc aggaacctgg agccaccgcc ggagccagcg tcatctctag ggtcactggc 4320
caggggactg cattctggtt tgggactttg cctatggaaa tgggaaaaat gaaattcctg 4380
ctaaggtgct tctatctctt tcagattcat gcattgaagg agagattttt tatactttat 4440
gttttatctt tctcagttat ttgcaagtga gtgtcctttt aaaaacacac ttcttcatgc 4500
ttttctttgt aaatgacaga tcgaagtata ggttacatca aaaccctacc atcctgagaa 4560
gagttatggt tctattatag cagacgtcag ccacacagcc tatgtgacaa taaccttaga 4620
gtcctgtgtt ttgtttttgt gtgttgtgag attttaatct tttttttttt cggtgagtct 4680
ggccatttct ataatgccag gtgggaagcc aggctgcggg tgttagggtg ggaatctgcc 4740
cggcgtctct ggcaccctcc ctgccatcct cagtgcggct gctgttctcc tgtccggtgc 4800
tgtggctcca ttccaaaggg gcacctggat atttatattt gctgaagttt tataataaag 4860
tttatatggt acagtgaaaa aaaaaaaa 4888
<210> 20
<211> 1074
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7483438CB1
<400> 21
gccgtgcaca acaccaagcg catggactct gaccccagtg cagtgtctgt agggaagagg 60
agccatgggg cttcgggcag gccccatcct gcttctgctg ctgtggctgc tgccaggggc 120
ccattgggat gtgctgcctt cagaatgcgg ccactccaag gaggccggga ggattgtggg 180
aggccaagac acccaggaag gacgctggcc gtggcaggtt ggcctgtggt tgacctcagt 240
ggggcatgta tgtgggggct ccctcatcca cccacgctgg gtgctcacag ccgcccactg 300
cttcctgagg tctgaggatc ccgggctcta ccatgttaaa gtcggagggc tgacaccctc 360
actttcagag ccccactcgg ccttggtggc tgtgaggagg ctcctggtcc actcctcata 420
ccatgggacc accaccagcg gggacattgc cctgatggag ctggactccc ccttgcaggc 480
ctcccagttc agccccatct gcctcccagg accccagacc cccctcgcca ttgggaccgt 540
gtgctgggta aacgggctgg gggaggtggc tgtgcccctc ctggactcga acatgtgtga 600
gctgatgtac cacctaggag agcccagcct ggctggccag cgcctcatcc aggacgacat 660
gctctgtgct ggctctgtcc agggcaagaa agactcctgc cagggtgact ccggggggcc 720
gctggtctgc cccatcaatg atacgtggat ccaggccggc attgtgagct ggggattcgg 780
ctgtgcccgg cctttccggc ctggtgtcta cacccaggtg ctaagctaca cagactggat 840
tcagagaacc ctggctgaat ctcactcagg catgtctggg gcccgcccag gtgccccagg 900
atcccactca ggcacctcca gatcccaccc agtgctgctg cttgagctgt tgaccgtatg 960
cttgcttggg tccctgtgaa ccatgagcca tggagtccgg gatccccttt ctggtaggat 1020
tgatggaatc taataataaa aactgtaggt tttttatgtg taaaaaaaaa aaaa 1074
<210> 21
<211> 3573
<212> DNA
<213> Homo Sapiens
30/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
<220>
<221> misc_feature
<223> Incyte ID No: 7246467CB1
<400> 22
caagaattcg gcacaggggt tctgtatccc tacttccatt accctcggct cctcccactc 60
ctcgggggct ccgtgctttc cgcgggtctg tccgggggct ccggaccctc ggcgcacgtg 120
agttgatggc ttccggagaa ctggcatagc tgcagaatat gagtagtgtc ccaagaagag 180
tgctttgcct ttgcgacaag gatcagaata aagttatttg ccatacagta accagagacc 240
tccaactagg ggcccccaaa ctgtatcctg cctgtagaac ctgccaggta aaggtatatt 300
tttgcttttt aatttagcca gaagcaattt ttaaagaaaa tatgtctcct ctgaagatac 360
atggtcctat cagaattcga agtatgcaga ctgggattac aaagtggaaa gaaggatcct 420
ttgaaattgt agaaaaagag aataaagtca gcctagtagt tcactacaat actggaggaa 480
ttccaaggat atttcagcta agtcataaca ttaaaaatgt ggtgcttcga cccagtggag 540
cgaaacaaag ccgcctaatg ttaactctgc aagataacag cttcttgtct attgacaaag 600
taccaagtaa ggatgcagag gaaatgaggt tgtttctaga tgcagtccat caaaacagac 660
ttcctgcagc catgaaaccg tctcaggggt ctggtagttt tggagccatt ctgggcagca 720
ggacctcaca gaaggaaacc agcaggcagc tttcttactc agacaatcag gcttctgcaa 780
aaagaggaag tttggaaact aaagatgata ttccatttcg aaaagttctt ggtaatccgg 840
gtagaggatc gattaagact gtagcaggaa gtggaatagc tcggacgatt ccttctttga 900
catctacttc aacacctctt agatcagggt tgctagaaaa tcgtactgaa aagaggaaaa 960
gaatgatatc aactggctca gaattgaatg aagattaccc taaggaaaat gattcatcat 1020
cgaacaacaa ggccatgaca gatccctcca gaaagtattt aaccagcagt agagaaaagc 1080
agctgagttt gaaacagtca gaagagaata ggacatcagg tgggctttta cctttacagt 1140
catcatcctt ttatggtagc agagctggat ccaaggaaca ctcttctggt ggcactaact 1200
tagacaggac taatgtttca agccagactc cctctgccaa aagaagtttg ggatttcttc 1260
ctcagccagt tcctctttct gttaaaaaac tgaggtgtaa ccaggattac actggctgga 1320
ataaaccaag agtgcccctt tcctctcacc aacagcagca actgcagggc ttctccaatt 1380
tgggaaatac ctgctatatg aatgctattc tacaatctct attttcactc cagtcatttg 1440
caaatgactt gcttaaacaa ggtatcccat ggaagaaaat tccactcaat gcacttatca 1500
gacgctttgc acacttgctt gttaaaaaag atatctgtaa ttcagagacc aaaaaggatt 1560
tactcaagaa ggttaaaaat gccatttcag ctacagcaga gagattctct ggttatatgc 1620
agaatgatgc tcatgaattt ttaagtcagt gtttggacca gctgaaagaa gatatggaaa 1680
aattaaataa aacttggaag actgaacctg tttctggaga agaaaattca ccagatattt 1740
cagctaccag agcatacact tgccctgtta ttactaattt ggagtttgag gttcagcact 1800
ccatcatttg taaagcatgt ggagagatta tccccaaaag agaacagttt aatgacctct 1860
ctattgacct tcctcgtagg aaaaaaccac tccctcctcg ttcaattcaa gattctcttg 1920
atcttttctt tagggccgaa gaactggagt attcttgtga gaagtgtggt gggaagtgtg 1980
ctcttgtcag gcacaaattt aacaggcttc ctagggtcct cattctccat ttgaaacgat 2040
atagcttcaa tgtggctctc tcgcttaaca ataagattgg gcagcaagtc atcattccaa 2100
gatacctgac cctgtcatct cattgcactg aaaatacaaa accacctttt acccttggtt 2160
ggagtgcaca tatggcaatg tctagaccat tgaaagcctc tcaaatggtg aattcctgca 2220
tcaccagccc ttctacacct tcaaagaaat tcaccttcaa atccaagagc tccttggctt 2280
tatgccttga ttcagacagt gaggatgagc taaaacgttc tgtggccctc agccagagac 2340
tttgtgaaat gttaggcaac gaacagcagc aggaagacct ggaaaaagat tcaaaattat 2400
gcccaataga gcctgacaag tctgaattgg aaaactcagg atttgacaga atgagcgaag 2460
aagagcttct agcagctgtc ttggagataa gtaagagaga tgcttcacca tctctgagtc 2520
atgaagatga tgataagcca actagcagcc cagataccgg atttgcagaa gatgatattc 2580
aagaaatgcc agaaaatcca gacactatgg aaactgagaa gcccaaaaca atcacagagc 2640
tggatcctgc cagttttact gagataacta aagactgtga tgagaataaa gaaaacaaaa 2700
ctccagaagg atctcaggga gaagttgatt ggctccagca gtatgatatg gagcgtgaaa 2760
gggaagagca agagcttcag caggcactgg ctcagagcct tcaagagcaa gaggcttggg 2820
aacagaaaga agatgatgac ctcaaaagag ctaccgagtt aagtcttcaa gagtttaaca 2880
actcctttgt ggatgcattg ggttctgatg aggactctgg aaatgaggat gtttttgata 2940
tggagtacac agaagctgaa gctgaggaac tgaaaagaaa tgctgagaca ggaaatctgc 3000
ctcattcgta ccggctcatc agtgttgtca gtcacattgg tagcacttct tcttcaggtc 3060
attacattag tgatgtatat gacattaaga agcaagcgtg gtttacttac aatgacctgg 3120
aggtatcaaa aatccaagag gctgccgtgc agagtgatcg agatcggagt ggctacatct 3180
tcttttatat gcacaaggag atctttgatg agctgctgga aacagaaaag aactctcagt 3240
cacttagcac ggaagtgggg aagactaccc gtcaggcctc gtgaggaaca aactcctggg 3300
ttggcagcat gcactgcata tttgttactg ctgcccacct cacctttcct ctgctgaagg 33&0
agaatttgga attctacttg atgcgggagc aacaaacagc tcagggccaa accaaaagac 3420
aaaaattgga gtaacgtaga atgctccatg ctattttatg gaaactttgg tctcacatcc 3480
31/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
gtagctgatt~atcctctttt tctcctatga gtggcacttc ttttgtctta ggaatacctg 3540
ttgtacatct gtctccgtgt tgtgtttttt ccc 3573
<210> 22
<211> 4659
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7997881CB1
<400> 23
ggcggcgggc gcggcgctga cccggaggcg gcggcggcgg tgcccggatg gaggcacgtc 60
attgtacccc cgccgggggg ctgggctgtg tgcggcggcg gcggcggcgg ccgaggggga 120
tggagcgagc gccgagccgg gtcagagttg aacaatgacc atagttgaca aagcttctga 180
atcttcagac ccatcagcct atcagaatca gcctggcagc tccgaggcag tctcacctgg 240
agacatggat gcaggttctg ccagctgggg tgctgtgtct tcattgaatg atgtgtcaaa 300
tcacacactt tctttaggac cagtacctgg tgctgtagtt tattcgagtt catctgtacc 360
tgataaatca aaaccatcac cacaaaagga tcaagcccta ggtgatggca tcgctcctcc 420
acagaaagtt cttttcccat ctgagaagat ttgtcttaag tggcaacaaa ctcatagagt 480
tggagctggg ctccagaatt tgggcaatac ctgttttgcc aatgcagcac tgcagtgttt 540
aacctacaca ccacctcttg ccaattacat gctatcacat gaacactcca aaacatgtca 600
tgcagaaggc ttttgtatga tgtgtacaat gcaagcacat attacccagg cactcagtaa 660
tcctggggac gttattaaac caatgtttgt catcaatgag atgcggcgta tagctaggca 720
cttccgtttt ggaaaccaag aagatgccca tgaattcctt caatacactg ttgatgctat 780
gcagaaagca tgcttgaatg gcagcaataa attagacaga cacacccagg CCaccactCt 84O
tgtttgtcag atatttggag gatacctaag atctagagtc aaatgtttaa attgcaaggg 900
cgtttcagat acttttgatc catatcttga tataacattg gagataaagg ctgctcagag 960
tgtcaacaag gcattggagc agtttgtgaa gccggaacag cttgatggag aaaactcgta 1020
caagtgcagc aagtgtaaaa agatggttcc agcttcaaag aggttcacta tccatagatc 1080
ctctaatgtt cttacacttt ctctgaaacg ttttgcaaat tttaccggtg gaaaaattgc 1140
taaggatgtg aaataccctg agtatcttga tattcggcca tatatgtctc aacccaacgg 1200
agagccaatt gtctacgtct tgtatgcagt gctggtccac actggtttta attgccatgc 1260
tggccattac ttctgctaca taaaagctag caatggcctc tggtatcaaa tgaatgactc 1320
cattgtatct accagtgata ttagatcggt actcagccaa caagcctatg tgctctttta 1380
tatcaggtcc catgatgtga aaaatggagg tgaacttact catcccaccc atagccccgg 1440
ccagtcctct ccccgccccg tcatcagtca gcgggttgtc accaacaaac aggctgcgcc 1500
aggctttatc ggaccacagc ttccctctca catgataaag aatccacctc acttaaatgg 1560
gactggacca ttgaaagaca cgccaagcag ttccatgtcg agtcctaacg ggaattccag 1620
tgtcaacagg gctagtcctg ttaatgcttc agcttctgtc caaaactggt cagttaatag 1680
gtcctcagtg atcccagaac atcctaagaa acaaaaaatt acaatcagta ttcacaacaa 1740
gttgcctgtt cgccagtgtc agtctcaacc taaccttcat agtaattctt tggagaaccc 1800
taccaagccc gttccctctt ctaccattac caattctgca gtacagtcta cctcgaacgc 1860
atctacgatg tcagtttcta gtaaagtaac aaaaccgatc ccccgcagtg aatcctgctc 1920
ccagcccgtg atgaatggca aatccaagct gaactccagc gtgctggtgc cctatggcgc 1980
cgagtcctct gaggactctg acgaggagtc aaaggggctg ggcaaggaga atgggattgg 2040
tacgattgtg agctcccact ctcccggcca agatgccgaa gatgaggagg ccactccgca 2100
cgagcttcaa gaacccatga ccctaaacgg tgctaatagt gcagacagcg acagtgaccc 2160
gaaagaaaac ggcctagcgc ctgatggtgc cagctgccaa ggccagcctg ccctgcactc 2220
agaaaatccc tttgctaagg caaacggtct tcctggaaag ttgatgcctg ctcctttgct 2280
gtCtCtCCCa gaagacaaaa tcttagagac cttcaggctt agcaacaaac tgaaaggctc 2340
gacggatgaa atgagtgcac ctggagcaga gaggggccct cccgaggacc gcgacgccga 2400
gCCtCagCCt ggCagCCCCg ccgccgaatc cctggaggag ccagatgcgg ccgccggcct 2460
cagcagcacc aagaaggctc cgccgccccg cgatcccggc acccccgcta ccaaagaagg 2520
cgcctgggag gccatggccg tcgcccccga ggagcctccg cccagcgccg gcgaggacat 2580
cgtgggggac acagcacccc ctgacctgtg tgatcccggg agcttaacag gcgatgcgag 2640
cccgttgtcc caggacgcaa aggggatgat cgcggagggc ccgcgggact cggcgttggc 2700
ggaagccccg gaagggttga gtccggctcc gcctgcgcgg tcggaggagc cctgcgagca 2760
gccactcctt gttcacccca gcggggacca cgcccgggac gctcaggacc catcccagag 2820
cttgggcgca cccgaggccg cagagcggcc gccagctcct gtgctggaca tggccccggc 2880
cggtcacccg gaaggggacg ctgagcctag ccccggcgag agggtcgagg acgccgcggc 2940
gccgaaagcc ccaggccctt ccccagcgaa ggagaaaatc ggcagcctca gaaaggtgga 3000
32/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
ccgaggccac taccgcagcc ggagagagcg ctcgtccagc ggggagcccg ccagagagag 3060
caggagcaag actgagggcc accgtcaccg gcggcgccgc acctgccccc gggagcgcga 3120
ccgccaggac cgccacgccc cggagcacca ccccggccac ggcgacaggc tcagccctgg 3180
cgagcgccgc tctctgggca ggtgcagtca ccaccactcc cgacaccgga gcggggtgga 3240
gctggactgg gtcagacacc actacaccga gggcgagcgt ggctggggcc gggagaagtt 3300
ctaccccgac aggccgcgct gggacaggtg ccggtactac catgacaggt acgccctgta 3360
cgctgcccgg gactggaagc ccttccacgg cggccgcgag cacgagcggg ccgggctgca 3420
cgagcggccg cacaaggacc acaaccgggg ccgtaggggc tgcgagccgg cccgggagag 3480
ggagcggcac cgccccagca gcccccgcgc aggcgcgccc cacgccctcg ccccgcaccc 3540
cgaccgcttc tcccacgaca gaactgcact tgtagccgga gacaactgta acctctctga 3600
tcggtttcac gaacacgaaa atggaaagtc ccggaaacgg agacacgaca gtgtggagaa 3660
cagtgacagt catgttgaaa agaaagcccg gaggagcgaa cagaaggatc ctctagaaga 3720
gcctaaagca aagaagcaca aaaaatcaaa gaagaaaaag aaatccaaag acaaacaccg 3780
agaccgcgac tccaggcatc agcaggactc agacctctca gcagcgtgct ctgacgctga 384
cctccacaga cacaaaaaaa aagaagaaga aaaagaagag acattcaaga aaatcagagg 3900
actttgttaa agattcagaa ctgcacttac ccagggtcac cagcttggag actgtcgccc 3960
agttccggag agcccagggt ggctttcctc tctctggtgg cccgcctctg gaaggcgtcg 4020
gacctttccg tgagaaaacg aaacacttac ggatggaaag cagggatgac aggtgtcgtc 4080
tctttgagta tggccagggt gattgaaaac tcagcctcaa aacaaaaaat tcactagtta 4140
tgattcaacg cgttcaacag aagccatccc cagcccagct taaattataa agatagacaa 4200
taactctgtt ccaatctgcg tggtgcttct ttagtaaata ctgtacagat tttaccatgg 4260
agaacttttt ttttagtttt taccttttct taattaccct tattccgaat ggacgaacac 4320
tttctaccac tgctgaccat tgtaaaatac cgtgtatata aatcccattg aaataatgcc 4380
ctggaataga acatctcaaa tgctgcttaa ttacagactc aggtcgatta cttgtatttc 4440
atgtaatgtt cctccaagtt agacatctgg tgcaagacca accgggagac catggaattg 4500
tcaaaagtac aaactgacag tgtgtatatt taatttaaag acttatttaa aaactcacaa 4560
gctctcacct agactttgga gagcagtctg ttttctgtaa tgtctgatac tagaaactaa 4620
tttgcttatt ttagttgtat tcaagatttg aagatgtat 4659
<210> 23
<211> 3711
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte TD No: 7484378CB1
<400> 24
atggagccca ctgtggctga cgtacacctc gtgcccagga caaccaagga agtccccgct 60
ctggatgccg cgtgctgtcg agcggccagc attggcgtgg tggccaccag ccttgtcgtc 120
ctcaccctgg gagtcctttt gggaggaatg aacaactcca gacacgctgc cttaagagct 180
gcaacactcc ctgggaaggt ctacagcgtc actcctgaag caagcaagac cacgaaccca 240
ccagaaggaa gaaattccga acacatccga acatcagcaa gaacaaactc cggacacacc 300
atctttaaga aatgtaacac tcagcccttc ctctctacac agggcttcca cgtggaccac 360
acggccgagc tgcggggaat ccggtggacc agcagtttgc ggcgggagac ctcggactat 420
caccgcacgc tgacgcccac cctggaggca ctgctgcact ttctgctgcg acccctccag 480
acgctgagcc tgggcctgga ggaggagcta ttgcagcgag ggatccgggc aaggctgcgg 540
gagcacggca tctccctggc tgcctatggc acaattgtgt cggctgagct cacagggaga 600
cataagggac ccttggcaga aagagacttc aaatcaggcc gctgtccagg gaactccttt 660
tcctgcggga acagccagtg tgtgaccaag gtgaacccgg agtgtgacga ccaggaggac 720
tgctccgatg ggtccgacga ggcgcactgc gagtgtggct tgcagcctgc ctggaggatg 780
gccggcagga tcgtgggcgg catggaagca tccccggggg agtttccgtg gcaagccagc 840
cttcgagaga acaaggagca cttctgtggg gccgccatca tcaacgccag gtggctggtg 900
tctgctgctc actgcttcaa tgagttccaa gacccgacga agtgggtggc ctacgtgggt 960
gcgacctacc tcagcggctc ggaggccagc accgtgcggg cccaggtggt ccagatcgtc 1020
aagcaccccc tgtacaacgc ggacacggcc gactttgacg tggctgtgct ggagctgacc 1080
agccctctgc ctttcggccg gcacatccag cccgtgtgcc tcccggctgc cacacacatc 1140
ttcccaccca gcaagaagtg cctgatctca ggctggggct acctcaagga ggacttccgt 1200
aagcatcttc ctcggcctgc aatggtcaag ccagaggtgc tgcagaaagc cactgtggag 1260
ctgctggacc aggcactgtg tgccagcttg tacggccatt cactcactga caggatggtg 1320
tgcgctggct acctggacgg gaaggtggac tcctgccagg gtgactcagg aggacccctg 1380
gtctgcgagg agccctctgg ccggttcttt ctggctggca tcgtgagctg gggaatcggg 1440
33/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
tgtgcggaag cccggcgtcc aggggtctat gcccgagtca ccaggctacg tgactggatc 1500
ctggaggcca ccaccaaagc cagcatgcct CtggCCCCCa CCatggCtCC tgcccctgcc 1560
gcccccagca cagcctggcc caccagtcct gagagccctg tggtcagcac ccccaccaaa 1620
tcgatgcagg ccctcagtac cgtgcctctt gactgggtca ccgttcctaa gctacaagaa 1680
tgtggggcca ggcctgcaat ggagaagccc acccgggtcg tgggcgggtt cggagctgcc 1740
tccggggagg tgccctggca ggtcagcctg aaggaagggt cccggcactt ctgcggagca 1800
actgtggtgg gggaccgctg gctgctgtct gccgcccact gcttcaacca cacgaaggtg 1860
gagcaggttc gggcccacct gggcactgcg tccctcctgg gcctgggcgg gagcccggtg 1920
aagatcgggc tgcggcgggt agtgctgcac cccctctaca accctggcat cctggacttc 1980
gacctggctg tcctggagct ggccagcccc ctggccttca acaaatacat ccagcctgtc 2040
tgcctgcccc tggccatcca gaagttccct gtgggccgga agtgcatgat ctccggatgg 2100
ggaaatacgc aggaaggaaa tgccaccaag cccgagctcc tgcagaaggc gtccgtgggc 2160
atcatagacc agaaaacctg tagtgtgctc tacaacttct ccctcacaga ccgcatgatc 2220
tgcgcaggct tcctggaagg caaagtcgac tcctgccagg gtgactctgg gggccccctg 2280
gcctgcgagg aggcccctgg cgtgttttat ctggcaggga tcgtgagctg gggtattggc 2340
tgcgctcagg ttaagaagcc gggcgtgtac acgcgcatca ccaggctaaa gggctggatc 2400
ctggagatca tgtcctccca gccccttccc atgtctcccc cctcgaccac aaggatgctg 2460
gccaccacca gccccaggac gacagctggc ctcacagtcc cgggggccac acccagcaga 2520
cccacccctg gggctgccag cagggtgacg ggccaacctg ccaactcaac cttatctgcc 2580
gtgagcacca ctgctagggg acagacgcca tttccagacg ccccggaggc caccacacac 2640
acccagctac cagactgtgg cctggcgccg gccgcgctca ccaggattgt gggcggcagc 2700
gcagcgggcc gtggggagtg gccgtggcag gtgagcctgt ggctgcggcg ccgggaacac 2760
cgttgcgggg ccgtgctggt ggcagagagg tggctgctgt cggcggcgca ctgcttcgac 2820
gtctacgggg accccaagca gtgggcggcc ttcctaggca cgccgttcct gagcggcgcg 2880
gaggggcagc tggagcgcgt ggcgcgcatc tacaagcacc cgttctacaa tctctacacg 2940
ctcgactacg acgtggcgct gctggagctg gcggggccgg tgcgtcgcag ccgcctggtg 3000
cgtcccatct gcctgcccga gcccgcgccg cgacccccgg acggcacgcg ctgcgtcatc 3060
accggctggg gctcggtgcg cgaaggaggc tccatggcgc ggcagctgca gaaggcggcc 3120
gtgcgcctcc tcagcgagca gacctgccgc cgcttctacc cagtgcagat cagcagccgc 3180
atgctgtgtg ccggcttccc gcagggtggc gtggacagct gctcgggtga cgctggggga 3240
cccctggcct gcagggagcc ctctggacgg tgggtgctaa ctggggtcac tagctggggc 3300
tatggctgtg gccggcccca cttcccaggt gtctataccc gggtggcagc tgtgagaggc 3360
tggataggac agcacatcca ggagtgacca ccacgtgact gcccaggccg agactctacg 3420
tgaaagcaac aggagcagca ggccacccaa caccccacgc gccaccgtac cctacccaag 3480
gacgggtgtg ggggggctgt gggtcatggg gatgcatctt tgggtaccac cctttagttc 3540
caataaacac agcccctcca ccctagctca ctggctcagc acctcagtgt cacagcgaag 3600
gaccacatgc atggtgctcc accaggaccc ggggtggcac taaggggaaa gatggacttc 3660
tcccaaccca ggggaggctg agaccctccg agctggggtt ccagggacac g 3711
<210> 24
<211> 2017
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte 2D No: 7473143CB1
<400> 25
tgcagtgcaa gagtgtggca gatacaaagg acagaaacag gcaggatttg gctggaaagc 60
tggtggatat gaagctggga ggtcactaag ggccaggcca tgtggaaagc cttgtatact 120
ttaattttcc ttattgaaga gcaaggagga gccattgaat gtttggggca tttgggaggt 180
tggcatgacc tcaccacctt ctgcgtgcag tgtgaagaac agattggcaa ggaccagagc 240
aaatgtggct gaccagttag gagttaatac ggcagtttag gaatgagctt ggtgtagggt 300
ggggacagac ggagatagag atagagtggt agattagcca tggggttgtg aagaagagga 360
agcttctagg tgagccttac ttagataaag agatggaggc atgattccat tcactgagtt 420
ggggggtagg caacagaaga ggagagagtg ggtgggggga catcgagagc atcccaaagg 480
ggtgatgggc ctggcccaca gagggatggc tggcctggat catgacgttg tgagtaacca 540
atgcacaagt gggaagtccc ccaaatcgga gagaggagca gaggccttgg cacggagact 600
gaaaggaggc agagaaagag caggagcagg aaaggagtat ggtattgtgg gaggaagctc 660
agggcattgc tgctcaaagt gtggtcccac agagggcatc atcacatcac cagggagcat 720
ggtgggaagg cagtccctcc agctccaccc cggtgtcgat ctgaatctcc atttgagaca 780
gattccccag gtgatgcgtg tgcacagcca gaactgcacg ttccaactgc acggtcccaa 840
34/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
tgggacagtt gagagcccag ggttcccata tggctacccc aattacgcca actgcacgtg 900
gaccatcacc gcggaagagc agcacagaat ccagcttgtg ttccagtcct ttgccctgga 960
agaggacttt gatgtcctgt cggtgtttga tggtccaccc cagccagaga atctgcgtac 1020
gaggctcaca ggctttcagc tgccagccac cattgttagt gcagccacca ccctctctct 1080
gcgcctcatc agcgactatg cagtcagtgc ccaaggcttc cacgccacct atgaagttct 1140
ccccagccac acatgtggga acccagggag gctgcccaat ggcatccagc agggttcaac 1200
cttcaacctc ggtgacaagg tccgctacag ctgcaacctt ggcttcttcc tggagggcca 1260
cgccgtgctc acctgccacg ctggctctga gaacagcgcc acgtgggact tccccctgcc 1320
ttcctgcaga gctgatgatg cctgtggtgg gaccctgcgg ggccagagtg gcatcatctc 1380
cagcccccac ttcccctcgg agtaccataa caatgccgac tgcacatgga ccatcctggc 1440
tgagctgggg gacaccatcg ccctggtgtt tattgacttc cagctggagg atggttacga 1500
ctttctggaa gtcactggga cagaaggctc ctccctctgg ttcaccggag ccagcctccc 1560
agcccccgtt atcagcagca agaactggct gcgactgcac ttcacatcgg atggcaacca 1620
ccggcagcgc ggattcagtg cccaatacca agtcaagaag caaattgagt tgaagtctcg 1680
aggtgtgaag ctgatgccca gcaaagacaa cagccagaag acgtctgtgt gtttccacct 1740
cactcctcgt gcctgtctat ctttgtcatc tctgttgccg tgtgtctaaa tcctattagc 1800
tcagaaggtc catgttcgat gccacctctt ccaggcagcc tcacatgcgg gtgcatcctt 1860
catccctccc cactgtggtc ccacagtccg cttccgtggt ttatgtcctc actcaactgg 1920
aaactccttg aggacagtgg tcttatctga ctacctttcg catttccatg gtatccaaat 1980
aaagccttgt acacagtaaa aaaaaaaaaa aaaaaaa 2017
<210> 25
<211> 2646
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4382838CB1
<400> 26
tccttctgga tgttgtggtc agaaagagta cggccatctt acagctgcat tgccaataat 60
aatgtgggaa accctgcaaa aaagtccacc aacatcattg tgagagcatt aaaaaaagga 120
cgattttgga tcacaccaga tccttatcac aaagatgaca acatccagat tggccgtgag 180
gtgaaaatat cttgccaagt agaagctgtt ccttctgagg aggtaacatt tagttggttt 240
aaaaatggtc gtccattaag aagttctgag cggatggtca ttacacagac tgatcctgat 300
gtctctccgg gaacaacaaa cttggacatc attgatttaa aattcacgga ttttgggacg 360
tacacatgtg tagcatctct gaagggagga ggaatatctg atatcagtat cgatgttaat 420
atatccagca gcacagttcc acccaatctg actgttccac aggaaaaatc accattggtc 480
accagagaag gagacacaat agaactgcaa tgtcaagtaa ctggcaaacc taaaccaatc 540
atcctttggt ctagagcgga taaagaagtt gcaatgcctg atggatcaat gcaaatggag 600
agttatgatg gaacactgag gattgtgaat gtatctaggg aaatgtcagg aatgtacaga 660
tgtcagacca gccaatacaa tggatttaac gtgaaaccaa gggaagcctt ggtgcagctc 720
atcgttcagt atccccctgc agtggaacca gcattcttgg aaatccggca aggacaggat 780
cgaagtgtca ctatgagttg cagagtactg agagcctatc caatacgggt gctgacctat 840
gagtggcgct tgggcaataa attattacgg acgggtcaat ttgactctca ggaatacaca 900
gagtacgctg tgaagagtct ttccaatgaa aactatgggg tttataactg tagcatcata 960
aatgaagctg gagctgggag atgcagcttt cttgttacag gaaaggccta tgctccagaa 1020
ttctattatg atacctacaa tccagtatgg cagaacagac accgtgttta ttcttacagt 1080
ctacagtgga cacagatgaa tcctgatgca gtggatcgga ttgttgcata ccggttgggc 1140
atcaggcagg ctggacagca gcgctggtgg gagcaggaga ttaaaataaa tgggaatatt 1200
caaaagggag aattaattac atataacttg acagagctaa ttaaaccaga agcttatgaa 1260
gtccgactga ctcctctcac caaatttggt gaaggagatt caacaattcg ggtgatcaaa 1320
tatagtgctc ctgtaaatcc tcatttgaga gaatttcatc gtggatttga agatggtaat 1380
atttgtttgt tcactcaaga tgatacagat aattttgact ggacaaagca aagtacagca 1440
acaagaaata caaaatatac tcctaataca ggacctaatg ctgaccgtag tggctccaaa 1500
gaaggttttt atatgtacat tgagacatca cgacccagat tggaaggcga aaaggctcga 1560
cttcccagcc ctgttttcag catagctccc aaaaaccctt atggacccac aaacactgca 1620
tattgtttca gcttctttta tcacatgtat ggacaacata taggtgtctt aaatgtttat 1680
ctacgtttga aagggcaaac aacaatagag aatccactgt ggtcttcaag tgggaataaa 1740
ggacaaagat ggaatgaggc tcatgttaat atatacccaa ttacttcatt tcagctcatt 1800
tttgaaggta tccgaggtcc tggaatagaa ggtgacattg ctattgatga tgtatcaatt 1860
gcagaaggag aatgtgcaaa acaagaccta gcaactaaga attccgttga tggtgctgtt 1920
35/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
gggattttgg ttcatatatg gctttttccc attatcgtcc tcatctctat cttaagtcct 1980
cgaaggtgac cttatcctgg cagaggctat aaaagattca ccaggcactg gcatgaagaa 2040
agagtctttg taaatggaca ttgaacaaac aaactaccaa agattcctcc actgactact 2100
gactcaaaaa taaaataata aaaacaaatt tttttaagcg ctggggataa aaagacatca 2160
tggaagtata acttattcca gactaaacat aaaagataat cttgacctga gtagagaaga 2220
gaccttcagg tgcttttgtg gctaaaaaga ttacagcgtc atctggttga actctggaaa 2280
aaaaaaaaaa aaaatgaaaa aaagaaaaaa aaaagagcta tagaaatcct tgtcaaagca 2340
caaagtcatg gctggttttg tttcaaatga atagtttgct tgttaccatg gaaacctaat 2400
ggcctgccaa caaaaacctc actgtaaaca gggtacgtga agagctggca tttattttcc 2460
ttacgagaag gttttcgtag agaattaaat aaatgtaggc ccttttacct ttggctgtta 2520
cccttccttg aaaataaacc cgacttcgat ttttttaaag cttcctgttt tttacccacc 2580
tttttcccca tccccccctt attattatta ttattaatac cctggggtaa ggttgagtaa 2640
cataac 2646
<210> 26
<211> 2088
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6717888CB1
<400> 27
atgggacctg cctgggtcca ggaccccttg acaggtgctc tctggctgcc tgtcctctgg 60
gcactcttgt cccaggtcta ttgttttcat gacccaccag gatggcgctt cacttcctca 120
gaaattgtga tccccaggaa agtgccccac aggaggggtg gagttgagat gccagaccag 180
ctctcttaca gcatgcattt ccggggccaa agacacgtga ttcacatgaa gctcaagaag 240
aacatgatgc ccagacattt acctgttttt actaataatg accaaggggc catgcaggag 300
aactaccctt ttgtcccacg agactgttac tacgattgct acctggaagg ggttcctggg 360
tctgtggcca cattggacac ctgccgtgga ggtctgcgtg gcatgctgca ggtggatgac 420
ctgacttatg aaatcaaacc cctggaggct ttttccaaat ttgagtatgt agtatctctg 480
cttgtgtcag aagaaagacc aggagaggtc agtagatgta agactgaagg ggaagagata 540
gatcaagaat ctgaaaaggt aaaactggct gaaactccca gagaaggcca cgtttatttg 600
tggaggcatc atagaaaaaa cttgaaactt cactacacag ttactaatgg attattcatg 660
cagaacccta atatgtcaca cataatagag aatgtagtga ttattaacag catcatacat 720
accattttca aaccagttta tttaaatgtc tatgtacgtg ttttgtgcat atggaatgat 780
atggatatag taatgtataa catgcctgcc gacctggttg taggagagtt tggttcgtgg 840
aaatattatg aatggttttc acaaattcca catgatacct cagttgtttt tacatcaaat 900
cgacttggaa acactcctcg ttgtggagac aagatcaaaa atcagaggga agaatgtgac 960
tgtggctccc ttaaagattg tgccagtgat agatgttgtg agacctcttg taccctttct 1020
cttggcagtg tttgcaatac aggactttgc tgccataagt gtaaatatgc tgcccctgga 1080
gtggtttgca gagacttggg tggtatatgt gatctaccgg aatactgtga tgggaaaaag 1140
gaagagtgtc caaatgacat ctacatccag gatggaaccc catgttcagc agtatctgtt 1200
tgtataagag gaaactgcag tgaccgtgat atgcagtgtc aagccctttt tggctaccaa 1260
gtgaaagacg gttccccagc gtgctatcga aaattgaata ggattggtaa ccgatttgga 1320
aactgtgggg ttattctacg gcgaggggga agtagacctt ttccatgtga agaagatgat 1380
gttttttgtg gaatgttgca ctgtagccgt gtcagccaca ttcccggtgg aggtgagcac 1440
actacatttt gtaatatatt agtacacgac ataaaagaag aaaaatgctt tggctatgaa 1500
gcacaccagg ggacagactt gccagaaatg gggctggtag tggatggtgc aacctgtggc 1560
ccagggagct actgtcttaa acgcaattgt actttttatc aagacetgca ttttgagtgt 1620
gatcttaaaa catgcaatta caaaggagta tgtaacaaca aaaaacattg tcattgtctg 1680
catgagtggc aaccaccaac atgtgaactg agaggaaaag gaggtagtat agatagtggc 1740
cctctacctg acaaacaata tcgtattgca ggcagcatac ttgtaaatac aaaccgagca 1800
ctagttttaa tatgtattcg ttacatcctt tttgtggttt cgcttctctt tggtggcttt 1860
tcacaagcaa tacaatgtta gggaagagaa aggaaaagag cccacacatg gagtaaatta 1920
cattgacact tactgggaga tataatcaat agtcactctg acaattacat catcttttag 1980
caattctgat gtcatcttga aataaaatcc cttggcaatt taaaaaggtc tgtgtgttta 2040
aatttactta acatttcatg tctggtcaca ttctcaatac ttctatag 2088
<210> 27
<211> 1890
<212> DNA
36/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472044CB1
<400> 28
atgctgctgg ctgtgctgct gctgctaccc ctcccaagct catggtttgc ccacgggcac 60
ccactgtaca cacgcctgcc ccccagcgcc ctgcaagtct tcactctcct cttgggggca 120
gagactgtgt tgggccgcaa cctagactac gtttgtgaag ggccgtgcgg cgagaggcgt 180
ccgagcactg ccaatgtgac gcgggcccac ggccgcatcg tggggggcag cgcggcgccg 240
cccggggcct ggccctggct ggtgaggctg cagctcggcg ggcagcctct gtgcggcggc 300
gtcctggtag cggcctcctg ggtgctcacg gcagcgcact gctttgtagg ctgccgctcg 360
acccgcagcg ccccgaatga gcttctgtgg actgtgacgc tggcagaggg gtcccggggg 420
gagcaagcgg aggaggtgcc agtgaaccgc atcctgcccc accccaagtt tgacccgcgg 480
accttccaca acgacctggc cctggtgcag ctgtggacgc cggtgagccc ggggggatcg 540
gcgcgccccg tgtgcctgcc ccaggagccc caggagcccc ctgccggaac cgcctgcgcc 600
atcgcgggct ggggcgccct cttcgaagac gggcctgagg ctgaagcagt gagagaggcc 660
CgtgttCCCC tgctcagcac cgacacctgc cgaagagccc tggggcccgg gctgcgcccc 720
agcaccatgc tctgcgccgg gtacctggcg gggggcgttg actcgtgcca gggtgactcg 780
ggaggccccc tgacctgttc tgagcctggc ccccgcccta gagaggtcct gttcggagtc 840
acctcctggg gggacggctg cggggagcca gggaagcccg gggtctacac ccgcgtggca 900
gtgttcaagg actggctcca ggagcagatg agcgcctcct cctccagccg cgagcccagc 960
tgcagggagc ttctggcctg ggaccccccc caggagctgc aggcagacgc cgcccggctc 1020
tgcgccttct atgcccgcct gtgcccgggg tcccagggcg cctgtgcgcg cctggcgcac 1080
cagcagtgcc tgcagcgccg gcggcgatgc gagctgcgct cgctggcgca cacgctgctg 1140
ggcctgctgc ggaacgcgca ggagctgctc gggcctcgtc cgggactgcg gcgcctggcc 1200
cccgccctgg ctctccccgc tccagcgctc agggagtctc ctctgcaccc cgcccgggag 1260
ctgcggcttc actcaggctg ccctgggctg gagcccctgc gacagaagtt ggctgccctg 1320
cagggggccc atgcctggat cctgcaggtc ccctcggagc acctggccat gaactttcat 1380
gaggtcctgg cagatctggg ctccaagaca ctgaccgggc ttttcagagc ctgggtgcgg 1440
gcaggcttgg ggggccggca tgtggccttc agcggcctgg tgggcctgga gccggccaca 1500
ctggctcgca gcctcccccg gctgctggtg caggccctgc aggccttccg cgtggctgcc 1560
ctggcagaag gggagcccga gggaccctgg atggatgtag ggcaggggcc cgggctggag 1620
aggaaggggc accacccact caaccctcag gtaccccccg ccaggcaacc ctgagccatg 1680
tctgggcccc cagcccctgg ggaggaccta ctgctcccag gggctgagag gggttcggga 1740
gcataatgac aaactgtcgc tgccccagtg gctgggtgtg tgtgggtggg atggggtggg 1800
ggtcctgggc cccccgtgtc ttcccaggtt tacaatcaga gaatcacagc tgctttaata 1860
aatgttattt ataataaaaa aaaaaaaaaa 1890
<210> 28
<211> 2984
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477384CB1
<400> 29
agtgaagacg actgtcttat ctgactgtag gcacagagag tgcgccgcga gagggcggct 60
cctcaccgtc aggcgccggc aggtcgcgtt ctctgctggc cgacgcccga aggcgccgaa 120
tgggggggcc ctgccgagct cccttacagc cccaatgtgc gcgccgccgg gaggcttggg 180
cacgcaggca ccgccggcgg ggggcggggc gaaggcggcg gggcggggca ccagctgcgc 240
gcgcggggcg ggggcggggg cgggggcggg gcgcgctgcg tggtcccggc cggccctggg 300
ctcctccccc tcccgcgccc aggccagcgg cgggcccagc tcctcccccg actcggtctc 360
tCtCCCCtCC CCtCCgCCCg gCagttCCtC CCtCCCgCCg ccgcctcttc ctcggtgagg 420
cgctcttcca gcgggcaggc agcatggcgg ccgtggagac gcgggtgtgc gagacagacg 480
gctgcagcag tgaggccaag ctccagtgtc ccacttgcat caagctgggc atccagggct 540
cgtacttctg ctcgcaggaa tgttttaaag gaagttgggc tactcacaag ttactacata 600
agaaagcaaa agatgaaaag gcgaagcgag aagtgtcttc ctggactgtg gaaggtgata 660
ttaatactga cccatgggca ggttatcgat atactggtaa actcagacca cattatccac 720
tgatgccaac aaggccagtg ccaagttata ttcaaagacc agattatgct gatcatccct 780
37/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
taggaatgtc tgaatctgaa caggctctta aaggtacttc tcagattaaa ttactctcat 840
ctgaagatat agaagggatg cgacttgtat gtaggcttgc tagagaagtt ttggatgttg 900
ctgccggcat gattaaacca ggtgtaacta ctgaagaaat agatcacgct gtacacttag 960
catgtattgc aagaaattgc tacccttctc ccctgaatta ttataatttc ccaaagtctt 1020
gttgtacctc agtgaatgaa gtcatttgcc atggaatacc agacagaagg cccttacaag 1080
aaggtgacat tgttaatgtg gatatcactc tttatcgcaa tggttatcat ggggacctga 1140
atgagacatt ttttgttgga gaagtggatg atggagcacg gaaacttgtt cagaccacat 1200
atgagtgcct gatgcaagcc attgatgcag tgaagcctgg tgttcggtac agagaattgg 1260
gaaacattat ccagaagcat gcccaagcaa atgggttttc agttgttcga agctattgtg 1320
ggcatggaat ccacaagctt tttcatacag ctcccaatgt accccactat gctaaaaata 1380
aagcagttgg agtgatgaag tcgggccatg tatttacaat tgagccaatg atttgtgaag 1440
gcggatggca ggatgaaacc tggccagatg gttggactgc ggtgacaaga gacggaaagc 1500
ggtctgctca gtttgagcac accctcctgg tcacagacac tggctgtgaa atcctaaccc 1560
ggcgacttga cagtgcacgg cctcacttca tgtctcaatt ttaatttctc ccaagatggc 1620
acatctcagt accttcttac tgtgctatgc attttattga gagtacagaa aggaagagga 1680
accttttttt aatcacttgt tttgttttga ctatagataa gaaaggacta cagcatttga 1740
tgtgtgtcct caagaacttg tcttgggtct gaaaaagctg agaagaataa aggaaacatt 1800
gctcaactct tcagccccct ccccctgcac acctgttttc tcatttgccc tttgagcact 1860
tttacttaaa cttgcttgta gttgctttta tcactgccgc aaaacagcca tcaagagcca 1920
tctgctttcc aggtgaacat tggaaatgag aatctttgaa acttagcaat atgtgttgca 1980
ccagattttt taaattatat atatggaaat atatatgtat acattttaag ttctgtatac 2040
ataattacca aacactatgt gacctggagt ttgtgttgtt tctgctctga caggtttata 2100
tgttcttaca aatggatcca tagtttgcag tgatttaatt cctggttggg atttggcctc 2160
ccctctcccc catgctaatt atttaccctt gtaattgtgc atagggaagc actcacccaa 2220
tgagactttc tccaatgtgg actctgtgtg tcagtgaatg aatgtagtaa aattcacttt 2280
ggaaggttat caggctttta aaaatctagt ttatggcaaa aatagccatt ttccaagtgg 2340
tggctgactg ttgcagggaa tgagaatttc ataatacact gctatttcag acctctgttt 2400
ggtcagaaat ggaaaagaaa aagccccctt tcttcccttt tctgttttac ttcaagggca 2460
taccttggag gtgctcagag aagcgtgaag tttgcactat ggtggaggat ggggaaagag 2520
ttctaaagtg tctccagctg tgaacccagg aggtcaagtg ggctattaaa atctaacgtt 2580
gagtaaatgt gatagtgatg agaaaggaat tttgtgtact gtaaccttgc agtagagatg 2640
cagctgtcct tcgtgtgtgg aaacacacct ctcctttaca tagttgggaa cctcattaga 2700
aatgacctca gctgccccat atctacgttc ctttcagcag ttgtccaagt aggagtgtat 2760
ccagtgaaga catatcaaat cacaaagtca ttgtcattag agtgtacttg attactgggc 2820
atccttgtaa tataatttca taccactgac acattatact tgtaagagaa catctttccc 2880
agagtgcctc agaccttatt gctttaaaat ataataatgt tttcattact tttattattt 2940
gaatgattta gtaaagttga ctgaatctgg tatagacttt ggga 2984
<210> 29
<211> 2255
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7077175CB1
<400> 31
ccacagtgtg gatgcccctt gaggatgtca cactcatgag acgccagaca caaaacgcca 60
cacagtgtgt aatcccattt ccatgaaatg tccaggtcag gccagtgcac agacacaggc 120
agcgggtgtg tgggcagcgg ggctggagaa ggggacgggg agtgaccgct gagggggaca 180
ggcttctttt agtggggatg aacgttctaa aattggacac attggtggtg gcacagctgt 240
ggagatatga aaacgcgaaa cctacgggtg agctgggtga accttatgag gcgggaatta 300
actgctccgg ctctggcgct gaggaaaagg aggaccggag gatggcgatc atctgggccg 360
tgccctccac atctgtgtcc tgggaacaga cttctagaaa aacccaaatc aggaaaaagc 420
ggccagctcc acgctgcaaa cagctgggca ccaggcagag agtgttacca gtggtcaagc 480
cagaggtgct gcagaaagcc actgtggagc tgctggacca ggcactgtgt gccagcttgt 540
acggccattc actcactgac aggatggtgt gcgctggcta cctggacggg aaggtggact 600
cctgccaggg tgactcagga ggacccctgg tctgcgagga gccctctggc cggttctttc 660
tggctggcat cgtgagctgg ggaatcgggt gtgcggaagc ccggcgtcca ggggtctatg 720
cccgagtcac caggctacgt gactggatcc tggaggccac caccaaagcc agcatgcctc 780
tggcccccac catggctcct gcccctgccg cccccagcac agcctggccc accagtcctg 840
agagccctgt ggtcagcacc cccaccaaat cgatgcaggc cctcagtacc gtgcctcttg 900
38/39
CA 02425829 2003-04-08
WO 02/38744 PCT/USO1/51034
actgggtcac cgttcctaag ctacaagaat gtggggccag gcctgcaatg gagaagccca 960
cccgggtcgt gggcgggttc ggagctgcct ccggggaggt gccctggcag gtcagcctga 1020
aggaagggtc ccggcacttc tgcggagcaa ctgtggcggg ggaccgctgg ctgctgtctg 1080
ccgcccactg cttcaaccac acgaaggtgg agcaggttcg ggcccacctg ggcactgcgt 1140
ccctcctggg cctgggcggg agcccggtga agatcgggct gcggcgggta gtgctgcacc 1200
ccctctacaa ccctggcatc ctggacttcg acctggctgt cctggagctg gccagccccc 1260
tggccttcaa caaatacatc cagcctgtct gcctgcccct ggccatccag aagttccctg 1320
tgggccggaa gtgcatgatc tccggatggg gaaatacgca ggaaggaaat gccaccaagc 1380
ccgagctcct gcagaaggcg tccgtgggca tcatagacca gaaaacctgt agtgtgctct 1440
acaacttctc cctcacagac cgcatgatct gcgcaggctt cctggaaggc aaagtcgact 1500
cctgccaggg tgactctggg ggccccctgg cctgcgagga ggcccctggc gtgttttatc 1560
tggcagggat cgtgagctgg ggtattggct gcgctcaggt taagaagccg ggcgtgtaca 1620
cgcgcatcac caggctaaag ggctggatcc tggagatcat gtcctcccag ccccttccca 1680
tgtctccccc ctcgaccaca aggatgctgg ccaccaccag ccccaggacg acagctggcc 1740
tcacagtccc gggggccaca cccagcagac ccacccctgg ggctgccagc agggtgacgg 1800
gccaacctgc caactcaacc ttatctgccg tgagcaccac tgctagggga cagacgccat 1860
ttccagacgc cccggaggcc accacacaca cccagctacc aggtaccggg agagacggag 1920
ggatccctgg gagtggaggg tcccatgtta atcagcctgg gctgcctaac aagacataac 1980
gtcgtccact ttgggaggcc gaggcgggcg gatcaagagg tcaggagatc gagaccatcc 2040
tggcgaacac ggtgaaacct tgtctctact aaaaaaatac aaaaaattag ccaggcgtgg 2100
tggtgggcgc ctgtagtccc aactacgcgg gaggctaagg caggagaatg gcatgaagcc 2160
gggaggcgga gcttgcagtg agctgcatgc cactgcactc cagcctggca acaagcgaaa 2220
ctccgtctca aaaaagaaaa agacataacg gcctc 2255
<210> 30
<211> 1250
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7480124CB1
<400> 32
ccgtcatggg cccactcggg ccctctgccc tgggccttct gctgctgctc ctggtggtgg 60
cccctccccg ggtcgcagca ttggtccaca gacagccaga gaaccaggga atctccctaa 120
ctggcagcgt ggcctgtggt cggcccagca tggaggggaa aatcctgggc ggcgtccctg 180
cgcccgagag gaagtggccg tggcaggtca gcgtgcacta cgcaggcctc cacgtctgcg 240
gcggctccat cctcaatgag tactgggtgc tgtcagctgc gcactgcttt cacagggaca 300
agaatatcaa aatctatgac atgtacgtag gcctcgtaaa cctcagggtg gccggcaacc 360
acacccagtg gtatggggtg aacagggtga tcctgcaccc cacatatggg atgtaccacc 420
ccatcggagg tgacgtggcc ctggtgcagc tgaagacccg cattgtgttt tctgagtccg 480
tgctcccggt ttgccttgca actccagaag tgaaccttac cagtgccaat tgctgggcta 540
cgggatgggg actagtctca aaacaaggtg agacctcaga cgagctgcag gaggtgcagc 600
tcccgctgat cctggagccc tggtgccacc tgctctacgg acacatgtcc tacatcatgc 660
ccgacatgct gtgtgctggg gacatcctga atgctaagac cgtgtgtgag ggcgactccg 720
ggggcccact tgtctgtgaa ttcaaccgca gctggttgca gattggaatt gtgagctggg 780
gccgaggctg ctccaaccct ctgtaccctg gagtgtatgc cagtgtttcc tatttctcaa 840
aatggatatg tgataacata gaaatcacgc ccactcctgc tcagccagcc cctgctctct 900
ctccagctct ggggcccact ctcagcgtcc taatggccat gctggctggc tggtcagtgc 960
tgtgaggtca ggatacccac tctaggattc tcatggctgc acaccctgcc ccagcccagc 1020
tgcctccaga cccctaagca tctcctgtcc tggcctctct gaagcagaca agggccacct 1080
atcccggggg tggatgctga gtccaggagg tgatgagcaa gtgtacaaaa gaaaaaaggg 1140
aagggggaga ggggctggtc agggagaacc cagcttgggc agagtgcacc tgagatttga 1200
taagatcatt aaatatttac aaagcaaaaa aaaaaaaaaa aaaaaaattg 1250
39/39
